DBT-HRD

Indian Council of Agricultural Research Ministry of Science and Technology

Central Marine Fisheries Research Institute Department of Biotechnology

CMFRI Training Manual Series No.15/2018

Training Manual
In the frame work of the project:
DBT sponsored Three Months National Training in

Molecular Biology and Biotechnology

for Fisheries Professionals 2015-18
Training Manual
In the frame work of the project:
DBT sponsored Three Months National Training in

Molecular Biology and Biotechnology

for Fisheries Professionals 2015-18
 Training Manual
This is a limited edition of the CMFRI Training Manual provided to
participants of the “DBT sponsored Three Months National Training
in Molecular Biology and Biotechnology for Fisheries Professionals”
organized by the Marine Biotechnology Division of Central Marine
Fisheries Research Institute (CMFRI), from 2nd February 2015 - 31st
March 2018.

Principal Investigator
Dr. P. Vijayagopal

Compiled & Edited by

Dr. P. Vijayagopal
Dr. Reynold Peter
Central Marine Fisheries Research Institute

Assisted by
Aditya Prabhakar
Swetha Dhamodharan P V

ISBN 978-93-82263-24-1
CMFRI Training Manual Series No.15/2018

Published by
Dr A Gopalakrishnan
Director, Central Marine Fisheries Research Institute (ICAR-CMFRI)
PB.No:1603, Ernakulam North P.O, Kochi-682018, India.

2
Foreword

Central Marine Fisheries Research Institute (CMFRI), Kochi along with CIFE, Mumbai and CIFA, Bhubaneswar
within the Indian Council of Agricultural Research (ICAR) and Department of Biotechnology of Government
of India organized a series of training programs entitled “DBT sponsored Three Months National Training in
Molecular Biology and Biotechnology for Fisheries Professionals”. The scope of this training is to promote
development of trained human resource for application of molecular tools to research problems in fisheries
and aquaculture, to help them adapt to such facilities and work programs and to include analyses that comply
with worldwide regulatory acts in the field of biotechnology.

At present, mostly traditional methods are being used in the fisheries sector and only a few researchers are
applying molecular methodologies. The emphasis of this training program is to enhance the capabilities of
research personnel already employed in fisheries Institutes, Universities and Colleges, but working in the area
of marine biology/fisheries/aquaculture. The contents of this training course are intended to teach molecular

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

techniques to laboratory personnel with a good level of analytical knowledge, but with no or little expertise
in this specific domain.

The 3 months training program comprises of theory classes with hands-on practical sessions and research
work. All basic molecular biology, genetic engineering and molecular genetics techniques are included in
the course along with their applications in various aspects of aquaculture and fisheries. Each institute offers
specialized modules based on areas of expertise. The course content at CMFRI is designed to cover all kinds of
applications of molecular methods in fisheries. The emphasis is on hands-on experience and skill development
of the participants. Technical details were provided to trainees as oral presentations and brief written outlines.
Aware of the need for a permanent source of information, the Marine Biotechnology Division of CMFRI
developed this manual as background information for course participants and is intended to provide the
theoretical and practical information on methodologies and protocols currently used, which describes some
of the techniques used in our laboratory.

It is our hope that the structure and content of this manual will help course participants (as well as other users)
in the diffusion and dissemination of the acquired skills in the context of the different working environments
according to needs. This manual aims to complement existing information in the specialised literature.

Dr. P. Vijayagopal supervised the preparation of this manual and the contributors are mentioned in the Table of
contents. A special recognition and acknowledgment to all personnel who, even not individually mentioned,
contributed to the successful preparation of the manual. Thanks are also extended to Dr. Reynold Peter, Ms.
Adithya C. and Ms. Swetha Damodharan P.V. for their support for the preparation of this manual.

Dr. A. Gopalakrishnan
Director, CMFRI

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 Contents

Marine Biology
Introduction to Marine Biodiversity� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 11
K. K. Joshi

Introduction to Mariculture� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 21
G. Gopakumar

Introduction to Marine Aquariculture � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 29

K. Madhu and Rema Madhu

An Overview of the Fish Diversity of Indian Waters � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 35

Rekha J. Nair and S. Dinesh Kumar

Crustacean Diversity � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 67
S. Lakshmi Pillai and G. Maheswarudu

Molluscan Diversity � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 76
Geetha Sasikumar, V. Venkatesan and K. S. Mohamed

Bryozoa – Taxonomy and Diversity � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 92

N. Nandini Menon

Seaweeds and Marine Biotechnology � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 99

P. Kaladharan

Seagrass Diversity � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 104

M. P. Prabhakaran

Molecular Biology
Central Marine Fisheries Research Institute

Standard Operating Procedure (SOP)� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 109

M. P. Paulton

Principles of Isolation, Purification and Analysis of Nucleic Acids� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 113

M. P. Paulton

Polymerase Chain Reaction and its various modifications� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 116

P. C. Thomas

Quantitative Genetics� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 129

V. Srinivasa Raghavan

Marker Assisted Selection � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 135

V. Srinivasa Raghavan

4
Cryopreservation of fish spermatozoa and its Application in Aquaculture and Conservation� � � � � � � � � � � � � 144
V. S. Basheer and A. Gopalakrishnan

Cytogenetics and its applications in fishes � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 148

Basdeo Kushwaha, Ravindra Kumar and N. S. Nagpure

Molecular Taxonomy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 155

Reynold Peter

An overview of the basic concepts and principles of Population Genetics � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 160

N. S. Jeena

Protein Isolation and purification by different chromatographic techniques. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 167

M. A. Pradeep and Esha Arshad

Genes as Molecular Guardians in Environment Management and Aquaculture � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 170

M. P. Paulton

Molecular Systematics � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 174

Sandhya Sukumaran

The science of ‘omics’ – Genomics, Proteomics and Metabolomics � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 180

M. A. Pradeep, S. R. Krupesha Sharma and Esha Arshad

Functional Genomics� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 190

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

M. P. Paulton

Population Genomics of Fishes � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 193

Sandhya Sukumaran

Next Generation Sequencing and RAD sequencing � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 198

Sandhya Sukumaran

Software Packages used in Population Genetics � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 202

Sandhya Sukumaran, N. S. Jeena, Reynold Peter and Wilson Sebastian

General Methods of Tissue Culture� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 206

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In vitro Culture of Finfish Cells – Principle and its Applications� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 213

T. Raja Swaminathan and V. S. Basheer

Methods for examination of Cell Culture � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 220

V. Srinivasa Raghavan

Tissue Culture–Marine Invertebrates � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 224

C. P. Suja

Molecular markers in Population Genetics� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 228

K. A. Sajeela

Recombinant DNA technology and Molecular Cloning � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 233

Reynold Peter

5
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Fish Health Management � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 239
N. K. Sanil and K. K. Vijayan

Livestock Disease Surveillance� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 245

M. R. Gajendragad

Disease Diagnostic Techniques in Aquaculture� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 252

K. V. Rajendran

Diseases in Fish Hatcheries � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 259

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Autopsy Procedure in Fish � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 265

S. R. Krupesha Sharma and N. K. Sanil

Bacterial Diseases of Marine Fish and Shellfish� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 267

S. R. Krupesha Sharma, M. A. Pradeep and N. K. Sanil

Viral Diseases of Marine Fish and Shellfish� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 274

S. R. Krupesha Sharma, M. A. Pradeep and N. K. Sanil

Microbiological Staining Techniques� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 278

S. R. Krupesha Sharma and M.A. Pradeep

Histopathology � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 280
N.K. Sanil
Electron Microscopy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 286
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Fish Immunological Techniques � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 291

K. J. Reshma

Hybridoma Technology and its Use in Disease Diagnosis and Therapy � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 298
K. Pani Prasad

Antibiotic Susceptibility Test -Applications in Fisheries Science� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

T. G. Sumithra

Immunization of Fish: A Tool for Aquaculture Health Management� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 305

Central Marine Fisheries Research Institute

T. G. Sumithra

Polyphasic Taxonomy as a Consensus Methodology for Bacterial Identification� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 308

Anusree V. Nair

Marine Chemistry
Laboratory- Safety and Hazards� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 315
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General Biochemical Methodologies � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 328

Kajal Chakraborty

Introduction to Marine Bio-prospecting� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 335

Kajal Chakraborty

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Marine Organisms –Treasure House of Valuable Products and their Chemical Perspectives � � � � � � � � � � � � � � � 353
I. Rajendran and P. Vijayagopal

Classification of Organic Compounds with Reference to Natural Products� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 361

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Physical and Chemical Methods for Structural Elucidation and Identification of Organic
Compounds � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 366
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General Methods of Isolation Procedures and Separation Methods for Organic Compounds � � � � � � � � � � � � 371
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Minipreparation of Plasmid DNA by Alkaline Lysis with SDS � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 498
Restriction Enzyme digestion � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 501
Ligation � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 503
Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis (SDS PAGE)� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 504
Tricine–SDS Poly Acrylamide Gel Electrophoresis (Tricine–SDS-PAGE) � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 509
Central Marine Fisheries Research Institute

Separation of DNA in Polyacrylamide Gels � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 511

Visualization of DNA in Polyacrylamide gels using silver staining � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 513
Production of Antibodies� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 514
Author Index � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 515

8
Marine Biology
Introduction to Marine Biodiversity
K. K. Joshi
Principal Scientist & Head
Marine Biodiversity Division, CMFRI, Kochi
e-mail:[email protected]

Introduction and amenity services. Provisioning services means

The Convention on Biological Diversity (CBD) defined the products obtained from ecosystem in the form
biodiversity as being the variability among living of food, natural products, fuel wood, medicines,
organisms from all sources including, among others, genetic and ornamental resources, energy resources,
terrestrial, marine and other aquatic ecosystems and and product from bioprospecting. Regulating services
the ecological complexes of which they are part; this include the shoreline stabilization, flood prevention,
includes diversity within species, between species and storm protection, climate regulation, hydrological
of ecosystems. services, nutrient regulation, carbon sequestration,
detoxification of polluted waters and waste disposal.
An ecosystem is a dynamic complex community Supporting services are mainly the habitat provision,
of plant, animal, microorganism and the non- nutrient cycling, migration, seed dispersal, primary
living environment interacting as a functional unit. productivity and soil formation. Cultural and amenity

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Biological components are crucial in proper ecosystem services include the culture, tourism and recreation.
functioning, which provides essential ecosystem
services to human beings. Ecosystem diversity is the Provisioning services
variation of different biological communities and their
interaction with the biotic and abiotic environment. Food provisioning in the form of fish landings and
aquaculture products is one of the most important
Biological diversity comprises species, genetic and services obtained from the marine ecosystems.
ecosystem diversity. Species diversity is the diversity of Mangroves are important in supporting fisheries
all the species on earth from single celled bacteria and due to their function as fish nurseries. Mangroves
protists to the species of the multicellular kingdom. help to increase fish production in the inshore
Diversity in species shows the variation of species waters near to it. Coral reefs also provide services
due to evolutionary and ecological adaptations like protection of breeders and larvae for the better
of the species to the entire geographical range. survival and recruitment success of the resources. They
Genetic diversity is the variation within species due form an important source of fisheries products for
to geographical separation and intraspecific variation coastal residents and export markets. The coral reefs
within the population. of the Gulf of Mannar, Andaman Nicobar Islands,
Lakshadweep Islands and Gulf of Kutch contribute
Ecosystem services from Marine substantially to the total marine finfish catch of India.
Other ecosystems like rocky intertidal, near shore
and Coastal Ecosystems
mudflats, seagrass beds, mud bank areas, seamounts,
Marine ecosystems provide a wide variety of services brackish water, lagoons, estuaries, marshy areas and
to nature which is essential for the well-being of beaches also helps in the production of fish as food
the human population. The ecosystem services in one way or another.
are classified into four i.e. provisioning services,
regulating services, supporting services and cultural The total marine fish landings from India were

11
 estimated at 3.63 million tonnes during 2016. share about 3.0 lakh tonnes which forms about
Fisheries sector plays an important role in the Indian 64% of the total value realized. Increased export
Economy, contributing about 1% to the national demand often leads to expansion of mariculture
GDP. The sector provides livelihood to about 4 practices. Coastal areas provide the foundation for
million fisher folk population along the coastal the marketers which produce fisheries products
line of 8129 Km. The value of total marine fish from prawn, crab and fish. The factors affecting
landings at landing center level was estimated at Rs. the marine fish production are the overexploitation,
48381 crore during 2016. Since 1950 the marine species extinctions and use of destructive methods
fish production in India has gradually increased of fishing. The proportion of marine fish stock s that
from mere 5.8 lakh tonnes (1950) to 3.63 million are over exploited and depleted are increasing over
tonnes (2016) showing six fold increase. History of the last 30 years. It is reported that 133 extinctions
the development of the Indian fisheries sector has of regional and global marine species occurred over
gone through three phases. First phase (>1965) the last 30 years. The major cause of the extinction
is characterized by non-mechanized indigenous was overexploitation (55%) and rest of habitat loss
crafts and gears and the landings remained and other reasons.
below one million tonnes during this phase.
Second phase (1965-1985) showed an increase Bio prospecting is the exploration of biodiversity for
in the mechanization of crafts, use of advanced new biological resources of social and economic value.
gear materials, introduction of motorization of It yielded several products from species in marine
traditional crafts, and expansion of export trade. and coastal ecosystems. Coral reefs are important
Third phase (1986-2015) is featured intensification reservoirs of natural bioactive products many of which
of mechanization, motorization of traditional exhibit structural features not found in the terrestrial
crafts, multi-day and multi-gear stay over fishing natural products. The pharmaceutical industry has
and introduction of deep sea fishing. A total of discovered several potentially useful substances
about 2000 marine species is caught from the among sponges, jellyfish and Mollusca.
Indian seas. They are categorized into 29 resource
groups. Out of the 26 groups studied by CMFRI, Regulating services
20 were found to be under the abundant category,
four under less abundant category and one each Regulating services are the benefits people obtained
under declining, depleted and collapsed category. from the regulation of ecosystem processes, including
Elasmobranchs, threadfins, ribbonfishes, mullets air quality maintenance, erosion control, regulation
and flatfishes are the four resource groups falling of human diseases and water purification. The
under less abundant category. Big-jawed jumper mangroves, sea grass, coral reefs, rocky intertidal,
falls under declining category, flying fishes under mudflats, and deltas play key role in shoreline
depleted category and unicorn cod is the one that stabilization, protection from storms, floods and soil
Central Marine Fisheries Research Institute

falls under collapsed category (Sathianandan et al., erosion, processing pollutants and stabilizing land
2011). Many of the marine and coastal ecosystems in the event sea level rise. Mangroves have great
provide coastal communities with construction capacity to absorb heavy metals and other toxic
materials and building materials from the mining substances, coral reefs buffer land from storms and
of coral reefs. Mangroves provide coastal and prevent beach erosion. Estuaries, lagoons, marshes,
Island community with building materials for brackish water areas play a key role in maintaining
boat construction. hydrological balance and filtering water of pollutants.
Marine ecosystems play significant roles in climate
According to Marine Products Export Development regulation. CO2 is continuously exchanged between
Authority export of marine products from India the atmosphere and ocean; it dissolves in surface
reached an all-time high of Rs. 30213 crores for waters and is then transported to the deep ocean.
quantity of 9.8 lakh tonnes (2014). Among the Marine plants fix CO2 during photosynthesis in the
products exported, shrimp product formed the major ocean and return it via respiration.

12
Supporting Services value. Stretches of beach, rocky cliffs, estuarine and
coastal marine waterway are the places where people
Many species use coastal areas like estuaries, make frequent visits for sightseeing and recreation.
mangroves, sea grasses as nurseries. Estuaries are Some species are of considerable cultural importance,
particularly important as nursery areas for fisheries for example cultural significance of Turbinella pyrum
and other species and they form one of the strongest (Sacred Chank) as it is used temples and bangle
linkages between coastal, marine and freshwater industry. The sea shores are also of great spiritual
ecosystems and the ecosystem services they provide. importance as so many temples are situated along the
The success of the prawn fishery mainly depends coast. Marine and coastal ecosystems are areas that
on the migration of prawns through the estuary. received attention through research. Rocky intertidal
Mangroves provide nursery for many species as habitats main focus of research and provided some
well as give links to sea grass beds with associated of the foundation principles of ecology.
coral reefs. Decline in the area of mangroves can
interrupt these linkages and cause biodiversity loss Status of Marine Resources
which results in lower productivity from the reef
and sea grass beds. The ecosystem goods and services provided by
the fauna and flora and the interrelationship
Sea grasses are important in providing nursery between the biodiversity and ecological processes
areas and it provides habitat for coral reef fishes are the fundamental issues in the sustainability
and invertebrates. It is an important source of food and the equilibrium of the ecosystem. Ecosystem
for many species of coastal and marine species. Sea goods from the marine realm included the fin
horses, sea cow, and turtles are the species associated fishes, crustaceans, mollucans and seaweeds.
with sea grass beds. Kelp beds and other Macro The important flora and fauna falling to the two

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

algae provide nursery habitat for some species. They major kingdoms such as Animal and Plant kingdom
support many fish species and invertebrates like sea recorded from the Indian region and their present
urchins. Estuaries provide a range of habitats to status are discussed below.
sustain diverse flora and fauna. There are many
more estuarine dependent species than estuarine Kingdom: Plantae
resident species. Mudflats are also critical habitat for
migrating shorebirds and many marine organisms Mangroves: Mangroves trees up to medium size and
including the commercially important clam species. shrubs that grow in saline coastal sediment habitats
Sea mounts forms another important habitat in the tropics and subtropics. Asia has the largest
provides nursery to several species of fishes. All of amount (42%) of the world’s mangroves (Kathiresan
the these ecosystems the beaches, sandy shores, and Rajendran, 2005).
dune systems, salt marshes, estuaries, and mudflats
provide feeding and nesting habitats to numerous Seagrasses: Sea grasses are flowering plants from
species of birds, fishes, molluscs, crustaceans, and one of four plant families (Posidoniaceae, Zosteraceae,
other organisms. Hydrocharitaceae, or Cymodoceaceae), all in the order
Alismatales grow in marine fully saline environments.
Cultural and Amenity Services A total of 14 species of sea grasses in six genera
is reported from Indian seas (Venkataraman and
Cultural services include tourism and recreation, Wafer, 2005).
aesthetic and spiritual services, traditional knowledge
and education and research services. Most important Macro algae (Sea weeds): Sea weeds are large
cultural services provided by coastal and marine multicellular plants that resemble vascular plants
ecosystems are tourism and recreation. Natural but lack the complex array of tissues used for
amenities are highly valued by people and contribute reproduction and water transport. They are found in
to human welfare, thus providing significant economic red (Rhodophyta), green (Chlorophyta) and brown

13
 (Phaeophyta) divisions. A total of 1010 species of macro cannot survive outside the colony. The Portuguese
algae has been reported from India. They are most Man of War (Physalia physalis) and Crambionella
abundant along the Gujarat, Kerala and TamilNadu stulhamani are important jelly fish species. About
coasts and around the Andaman and Lakshadweep 116 species of hydrozoans belonging to 13 families
Islands. A large number of seaweed species known have been reported from India.
from the Indian seas are edible and serve various
industrial purposes. The edible seaweeds are known Class: Anthozoa
to be rich in protein (20 to 25%) Carbohydrates (16
to 24%), lipids (6 to 11%) vitamins and amino acids. Octocorallia (Soft corals): Octocorallia is belonging
to the subclass of Anthozoa. It includes the blue
Kingdom: Animalia coral, soft corals, sea pens and gorgonians (sea fans
and sea whips) within three orders: Alcyonacea,
Helioporacea, and Pennatulacea. Their life cycle
Phylum: Porifera (Sponges) includes a motile phase as plankton and later a
Sponges are multicellular organisms which have sessile phase. About 300 species of soft corals have
bodies have pores and channels, allowing water been reported from India. Gorgonids are abundant
to circulate through them, consisting of jellylike in the Gulf of Mannar and distributed almost all
mesohyl sandwiched between two thin layers of along the Indian coasts including Andaman Sea.
cells. About 519 species of sponges are known to About 22 species belonging to 7 families and 15
occur in the Indian seas. About 34 species of coral genera were reported from India (Thomas, 1996
boring sponges have been reported from the Gulf of b). The biomedical versatility of the gorgonids,
Mannar and Island system of India (Thomas, 1996 popularly known as the sea fans, attracted great
a).Sponges are the major components of the benthic attention to this resource. The four species which
fauna and are distributed from the intertidal to the have already shown symptoms of depletion include
hadal depths and are a potential source of many new Echinomuricea indica, Heterogorgia flabellum,
bioactive compounds. In India out knowledge of the Echinogorgia complexa and Gorgonella umbraculum
identity, biology, availability, population structure and (Thomas and Ranimary George, 1987).
possibilities of commercial exploitation of sponges is
meagre and requires prioritization. Ceriantharia (Tube – dwelling anemones):
Tube-dwelling anemones, which are similar to sea
Ctenophora (Comb jellies): Ctenophora are live in anemones, but belong to the subclass of anthozoans.
marine waters and distinctive feature is the groups They are solitary, living buried in soft sediments.
of cilia (comb) they use for swimming. They are the Tube anemones live and can withdraw into tubes,
largest animals that swim by means of cilia. A total of which are made of a fibrous material, which is made
20 species of comb jellies has been reported from India. from secreted mucus and threads of nematocyst
Central Marine Fisheries Research Institute

like organelles known as ptychocysts. The diversity

Phylum: Cnidaria included about 20 species in India.

Class: Scyphozoa (True jellyfish) Actiniaria (Sea anemones): The Actiniaria belongs
Scyphozoa is referred as the true jellyfish. Their to the class Anthozoa which includes the true sea
stings may cause skin rashes, muscle cramps, or even anemones. They are water-dwelling, predatory
death. A total of 30 species of Scyphozoans has been animals. They have large polyps that allow for
reported from India. digestion of larger prey and also lack a medusa stage.
They are related to corals, jellyfish, tube-dwelling
Class: Hydrozoa (Jelly fish) anemones, and Hydra. Sea anemone Heteractis
Hydrozoans are small, predatory animals, some solitary magnifica Quoy and Gairmad, 1833 is associated
and some colonial, and marine. The colonies are large, with clown fish. Actiniarian diversity included about
and in some cases the specialized individual animals 30 species in India.

14
Corallimorpharia (Coral anemones): Phylum: Echiura (Spoon worms)
Corallimorpharia is closely related to the true sea
anemones (Actiniaria). The tentacles are usually short The Echiura are a small group of marine animals.
and arranged in rows radiating from the mouth. They lack the segmented structure found in other
They resemble the stony corals, except for the Annelids of this group. Recent studies show they
absence of a stony skeleton. They occur in a wide may be included in the phylum Annelida. About
range of marine habitats, and are associated with 43 species under 14 genera have been reported
phase shifts in coral reefs that change from hard- from India.
coral dominated to soft-coral dominated. Diversity
of Corallimorpharia includes about 10 species along Phylum: Sipuncula (Peanut worms)
the Indian coast.
The Sipuncula are bilaterally symmetrical worms
Zoanthidea (Mat anemones): Zoanthids are and contains about 144-320 species. They live in
commonly found in coral reefs, the deep sea and shallow waters, either in burrows or in discarded
many other marine environments around the world. as molluscan shells. Some bore into solid rocks to
They may be in the form individual polyps, attached make a shelter for themselves. About 35 species under
by a fleshy stolon or a mat that can be created from 10 genera have been reported from India. They are
small pieces of sediment, sand and rock. A total of 8 concentrated mainly along the Andaman and Nicobar
species of Zoanthids has been reported from India. Islands, Lakshadweep Islands, Gulf of Mannar and
Gulf of Kutch.
Scleractinia (Reef building corals): Scleractinia are
marine corals that form a hard skeleton. Most of the Phylum: Annelida
modern coral reefs are formed by scleractinians. About

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

200 species Scleractinia from the diversity of India Class: Polychaeta (Clam worms)
(Pillai, 1996).
The Polychaeta are generally marine, and belong
Antipatharia (Black corals): Black corals are tree- to phylum Annelida. The body has a pair of fleshy
like corals related to sea anemones and found in protrusions called parapodia that bear many bristles,
deeper depths. There are about 230 known species called chaetae, which are made of chitin. The annelid
of black corals in 42 genera of this 10 species occur worm diversity includes about 300 species in India
in India (Pillai, 1996). Though black coral's living (Venkataraman and Wafer, 2005).
tissue is brilliantly coloured, it takes its name from the
distinctive black or dark brown colour of its skeleton. Class: Clitella

Phylum: Platyhelminthes Oligochaeta (Earthworms): The Oligochaeta is

different types of aquatic and terrestrial worms.
(Flat worms)
Earthworms are semi aquatic or fully aquatic. There
Platyhelminthes are bilaterally symmetrical, are several interstitial marine worms. About 10 species
unsegmented, soft-bodied invertebrate worms. reported from India.
They don’t have body cavity, and circulatory and
respiratory systems, which made them to in a Phylum: Nemertea (Ribbon worms)
flattened shape. The digestive cavity has only one
opening for both the ingestion and evection as a Nemertea (Nemertini, Nemertinea, Rhynchocoela) is
result; the food cannot be processed continuously. a group of invertebrate animals and known as
About 100 species of flatworms have been reported ribbon worms or proboscis worms. They have an
from India. Research on the Platy helminthes of unsegmented body, thin and elongated with no
India is less as compared to the Annelids of India differentiated head. Ribbon worm diversity includes
(Venkataraman and Wafer, 2005). about 60 species in India.

15
 Phylum: Arthopoda Order: Stomatopoda (Mantis shrimp)

Subphylum: Chelicerata Stomatopods are marine crustaceans and they

occur in a variety of different colours, from
Class: Merostomata
shades of browns to bright neon colours. These
Horseshoe crab: Horseshoe crab belonging to aggressive and typically solitary sea creatures spend
the family Limulidae and known as Living Fossil most of their time hiding in rock formations or
because their origin is 450 million years ago. The burrowing indicate passageways in the sea bed.
marine king crab of the species Tachypleus gigas and Unlike most crustaceans, stomatopods hunt, chase,
Carcnoscorpius rotundicauda occurs in the deltaic and kill their prey.  Most species live in tropical
regions of Ganges and Mahanadi along the northeast and subtropical seas, although some live in
coast. They are considered as living fossil and hence temperate seas. The stomatopod diversity includes
care should be taken to preserve them in the nature. about 30 species along the Indian coast.
Recently because of biotic interference there has been
a decline in the numbers of these animals in Orissa. Order: Decapoda
The chemical reagent lysat is produced from the blood
of this crab. This medicine has got a wide usage in Dendrobranchiata (Shrimp, prawns): Dendro-
the treatment of several diseases. branchiata are decapod crustaceans, known as
shrimp or prawns. There are 540 extant species in
Subphylum: Crustacea seven families. They differ from related animals, such
as Caridea and Stenopodidea, from the branching
India is endowed with rich diversity of crustaceans form of the gills and by the fact that they do not
and several of them supporting commercial fisheries brood their eggs, but release them directly into the
since ancient times. water. They are widely fished and farmed for human
consumption. About 10 species have contributed to
Class: Maxillopoda the diversity in India.
Cirripedia (Barnacles): They have a calcareous shell
composed of several pieces. They are known as curl Caridea (Caridean shrimp): The Caridean shrimp is
footed because of their curved legs. A total of 36 an infraorder of shrimp within the order Decapoda.
species of cirripedia have been reported from India. They are found widely around the world in both
fresh and salt water. Carideans are found in every
Class: Malacostraca kind of aquatic habitat, with the majority of species
Order: Amphipoda (Land hoppers) being marine. About 150 specimens included in the
caridean shrimp diversity of India.
Amphipoda is have no carapace and generally with
Central Marine Fisheries Research Institute

laterally compressed bodies. Amphipods range Palinura (Lobsters): Lobsters have a cylindrical, sub
in size from 1 to 340 millimeters and are mostly ovoid or dorso-ventrally compressed carapace and
detritivorus or scavengers. They live in marine aquatic flattened abdomen. The group includes the spiny
environments. A total of 132 species of Amphipods lobsters and slipper lobsters. The abdomen is flattened.
belonging to 54 genera has been reported from India.
Thalassinidea (Ghost shrimps, mud shrimps):
Order: Isopoda (Pill bugs, sow bugs) Thalassinidea include crustaceans, which live in burrows
in muddy bottoms of the sea. Thalassinids typically live
The Isopoda are small crustaceans with seven in deep and sometimes complex burrows. Shallow
pairs of legs in the size of above 300 micrometers. water local species typically remain deep in the burrow
They have dorso-ventrally, flattened body, without and suspension feed (filtering plankton and organic
carapace. There are about 33 species belonging to particles from the water) by beating their pleopods to
13 genera have been reported from India. create a current. About 20 species of Thalassinides has

16
been reported from the Indian Ocean. human consumption and some species are used in
aquaculture systems. Sea cucumbers serve a useful
Anomura (Hermit crabs, sand crabs): Anomura purpose in the marine ecosystem as they help
is a group of decapod crustaceans, including hermit recycle nutrients, breaking down detritus and other
crabs and others. All true crabs are in the sister group organic matter after which bacteria can continue
to the Anomura. A total of 20 species Anomuran the degradation process. About 150 species of sea
crabs has been reported from India. cucumber have been reported from India. About 12
species of sea cucumber are economically important
Brachyura (Crabs): Crabs are decapod crustaceans and have commercial value (James, 1996).
with a typically very short tail, usually entirely hidden
under the thorax. A total of 250 species of crabs has Class: Asteriodea (Starfish)
been reported from Indian coast.
Starfish is among the most familiar and diverse group
Phylum: Mollusca of marine invertebrates. They have a central disc and
five arms, and some species have more than five arms.
Molluscs in general had a tremendous impact on The ochre sea star (Pisaster ochraceus) and the reef
Indian tradition and economy and were popular sea star (Stichaster australis) are widely known as
among the common man as ornaments, currency, examples of the keystone species concept in ecology.
as a part of spiritual activities even at the inception A total of 180 species of starfishes belonging to 81
of human culture and civilization. A total of 3271 genera have been reported from India.
species of molluscs distributed among 220 families
and 591 genera, of which 1900 are gastropods, 1100 Class: Ophiuroidea (Brittle stars)
bivalves, 210 cephalopods, 41 polyplacophora and

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

20 scaphopods. Among these 8 species of oysters, 2 They have a disk and generally have five long, slender,
species of mussels, 17 species of clams, 3 species of whip-like arms which may reach up to 60 centimeters
pearl oysters, 3 species of giant clams, 1 species of in length on the largest specimens. A total of 150
windowpane oyster and gastropods such as Sacred species of brittle stars belonging to 79 genera have
Chank, Trochus, Turbo and 15 species of Cephalopods been reported from India.
are exploited from the Marine sector of India. The
species like Cassis cornuta, Charonia tritonis, Conus Class: Crinoidea (Sea lilies)
milneedwardsi, Cypraecassis rufa, Nautilus pompilius,
Hippopus hippopus, Tridacna maxima, Tridacna They live both in shallow water and in depths up
squamosa etc. are the some of the molluscs protected to 6,000 meters. Sea lilies in their adult form are
under the Wildlife (Protection) Act, 1972 Schedule I. attached to the sea bottom by a stalk. They have
a mouth on the top surface that is surrounded by
Phylum: Echinodermata feeding arms.  Crinoids usually have a stem used to
attach them to a substrate, but many live attached
Class: Echinoidea (Sea urchin) only as juveniles and become free-swimming as
adults. A total of 95 species belonging to 43 genera
Sea urchins move slowly, feeding mostly on algae. Sea have been reported from India.
otters, wolf eels, triggerfish, and other predators feed
on them. A total of 60 species of sea urchins was Phylum: Phoronida
showed occurrence in the Indian seas.
(Horseshoe worms)
Class: Holothuroidea (Sea cucumbers) They live in most of the oceans and seas, including the
Total number of holothurians species is 1,250 in Arctic Ocean but excluding the Antarctic Ocean, and
the world and maximum number being in the between the intertidal zone and about 400 meters down.
Asia Pacific region. Many of these are utilized for About 5 species of phoronids were reported from India.

17
 Phylum: Brachiopoda (Lamp shells) Phylum: Chordata
Class: Thaliacea (Pelagic tunicates)
They have hard shells on the upper and lower
surfaces, unlike the left and right arrangement Thaliaceans are free-floating for their entire lifespan.
in bivalve molluscs. There are two types are They include both solitary and colonial species.
recognized, articulate and inarticulate. Articulate Thaliaceans have 30% carbon by mass. Therefore,
brachiopods have toothed hinges and simple opening their dense bodies sink to the bottom of the oceans
and closing muscles, while inarticulate brachiopods when they die and this may be a major part of the
have untoothed hinges and a more complex system of worldwide carbon cycle. A total of 40 species was
muscles used to keep the two halves aligned. About reported from India.
5 species of Brachiopods were found in India.
Class: Ascidiacea (Sea squirts)
Phylum: Bryozoa (Moss animals)
Ascidians are found all over the world, usually
They are known as Polyzoa, Ectoprocta or moss in shallow water with salinities over 2.5% the
animals are aquatic invertebrate  animals. Size range members of the Thaliacea and Larvacea swim
from 0.5 millimeters long, and are filter feeders. Over freely like plankton, sea squirts are sessile animals.
4,000 living species are known. One genus is solitary A total of 50 species belonging 21 genera have
and the rest colonial. There is a rich biodiversity in India been reported from India against 2000 species of
with about 500 species are reported so far. Several Asidian in the world.
collections and descriptions in the past, enriched the
knowledge about the Bryozoans occurring along the Class: Pisces
Indian coast (Venkataraman and Wafer, 2005). Elasmobranchs: The elasmobranchs consists of
sharks, sawfishes, rays, skates and guitar fishes.
Phylum: Hemichordata The protected elasmobranchs as per the Wildlife
(Protection) Act, 1972, Schedule I are Rhincodon typus
(Acorn worms)
(Whale shark), Anoxyprisits cuspidate (Pointed saw
Acorn worms are solitary live in burrows and are fish), Prisitis microdon (Large tooth sawfish), Prisitis
deposit feeders, and species are filter feeders. About zijsron (Long comb sawfish), Carcharhinus hemiodon
12 species of hemichordates have been reported (Pondicherry shark), Glyphis gangeticus (Ganges shark),
from India as compared to global species of 102. Glyphis glyphis (Speer tooth shark), Himantura fluviatilis
The balanoglossus, Phychodera fluva is a unique living (Gangetic sting ray), Rhyncobatus djiddensis (Giant
fossil that links vertebrates and invertebrates occurs guitarfish) and Urogymnus asperimus (Thorny ray).
in the Gulf of Mannar area of India.
Ornamental fish: The Gulf of Mannar, Palk bay, Gulf
Central Marine Fisheries Research Institute

Phylum: Chaetognatha of Kutch, South West coast and the Lakshadweep

and Andaman group of Islands are known to be
(Arrow worms)
rich in Ornamental fishery. The Wrasses, damsel fish,
Arrow worms are predatory marine worms that Surgeon, Butterfly fish, Moorish idol, Squirrel fish,
form a major component of plankton worldwide. Trigger fish, Rabbit fish, Parrot fish, Angels, Goat
About 20% of species are benthic, and can attach fish and Puffer fish are the major aquarium fishes
to algae and rocks. They range in size from 2 to 120 represented by about 180 species (Murty et al., 1989;
millimeters. A total of 30 species have been reported Murty, 2002. CITES have listed all the sea horse in
from India. They are abundant all along the Indian the Appendix I to stop the trade of these organisms.
coast. Extensive studies along the Malabar Coast, Indian wild Life Act 2002 also protects the sea horse
Vishakhapatnam Coast, Andhra coast revealed by putting them in Schedule list I. Dried sea horse
the presence of 30 species occurring in India has got a high demand in Singapore and China for
(Venkataraman and Wafer, 2005). making soup and for medicinal purposes.

18
Class: Reptilia Order: Crocodilia
Marine reptiles: Marine reptiles are air-breathing,
ectothermic, poikilothermic vertebrates. Their skin is Salt water crocodiles: Salt water crocodile, Crocodylus
covered with dry scales and lays their egg on land. porosus (Schneider, 1801) is the largest reptile in the
Out of the 700 living species only few species of present world with about six meter lengths and up
snakes, turtles, and crocodiles are seen in the ocean. to one metric ton weight. They can live in saltwater,
but usually occurs in mangrove swamps, estuaries,
Order: Chelonia deltas, lagoons and lower stretches of rivers. The
species is endangered due to hunting, loss of habitat
Sea Turtles: Five species of sea turtles were reported and breeding sites. Marsh crocodile or Indian swamp
in India which include, Olive Ridley (Lepidochelys crocodile is Crocodylus palustris (Lesson, 1831) found
olivacea) Green Turtle (Chelonia mydas), Leather back in rivers, swamps, lakes and saltwater lagoon. Indian
(Dermocheylus olivacea), Hawksbill (Eretmocheylus Gharial is Garialis gangeticus (Gmelin, 1789) are found
imbricate) and Logger head (Caretta caretta). Green mainly in river Ganga, Brahma putra and Mahanadi.
turtles (Chelone mydas) are found in coastal water
and feed mainly as sea grasses and sea weeds. The Class: Aves
hawksbill turtle (Erectmochelys imbricata) feed on
encrusting animals like sponges, sea quirts, barnacles Sea birds: Sea birds are long lived, with very low
and sea weeds. The largest sea turtle the leather back natural mortality. These biological traits and human
(Dermocheylus coriacea) have a series of shells and induced adult mortality potentially damaging
an oceanic species. They have scissor-like jaws for population decreases and collapse of the population.
capturing and they feed on jelly fish. Other species Ecosystem services that birds provide are mainly
feed on soft, bottom invertebrates like sponges, indirect and supporting services. It includes trophic

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

soft corals, jelly fishes and crabs. Prey-predator effects, seed dispersal, nutrient cycling, aesthetics,
relationship in the ecosystem is one of the important and recreation. Also most of the tropical sea bird
factors in limiting as well as proliferation of organisms species feed in association with tuna stocks which
due to the decline of the one of the components derive their prey to the surface, thereby bringing
in the trophic relations (Joshi, 2012). All species of within the reach of the sea birds. The depletion of
marine turtles are in the endangered category, and the tuna stocks could therefore have impacts on
are therefore, protected under the Indian Wildlife their dependent species like sea birds. The cascade
Act, 1972. effects of reduce tuna or shark on the ecosystem are
not known.
Order: Squamata
Common birds are Grey heron (Ardea cinerea),
Sea snakes: Sea snakes occur in the tropical and Pond heron (Ardeola grayii), Large erget (Egretta
sub-tropical waters of the Indian Ocean from the alba), Little erget (E. garzetta), Painted stork (Ibis
east coast of Africa to Australia. They occur in leucocephalus), Spoon bill (Platalea leucocordia),
shallow coastal waters, estuary, lakes and fresh Flamingo (Phoenicopterus roseus), Parian kite (Milvus
water in the rivers away from the sea. They feed nigrans), Golden plover (Pluvuailis dominica), Black
on fish, fish eggs, crustacean and tuna. The genus headed Gull (Larus ridibundus), Gull billed Tern
Laticauda is oviparous and all other sea snakes (Geolchelidon nilotica), Caspian tern (Hydroprogne
are viviparous. The Sea snake bite is dangerous caspia), Little tern (Sterma abilfrons) and Sandwich tern
and it is neurotoxic like terrestrial snakes like krait and (Sterna sandvicensis), Three species of Albatross
and cobra. There are about 80 species, sea snakes are endangered (IUCN) two species near threatened
belonging to three families inhabiting the world and one is critically endangered (IUCN). Sea birds
oceans and estuaries. In Indian waters, about 22 occur along the Gulf of Kutch, Gulf of Mannar, Chilka
species of marine snakes belonging to three families Lake, Coringa Wild life Sanctuary and the Sundarbans,
have been documented. Islands of Laccadive such as Pitti and Batapari are the

19
 colonies of sea birds. Sundarbans is important staging sustainable use of biodiversity not only for the present
and wintering area of gulls and terns. generation but for the future generations. Despite
the huge role of marine ecosystems and organisms
Class: Mammalia very little research has been done to the ecosystem-
species interactions. A thorough knowledge about
Dolphins: The species diversity of dolphins in India the comprehensive taxonomy of the marine organism
is one among the richest in the world. A total of from Phytoplankton to Marine mammals will form the
five species, dolphins was recorded from our seas. basis of such ecosystem-species interaction research.
Important species are Stenella longirostris (Spinner Further, human activities are the major causes for the
dolphin), Sousa chinensis (Humpback dolphin), loss of biodiversity and degradation of marine and
Delphinus delphis (Common dolphin), Tursiops coastal habitats, which needs immediate attention
truncatus (Bottlenose dolphin) and Risso’s dolphin and comprehensive action plan to conserve the
(Grampus griseus). biodiversity for living harmoniously with nature.

Whales: Whales constitute the most dominant groups Suggested readings

of marine mammals. They usually occupy in the
temperate and polar oceanic waters, they migrate James, D.B. 1996. Conservation of Sea cucumbers. In Marine
to tropical waters for breeding and avoid extreme Biodiversity Conservation and Management (Eds : N. G. Menon
and C.S.G. Pillai ), Central Marine Fisheries Research Institute,
climatic conditions during certain seasons. Whales Cochin PP 80-88.
are classified into Odontoceti (toothed whales) and Joshi, K. K. 2012. Marine biodiversity and the conservation of the
important marine organisms. In: Marine biodiversity status,
mysticeti (baleen whales). All the Cetaceans are opportunities and challenges. Ramachandran, A and Joseph,
included in the list of protected animals. A total of Aneykutty, (eds.) CUSAT, Kochi, pp. 35-78.
about 10 species has been reported from the Indian Kathiresan, K. and N. Rajendran. 2005. Mangrove ecosystem of
the Indian Ocean region. Indian Journal of Marine Sciences,
seas. They are Indopacetus pacificus (Longman’s 34 (1) 104 -113.
Beaked whale), Balaenoptera borealis, Balaenoptera Murty, V. Sriramachandra. 2002. Marine ornamental fish resources
of Lakshadweep. CMFRI Special Publication, 72. pp. 1-134.
musculus, Balaenoptera acutorostrata, Pseudorca Pillai, C. S. G. 1996. Coral reefs of India Their conservation and
crassidens, Physeter macrocephalus, Ziphius carvirostris Management in Marine Biodiversity Conservation Management
and Balaenoptera sp. ( Eds. Menon N. G. and C. S. G. Pillai CMFRI, pp. 16-31.
Raje, S. G. and Sivakami, S and Mohanraj, G and Manojkumar,
P. P. and Raju, A and Joshi, K. K. 2007. An atlas on the
Sea Cow: The sea cow, Dugong dugon inhabits in Elasmobranch fishery resources of India. CMFRI, Special
Publication, 95. pp. 1-253.
the Gulf of Mannar and Palk bay area and is included Sathianandan, T .V. Jayasankar, J, Somy Kuriakose, Mini, K. G. and
in the List of protected animals as per the Wildlife Wilson T. Mathew. 2011. Indian marine fishery resources:
(Protection) Act, 1972 Schedule I. optimistic present, challenging future. Indian Journal of
Fisheries, 58 (4). pp. 1-15.
Thomas, P.A. 1996 b. The Gorgonid resources and their
Conclusion Conservation in India. In Marine Biodiversity Conservation
Central Marine Fisheries Research Institute

and Management (Eds: N. G. Menon and C.S.G. Pillai ), Central

Marine Fisheries Research Institute, Cochin PP 32-40.
The marine organisms provide ecosystem services, Thomas, P. A. and Ranimary George. 1987. Gorgonid resources
but are not valued properly or unappreciated. It of India. Marine Fisheries Information Service T& E Series,
74: 1-13.
has necessary to ecosystem services been better Venkataraman, K. and Mohideen Wafar. 2005. Coastal and marine
studied and valued properly for the conservation and biodiversity of India. Indian Journal of Marine Sciences, 34
(1): 57-75.

20
Introduction to Mariculture
G. Gopakumar
Emeritus Scientist
Vizhinjam Research Centre of CMFRI, Thiruvananthapuram
e-mail:[email protected]

It is well accepted the hunger is the most vital issue last 50 years. Estimated additional 20-30 million tons
of mankind. The global population has grown are required to meet demand by 2020. The per capita
from 1.5 billion in 1900 to 7 billion now and is consumption has increased from 11.5kg in 1970 to
expected to reach about 10 billion by the year 2050. 12.5kg in 1980 to 14.4kg in 1990 to 19.2kg in 2012.
It is estimated that about 925 million are under-
nourished with the major chunk at the Asia-Pacific A global review of the marine capture fisheries
Region. Micronutrient deficiencies affect > 2 billion; scenario reveals that 80% of the world’s fish stocks
250 million children at risk of vitamin A deficiency; for which assessment information is available are
equal number suffer from deficiency of minerals reported as fully exploited and thus requiring effective
(iron, zinc, calcium, etc.). Hence food and nutritional and precautionary management. The maximum wild
security assumes highest priority for future prosperity capture fisheries potential from world’s oceans have
of mankind. Food security is not just producing food, almost been exploited and a more closely controlled
but also providing access to food and is linked with approach to fisheries management is required. The

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

poverty and rural development. It is evident that current marine capture fisheries scenario in India is
economic access to food is only when households also characterized by increased and excessive fishing
generate sufficient income. In this context, Fisheries effort, overexploitation of certain resources from
and Aquaculture is a major sector producing not only the inshore grounds and increased conflicts among
food but also generating employment opportunities the different stakeholders in the sector. Due to the
leading to accessibility of food. larger dependency on inshore fisheries over the years,
the production from nearshore waters has reached
Fish is considered as the “Rich food for Poor” and the asymptotic level and hence ensuring sustainability is
cheapest source of animal protein. It provides over inevitable in our marine fisheries policy.
20% of animal protein to 2.6 billion people globally.
In developed countries it contributes 13%, while in Aquaculture the farming and husbandry of aquatic
developing countries > 30% of the animal protein. animals is the promising area for increasing aquatic
Fish forms the major source of animal protein in food production in future years. It is the fastest growing
regions where animal protein in diets is below world food production sector with annual growth of >6% in
average. It provides at least half of animal protein last two decades. It increased from <1 million tonne in
intake for 400 million poor in S. Asia & Africa Fish is 1950 to 55 million tonnes in 2009. About 80% of the
a rich source of protein, essential fatty acids, vitamins production comes from 20 million small-holder farms
and minerals. Some fishes are high in calcium, zinc, (<2ha) in developing countries. The environmental
vitamin A and iron. Globally over 540 million (8% of demands for unit biomass of protein produced are lower
population) are involved in fisheries & aquaculture; as compared to poultry, piggery and beef. Aquaculture
the growth in the sector is more than population & provides primary source of income. Aquaculture can be a
employment in traditional agriculture. The present starting point for alleviation of poverty in rural areas. Due
global production of food fish is estimated as 158 to all these advantages aquaculture has been growing
million tonnes (capture and culture). The demand for rapidly - faster than any other food production sector
fish has increased at twice population growth over - over the past three decades, and is continuing. It is

21
 clear that the bulk of fish required to feed the world in districts of Tamilnadu producing about 17000 tonnes
the coming decades will come from aquaculture. It is also wet weight annually.
time to put “nutrition security” at par with “food security”.
Fish, wherever it comes from, is a global commodity of The Central Marine Fisheries Research Institute
key significance due to its potential to improve human (CMFRI) has been pioneering in the development and
health and nutrition, its accessibility by the poor, and the standardization of several commercially viable coastal
low environmental impact of its production compared aquaculture technologies viz. mussel farming, oyster
to that of other animal source foods. farming, sea cage farming of marine finfish, seaweed
faming, clam culture, pen culture, integrated farming,
Relevance of Mariculture pearl oyster culture and pearl production. Many of these
technologies are very simple, eco-friendly and use only
It is widely accepted that the exploited marine fisheries locally available infrastructure facilities for construction
in India has reached a maximum sustainable level of farm, feed and seed and hence the entire farming
as reflected in the recent total fish production. The can be practiced by traditional fishermen.
exploitation pattern over a long term is indicative
of the fact that the additional demand for marine Brackish water shrimp farming
fish in future years cannot be met from the capture
and the lessons learnt
fisheries. In this context, mariculture – the farming
and husbandry of marine plants and animals in the Brackish water shrimp farming started in a big
marine environment- is a promising sector by which way in India in the early 90s especially in the
the additional marine fish requirement can be met coastal districts of Andhra Pradesh and Tamil
in the future years. It is also the fastest growing sub- Nadu. So far, shrimp remains as the single largest
sector of aquaculture. At global level, mariculture and maximum value earner among the seafood
produces many high value finfish, crustaceans, exported from the country. Shrimp farming in
and molluscs like oysters, mussels, clams, cockles India, till 2008, was synonymous with the mono
and scallops. In 2012 mariculture has contributed culture of tiger shrimp, Penaeus monodon.  Since
around 24.7 million tonnes of foodfish globally which 1995, culture of P. monodon is affected by White
formed about 35.7%of the aquaculture production. Spot Syndrome Virus (WSSV) and the development
(World aquaculture production was 90.4 million of shrimp farming has become stagnant. Most
tonnes in 2012 – contributed 42.2% to the total of the Southeast Asian countries like Thailand,
fish production, supplied 9.4kg of foodfish per Vietnam and Indonesia shifted to culture of the
person). Molluscs dominated the global mariculture exotic white leg shrimp, Litopenaeus vannamei.
production (60.3%) followed by finfish (22.5%), The successful development of Specific Pathogen
crustaceans (15.9%) and others (1.3%). In addition Free (SPF) and Specific Pathogen Resistant (SPR)
about 23.8 tonnes of macro algae and seaweeds broodstock of L. vannamei also favoured the
Central Marine Fisheries Research Institute

were also produced by mariculture. large scale expansion of its farming. However, in
India, pilot-scale introduction of L. vannamei was
Indian scenario of Mariculture initiated in 2003 and after risk analyses large-scale
introduction was permitted in the year 2009. Of late
Research and Development
L. vannamei farming is being threatened by outbreak
In India the potential of mariculture production largely of new diseases namely Early Mortality Syndrome
remains untapped. The mariculture activities are (EMS), Acute Pancreatic and Haematopoietic
confined only to coastal brackish water aquaculture, Necrosis Syndrome (APHNS) and many viral diseases.
chiefly shrimp farming. The other coastal aquaculture The very fact is that these diseases are common to
activities are green mussel farming which is confined many of the shrimp species, the aqua farmers are
to Malabar coast in Kerala producing around 15,000 now desperately looking for an additional species
tonnes and seaweed farming along Ramanathapuram, for farming. Hence, species diversification with
Puthukottai, Tanjore, Tuticorin and Kanyakumari viable finfish can be one of the best options for a

22
long term solution for sustaining the aquaculture Seed Production and Farming of
sector. The major constraints for initiating and Silver pompano
developing marine finfish farming in the country
is the lack of seed production technologies for Among the many high value marine tropical finfish
suitable high value species and the non-availability that could be farmed in India, the silver pompano,
of commercially viable farming techniques. Now, Trachinotus blochii is one of the topmost, mainly due
with the development of indigenous technology to its fast growth rate, good meat quality and high
for seed production and farming of cobia and silver market demand. The silver pompano is caught only
pompano by CMFRI, and seabass by CIBA and RGCA, sporadically in the commercial fishery and hence its
there is great scope for the aqua farmers to diversify availability is rather scarce. It is a much sought after
their aquaculture practices. species and hence the demand can only be met through
aquaculture. The farming can be successfully carried out
Marine Finfish in ponds, tanks and floating sea cages. The species is
pelagic, very active and is able to acclimatize and grow
well even at a lower salinity of about 10 ppt and hence
Seed Production and farming is suitable for farming in the vast low saline waters of
of Cobia our country besides its potential for sea cage farming.
Fast growth rate, adaptability for captive breeding,
low cost of production, good meat quality and high CMFRI has successfully developed and standardised
market demand are some of the attributes that make the broodstock development, induction of spawning,
cobia, Rachycentron canadum an excellent species larviculture and fingerling production of silver
for aquaculture. In recent years the seed production pompano for the first time in India. The first farming
and farming of cobia is rapidly gaining momentum demonstration from the hatchery produced seed was

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

in many Asian countries. Envisaging the prospects of carried out in a coastal aquaculture pond at Anthervedi
cobia farming in India, CMFRI has developed for the Village, East Godavari District, Andhra Pradesh. The
first time in the country the broodstock development, growth performance, survival and productive capacity
breeding and seed production of cobia and several of silver pompano, Trachinotus blochii, were evaluated
successful seed production trials were conducted and in a brackishwater pond. A total of 3,400 fingerlings
the technology is now standardised at its Mandapam of silver pompano (30.59 ± 0.24 mm mean length
Regional Centre. and 2.00 ± 0.04 g mean weight) were stocked into
a one acre pond (0.4047 hectare) with salinity of 8 ±
The farming protocols for the hatchery produced 1.2 ppt. The salinity gradually raised to 24 + 1.8 ppt
cobia fingerlings in sea cages with different feeding during the farming period due to high saline intake
strategies were developed, tested and validated. Based water. Fishes were fed with extruded floating pellet
on the trials, an economically viable farming model feed containing 30% to 50% crude protein and 6 %
has been evolved. This farming method has been to 10 % crude fat. After 240 days culture, 1305kg
adopted by private entrepreneurs, fishermen groups of silver pompano were harvested and the survival
and farmers. In a recent demonstration conducted at rate was 91.32%. The mean length of the harvested
Mandapam, nursery reared juveniles were transferred fishes was 296.88 ± 6.27mm and mean body weight
to the grow-out sea cages. The stocking density was 464.65 ±10.25 g. The absolute growth was 1.93 g/
maintained at 3.0-5.0kg/m3 or 750 nos of juvenile day and specific growth rate was 2.27%/day. Based on
cobia per cage of 6m diameter and 3 metre depth. the experience gained from the above demonstration,
The entire grow-out culture was carried out for a farming protocols were evolved.
period of 6- 7 months. The juveniles reached an
average weight of 1.0kg in 4 months and 2.5 – 3.0kg Ornamental Fish Culture
in 6- 7 months. The grow-out fishes could attain an
average weight of 7.0kg with a maximum weight of The marine ornamental fish trade has been expanding
8.0kg in one year. in recent years and has grown into a multimillion

23
 dollar enterprise. The ornamental animals are the is the only option for the development of a long
highest valued products that are mostly harvested term sustainable trade. The Central Marine Fisheries
from coral reef environments. The global marine Research Institute (CMFRI) has been focusing on this
ornamental trade is estimated at US$ 200-330 vital aspect for the past few years. The Institute was
million. The trade is operated throughout the tropics. able to develop hatchery production methods of the
Philippines, Indonesia, Solomon Islands, Sri Lanka, following species of ornamental fishes which are in
Australia, Fiji, Maldives and Palau supplied more than high demand in the international trade.
98% of the total number of marine ornamental fish
exported in recent years. It is a multi-stakeholder Table 1. Marine Ornamental Fishes
industry ranging from specimen collectors, culturists, Amphiprion percula Orange clown
wholesalers, transhippers, retailers, and hobbyists A. ocellaris False clown
A.sebae Sebae clown
to researchers, government resource managers and
A.nigripes Maldive’s clownfish
conservators and hence involves a series of issues
A.ephippium Red saddleback clownfish
to be addressed and policies to be formulated for A.perideraion Pink skunk
developing and expanding a sustainable trade. It is A.clarkii Clark’s anemonefish
well understood that a long term sustainable trade Premnas biaculeatus Maroon clown(spine cheek
of marine ornamental fishes can be developed only anemonefish)
Pomacentrus cearuleus Blue damsel
through the development and commercialization
P.pavo Peacock damsel
of hatchery production technologies for the species Dascyllus trimaculatus Three spot damsel
which are in high demand in the trade. Dascyllus aruanus Humbug damsel
Chromis viridis Bluegreen damsel

Global scenario Neopomacentrus nemurus

N.cyanomos
Yellowtail damsel
Filamentous tail damsel
Chrysiptera cyanea Sapphiredevil damsel
In recent years it has been reported that nearly 1500
species of marine ornamental fishes are traded globally The damaging fishing methods which destroy the
and most of these are associated with coral reefs. fragile corals and over harvesting of the species
Nearly 98% of the marine ornamental fishes marketed in demand are the vital problems associated with
are wild collected from coral reefs of tropical countries. the trade. It is widely accepted that the ultimate
Among the most commercially traded families of reef answer to a long term sustainable trade of marine
fishes, family Pomacentridae dominate, accounting for ornamental trade can be achieved only through the
nearly 43% of all fish traded. The family contains about development of hatchery production technologies. In
235 species worldwide. They are followed by species this context it is imperative to develop commercially
belonging to Pomacanthidae (8%), Acanthuridae (8%), viable seed production techniques of species
Labridae (6%), Gobiidae (5%), Chaetodontidae (4%), which are in demand. It is well accepted as an
Callionymidae (3%), Microdesmidae (2%), Serranidae environmentally sound way to increase the supply of
Central Marine Fisheries Research Institute

(2%) and Blennidae(2%). In recent years the blue green marine ornamentals by reducing the pressure on wild
damselfish (Chromis viridis), the clown anemone fish population and producing juvenile and market sized
(Amphiprion ocellaris), the whitetail Dascyllus (Dascyllus fish of wide variety of fish year round. In addition
aruanus), the sapphire devil (Chrysiptera cyanea) and hatchery produced fish are hardier and fair better
the three spot damsel (Dascyllus trimaculatus) are in captivity and survive longer. The methodologies
among the most commonly traded species. developed by CMFRI can be scaled up for commercial
level production and a hatchery produced marine
Hatchery production Technologies ornamental fish trade could be developed.

Indiscriminate exploitation of ornamental fishes Marine Finfish Brood bank

from the coral reef areas has been threatening the
long term sustainability of the trade. Hence hatchery The availability of required quantities of bio-secure
production of selected marine ornamental fishes seed is the major prerequisite for the initiation and

24
expansion of marine finfish farming in the country. years on a global basis and it is viewed by many
The major bottleneck in achieving commercial level stakeholders in the industry as the aquaculture system
seed production is the non-availability of a facility of the millennium. Cage culture has made possible
where the bio-secure broodstocks can be maintained the large-scale production of commercial finfish in
and controlled spawning can be obtained year many parts of the world and can be considered as
round. Broodstock management usually includes the most efficient and economical way of rising fish.
collection, selection and domestication of brooders The rapid growth of the industry in most countries
as well as control of maturation, spawning and can be attributed to (i) availability of suitable sites for
egg production. The broodstocks of large growing cage culture (ii) well established breeding techniques
fishes like cobia is mostly developed in sea cages. that yield a sufficient quantity of various marine and
However, the broodstock developed in sea cages freshwater fish juveniles (iii) availability of supporting
are susceptible to mortality due to the changes in industries such and feed, net manufactures, fish
the water quality of the cage site, disease problems processors etc. (iv)strong research and development
and impact of harmful algal blooms. In addition, the initiatives from institutions, governments and
broodstock developed in sea cages is not bio-secure universities and (v) the private sector ensuring
and hence can lead to spreading of diseases while refinement and improvement of techniques/ culture
farming is taken up on a commercial basis. If the systems, thereby further developing the industry.
broodstock can be maintained onshore in controlled
facilities the loss of broodstock can be minimised Commercial cage culture was pioneered in Norway in
and controlled breeding by manipulating the photo the 1970s with the rise and development of salmon
thermal regimes and spawning all through the year farming. As in terrestrial agriculture, the move
can be achieved. within aquaculture towards the development and
use of intensive cage farming systems was driven by

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Recirculating Aquaculture a combination of factors, including the increasing
competition faced by the sector for available resources
System (RAS)
(including water, land, labour, energy), the drive for
Closed-system aquaculture presents a new and increased productivity per unit area and the need for
expanding commercial opportunity. Recirculating the sector to access and expand into new untapped
aquaculture Systems (RAS) are tank-based systems open water culture sites such as lakes, reservoirs, rivers
in which fish can be grown at high density under and coastal brackish and marine offshore waters. The
controlled environmental conditions. They are closed- cage aquaculture sector has grown very rapidly during
loop facilities that retain and treat the water within the past 20 years and is presently undergoing rapid
the system. In an RAS, water flows from a fish tank changes in response to pressures from globalization
through a treatment process and is then returned to and growing demand for aquatic products. Total
the tank, hence the term Recirculating Aquaculture reported cage aquaculture production from 62
System. These systems use land based units to pump countries and provinces/regions from where data is
water in a closed loop through fish rearing tanks and available amounted to 2412167 tonnes (excluding
consist of a series of sub-systems for water treatment China) On the basis of the reported information,
which include equipment for solids removal, the major cage culture producers in 2005 included–
biological filtration, heating or cooling, dissolved Norway (652306 tonnes), Chile (588 060 tonnes),
gas control, water sterilization and photo-thermal Japan (272 821 tonnes), United Kingdom (135
control. Sustainable production of bio-secure fish 253 tonnes), Vietnam (126 000 tonnes), Greece
seed all through the year employing photo-thermal (76 577 tonnes), Turkey (78 924 tonnes), and the
conditioning is possible only by recirculating systems. Philippines (66 249 tonnes). Currently on a global
basis commercial cage culture has been restricted to
Sea cage farming the culture of high value, compound feed fed finfish
species, including salmon, Japanese amberjack,
The sea cage farming has been expanding in recent red sea bream, yellow croaker, European sea bass,

25
 gilthead sea bream, cobia and groupers. Cage culture Sea bass and spiny lobsters were the major groups
systems employed by farmers vary from traditional employed for farming. These demonstrations have
family owned cage farms (Asian countries) to modern created an awareness regarding the prospects of
commercial large scale salmon and trout cage farms sea cage farming in India. Many entrepreneurs,
in Northern Europe and Americas. The rapid rise and fishermen and farmers are coming forward to take
success of the salmon cage farming industry has been up this venture.
due to a combination of interlinked factors such as
the development and use of an easily replicated and Mussel Farming
cost effective technology (including hatchery seed
production), access to large areas of suitable waters, The Institute has developed technologies for culture
good species selection, market acceptability, increased of bivalves’ viz. raft method (in bays, inshore waters),
corporate investment and a good and supportive rack method (in brackishwater, estuaries) or long line
government regulatory environment. This can be method (open sea). These methods are commonly
taken as a model for cage culture development. adopted for mussel farming (Perna indica and P. viridis).
Mussel seeds of 15-25 mm size collected from intertidal
Marine cage farming is relatively new in Asia and and sub tidal beds are attached to coir/nylon ropes of
was developed initially in Japan for species such as 1-6 m length and enveloped by mosquito or cotton
yellowtail (Seriola quinqueradiata) and red sea bream netting. Seeds get attached to rope within a few days
Pagrus major. Over the last twenty years the cage while the netting disintegrates. The seeded ropes are
farming practice has spread almost throughout hung from rafts, racks or longlines. A harvestable size
Asia. The major cage farming countries are China, of 70-80 mm is reached in 5-7 months and production
Indonesia, Taiwan Province of China and Vietnam. A of 12-14kg mussel (shell on) per metre of rope can
large number of finfish species are farmed in cages be obtained. Innovations such as automatic seeding
in Asia viz. groupers, snappers, carangids, seabass machines and depuration protocols were also evolved.
and cobia. In most countries individual operations The farming of mussels is currently being practised
are not large, and often a clustering of farming commercially at Malabar Coast, Kerala.
activities, which is due to limited site availability in
coastal waters is seen. Edible Oyster Farming
When compared to many countries in the Asia- CMFRI has developed methods for edible oyster
Pacific Region, India is still in its infancy in sea cage (Crassostrea madrasensis) culture and has produced
farming. For the first time in India as part of R &D a complete package of technology, which is presently
a marine cage of 15 m diameter with HDPE frame being widely adopted by small scale farmers in shallow
was successfully launched in 2007and operated estuaries, bays and backwaters. In the adopted rack
at Visakhapatnam, in the east coast of India by and ren method, a series of vertical poles are driven
Central Marine Fisheries Research Institute

the Central Marine Fisheries Research Institute. into the bottom in rows, on top of which horizontal
Since then, a lot of innovations on designing and bars are placed. Spat collection is done mainly from
fabrication of cages and mooring systems were the wild on suitable cultch materials. Spat collectors
made which led to the development of better consist of clean oyster shells (5-6 Nos.) suspended on
designs of cages of 6m diameter with improved a 3 mm nylon rope at spaced intervals of 15-20 cm
mooring systems that can withstand rough sea and suspended from racks, close to natural oyster beds.
conditions. Subsequently demonstrations of cage Spat collection and further rearing is carried out at
farming were undertaken along different parts the same farm site and harvestable size of 80 mm is
of the Indian coast under a participatory mode reached in 8-10 months. Harvesting is done manually
with the local coastal fishermen. Successful sea with a production rate of 8-10 tonnes/ha. Oyster shells
cage farming demonstrations were conducted at are also in demand by local cement and lime industry.
Kanyakumari, Vizhinjam, Kochi, Mangalore, Karwar,
Veraval, Mandapam, Chennai and Balasore. Cobia, Mollucan shellfish (mussels, oysters and clams) are

26
much sought after and widely consumed throughout Way Forward
the world as gourmet food. But in India these
nutritious seafood have not found much acceptance. Seed availability is the major constraint for the
Currently farmed mussel and oyster production in initiation of commercial level farming of marine
India is between 15,000 and 20,000 tonnes. In finfishes and shellfishes. The huge demand for
order to create an awareness on the general public cobia and pompano seeds received at CMFRI from
on molluscan shellfish as a highly nutritious food, fish farmers and entrepreneurs is indicative of the
ShellCon 2014 was organised by CMFRI in which need of the sector. Hence there is an urgent need
shellfish food festival and programmes for the to establish marine finfish hatcheries by fisheries
popularisation of the consumption of molluscan development agencies /private sector to ensure
shellfish were conducted. the seed availability. In addition, it is required to
intensify research programmes for the development
Mabe Pearl Production of seed production techniques for at least one
dozen species of high value marine fishes. In this
CMFRI successfully developed and standardised a context, CMFRI has already taken up broodstock
simple technique for value added marine pearls, development and seed production of orange spotted
called mabe pearls. A mabe pearl is a dome shaped Grouper Epinephelus coioides, Indian Pompano
or image pearl produced by placing a miniature Trachinotus mookalee and Malabar Red Snapper
image against the side of the oyster shell interior. Lutjanus argentimaculatus. Initial success has already
The result is an exquisite pearly nacre coated image. been obtained in the broodstock development and
The main advantage is the very short gestation period seed production of E. coioides and T. mookalee at
(2 months) and the superior quality of the nacre of the Vishakapatnam Research Centre of CMFRI.
Indian pearl oyster Pinctada fucata. Broodstock development of L. argentimaculatus is

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

being pursued. If seed production technologies of
Seaweed Culture more species are available, the farmers will be able
to select the species as per the need of the locality.
Around 60 species of commercially important
seaweeds with a standing crop of one lakh tonne The commercial level farming of lucrative shellfish
occur along the Indian coast. Seaweed products like species like the sand lobster, Thenus unimaculatus and
agar, algin, carrageenan and liquid fertilizer are in the blue swimmer crab Portunus pelagicus can also
demand in global markets and some economically be practiced if hatchery produced seeds are available.
viable seaweed cultivation technologies have been CMFRI is able to succeed in the seed production of
developed in India. CMFRI has developed technology both the species and now research is being focused
to culture seaweeds by either vegetative propagation on the standardization of these techniques. Similarly
using fragments of seaweeds collected from natural seed production techniques are already developed by
beds or spores (tetraspores/ carpospores). It has the the Institute for edible oyster, pearl oyster and green
potential to develop in large productive coastal belts. mussel. The methods can be scaled to commercial
The rate of production of Gelidiella acerosa from level production as per the requirement of the sector.
culture amounts to 5 tonnes dry weight per hectare,
while Gracilaria edulis and Hypnea production is The development of farming systems especially
about 15 tonnes dry weight per hectare. Recently the sea cage farming deserves prime attention.
the culture of the carageenan yielding sea weed To promote sea cage farming in the country,
Kappaphycus alvarezii has become very popular due identification of suitable sites with proper depth,
to its fast growth and less susceptibility to grazing water quality and water current are required. Site
by fishes and is being cultivated extensively along the selection survey and identification of at least a dozen
Ramanathapuram, Pudukkottai, Tanjore, Tuticorin and sites suitable for cage farming by the entrepreneurs
Kanyakumari districts of Tamil Nadu producing about and farmers deserves urgent attention. Availability
17000 t wet weight annually. of logistic support for cage farming should be given

27
 careful consideration if a profitable business is to balanced systems for environmental stability ( bio-
be established. Cage farming has to be promoted mitigation) economic stability (product diversification
away from the human settlements, discharge points and risk reduction) and social acceptability (better
of industrial and municipal waste, so as to maintain management practices). IMTA is well recognized as
ideal water quality for sea farming. Further, policy a mitigation approach against the excess nutrients
for leasing the suitable sites, bank finance, and / organic matter generated by intensive aquaculture
governmental support through subsidy assistance activities especially in marine waters, since it
are the needs of the hour. incorporates species from different trophic levels
in the same system. In addition, it is also relevant
The bivalve farming which is already being practiced in the implementation of ecosystem approach to
at a few locations can be further expanded. The aquaculture (EAA) propagated by FAO. IMTA can
carrying capacity assessment, low value availability also increase the production capacity of a particular
of hatchery produced seed and the feasibility of open site. It is well understood that increasing use of
sea farming of bivalves require attention by the R&D coastal waters worldwide coupled with rapid growth
sector. Similarly the expansion of sea weed farming and expansion of mariculture demand for more
offers immense scope. A concerted effort by the sustainable practices and hence the concept of IMTA
developmental agencies for popularization of sea has much relevance and scope. The development
weed farming is warranted. of IMTA in marine and coastal environments, has
not been demonstrated as a viable enterprise in
On a global basis, the mariculture practices are India and hence there is an urgent need to impart
dominated by intensive monocultures which have front line demonstration on this potential sector of
led to sustainability problems, environmental mariculture to different stake holders.
degradation and consequent disease problems.
In this context, the idea of bio-mitigation of The development of commercial level seed production
the environment along with increased biomass technologies for a few species of high market value
production integrating commercially important finfish and shellfish, establishment of hatcheries by
species of different trophic levels is emerging as fisheries development agencies, identification of
an innovation in aquaculture. Integrated Multi appropriate cage/coastal farming sites, development
trophic aquaculture (IMTA) is the practice which of economically viable farming protocols, formulation
combines in appropriate proportions the cultivation of suitable grow-out feeds, health management
of fed aquaculture species (E.g. fin fish / shrimp) protocols, development of mariculture policies and
with organic extractive aquaculture species (e.g. appropriate marketing strategies can go a long way
shell / herbivorous fish) and inorganic extractive to promote mariculture as a substantial contributor
aquaculture species (e.g. seaweed) to create of sea food production of India.
Central Marine Fisheries Research Institute

28
Introduction to Marine Aquariculture
K. Madhu* and Rema Madhu
Principal Scientist
Mariculture Division, CMFRI, Kochi
e-mail: [email protected]

Introduction Considering many pressures currently faced by reefs, it

Aquarium keeping is amongst the most popular of is vital that ornamental fisheries are to be investigated
hobbies with millions of enthusiasts worldwide, and and monitored, and management strategies are need
the aquarium industry is also a multimillion dollar to be formulated to ensure that they are sustainable.
business in the world. Profit of marine aquarium This requires research, monitoring, training, use of
industry is threefold increase than freshwater. non-damaging collecting methods and adoption of
However, 90% of income from marine industry is conservation strategies for controlling catch, such as
from wild caught only. The main drawback of this reserves, quotas and closed seasons. Such measures
industry is difficulties endowed with various stages include limiting collecting effort, establishment of
in marine fish rearing under captive conditions. species-based or overall quotas, restrictions on rare
The acquisition of marine ornamental fish has and/or endemic species, temporary closures and
also greatly increased in recent years. Presently, 30 establishment of fishery break reserves. There are

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

million coral reef fish belonging to 1,000 species and also a number of possibilities for enhancing the
100 species of invertebrate are collected annually fishery, such as mariculture and construction of
to supply private and public aquaria around the artificial reefs. It is well understood that India has a
world. The majority of these specimens come from wealth of marine ornamental animals in our island
coral reefs and associated habitats, with about ecosystems of Lakshadweep and Andaman- Nicobar,
45 countries supplying the ornamental market. besides many areas of mainland. In the context of
Many fish collectors in tropical and subtropical the expanding global marine ornamental fish trade
countries are employing cyanide to stun tropical in recent years it appears that India has the potential
fish, making it easier to collect them, but widespread to develop a lucrative marine ornamental fish trade.
cyanide application harms coral reefs and their A critical assessment of the current global scenario
ecosystems and threatens the food source of the of marine ornamental trade can provide much
local population. Therefore, in the last few years, a insight into the complexities and conservational
number of scientists have studied the reproduction issues associated with trade, which will be of
of some of the species which are most commonly much relevance while formulating policies for the
used in the aquarium trade for the purpose of rearing development of a marine ornamental industry. The
and breeding them in captivity (Thresher, 1884; Philippines, Indonesia, Soloman Islands, SriLanka,
Holt, 2003; Olivotto et al., 2003, 2004). Marine Australia, Fiji, the Maldives and Palau supplied the
ornamental fish production is now considered as one major share of marine ornaments during the recent
of the most important trade in international markets. years. The United States, the United Kingdom, the
Due to the popularity of aquariums in households Netherlands, France and Germany were the most
in many parts of the world, aquariculture play a important countries of destination. It is well accepted
growing role in the international fish trade. The total that the trade developed from tank reared fish and
value of wholesale ornamental trade is estimated at other ornamentals is the final solution for a long
close to US$ 1 billion, and retail trade about US$ 3 term sustainable trade. The economic viability of
billion (Olivotto, 2005). ornamental fish production is more lucrative when

29
 compared to other mariculture species, due to their hatchery production of clown fish and few damsel
high unit value. fishes (Gopakumar et al., 2002, Ignatius et al., 2001,
Madhu and Rema Madhu, 2002) were reported
In India, till date no organized trade of marine first time in India with an objectives to generate
ornamentals has been initiated. But it is a fact scientific knowledge on ornamental fish maintenance,
that a great deal of illegal collection of marine behaviour, influence of social status on sex change,
ornamentals is in vogue in many parts of our reef pair formation, breeding, influence of lunar periodicity
ecosystem and this is a matter of great concern due in spawning, parental care, egg incubation and
to the indiscriminate nature of exploitation and eco hatching, developments of egg, larvae, and juveniles.
hostile methods of collection which damage the reef These investigations have resulted in the development
ecosystem. In addition to this, lack of knowledge on of hatchery technology for 20 species of marine
appropriate post-harvest husbandry practices leads ornamental fishes such as clown fishes Amphiprion
to large scale mortality of the collected animals. It is percula (True pecula/ clown anemone fish); A. ocellaris
time to evolve a marine ornamental fisheries policy for (Common Clown/ False clown aenemonefish) Rema
developing an organized trade of marine ornamentals et al.,2012; A. sandaracinos (Yellow Skunk Clown)
in many contries. It is felt that eventhough the ideal Rema and Madhu,2012 ; A. frenatus (Tomato clown)
situation is to develop a sustainable trade of marine Madhu et al., 2011, A. clarkii (Clark's Anemonefish)
ornamentals through tank reared species, it has to A. nigripes (Maldives Anemonefish ) (Madhu and
be admitted that development of commercial level Rema Madhu,2006; Madhu et al., 2006a, b, c; Rema
breeding technologies of all the species of demand Madhu, et al., 2007; Madhu et al., 2008; Madhu and
will take a very long time and if you have to wait till Rema, 2011) A. perideraion (Pink anemone fish) (Anil
then, we may fail to enter into this lucrative global et al., 2012), Amphiprion ephippium (redsaddle back
trade in the near future. A critical analysis of current anemone fish), A. sebae (Sebae clown) (Gopakumar,
global trade of the marine ornamentals from wild et al.,2007,2009); and Premnas biaculeatus (Maroon
collections reveals many ecological concerns which clown/ Spine cheek anemone fish) Madhu et al., 2012
require policy interventions. and dottyback Pseudochromis dilectus (Redhead
Dottyback) and Nemateleotris decora (Madhu and
Captive breeding of marine Rema, 2014). The species such as damsels Dascyllus
trimaculatus (Three spot damsel); D. aruanus (Striped
ornamentals in India
damsel); Pomacentrus caeruleus (Blue damsel); P. pavo
More than 200 varieties of export oriented marine (Sapphire or Peacock Damsel); Neopomacentrus
ornamental fishes are available in Indian waters, and nemurus (Yellow tail damsel); N. filamentosus
it is widely accepted that their wild collection from (Filamentous tail damsel); Chrysiptera cyanae
the flimsy reef ecosystem will lead to habitat damage (Sapphire devil); C. unimaculata (One spot damsel)
and overexploitation of the species which are in high and Chormis viridis (Green chromis) (Gopakumar, et
Central Marine Fisheries Research Institute

demand. In these scenario, development of a long al.,2007,2009, Syda Rao et al., 2010). At present
term sustainable trade of marine ornamental fishes tank reared species contribute only 1-2% of the trade.
through hatchery production is the only alternative. Culture of marine ornamental fish is well accepted as
The decline of exploited marine fishery resources due an environmentally sound way to increase the supply
to increasing fishing pressure, the setback in shrimp of such organisms by reducing the pressure on wild
farming due to disease outbreak and the impact of populations and producing juveniles of a wide variety
tsunami have adversely affected the livelihood of of species year round. In addition, hatchery produced
Indian coastal villagers, and an alternative livelihood fish are hardier which are survive better in captivity
option is felt very essential. Considering these and survive longer. The high unit value of ornamentals
situations, during the past few years, the Central makes them more commercially viable than marine
Marine Fisheries Research Institute (C.M.F.R.I.) has food fish culture. Hence in future, hatchery reared fish
intensified research activities on breeding and culture will become a significant part of marine ornamental
of marine ornamental fishes. The success in the fish trade in India and also globally.

30
Major requirements smaller one. A tank of the size 90cm x 40cm x 50cm
can be fabricated with 6mm glass and larger tanks
in Aquariculture of 500 litres and 1000 litres can be fabricated with
Filtration: Efficient filtration is mandatory in a marine glass plates of thickness 8mm and 12mm respectively.
aquarium. There are two basic types of contaminates A shallow tank is more advisable because the water
in aquarium water – suspended physical particles surface is the place where oxygen enters and the
and dissolved chemical compounds. The dissolved carbon-di-oxide leaves.
contaminants are created from the metabolic waste
materials of fish, invertebrates and plants, and also Lighting: The aquarium should not be installed in a
develop from the activity of bacteria on waste organic place where there is no much day light, in particular
matter produced in the tank. These dissolved chemical direct sunlight, because it may overheat the water of
compounds include ammonia, nitrite, nitrate, urea, the tank. Lighting is usually provided by a fluorescent
proteins, fatty acids, phenols, dyes and many tube or tubes with a reflecting hood. If you want to
other less abundant compounds. Filtration can be grow sea-anemones, sea-weeds and live corals, more
classified into three types. i) Mechanical filtration ii) light is needed and it must be provided in sufficient
Chemical filtration & iii) Biological Filtration (Under intensity by special lamps which can emit lights of red
gravel filtration) and blue wave lengths of visible spectrum. The light
should be on for at least 12 hours per day.
Adequate aeration: The purpose of aeration is
not only for oxygen but also keep the water moving Temperature: As temperature fluctuations may be
and exchanging gases with the air. This occurs in the detrimental to many species, it is important to have
surface, not between the bubbles and the water unless a heater / thermostat combination submersible and
these are very dense. Aeration is often combined with guaranteed suitable for salt water in the tanks. Power

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

filtration but it is better to provide air stones to add of heaters should be 100 watt for a 100 litre tank,
the effects of filters. Air stones come in all shapes 150 watt for a 200 litre tank and 200 watt for 300
and sizes, but what is most important is that they litre tank. A tropical marine tank generally needs a
should give medium sized bubbles between ½ and temperature range of 25 – 28ºC.
1 mm in diameter and these should move the water
most efficiently. Very fine bubbles are good but form Quality water: Quality seawater should have
a mist in open water. A good brand of diaphragm dissolved salts, trace elements and dissolved gases.
pump with a volume control can be used for both Only seven salts viz., sodium chloride, magnesium
air stones and filters. chloride, magnesium sulphate, calcium sulphate,
potassium sulphate, calcium carbonate and potassium
Formulation of synthetic sea water salts: There or sodium bromide make up over 99.5% of all the
are a number of sea salts readily available in the conservative salts in sea water. The remaining 0.5%
market. Synthetic seawater differs from natural of the inorganic solids is made up of at least 60
seawater in which the concentrations of the major elements found in such tiny amounts that they are
inorganic salts are not exactly the same, inorganic called trace elements. Eventhough the trace elements
trace elements are not the same in number or are present in extremely small amounts, some of them
concentration and there are no dissolved organics. especially zinc, copper, iodine, strontium, vanadium,
cobalt, molybdenum and arsenic are essential to many
Availability of suitable feeds: (Live and artificial). living organisms. The dissolved organic substances are
compounds such as amino acids, proteins, enzymes,
Treatment facilities: for common diseases of vitamins and pigments. Inshore water carries a greater
marine fishes. load of dissolved organics than clear offshore waters.

Size of the tanks: A large tank is more stable in Seawater treatments: The best way to treat the
the constitution and temperature of water than a water after collection is to store it in the dark to

31
 2-3 weeks prior to using it in the aquarium. Another can either be collected form the coral reef habitat
treatment of collected seawater is chlorination and or can be purchased from the pet shop depending
de- chlorination. Chlorine kills all lives in the collected upon the availability. In case mated pairs are not
water including bacteria and oxidizes the organic available, the fishes having different size groups
matter dissolved in natural seawater, including toxins. can be collected and made to pair under captive
For chlorination and de-chlorination we require condition through pair formation. In order to make
bleaching powder, sodium thiosulphate and a test breeding pairs form the juveniles groups, fishes
kit for chlorine. can be collected from the wild and transported to
the laboratory in healthy condition. In the case of
New tank syndrome clownfishes, fishes and sea anemones should be
kept in separate plastic transportation bags during
A new tank which has been carefully set up and transportation. For the pair formation, five fishes of
fishes are introduced without maturation of the each sex of different size groups need to be stocked
tank will become unhealthy after a few days and together along with single host sea anemone in a
fish mortality will result. Matured tanks will have 500 L FRP tanks fitted with biological filter to reduce
a balanced bacterial population with the nitrogen the aggression. The pair formation tanks need to
cycle proceeding satisfactorily, and hence generation maintained in the hatchery where an incident
of ammonia or nitrites will be less. As ammonia light intensity of 2500 to 3000 lux was available
is produced, it is converted rapidly into nitrites as the sea anemones require sunlight for its better
which in turn are converted into nitrates which survival under laboratory condition. The fishes and
are comparatively nontoxic. In a new tank there anemones should be fed two times per day with
will not be an inadequate population of any type wet feeds such as meat of shrimp, mussel and clam
of bacteria and the first thing is the growth of at the rate of 15% of their body weight and live
bacteria which decompose organic matters such as feeds like Brachionus plicatilis, artemia nauplii and
fish faeces, uneaten food or any kind of decaying adult artemia. Environmental parameters such as
matter. The end product of such decomposition is temperature 26 to 29º C, salinity 33 to 36 ppt,
ammonium hydroxide. dissolved oxygen 4.6 to 6.2 ml/L and pH 8.1 to
8.9 are need to be maintained in all the rearing
Aquarium Accessories tanks. After a period of 3 to 4 months rearing in
the pair formation, in each tank one pair grew
• Powered Canister Filters ahead of others and became the spawning pair. As
• Protein Skimmer the newly formed pairs will be very aggressive and
• Denitrator spending time for fleeing the other subordinates
• Ozonizer rather than reproductive activity, it is very essential
• UV Sterilizers: to stock each breeding pairs in separate broodstock
Central Marine Fisheries Research Institute

• Other auxiliary equipments (Test kits for measuring tanks (250 to 500 L capacity) with single healthy
salinity, pH, ammonia, nitrite and nitrate pair and host sea anemone. An ideal tank would
concentration; hand nets, siphons, etc.) be a 3ft x 2 ft x 2 ft with a layer of coral sand on
the bottom, a few live rocks, a healthy anemone,
Rearing/ breeding of fishes bright lighting and good filtration, preferably an
efficient protein skimmer to reduce the ammonia
in aquarium and organic materials from the fish. A trickle filter
could be used with regular water changes to keep
Pair formation and the nitrates low enough for the anemone to do
broodstock developments: well. Since the gonad development and spawning
of fishes are influenced by moon phases, the
The basic requirement is to have a sufficient broodstocks/ spawning tanks need to be kept in
number of broodstocks or breeding pairs which an apt place where the fish receive a regular day/

32
night lighting cycle (moon phase). An anemone is within a period of 4 to 6 months rearing under
generally not required to breed clownfish under captive condition if the broodstoks are provided
captive condition. The pairs formed through pair nutritious food and provided suitable rearing
formation should then be transferred to separate conditions. Few days prior to spawning, the male
glass aquaria for broodstock development. The selected a suitable site near to sea anemone for
broodstocks need to be fed with wet feeds such laying the egg and cleared algae and debris with
as meat of green mussel, shrimp, clam and fish its mouth and on the day of spawning both the
egg mass, and can also be provided formulated parents spent considerable time for the cleaning
feeds enriched with vitamins, minerals and algal of site which indicated that spawning may occur
powder at the rate of 10% of their body weight within few hours. Under laboratory condition, the
and supplied at an interval of every 3 hrs during spawning can be obtained between 0500 hrs to
day time. Apart from these, the broodstocks were 1530 hrs during day time and the spawning lasted
also fed with enriched rotifer 800 to 1000 nos/ml for one hr to one and a half hour. Each female lays
and artemia nauplii (200-400 nos/ml) and adult 300 to 1000 capsule shaped eggs at every 12 to 15
artemia (3 to 5 nos/ ml) every day. Provision of days interval depending on the species of clown fish,
enriched live feeds which apparently improved egg size of fish and previous experience. Generally the
quality and hatchability than the brooders fed with egg size of clown fishes ranges between 1.5 mm
non-enriched live feeds. to 3.0 mm in length with a width of 0.8 to 1.84
mm and adhered to the provided substratum with
Water quality maintenance stalk. An average of two spawning per lunar month
per pair resulting in an estimated annual fecundity
As a measure for this, the seawater need to be filtered of 7200 to 24000 eggs/ breeding pair/ year can be
through a series of sand filters before being taken to obtained under laboratory condition. Parental care

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

the rearing tanks. The temperature in all the breeding and egg morphology As parental care is inevitable
tanks need to be maintained between 26 to 30ºC, for hatching out of the larvae, the parents should
and level of dissolved oxygen (4.8 to 6.3 ml/L), pH be allowed to remain in the parental tank itself till
(8.0 to 8.9), salinity (32 to 36 ppt) and the water hatching. During incubation period, both the parents
needs to be recirculated to ensure water movement carefully look after the eggs during day time and it
and provided good water quality with the aid of a involved two basic activities viz. fanning by fluttering
specially devised filter system during the period of the pectoral fins and mouthing to remove the dead
rearing. Once in a week 25% of the water should or weakened eggs and dust particles.
be exchanged to avoid stress like a rapid increase in
plasma corticol concentration, depression of gonadal Egg hatching and larval rearing of
streroidogenesis, and subsequent development of
clown fishes
gonadal atresia.
On the expected day of hatching, two hours before
Substrate for egg deposition sunset, the eggs along with substratum were
transferred from the parental tank to hatching tanks
As many coral fishes are laying attached eggs, it is very (100 L) and provided with complete darkness for
essential to provided suitable substratum preferably accelerating the hatching. The larvae broke the egg
tiles or earthen pots or shells of edible oyster or PVC capsule and the hatchling emerged tail first and the
pipes which will also be helpful for the transfer of hatching occurred soon after sunset and the peak
deposited egg without any mechanical injury to hatching took place between 1900 to 2030 hrs under
hatching tank. darkness. The newly hatched larvae measured 3 to
4mm in length and each had a transparent body, large
Breeding of clownfishes eyes, visible mouth, and a small yolk sac and remained
at the bottom of the tank for a few seconds and soon
After broodstock rearing, each pair will start breeding after became free swimming. The larval rearing can

33
 be carried out under green water system and feeding Mar. Fish. Infor. Serv. T&E Ser. No. 188:1-5, C.MF.R.I., Kochi.
Madhu, K., Rema Madhu, G. Gopakumar, C.S. Sasidharan and
with super small rotifer B. rotundiformis and newly K.M.Venugopalan,2006b. Breeding, larval rearing and seed
hatched artemia nauplii. The larval period of clown production of maroon clown Premnas biaculeatus under
fishes generally last for maximum of 20 days and then captive conditions Mar. Fish. Infor. Serv. T&E Ser. No.190,
pp1-5. C.MF.R.I., Kochi.
after most of the fry resembled juvenile adult fish and Madhu, K., Rema Madhu, G. Gopakumar, M. Rajagopalan, C.S.
began to shift from partially pelagic to epibenthic Sasidharan and K.M.Venugopalan, 2006c. Success in captive
breeding of maroon clown Premnas biaculeatus (Bloch, 1790),
and started eating minced shrimp, fish flesh, mussel Fishing Chimes, Vol.27 (2): 30&57
meat, clam meat and formulated diets. Madhu, K. and Rema Madhu, 2002. Successful Breeding of
Common clown fish Amphiprion percula under captive
conditions in Andaman and Nicobar Islands, Fishing Chimes,
Suggested readings December 2002, 22 (9): 16-17.
Madhu, K. Rema Madhu and G. Gopakumar, 2008. Hatchery
production of the Spine-Cheek Anemone fish Premnas
Anil, M. K., Santhosh, B. Prasad, B. O. and Rani Mary George,2012 biaculeatus (Bloch, 1790) Tropical Fish Hobbyist, USA January
Broodstock development and breeding of black-finned Vol LVI (5): 104- 108.
anemone fish Amphiprion nigripes Regan, 1908 under captive Madhu, K., Rema Madhu and T. Retheesh, 2012. Broodstock
conditions, Indian J. Fish., 59(1) : 77-82. development, breeding, embryonic development and
Gopakumar, G., G. Sriraj, T.T. Ajithkumar, T.N. Sukumaran, B. larviculture of spine-cheek anemonefish, Premnas biaculeatus
Raju, C. Unnikrishnan, P. Hillari, V.P. Benziger, 2002. Breeding (Bloch, 1790), Indian J. Fish., 59(1): 65-75.
and larval rearing of three species of damsel fishes (Family Madhu, K., Rema Madhu, Grace Mathew and T. Retheesh, 2011.
Pomacentridae). Mar.Fish. Infor. Ser. (T&E), 171: 3-5. Captive breeding of Tomato clownfish Amphiprion frenatus,
Gopakumar, G., Madhu, K. and Rema Madhu, 2007. Seed Tropical Fish Hobbyist, USA November, Vol LX (3): 80-84.
production of marine ornamental fishes for trade. C.MF.R.I. Olivotto, I., Cardinali, M., Barbaresi, L., Maradonna, F., Carnevali,
Newsletter (April to June) pp 1-3. O., 2003. Coral reef fish breeding: the secrets of each species.
Gopakumar G. Boby Ignatius, I. Santhosi and N. Ramamoorthy, Aquaculture 224, 69– 78.
2009 Controlled Breeding and Larval Rearing Techniques, of Olivotto, I., Yasumasu, S., Gioacching, G., Maradonna, F., Cionna, C.,
Marine Ornamental Fishes, Asian Fisheries Science 22 (): 797- Carnevali, O., 2004. Cloning and expression of high choriolytic
804. enzyme, a component of the hatching enzyme system, during
Holt, G.J., 2003. Research on culturing the early life history stages embryonic development of the marine ornamental teleost,
of marine ornamental species. In: Cato, J.C., Brown, C.L. Chrysiptera parasema. Mar. Biol. 145, 1235–1241.
(Eds.), Marine Ornamental Species: Collection, Culture and Olivotto, I., Alessio, Z., Arianna, R., Beatrice Migliarini B., Matteo
Conservation. Iowa State Press pp. 251– 254. A., Carnevali, O., 2005. Breeding, rearing and feeding studies
Ignatius, B., G. Rathore, D. Kandasamy and A.C.C. Victor 2001 in the cleaner goby Gobiosoma evelynae. Aquaculture,
Spawning and larval rearing techniques for tropical clown fish 250,175– 182.
Amphiprion sebae under captive conditions. J. Aqua. Trop., Rema Madhu, Madhu, K. and T. Retheesh, 2012. Life history
16(3): 653-662. pathways in false clown Amphiprion ocellaris Cuvier, 1830:
Madhu, K., Rema, M., 2014.Captive spawning and embryonic A journey from egg to adult under captive condition. J. Mar.
development of marine ornamental purple firefish Biol. Ass. India, 54 (1), 77-90.
Nemateleotris decora (Randall & Allen, 1973). Aquaculture Rema Madhu and K. Madhu, 2012. Lunar spawning rhythm
424–425, 1–9. in orange anemone fish Amphiprion sandaracinos
Madhu, K. and Rema Madhu, 2011. Breeding and larval rearing (Pomacentridae) under captive condition, J. Aqua. Trop. Vol.
techniques for protandric tropical clownfish Amphiprion 27, No. (1-2) pp 1-12.
percula under captive condition J. Aqua. Trop. Vol. 26, No. Rema Madhu, K. Madhu and G. Gopakumar 2007. Success in
(3-4) pp 143-160. captive breeding of redhead Dottyback Pseudochromis dilectus,
Madhu, K. and Rema Madhu, 2006. Hatchery seed production of C.M.F.R.I. Newsletter (October to December) pp 2-4.
Marine Ornamental Clown fishes: A Remunerative option for
Central Marine Fisheries Research Institute

Syda Rao, G., V.P. Vipin Kumar, Shyam S. Salim, R. Sathyadhas, R.

livelihood in India,( in Hindi) In: National Official language Narayanakumar, Kajal Chakraborty, P. Vijayagopal, K.K.Vijayan,
Seminar Livelihood issues in Fisheries and Aquaculture Special K. Madhu, Rema Madhu, G.Gopakumar and K.K. Philipose,
Publication No. 90pp 51-55 2010. A manual on Entrepreneur-Ready Technologies of
Madhu, K. Rema Madhu, L. Krishnan, C.S. Sasidharan and CMFRI., pp. 1-39.
K.M.Venugopalan 2006 a Spawning and larval rearing of Thresher, R.E., 1984.Reproduction in reef fishes. Tropical Fish
False clown, Amphiprion ocellaris under captive condition. Hobbyist Publications Inc., Neptune City, New Jersey, 306–312.

34
An Overview of the
Fish Diversity of Indian Waters
Rekha J. Nair* and S. Dinesh Kumar
Principal Scientist
Demersal Fisheries Division, CMFRI, Kochi
e-mail: [email protected]

There are 20,000-30,000 species of fish in a ornamental fish category. At present, of the recorded
multitude of diverse marine aquatic ecosystems 24,000 species of finfishes in the world, about 2364
worldwide, and in freshwater environments many species are known to occur in India (www. fishbase.
new fish species continue to be ‘discovered’ by org). Rao (2009) recorded 1371 species in 77 families
science. About 22000 species of fishes have been from the Andaman and Nicobar islands.
recorded in the world; of which, about 11% are
found in Indian waters. Out of the 2200 species so Fish biodiversity
far listed, 73 (3.32%) belong to the cold freshwater
regime, 544 (24.73%) to the warm fresh waters The Indian fish fauna is divided into two classes,
domain, 143 (6.50%) to the brackish waters and viz., Chondrichthyes and Osteichthyes. The
1440 (65.45%) to the marine ecosystem. Chondrichthyes are represented by 131 species under

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

67 genera, 28 families and 10 Orders in the Indian
Fishing is one of the oldest human activities and it region. The Indian Osteichthyes are represented by
developed gradually, when our ancestors moved from 2,415 species belonging to 902 genera, 226 families
the collection of plants and animals to hunting by using and 30 orders.
tools and weapons. The oldest fishing implements so
far identified are harpoons, found in the territory of
Congo, and dating about 90,000 years. Interestingly, Classification and Diversity
these harpoons were found associated with the bones
of a species of now extinct giant catfish. In India too, Class Actinoptergyii-the ray
it is believed that the development of fishing must
finned fishes
have been parallel. There are reports that fishes were
grown in reservoirs as early as 320 BC. There are Numerically, actinopterygians are the dominant
several evidences of fish capture and culture since class of vertebrates, comprising nearly 99% of
then. There were evidences to indicate over-fishing the over 30,000 species of fish. Traditionally
in the River Ganges as early as 1785. Russell made actinopterygians have been divided into the
the first systematic study of the Indian fish fauna subclasses Chondrostei and Neopterygii. Neopterygii,
from 1785 to 1789 AD. Sir Francis Day studied the in turn, have been divided into the
systematics of Indian fishes for over 20 years and listed infraclasses Holostei and Teleostei.
351 genera and 1418 species of marine, brackish water
and freshwater fishes in 1868. Later, Alcock added Important characters of the class are
86 new genera and 200 species to the list. Jones and • Scales ganoid, cycloid, or ctenoid
Kumaran (1980) recorded 603 species of fish from the • Spiracles usually absent
Laccadive archipelago. Of the 603 species of marine • Pectoral radials attached to the scapula-coracoid
fishes belonging to 126 families that are reported complex except in Polypteriformes
from the islands, at least 300 species belong to the • Interopercle and branchiostegal rays usually present

35
 Infraclass teleostei Inferior: Bottom feeding fish generally have inferior
or sub-terminal mouths. Mouths located under the
Teleost are the most species rich and diversified group fishes head that are adapted for scavenging or grazing
of all the vertebrates on algae, molluscs or bottom dwelling invertebrates.

Though teleosts are a group wide variation is noticed Protrusible: Protrusible or protractile mouth in fish
in the parts of the fish. is a structural arrangement of the jaws that enables
the animal to extend the mouth at will. When fully
Mouth protruded, the cavity of the mouth is enlarged to form
a funnel-like space facilitating the uptake of food.
Fishes which feeds on small invertebrates in hidings
has protrusible mouth.
1 2
Teeth
These serve as a very important taxonomic character.
Generally, five types of teeth are recognised in
fish based on their structure-cardiform, villiform,
caniniform, incisiform and molariform.

3 4

5
1. Terminal Teeth types: The following teeth patterns are
2. Sub Terminal encountered in the fishes seen in tropical waters.
3. Superior
4. Inferior Canine teeth: They are sharp, highly pointed teeth
5. Protrusible seen in predatory fishes which are seen to attack
and hold prey in their sharp teeth. The teeth are also
Different mouth patterns are noticed in fish. They are used to tear off flesh from the prey. Sharks are best
Central Marine Fisheries Research Institute

examples of fishes with canine teeth.

Terminal: Fish with a terminal mouth position have
a mouth in the middle, or center of the head. These Incisor teeth: Incisors are used for cutting and they
fish are mostly predators who either chase their food come in variety of shapes. These are flattened tooth
or feed on what is seen in front of them. The terminal with chisel like or saw edges.
mouth position is the “normal” position of mouth for
most of the fishes inhabiting the middle levels of the Molar teeth: These are blunt, rounded, broad tooth
water column of oceans or lakes. adapted for crushing and grinding shellfish. They are
generally found in bottom dwelling fish.
Superior: This kind of fish has scoop-like mouth
which is designed to feed on prey that swims above Villiform teeth: Villiform teeth are elongated teeth
the fish (on the surface of the water), such as insects they are very long, slender and crowded having
or plankton. the appearance of velvet or fine bristles of a brush.

36
They are more common on deep see fishes used for caudal fin encountered in the present study are
stabbing and direction.
Heterocercal: the vertebrae extend into the upper
Dental plates: Teeth fused to form beak like plates. lobe of the tail, making it longer. Eg., sharks.

There are four different types of fish scale, each with Homocercal: the vertebrae extend for a very short
their own characteristics and variations. distance into the upper lobe of the fin, but the fin
appears superficially symmetric. Most modern fishes
Placoid Scales: Placoid scales are formed of a are homocercal tailed fishes. In homocercal tail
rectangular base plate that is embedded within the skin patterns, the following subpatterns are noticed.
of the fish and some of spine externally. The interior
of the scale is a pulp that receives blood from the Emarginate: ending in a slight inward curve
fish's vascular system, while the outside is made of an Lunate: ending in crescent shape
enamel-like substance called vitrodentine. The shape Forked: ending in two prolonged edges
of the spines can vary greatly depending on species. Deeply forked: with the caudal fin deeply cut
However, almost all give the fish a rough texture. Sharks into two
and rays are examples of fish with placoid scales. Truncate: ending in vertical edge
Rounded: ending in round shape
Cosmoid Scales: Cosmoid scales have evolved from Rhomboid: ending in rhomboid shape.
placoid scales fusing together. This is because cosmoid Notched or double emarginate: with two inward
scales have two base plates and similar external spines curves and a central point.
composed of vitrodentine. The base plates are made Pointed: trailing to a final point.
from bone and new bone is added as the fish grows.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Lungfishes and coelacanths have cosmoid scales. The list presented is tentative and adopted from
Froese and Pauly (2006)
Ganoid Scales: Ganoid scales have a bony base layer
similar to that of cosmoid scales. and are modified Order: Anguilliformes (Apodes)
cosmoid scales. However, they differ in that their
outer layer is made of an inorganic bone salt called The Order Anguilliformes, or true eels, contains 20
ganoine and that they are diamond-shaped and families and about 820 species. Species are usually
interconnected. Between ganoid scales are peg-and- elongate and slender, with single dorsal and anal fins
socket joints that articulate. Ganoid scales are found that are continuous with the caudal fin (if present) in
on sturgeons, bowfish, paddlefishes and gars. most species. All species lack fins and skeleton while
some groups lack pectoral fins. Scales are usually
Cycloid and Ctenoid Scales: Cycloid and ctenoid scales absent, or if present, are cycloid and embedded in
have different shapes but the same composition and skin. All have leptocephalus larvae.
positioning. Both are composed of collagen and calcium
carbonate, rather than bone, and both are overlapping. Most true eels are predators and belong to
This means that they are more flexible than the other types one of three families Congridae (Conger eels),
of scales. While the edges of cycloid scales are smooth, Muraenidae (Moray Eels) and Ophichthidae (snake
those of ctenoid scales have tiny teeth-like protrusions eels and worm eels). Some species are excellent
called ctenii, giving them a rougher texture. The majority food fish and form the basis of very important
of bony fish have cycloid or ctenoid scales. commercial fisheries.

Caudal Fin types In August 2011, a new family, the Protoanguillidae,

comprising a single genus and species, with a separate
The caudal fin is the tail fin, located at the end of the caudal fin and many other primitive characters
caudal peduncle and is used for propulsion. Types of was described. Johnson et al. (2011) reports that

37
 based on morphological and molecular data this Family Moringuidae (Worm or spaghetti eels)
species is the most primitive living member of the
order Anguilliformes Small eels with a maximum size of 50 cm. Body
moderately or very elongate, cylindrical except near
Family Anguillidae (Freshwater eels) tip of tail. Scales absent, eyes small covered with skin.

Members in this family undertake long migrations Species recorded from India
to breed offshore in the deep water (catadromous); • Moringua abbreviata
body elongate, one genus with 15 species. In India, • Moringua bicolor-Bicolor spaghetti eel
2 species has been reported. • Moringua javanica-Java spaghetti eel
• Anguilla bengalensis bengalensis-Indian longfin • Moringua microchir-Lesser thrush eel
eel, Indian mottled eel
• Anguilla bicolor bicolor-Shortfin eel, Indonesian Family Muraenesocidae (Pike congers)
shortfin eel
Elongated body with large eyes, covered with skin;
Family Colocongridae (Short tail Eels) dorsal fin origin over or slightly before the pectoral
base. Teeth well developed; conspicuous lateral line
Body stubby, snout blunt, pectoral fins present. One
genus, Coloconger with about 5 species; one species Four genera, Congresox, Cynoponticus, Muraenesox
reported from India and Sauromuraenesox with about eight species
• Coloconger raniceps-Froghead eel recorded worldwide. Four species recorded from India.

Family Congridae (Conger and garden Species recorded from India

eels) • Congresox talabon-Yellow pike conger
• Congresox talabonoides-Indian pike conger
Body elongate, lack scales, possess pectoral fins. These feed • Muraenesox bagio-Common pike conger, pike eel
on crustaceans and small fish and do not migrate to breed. • Muraenesox cinereus-Dagger-tooth pike conger

Three subfamilies with 32 genera and roughly 160 Family Muraenidae (Moray eels)
species; 11 species recorded from Indian waters.
Efficient predators on reefs and rocky shores.
Subfamily Heterocongrinae (Garden Eels) with 2 Smaller sized eels like Gymnothorax are reported to
genera-Gorgasia and Heteroconger be involved in ciguatera fish poisoning. This is said
to be due to the eels feeding on a ciguatoxic fish
Subfamily Bathymyrinae with about 5 genera- mainly plant eaters feeding on a particular algae.
Central Marine Fisheries Research Institute

Ariosoma, Bathymyrus, Chiloconger, Parabathymyrus About 15 genera with about 185 species reported
and Paraconger. worldwide; 35 species said to occur in India.

Subfamily Congrinae with about 25 genera. Family Muraenidae is divided into 2 subfamilies-
sub family Uropterygiinae with four genera
Species recorded from India Anarchias, Channomuraena, Scuticaria and
• Ariosoma anago Uropterygius and subfamily Muraeninae with about
• Bathymyrus echinorhynchus  11 genera, Echidna, Enchelycore, Enchelynassa,
• Conger cinereus-Longfin African conger Gymnomuraena, Gymnothorax, Siderea, Strophidon
• Gorgasia maculata-Whitespotted garden eel and Thyrsoidae
• Promyllantor purpureus 
• Uroconger lepturus-Slender conger Species recorded from India
• Xenomystax trucidans  • Anarchias allardicei-Allardice's moray

38
• Anarchias cantonensis-Canton Island moray when broken. Mouth large. Family represented
• Echidna delicatula-Mottled moray by six genera, Facciolella, Hoplunnis, Nettastoma,
• Echidna leucotaenia-Whiteface moray Nettenchelys, Saurenchelys and Venefica, with about
• Echidna nebulosa-Snowflake moray 38 species.
• Echidna polyzona-Barred moray
• Enchelynassa canina-Viper moray Species recorded from India
• Gymnomuraena zebra-Zebra moray • Nettenchelys taylori 
• Gymnothorax buroensis-Vagrant moray
• Gymnothorax enigmaticus-Enigmatic moray Family Ophichthidae (Snake eels)
• Gymnothorax favagineus-Laced moray
• Gymnothorax fimbriatus-Fimbriated moray Ophichthids are found worldwide in tropical to
• Gymnothorax flavimarginatus-Yellow-edged moray warm temperate waters. They inhabit a wide range
• Gymnothorax hepaticus-Liver-colored moray eel of habitats, from coastal shallows, and even rivers, to
• Gymnothorax javanicus-Giant moray depths of above 750 m (2,460 ft). Most species are
• Gymnothorax meleagris-Turkey moray bottom dwellers, hiding in mud or sand to capture
• Gymnothorax monochrous-Drab moray their prey of crustaceans and small fish, but some
• Gymnothorax monostigma-One-spot moray are pelagic. The family is supported by two subfamilies-
• Gymnothorax pictus-Peppered moray subfamily Myrophinae (worm eels) with 11 genera,
• Gymnothorax pseudothyrsoideus-Highfin moray Benthenchelys, Ahlia, Asarcenchelys, Glenoglossa,
• Gymnothorax punctatofasciatus-Bars'n spots moray Mixomyrophis, Muraenichthys, Myrophis, Neenchelys,
• Gymnothorax punctatus-Red Sea whitespotted moray Pseudomyrophis, Schismorhychus and Schultzidia and
• Gymnothorax reticularis  Subfamily Ophichthinae (Snake eels) with 41 genera
• Gymnothorax richardsonii-Richardson's moray

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

• Gymnothorax rueppellii-Banded moray Species recorded from India
• Gymnothorax thyrsoideus-Greyface moray • Bascanichthys deraniyagalai-Indian longtailed sand-
• Gymnothorax tile-Freshwater moray eel
• Gymnothorax undulatus-Undulated moray • Bascanichthys longipinnis 
• Scuticaria tigrina (native) Tiger reef-eel • Caecula pterygera-Finny snake-eel
• Strophidon sathete-Gangetic moray, slender • Callechelys catostoma-Black-striped snake eel
giant moray • Lamnostoma orientalis-Oriental worm-eel,
• Uropterygius concolor-Unicolor snake moray Oriental sand-eel
• Uropterygius macrocephalus-Needle-tooth moray • Lamnostoma polyophthalma-Ocellated sand-eel
• Uropterygius marmoratus-Marbled reef-eel • Leiuranus semicinctus-Saddled snake eel
• Muraenichthys schultzei-Maimed snake eel
Family Nemichthyidae (Snipe eels) • Myrichthys colubrinus-Harlequin snake eel
• Neenchelys buitendijki-Fintail serpent eel
Snipe eels are found in every ocean and generally • Ophichthus altipennis-Highfin snake eel
occupy depths of 300-600 m. Body elongate with • Ophichthus apicalis-Bluntnose snake-eel
extremely long, upper and lower jaws and large • Ophichthus cephalozona-Dark-shouldered snake eel
eyes. Three genera reported in the family Avocettina, • Pisodonophis boro-Rice-paddy eel
Labicthys, Nemichthys with about nine species • Pisodonophis cancrivorus-Longfin snake-eel
• Scolecenchelys macroptera-Slender snake eel
Species recorded from India • Skythrenchelys zabra-Angry worm eel
• Nemichthys scolopaceus-Slender snipe-eel • Xestochilus nebulosus-Nebulous snake eel

Family Nettastomatidae (Duckbill eels) Order Atheriniformes

Body elongate with slender tail which is regenerated The order has six families in 2 suborders -

39
 Suborder Atherinopsoidei Species recorded from India
• Chlorophthalmus agassizi-Shortnose greeneye
Family Atherinopsidae-new world silversides • Chlorophthalmus bicornis-Spiny jaw greeneye

11 genera and about 108 species in two subfamilies Family Ipnopidae-Deepsea tripod fishes

Suborder Atherinoidei The family Ipnopidae includes five genera

Bathymicrops, Bathypterois, Bathytyphlops,
Family Notocheiridae-Surf sardines Discoverichthys and Ipnops and 29 species of slender
deep-sea fishes (Nelson, 2006) distributed worldwide
Species recorded from India demersally in tropical and temperate seas, at depths
• Iso natalensis-Surf sprite between 476 and 6000 m (McEachran & Fechhelm
1998). Eyes minute or plate like, directed dorsally.
Family Atherinidae-Silversides
Species recorded from India
The Atherinidae is a large family of principally marine • Bathypterois atricolor-Attenuated spider fish
and euryhaline (living in varied salinities) fishes that is • Bathypterois guentheri-Tribute spiderfish
circumglobal in distribution. A few species are strictly • Bathypterois insularum
confined to fresh water.
Family Paraulopidae-Cucumber Fishes
Species recorded from India
• Atherinomorus duodecimalis-Tropical silverside One genes Paraulopus, with 10 species; no species
• Atherinomorus lacunosus-Hardyhead silverside recorded from Indian waters.
• Atherinomorus pinguis-Narrow-banded
hardyhead silverside Family Synodontidae-Lizardfishes
• Hypoatherina barnesi-Barnes' silverside
• Hypoatherina valenciennei-Sumatran silverside These are generally small with a slender cylindrical
body and head that superficially resemble those
Family Melanotaeniidae-rainbow fishes and of lizards. They have a dioecious mode of reproduction.
blue eyes Worldwide, four genera with about 57 species have
been recorded.
Seventeen genera with 113 species
recorded worldwide. In India 22 species have been reported in three genera-
Harpadon, Saurida, Trachinocephalus and Synodus
Order Aulopiformes
Central Marine Fisheries Research Institute

Subfamily Synodontinae (Lizard Fishes)

Suborder Chlorophthalmoidei Two genera, Synodus and Trachinocephalus with
includes 5 families about 37 species
Family Bathysauroididae-Bathysauroidids
Species recorded from India
One species Bathysauroides gigas (not reported • Synodus englemani-Engleman's lizardfish
from India) • Synodus indicus-Indian lizardfish
• Synodus jaculum-Lighthouse lizardfish
Family Chlorophthalmidae-Greeneyes • Synodus macrocephalus 
• Synodus macrops-Triplecross lizardfish
Large eye with teardrop-shaped pupil and distinctive • Synodus variegatus-Variegated lizardfish
lensless space anteriorly. A hermaphroditic species. • Trachinocephalus myops

40
Subfamily Harpadontinae- Species recorded from India
Bombay Ducks • Alepisaurus ferox-Longnose lancetfish
Two genera Harpadon and Saurida, with about 20 species
Order Batrachoidiformes
Species recorded from India
• Harpadon nehereus-Bombay duck Family Batrachoididae (Toadfishes)
• Saurida gracilis-Gracile lizardfish
• Saurida isarankurai-Shortjaw saury Species recorded from India
• Saurida longimanus-Longfin lizardfish • Allenbatrachus grunniens (native) Frog fish,
• Saurida micropectoralis-Shortfin lizardfish Grunting toadfish
• Saurida nebulosa-Clouded lizardfish • Austrobatrachus dussumieri (native) Flat toadfish
• Saurida tumbil-Greater lizardfish
• Saurida undosquamis-Brushtooth lizardfish Order Beloniformes
• Saurida wanieso-Wanieso lizardfish
Family Adrianichthyidae (Ricefishes)
Family Aulopidae (Flagfins)
Species recorded from India
Two genera Aulopus and Hime • Horaichthys setnai-Thready top-minnow,
Malabar ricefish
Family Pseudotrichonotidae (Sandiving • Oryzias carnaticus 
Lizardfishes) • Oryzias dancena 

Family Cimolichthyidae Family Belonidae (Needlefishes)

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one genus Cimolichthys Needlefishes have slender, stream-lined bodies, and
very long jaws filled with very sharp teeth. They
Family Enchodontidae are voracious predators and feed mostly on small
fishes. In most species, the upper jaw only reaches
With five genera Enchodus, Eurypholis, Palaeolycus, its full length in adulthood, so the juveniles have a
Parenchodus, Rharbichthys, Saurorhamphus half-beak appearance, with an elongated lower jaw,
but a much smaller upper one. During this stage of
Family Scopelarchidae their lifecycle, they eat plankton, switching to fish
once the beak fully develops. Needlefish reproduce
Pearleyes with four genera, Benthalbella, through mating and lay eggs. The male usually rides
Rosenblattichthys, Scopelarchoides and Scopelarchus the female on the waves as they mate.

Family Evermannellidae (Sabertooth 5 genera are recorded from India with 9 species
Fishes)
Species recorded from India
Three genera, Coccorella, Evermannella and
Odontostomps with seven species • Ablennes hians-Flat needlefish
• Platybelone argalus platyura-Keeled needlefish
Family Alepisauridae (Lancetfishes) • Strongylura incisa-Reef needlefish
• Strongylura leiura-Banded needlefish
Slender elongated body with a large mouth and strong • Strongylura strongylura-Spottail needlefish
teeth. Two genera, Alepisaurus and Omosudis reported • Tylosurus acus melanotus-Keel-jawed needle fish
worldwide (Nelson 2006). However as per Eschmeyer • Tylosurus crocodilus-Houndfish, Crocodile needlefish
(2015), only one genus Alepisaurus has been recorded. • Xenentodon cancila-Freshwater garfish

41
 Family Exocoetidae (Flyingfishes) • Hyporhamphus unicuspis -Simpletooth halfbeak
• Hyporhamphus xanthopterus -Red-tipped halfbeak
Elongate bodied fishes with cylindrical shape, • Oxyporhamphus micropterus
flattened ventrally in some species. Head short. Snout micropterus -Bigwing halfbeak
blunt. Worldwide, five subfamilies with 52 species in • Rhynchorhamphus georgii -Long billed half beak
8 genera has been reported. Represented in Indian • Rhynchorhamphus malabaricus -Malabar halfbeak
waters by 18 species in 6 genera. • Zenarchopterus buffonis-Buffon's river-garfish
• Zenarchopterus dispar -Viviparous half beak,
Species recorded from India Feathered river-garfish
• Cheilopogon abei-Abe's flyingfish • Zenarchopterus ectuntio -Ectuntio halfbeak
• Cheilopogon cyanopterus-Margined flyingfish • Zenarchopterus gilli -Viviparous halfbeak
• Cheilopogon furcatus-Spotfin flyingfish • Zenarchopterus pappenheimi -Bangkok halfbeak
• Cheilopogon intermedius  • Zenarchopterus striga -Hooghly halfbeak
• Cheilopogon nigricans-African flyingfish
• Cheilopogon spilopterus-Manyspotted flyingfish Order Beryciformes
• Cheilopogon suttoni-Sutton's flyingfish
• Cypselurus naresii-Pharao flyingfish The Order has 7 families with 29 genera and 144
• Cypselurus oligolepis-Largescale flyingfish species. All species are marine. Four families represented
• Exocoetus monocirrhus-Barbel flyingfish in Indian waters.
• Exocoetus volitans-Tropical two-wing flyingfish
• Hirundichthys coromandelensis- Family Berycidae (Alfonsinos)
Coromandel flyingfish
• Hirundichthys oxycephalus-Bony flyingfish Dorsal fin without notch, with 4-7 spines increasing
• Hirundichthys speculiger-Mirrorwing flyingfish in length from first to last, and 12-20 soft rays. 2
• Parexocoetus brachypterus-Sailfin flyingfish genera with about 9 species.
• Parexocoetus mento-African sailfin flyingfish
• Prognichthys brevipinnis-Shortfin flyingfish Species recorded from India
• Beryx decadactylus-Alfonsino
Family Hemiramphidae (Halfbeaks) • Beryx splendens-Splendid alfonsino
• Centroberyx rubricaudus-Red alfonsino
Elongate fishes with a prolonged lower jaw and a short
triangular upper jaw. Spines absent in fins. Represented Family Holocentridae (Squirrelfishes,
in Indian waters by 17 species in 7 genera. soldierfishes)

Species recorded from India Species with a long dorsal fin with spiny portion and
Central Marine Fisheries Research Institute

• Dermogenys brachynotopterus-Gangetic halfbeak soft rayed portion divided by a notch. Holocentrids

• Dermogenys pusilla-Freshwater halfbeak, (squirrelfish and soldierfish) are vocal reef fishes
Wrestling halfbeak whose calls and sound-producing mechanisms have
• Euleptorhamphus viridis-Ribbon halfbeak been studied in some species only. Worldwide, eight
• Hemiramphus archipelagicus-Jumping halfbeak genera with 78 species has been reported. In India,
• Hemiramphus far-Blackbarred halfbeak 18 species in 4 genera have been recorded.
• Hemiramphus lutkei -Lutke's halfbeak
• Hyporhamphus affinis -Tropical halfbeak Species recorded from India
• Hyporhamphus balinensis -Balinese garfish • Myripristis adusta-Shadowfin soldierfish
• Hyporhamphus dussumieri -Dussumier's halfbeak • Myripristis botche-Blacktip soldierfish
• Hyporhamphus limbatus-Congaturi halfbeak, • Myripristis hexagona-Doubletooth soldierfish
Hyporhamphus quoyi-Quoy's garfish • Myripristis murdjan-Pinecone soldierfish
• Hyporhamphus sindensis -Sind halfbeak • Neoniphon argenteus-Clearfin squirrelfish

42
• Neoniphon opercularis-Blackfin squirrelfish • Scoliodon laticaudus-Spadenose shark
• Neoniphon sammara-Sammara squirrelfish • Triaenodon obesus-Whitetip reef shark
• Ostichthys acanthorhinus 
• Ostichthys japonicus-Brocade perch Family Hemigaleidae (Weasel sharks)
• Sargocentron caudimaculatum-Silverspot squirrelfish • Chaenogaleus macrostoma-Hooktooth shark
• Sargocentron diadema-Crown squirrelfish • Hemipristis elongata-Elliot's grey shark,
• Sargocentron ittodai-Samurai squirrelfish Snaggletooth shark
• Sargocentron microstoma-Smallmouth squirrelfish
• Sargocentron praslin-Dark-striped squirrelfish Family Proscylliidae (Finback catsharks)
• Sargocentron punctatissimum-Speckled squirrelfish • Eridacnis radcliffei-Pygmy ribbontail catshark
• Sargocentron rubrum-Redcoat
• Sargocentron spiniferum-Sabre squirrelfish Family Scyliorhinidae (Cat sharks)
• Sargocentron violaceum-Violet squirrelfish • Apristurus investigatoris-Broadnose catshark
• Atelomycterus marmoratus-Marbled cat shark,
Family Monocentridae (Pinecone fishes) Coral catshark
• Cephaloscyllium silasi-Indian swellshark
The family contains just four species in two genera, • Halaelurus hispidus-Bristly catshark
one of which is monotypic. Worlwide two genera • Halaelurus quagga-Quagga catshark
with four species recorded. In India, one species in
one genus has been recorded. Family Sphyrnidae (Hammerhead
sharks)
Species recorded from India • Eusphyra blochii-Winghead shark
• Monocentris japonica -Japanese pinecone fish • Sphyrna lewini-Scalloped hammerhead

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• Sphyrna mokarran-Great hammerhead
Order Carcharhiniformes • Sphyrna zygaena-Round-headed hammerhead

Family Carcharhinidae (Requiem sharks) Family Triakidae (Houndsharks)

• Carcharhinus altimus-Bignose shark • Iago omanensis-Bigeye houndshark
• Carcharhinus amblyrhynchoides-Graceful shark • Mustelus mosis-Arabian smooth-hound
• Carcharhinus amboinensis-Pigeye shark
• Carcharhinus brevipinna-Spinner shark Order Clupeiformes
• Carcharhinus dussumieri-Whitecheek shark
• Carcharhinus falciformis-Blackspot shark, Silky shark Herrings are certainly among the most valuable
• Carcharhinus hemiodon-Pondicherry shark commercial fishes in the world, being important food
• Carcharhinus leucas-Bull shark fishes in many countries and serving as a chief source of
• Carcharhinus limbatus-Blacktip shark fish meal for animal feeds. The order Clupeiformes include
• Carcharhinus longimanus-Oceanic whitetip shark anchovies, herrings, sardines, shads, gizzard shads, wolf
• Carcharhinus macloti-Maclot's shark herrings and their relatives. The current classification by
• Carcharhinus melanopterus-Blacktip reef shark Nelson (2006), divided the Clupeoidei into four families:
• Carcharhinus sealei-Blackspot shark the Engraulidae with two subfamilies with 139 species in
• Carcharhinus sorrah-Sorrah, Spottail shark 16 genera, the Pristigasteridae with two subfamilies and
• Galeocerdo cuvier-Tiger shark 37 species in 9 genera, the Chirocentridae with 2 species
• Lamiopsis temminckii-Broadfin shark in one genus and the Clupeidae with five subfamilies with
• Loxodon macrorhinus-Sliteye shark 216 species in 66 genera.
• Negaprion acutidens-Sicklefin lemon shark
• Prionace glauca-Blue shark Family Chirocentridae (Wolf herring)
• Rhizoprionodon acutus-Milk shark
• Rhizoprionodon oligolinx-Grey dog shark Species recorded from India

43
 • Chirocentrus dorab-Dorab wolf-herring Family Engraulidae (Anchovies)
• Chirocentrus nudus-Whitefin wolf-herring
The anchovies are small herring-like fishes; but they
Family Clupeidae (Herring, shads and are easily distinguishable from the herrings by the fact
sardines) that their mouths are much larger and gape much
farther back, but are on the lower side of the head,
The family consists over 31 species in 16 genera. and are overhung by the upper jaw, which projects
like a short piglike snout in some species.
Species recorded from India
• Amblygaster clupeoides-Bleeker Though 16 genera with 139 speciesare recorded
smoothbelly sardinella worldwide, five genera with 35 species are recorded
• Amblygaster leiogaster -Smooth-belly sardinella from India.
• Amblygaster sirm-Spotted sardinella
• Anodontostoma chacunda -Chacunda Species recorded from India
gizzard shad • Coilia dussumieri-Goldspotted grenadier anchovy
• Dayella malabarica -Day's round herring • Coilia grayii-Gray's grenadier anchovy
• Ehirava fluviatilis-Malabar sprat • Coilia neglecta-Neglected grenadier anchovy
• Escualosa thoracata -White sardine • Coilia ramcarati-Ramcarat grenadier anchovy
• Gonialosa manmina -Ganges river gizzard shad • Encrasicholina devisi-Devis' anchovy
• Gudusia chapra-Indian river shad • Encrasicholina heteroloba-Shorthead anchovy
• Herklotsichthys • Encrasicholina punctifer-Buccaneer anchovy
quadrimaculatus -Bluestripe herring • Setipinna breviceps Shorthead hairfin anchovy
• Hilsa kelee -Kelee shad • Setipinna brevifilis (endemic) Short-hairfin anchovy
• Nematalosa galatheae-Galathea gizzard shad • Setipinna phasa (endemic) Gangetic hairfin anchovy
• Nematalosa nasus -Hairback, Bloch's • Setipinna taty-Scaly hairfin anchovy
gizzard shad • Setipinna tenuifilis-Common hairfin anchovy
• Opisthopterus tardoore -Tardoore • Stolephorus andhraensis-Andhra anchovy
• Sardinella albella -White sardinella • Stolephorus baganensis-Bagan anchovy
• Sardinella brachysoma-Deepbody sardinella • Stolephorus commersonnii-Commerson's anchovy
• Sardinella fimbriata -Fringescale sardinella • Stolephorus dubiosus-Thai anchovy
• Sardinella gibbosa -Goldstripe sardinella • Stolephorus indicus-Indian anchovy
• Sardinella jussieu -Mauritian sardinella • Stolephorus insularis-Hardenberg's anchovy
• Sardinella longiceps -Indian oil sardine • Stolephorus waitei-Spotty-face anchovy
• Sardinella melanura-Blacktip sardinella • Thryssa baelama-Baelama anchovy
• Sardinella sindensis -Sind sardinella • Thryssa dayi-Day's thryssa
Central Marine Fisheries Research Institute

• Spratelloides delicatulus-Delicate round herring • Thryssa dussumieri-Dussumier's thryssa

• Spratelloides gracilis -Silver-stripe round herring • Thryssa encrasicholoides-False baelama anchovy
• Tenualosa ilisha-Ilish • Thryssa gautamiensis-Gautama thryssa
• Tenualosa toli-Toli shad • Thryssa hamiltonii-Hamilton's thryssa
• Thryssa kammalensoides-Godavari thryssa
Family Dussumieriidae (Round herring) • Thryssa malabarica-Malabar thryssa
• Thryssa mystax-Moustached thryssa
Though two genera are recorded worldwide, one • Thryssa polybranchialis-Humphead thryssa
genus with two species are reported. • Thryssa purava-Oblique-jaw thryssa
• Thryssa setirostris-Longjaw thryssa
Species recorded from India • Thryssa spinidens-Bengal thryssa
• Dussumieria acuta-Rainbow sardine • Thryssa stenosoma-Slender thryssa
• Dussumieria elopsoides-Slender rainbow sardine • Thryssa vitrirostris-Orangemouth anchovy

44
Family Pristigasteridae (Pristigasterids) • Heptranchias perlo (native) Sharpnose
• Ilisha elongata-Elongate ilisha sevengill shark
• Ilisha filigera-Coromandel ilisha
• Ilisha kampeni-Kampen's ilisha Order Lamniformes
• Ilisha megaloptera-Bigeye ilisha
• Ilisha melastoma-Indian ilisha Family Alopiidae (Thresher sharks)
• Ilisha striatula-Banded ilisha • Alopias pelagicus (native) Pelagic thresher
• Pellona dayi-Day's pellona • Alopias superciliosus (native) Bigeye thresher
• Pellona ditchela-Indian pellona • Alopias vulpinus (native) Thintail thresher

Order Elopiformes Family Lamnidae (Mackerel sharks or

white shark)
Family Elopidae (Tenpounders) • Isurus oxyrinchus (native) Shortfin shark,
• Elops machnata (native) Ladyfish, tenpounder Shortfin mako

Family Megalopidae (Tarpons) Family Odontaspididae (Sand tigers)

• Megalops cyprinoides (native) Oxeye tarpon, Indo- • Carcharias taurus (native), Sand tiger shark
Pacific tarpon • Carcharias tricuspidatus (native) Blue nurse sand-
tiger, Indian sand tiger
Order Gadiformes
Order Lampriformes
Family Bregmacerotidae (Codlets)
• Bregmaceros mcclellandi (native) Spotted codlet Family Lophotidae (Crestfishes)

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• Eumecichthys fiski (native), Unicorn crestfish
Family Macrouridae (Grenadiers or rattails)
• Bathygadus furvescens (native) Family Veliferidae (Velifers)
• Caelorinchus flabellispinnis (native) • Velifer hypselopterus (native), Sailfin velifer
• Caelorinchus parallelus (native) Spiny grenadier
• Coryphaenoides hextii (native) Order Lophiiformes
• Coryphaenoides macrolophus (native)
• Coryphaenoides woodmasoni (native) Family Antennariidae (Frogfishes)
• Gadomus multifilis (native) • Antennarius coccineus (native), Scarlet frogfish
• Sphagemacrurus pumiliceps (native) • Antennarius hispidus (native), Shaggy angler
• Antennarius indicus (native), Indian frogfish
Order Gasterosteiformes • Antennarius nummifer (native), Spotfin frogfish
• Antennarius pictus (native), Painted frogfish
Family Pegasidae (Seamoths) • Antennarius striatus (native), Striated frogfish
• Eurypegasus draconis (native) Short dragonfish • Histrio histrio (native), Sargassum fish
• Pegasus laternarius (native)
• Pegasus volitans (native) Longtail seamoth Family Chaunacidae (Sea toads)
• Chaunax pictus (native), Pink frogmouth
Family Chanidae (Milkfish)
Family Diceratiidae (Double anglers)
Chanos chanos (native) Milkfish • Diceratias bispinosus (native), Two-rod anglerfish

Order Hexanchiformes Family Lophiidae (Goosefishes)

• Lophiodes gracilimanus (native)
Family Hexanchidae (Cow sharks) • Lophiodes mutilus (native), Smooth angler

45
 • Lophiomus setigerus (native), Blackmouth angler • Carapus mourlani (native), Star pearlfish
• Encheliophis gracilis (native), Graceful pearlfish
Family Ogcocephalidae (Batfishes) • Encheliophis homei (native), Silver pearlfish
• Dibranchus nasutus (native)
• Halicmetus ruber (native) Family Ophidiidae (Cusk-eels)
• Halieutaea coccinea (native) • Bassozetus glutinosus (native)
• Halieutaea indica (native), Indian handfish • Brotula multibarbata (native), Goatsbeard brotula
• Halieutaea stellata (native), Starry handfish • Dicrolene introniger (questionable), Digitate
cusk eel
Family Oneirodidae (Dreamers) • Enchelybrotula paucidens (native)
• Lophodolos indicus (native) • Glyptophidium argenteum (native)
• Holcomycteronus pterotus (native)
• Monomitopus conjugator (native)
Order Myctophiformes • Monomitopus nigripinnis (native)
• Neobythites steatiticus (native)
Family Myctophidae (Lanternfishes) • Porogadus trichiurus (native)
• Bolinichthys pyrsobolus (native) • Tauredophidium hextii (native)
• Benthosema petrotum Skinnycheek lantern fish
• Diaphus luetkeni (native)
• Diaphus splendidus (native) Order Orectolobiformes
• Hygophum reinhardtii (native), Reinhardt's
lantern fish Family Ginglymostomatidae (Nurse
• Myctophum affine (questionable), Metallic sharks)
lantern fish • Nebrius ferrugineus (native) Giant sleepy shark,
• Myctophum aurolaternatum (native), Tawny nurse shark
Golden lanternfish
• Myctophum indicum (native) Family Hemiscylliidae (Bamboo sharks)
• Myctophum spinosum (native), Spiny lantern fish • Chiloscyllium arabicum (native),
• Symbolophorus evermanni (native), Evermann's Arabian carpetshark
lantern fish • Chiloscyllium griseum (native) Grey bambooshark
• Chiloscyllium indicum (native)
Order Notacanthiformes Slender bambooshark
• Chiloscyllium plagiosum (native),
Family Halosauridae (Halosaurs) Whitespotted bambooshark
• Aldrovandia affinis (native), Gilbert's halosaurid fish • Chiloscyllium punctatum (native),
Central Marine Fisheries Research Institute

• Aldrovandia mediorostris (native) Brownbanded bambooshark

• Aldrovandia phalacra (native), Hawaiian
halosaurid fish Family Rhincodontidae (Whale shark)
• Halosaurus parvipennis (native) • Rhincodon typus (native) Whale shark, Whale shark

Order Ophidiiformes Family Stegostomatidae (Zebra sharks)

• Stegostoma fasciatum (native) Zebra shark, Zebra shark
Family Bythitidae (Viviparous brotulas)
• Dinematichthys iluocoeteoides (native), Yellow Order Osmeriformes
pigmy brotula
Family Alepocephalidae (Slickheads)
Family Carapidae (Pearlfishes) • Aulastomatomorpha phospherops (native)
• Carapus boraborensis (native), Pinhead pearlfish • Bathytroctes squamosus (questionable)

46
• Narcetes erimelas (native) • Zebrasoma veliferum
• Zebrasoma xanthurum (native), Yellowtail tang
Family Platytroctidae (Tubeshoulders)
• Platytroctes apus (native), Legless searsid Family Acropomatidae (Lanternbellies,
• Platytroctes mirus (native), Leaf searsid • Acropoma japonicum (native), Glowbelly
• Synagrops philippinensis (native)

Order Perciformes Family Ambassidae (Asiatic glassfishes)

• Ambassis ambassis (native) Commerson's
Family Acanthuridae (Surgeonfishes, glassy perchlet
tangs, unicornfishes) • Ambassis buton (native) Buton glassy perchlet
• Acanthurus gahhm, Black surgeonfish • Ambassis dussumieri (native) Malabar
• Acanthurus leucosternon (native), glassy perchlet
Powderblue surgeonfish • Ambassis gymnocephalus (native) Bald
• Acanthurus lineatus (native) Blue glassy perchlet
linned surgeonfish • Ambassis interrupta (native) Interrupta glassy
• Acanthurus mata (native), Elongate surgeonfish perchlet, Long-spined glass perchlet
• Acanthurus nigricans (native), • Ambassis kopsii (native) Singapore glassy
Whitecheek surgeonfish perchlet, Freckled hawkfish
• Acanthurus nigrofuscus (native), • Ambassis macracanthus (native), Estuarine
Brown surgeonfish glass perchlet
• Acanthurus nigroris (native), • Ambassis miops (native) Myops glassy perchlet,
Bluelined surgeonfish Flag-tailed glass perchlet

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• Acanthurus tennentii (native), • Ambassis nalua (native) Nalua-chanda,
Doubleband surgeonfish Scalloped perchlet
• Acanthurus thompsoni (native), • Ambassis urotaenia (native) Banded-tail glassy
Thompson's surgeonfish perchlet, Banded-tail glassy perchlet
• Acanthurus triostegus (native), • Chanda nama (native) Elongate glass-perchlet
Convict surgeonfish • Parambassis dayi (endemic) Day's glass fish
• Acanthurus xanthopterus (native), • Parambassis lala (native) Highfin glassy perchlet
Yellowfin surgeonfish • Parambassis ranga (native) Indian glassy fish
• Ctenochaetus striatus (native), • Parambassis thomassi (endemic) Western Ghat
Striated surgeonfish glassy perchlet
• Ctenochaetus strigosus (questionable), • Pseudambassis baculis (native) Himalayan
Spotted surgeonfish glassy perchlett
• Naso brachycentron (native),
Humpback unicornfish Family Ammodytidae (Sand lances)
• Naso brevirostris (native), Spotted unicornfish • Bleekeria kallolepis (native)
• Naso lituratus (misidentification),
Orangespine unicornfish Family Anabantidae (Climbing
• Naso tonganus (native), Bulbnose unicornfish gouramies)
• Naso tuberosus (native), Humpnose unicornfish • Anabas cobojius (native) Gangetic koi
• Naso unicornis (native), Bluespine unicornfish • Anabas testudineus (native) Climbing perch
• Naso vlamingii (native), Bignose unicornfish
• Paracanthurus hepatus (native), Family Apogonidae (Cardinalfishes)
Palette surgeonfish • Apogon coccineus (native), Ruby cardinalfish
• Zebrasoma flavescens (questionable), • Apogon fasciatus (native), Broad-
Yellow tang banded cardinalfish

47
 • Apogon fleurieu (native), Cardinalfish • Badis tuivaiei (native)
• Apogon fraenatus (native), Bridled cardinalfish • Dario dario (native) Scarlet badis
• Apogon guamensis (native), Guam cardinalfish
• Apogon holotaenia (native), Family Bathyclupeidae
Copperstriped cardinalfish • Bathyclupea hoskynii (native)
• Apogon kallopterus (native), Iridescent cardinalfish
• Apogon leptacanthus (native), Threadfin cardinalfish Family Blenniidae (Combtooth blennies)
• Apogon moluccensis (native), • Alticus kirkii (native) Kirk's blenny
Moluccan cardinalfish • Andamia reyi (native) Suckerlip blenny
• Apogon novemfasciatus, • Antennablennius bifilum (native) Horned rockskipper
Sevenstriped cardinalfish • Aspidontus tractus (native)
• Apogon oxina (native) • Blenniella leopardus (native)
• Apogon poecilopterus (native), Pearly- • Blenniella periophthalmus (native) Blue-
finned cardinalfish dashed rockskipper
• Apogon quadrifasciatus (native), • Cirripectes castaneus (native) Chestnut eyelash-
Twostripe cardinal blenny
• Apogon sangiensis (native), Sangi cardinalfish • Cirripectes filamentosus (native) Filamentous blenny
• Apogon savayensis (native), Samoan cardinalfish • Cirripectes perustus (native) Flaming blenny
• Apogon taeniophorus (native), Reef- • Cirripectes polyzona (native)
flat cardinalfish • Cirripectes quagga (native) Squiggly blenny
• Apogonichthys ocellatus (native), • Cirripectes stigmaticus (native) Red-streaked blenny
Ocellated cardinalfish • Cirripectes variolosus (questionable) Red-
• Archamia bleekeri (native) speckled blenny
• Archamia fucata (native), • Ecsenius midas (native) Persian blenny
Orangelined cardinalfish • Ecsenius pulcher (native)
• Cheilodipterus arabicus (native), Tiger cardinal • Enchelyurus kraussii (native) Krauss' blenny
• Cheilodipterus quinquelineatus (native), Five- • Entomacrodus striatus (native) Reef margin blenny
lined cardinalfish • Entomacrodus vermiculatus (native)
• Foa brachygramma (native), Weed cardinalfish Vermiculated blenny
• Fowleria aurita (native), Crosseyed cardinalfish • Exallias brevis (native) Leopard blenny
• Gymnapogon africanus (questionable), • Haptogenys bipunctata (native)
Crystal cardinal • Hirculops cornifer (native) Highbrow rockskipper
• Pseudamia gelatinosa (native), • Istiblennius dussumieri (native) Streaky rockskipper
Gelatinous cardinalfish • Istiblennius edentulus (native) Rippled rockskipper
• Rhabdamia cypselura (native), • Istiblennius lineatus (native) Lined rockskipper
Central Marine Fisheries Research Institute

Swallowtail cardinalfish • Istiblennius spilotus (native) Spotted rockskipper

• Rhabdamia gracilis (native), • Meiacanthus smithi (native) Disco blenny
Luminous cardinalfish • Mimoblennius atrocinctus (native)
• Omobranchus elongatus (native) Cloister blenny
Family Ariommatidae (Ariommatids) • Omobranchus fasciolatus (native) Arab blenny
• Omobranchus ferox (native) Gossamer blenny
Ariomma indica (native), Indian ariomma • Omobranchus obliquus (native)
• Omobranchus punctatus (native) Muzzled blenny
Family Badidae • Omobranchus zebra (native) Zebra blenny
• Badis assamensis (native) • Parablennius thysanius (native) Tasseled blenny
• Badis badis (native) Blue perch • Petroscirtes breviceps (native) Striped poison-fang
• Badis blosyrus (native) blenny mimic
• Badis kanabos (native) • Petroscirtes mitratus (native) Floral blenny

48
• Petroscirtes xestus (native) Xestus sabretooth blenny • Alepes melanoptera (native), Blackfin scad
• Plagiotremus rhinorhynchos (native) • Alepes vari (native), Herring scad
Bluestriped fangblenny • Atropus atropos (native), Cleftbelly trevally
• Plagiotremus tapeinosoma (native) Piano fangblenny • Atule mate (native), Yellowtail scad
• Salarias fasciatus (native) Jewelled blenny • Carangoides armatus (native), Longfin trevally
• Salarias reticulatus (sp. nov.)[6] • Carangoides chrysophrys (native), Longnose trevally
• Scartella emarginata (native) Maned blenny • Carangoides ciliarius (questionable)
• Xiphasia setifer (native) Hairtail blenny • Carangoides coeruleopinnatus (native),
Coastal trevally
Family Caesionidae (Fusiliers) • Carangoides dinema (native), Shadow trevally
• Caesio caerulaurea (native), Blue and gold fusilier • Carangoides ferdau (native), Blue trevally
• Caesio cuning (native), Redbelly yellowtail fusilier • C a r a n g o i d e s f u l v o g u t t a t u s   ( n a t i v e ) ,
• Caesio lunaris (native), Lunar fusilier Yellowspotted trevally
• Caesio teres (native), Yellow and blueback fusilier • Carangoides gymnostethus (native), Bludger
• Caesio varilineata (native), Variable-lined fusilier • Carangoides hedlandensis (native),
• Caesio xanthonota (native), Yellowback fusilier Bumpnose trevally
• Dipterygonotus balteatus (native), Mottled fusilier • Carangoides humerosus (native),
• Gymnocaesio gymnoptera (native), Slender fusilier Duskyshoulder trevally
• Pterocaesio chrysozona (native), Goldband fusilier • Carangoides malabaricus (native), Malabar trevally
• Pterocaesio digramma (questionable), Double- • Carangoides oblongus (native), Coachwhip trevally
lined fusilier • Carangoides orthogrammus (native), Island trevally
• Pterocaesio pisang (native), Banana fusilier • Carangoides plagiotaenia (native), Barcheek trevally
• Pterocaesio tessellata (native), One-stripe fusilier • Carangoides praeustus (native) Brown-backed

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

• Pterocaesio tile (native), Dark-banded fusilier trevally, Brownback trevally
• Carangoides talamparoides (native),
Family Callionymidae (Dragonets) Impostor trevally
• Bathycallionymus kaianus (native) • Caranx heberi (native), Blacktip trevally
• Callionymus carebares (native), Indian • Caranx hippos (questionable) Blacktailed trevally,
deepwater dragonet Crevalle jack
• Callionymus erythraeus (native), Smallhead dragonet • Caranx ignobilis (native) Giant kingfish,
• Callionymus fluviatilis (native) River dragonet, Giant trevally
River dragonet • Caranx lugubris (native), Black jack
• Callionymus japonicus (questionable) • Caranx melampygus (native), Bluefin trevally
• Callionymus kotthausi (native) • Caranx papuensis (native), Brassy trevally
• Callionymus margaretae (native), • Caranx sexfasciatus (native), Bigeye trevally
Margaret's dragonet • Caranx tille (native), Tille trevally
• Callionymus megastomus (native) • Decapterus macarellus (native), Mackerel scad
• Callionymus sagitta (native) Arrow headed • Decapterus macrosoma (native), Shortfin scad
dragonet, Arrow dragonet • Decapterus russelli (native), Indian scad
• Eleutherochir opercularis (native) Indian dragonet, • Elagatis bipinnulata (native), Rainbow runner
Flap-gilled dragonet • Gnathanodon speciosus (native), Golden trevally
• Megalaspis cordyla (native) Torpedo scad, Torpedo scad
Family Carangidae (Jacks and • Naucrates ductor (native), Pilotfish
pompanos) • Parastromateus niger (native) Brown pomfret,
• Alectis ciliaris (native), African pompano Black pomfret
• Alectis indicus (native), Indian threadfish • Scomberoides commersonnianus (native),
• Alepes djedaba (native), Shrimp scad Talang queenfish
• Alepes kleinii (native), Razorbelly scad • Scomberoides lysan (native) Double-spotted

49
 queenfish, Doublespotted queenfish • Chaetodon trifasciatus (native), Melon butterflyfish
• Scomberoides tala (native), Barred queenfish • Chaetodon vagabundus (native),
• Scomberoides tol (native), Needlescaled queenfish Vagabond butterflyfish
• Selar boops (native), Oxeye scad • Chaetodon xanthocephalus (native),
• Selar crumenophthalmus (native), Bigeye scad Yellowhead butterflyfish
• Selaroides leptolepis (native), Yellowstripe scad • Chelmon rostratus (native), Copperband butterflyfish
• Seriola lalandi (native), Yellowtail amberjack • Forcipiger longirostris (native),
• Seriola rivoliana (native), Almaco jack Longnose butterflyfish
• Seriolina nigrofasciata (native), Blackbanded trevally • Hemitaurichthys zoster (native), Brown-and-
• Trachinotus baillonii (native), Smallspotted dart white butterflyfish
• Trachinotus blochii (native), Snubnose pompano • Heniochus acuminatus (native) Pennant coral fish,
• Trachinotus botla (native), Largespotted dart Pennant coralfish
• Trachinotus mookalee (native), Indian pompano • Heniochus chrysostomus (native),
• Ulua mentalis (native), Longrakered trevally Threeband pennantfish
• Uraspis helvola (native), Whitemouth jack • Heniochus monoceros (native), Masked bannerfish
• Uraspis secunda (native), Cottonmouth jack • Heniochus pleurotaenia (native),
• Uraspis uraspis (native), Whitetongue jack Phantom bannerfish
• Heniochus singularius (native), Singular bannerfish
Family Centrogenyidae • Parachaetodon ocellatus (native),
• Centrogenys vaigiensis (native), False scorpionfish Sixspine butterflyfish

Family Centrolophidae (Medusafishes) Family Champsodontidae

• Psenopsis cyanea (native), Indian ruff • Champsodon capensis (questionable), Gaper
• Champsodon vorax (questionable)
Family Cepolidae (Bandfishes)
• Acanthocepola indica (native) Family Cichlidae (Cichlids)
• Etroplus maculatus Orange Chromide
Family Chaetodontidae (Butterflyfishes) • Etroplus canarensis (endemic) Canara pearlspot
• Chaetodon andamanensis (native) • Etroplus maculatus (native) Orange chromide
• Chaetodon auriga (native), Threadfin butterflyfish • Etroplus suratensis (native) Green chromide
• Chaetodon bennetti (native), Bluelashed butterflyfish • Oreochromis mossambicus (introduced)
• Chaetodon citrinellus (native), Speckled butterflyfish Mozambique cichlid
• Chaetodon collare (native), Redtail butterflyfish • Oreochromis niloticus (introduced), Nile tilapia
• Chaetodon decussatus (native), Indian
vagabond butterflyfish Family Cirrhitidae (Hawkfishes)
Central Marine Fisheries Research Institute

• Chaetodon falcula (native), • Cirrhitichthys aureus (native), Yellow hawkfish

Blackwedged butterflyfish • Cirrhitichthys bleekeri (native)
• Chaetodon kleinii (native), Sunburst butterflyfish • Cirrhitus pinnulatus (native), Stocky hawkfish
• Chaetodon lunula (native), Raccoon butterflyfish • Paracirrhites forsteri (native), Blackside hawkfish
• Chaetodon melannotus (native),
Blackback butterflyfish Family Coryphaenidae (Dolphinfishes)
• Chaetodon meyeri (native), Scrawled butterflyfish • Coryphaena hippurus (native), Common dolphinfish
• Chaetodon octofasciatus (native),
Eightband butterflyfish Family Datnioididae
• Chaetodon punctatofasciatus (questionable), • Datnioides polota (native) Four-barred tigerfish
Spotband butterflyfish
• Chaetodon speculum (native), Mirror butterflyfish Family Drepaneidae (Sicklefishes)
• Chaetodon trifascialis (native), Chevron butterflyfish • Drepane longimana (native), Concertina fish

50
• Drepane punctata (native) Spotted sicklefish • Gerres setifer (native) Small Bengal silver-biddy
• Pentaprion longimanus (native), Longfin mojarra
Family Echeneidae (Remoras)
• Echeneis naucrates (native), Live sharksucker Gobiidae (Gobies)
• Phtheirichthys lineatus (native), Slender suckerfish • Acentrogobius bontii (native)
• Remora osteochir (native), Marlin sucker • Acentrogobius caninus (native), Tropical sand goby
• Remora remora (native) Common remora • Acentrogobius cyanomos (native)
• Remorina albescens (native), White suckerfish • Acentrogobius griseus (endemic) Grey goby
• Acentrogobius masoni (native)
Family Eleotridae (Sleepers) • Acentrogobius viridipunctatus (native), Spotted
• Bostrychus sinensis (native), Four-eyed sleeper green goby
• Butis amboinensis (native), Olive flathead-gudgeon • Amblyeleotris gymnocephala (native),
• Butis butis (native) Duckbill sleeper Masked shrimpgoby
• Butis gymnopomus (native) • Amblygobius albimaculatus (native), Butterfly goby
• Butis koilomatodon (native), Mud sleeper • Amblyotrypauchen arctocephalus (native)
• Butis humeralis (native) Blackspot sleeper • Acentrogobius madraspatensis (native)
• Eleotris fusca (native) Dusky sleeperEleotris • Apocryptes bato (native)
lutea (native) Lutea sleeper • Apocryptodon madurensis (native)
• Eleotris melanosoma (native) Broadhead sleeper • Asterropteryx semipunctata (native), Starry goby
• Incara multisquamatus (native) Incara • Awaous grammepomus (native), Scribbled goby
• Odonteleotris macrodon (native) Gangetic sleeper • Awaous guamensis (native)
• Ophiocara porocephala (native) Flathead sleeper • Awaous melanocephalus (native), Largesnout goby
• Awaous ocellaris (native)

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Family Emmelichthyidae (Rovers) • Bathygobius cyclopterus (native), Spotted frillgoby
• Erythrocles schlegelli • Bathygobius fuscus (native), Dusky frillgoby
• Bathygobius niger (native), Black minigoby
Family Ephippidae (Spadefishes, • Bathygobius petrophilus (questionable)
batfishes and scats) • Bathygobius smithi (native)
• Ephippus orbis (native), Orbfish • Boleophthalmus dussumieri (native)
• Platax teira (native), Tiera batfish • Brachyamblyopus brachysoma (native)
• Brachygobius nunus (native) Bumblebee goby
Gempylidae (Snake mackerels) • Callogobius hasseltii (native), Hasselt's goby
• Epinnula magistralis (native), Domine • Callogobius seshaiyai (endemic)
• Gempylus serpens (native), Snake mackerel • Ctenogobiops crocineus (native),
• Neoepinnula orientalis (native), Sackfish Silverspot shrimpgoby
• Promethichthys prometheus (native), Roudi escolar • Paratrypauchen microcephalus (native) Comb goby
• Rexea bengalensis (native), Bengal escolar • Drombus globiceps (native) Bighead goby
• Rexea prometheoides (native), Royal escolar • Egglestonichthys melanoptera (native)
• Ruvettus pretiosus (native), Oilfish • Pseudogobiopsis oligactis (native)
• Eviota distigma (native), Twospot pygmy goby
Gerreidae (Mojarras) • Exyrias puntang (native), Puntang goby
• Gerres erythrourus (native), Deep-bodied mojarra • Favonigobius reichei (native), Indo-Pacific tropical
• Gerres filamentosus (native) Whiptail silver-biddy sand goby
• Gerres limbatus (native), Saddleback silver-biddy • Fusigobius neophytus (native), Common fusegoby
• Gerres longirostris (native) Strongspine silver-biddy • Glossogobius giuris (native) Tank goby
• Gerres macracanthus (native) • Glossogobius kokius (native)
• Gerres oblongus (native), Slender silverbiddy • Glossogobius mas (native)
• Gerres oyena (native) Common silvery-biddy • Pseudogobiopsis oligactis (native)

51
 • Gobiodon citrinus (native), Poison goby • Pleurosicya bilobata (native), Bilobed ghost goby
• Gobiodon rivulatus (native), Rippled coralgoby • Priolepis eugenius (native), Noble goby
• Gobiopsis canalis (native), Checkered goby • Priolepis inhaca (native), Brick goby
• Gobiopsis macrostomu (native), Longjaw goby • Psammogobius biocellatus (native) Sleepy goby
• Gobiopsis woodsi (native) • Pseudapocryptes elongatus (native)
• Gobiopterus chuno (native), Glass goby • Pseudogobius javanicus (native)
• Hemigobius hoevenii (questionable) • Pseudogobius gastrospinos
• Hetereleotris zonata (native), Goggles • Pseudogobius poicilosoma (native)
• Istigobius diadema (native) • Pseudotrypauchen multiradiatus (native)
• Istigobius ornatus (native), Ornate goby • Scartelaos cantoris (native)
• Istigobius spence (native), Pearl goby • Scartelaos histophorus (native), Walking goby
• Mahidolia mystacina (native) Smiling goby, Flagfin • Scartelaos tenuis (questionable), Indian Ocean
prawn goby slender mudskipper
• Obliquogobius cometes (native) • Schismatogobius deraniyagalai (native), Redneck goby
• Odontamblyopus roseus (native) • Sicyopterus griseus (native)
• Odontamblyopus rubicundus (native) • Sicyopterus microcephalus (native)
Rubicundus eelgoby • Silhouettea indica (native)
• Oligolepis acutipennis (native) Sharptail goby • Stenogobius gymnopomus (native)
• Oligolepis cylindriceps (native) • Stenogobius laterisquamatus (questionable)
• Oxuderces dentatus (native) • Stigmatogobius minima (native)
• Oxyurichthys dasi (native) • Stigmatogobius sadanundio (native)
• Oxyurichthys formosanus (native) • Taenioides anguillaris (native) Anguilla eelgoby
• Oxyurichthys microlepis (native) Maned goby • Taenioides buchanani (native) Burmese gobyeel
• Oxyurichthys ophthalmonema (native), • Taenioides cirratus (native) Hooghly gobyeel
Eyebrow goby • Taenioides gracilis (native), Slender eel goby
• Oxyurichthys paulae (native) Jester goby • Trimma annosum (native), Greybearded pygmy goby
• Oxyurichthys tentacularis (native) • Trypauchen vagina (native) Burrowing goby
• Aulopareia ocellatus (native) • Trypauchenichthys sumatrensis (native)
• Parachaeturichthys polynema (native) Taileyed goby • Valenciennea muralis (native), Mural goby
• Paragobiodon echinocephalus (native), • Valenciennea sexguttata (native), Sixspot goby
Redhead goby • Valenciennea strigata (native), Blueband goby
• Parapocryptes rictuosus (native) • Yongeichthys nebulosus
• Parapocryptes serperaster (native) • Yongeichthys tuticorinensis (native)
• Periophthalmodon schlosseri (native),
Giant mudskipper Haemulidae (Grunts)
Central Marine Fisheries Research Institute

• Periophthalmodon septemradiatus (native) • Plectorhinchus pictum (native), Painted sweetlips

• Periophthalmus argentilineatus (native), • Plectorhinchus albovittatus (native), Two-
Barred mudskipper striped sweetlips
• Periophthalmus barbarus (questionable), • Plectorhinchus chubbi (native), Dusky rubberlip
Atlantic mudskipper • Plectorhinchus diagrammus (questionable),
• Periophthalmus chrysospilos (native) Striped sweetlips
• Periophthalmus minutus (native) • Plectorhinchus gibbosus (native), Harry hotlips
• Periophthalmus novemradiatus (native) • Plectorhinchus lineatus (native),
Pearse's mudskipper Yellowbanded sweetlips
• Periophthalmus waltoni (questionable), • Plectorhinchus picus (native), Painted sweetlip
Walton's mudskipper • Plectorhinchus schotaf (native), Minstrel sweetlip
• Periophthalmus weberi (questionable) • Pomadasys argenteus (native) Silver grunt
Weber's mudskipper • Pomadasys argyreus (native) Bluecheek silver grunt

52
• Pomadasys commersonnii (native) Spotted grunter • Coris aygula (native), Clown coris
• Pomadasys furcatus (native), Banded grunter • Coris formosa (native), Queen coris
• Pomadasys guoraca (native) • Coris gaimard (questionable), Yellowtail coris
• Pomadasys kaakan (native), Javelin grunter • wrasse
• Pomadasys maculatus (native), Saddle grunt • Gomphosus caeruleus (native), Green
• Pomadasys multimaculatum (native), Cock grunter birdmouth wrasse
• Pomadasys olivaceus (native), Olive grunt • Gomphosus varius (native), Bird wrasse
• Pomadasys stridens (native), Striped piggy • H a l i c h o e r e s hortulanus (native),
Checkerboard wrasse
Istiophoridae (Billfishes) • Halichoeres marginatus (native), Dusky wrasse
• Istiophorus platypterus (native) Sailfish, Indo- • Halichoeres nebulosus (native), Nebulous wrasse
Pacific sailfish • Halichoeres nigrescens (native), Bubblefin wrasse
• Istiompax indica (native) Short nosed sword fish, • Halichoeres scapularis (native), Zigzag wrasse
Black marlin • Halichoeres timorensis (native), Timor wrasse
• Makaira mazara (native), Indo-Pacific blue marlin • Halichoeres zeylonicus (native), Goldstripe wrasse
• Tetrapturus angustirostris (native), Shortbill spearfish • Hemigymnus fasciatus (native), Barred thicklip
• Hemigymnus melapterus (native), Blackeye thicklip
Kraemeriidae (Sand darters) • Hologymnosus annulatus (native), Ring wrasse
• Kraemeria samoensis (native), Samoan sand dart • Hologymnosus doliatus (native), Pastel ringwrasse
• Iniistius pavo (native), Peacock wrasse
Kuhliidae (Aholeholes) • Labroides dimidiatus (native), Bluestreak
• Kuhlia mugil (native) Barred flagtail cleaner wrasse
• Kuhlia rupestris (native) Rock flagtail • Leptojulis cyanopleura (native), Shoulder-

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

spot wrasse
Kurtidae (Nurseryfishes) • Macropharyngodon meleagris (native),
• Kurtus indicus (native) Indian humphead, Indian Blackspotted wrasse
hump head • Novaculichthys taeniourus (native),
Rockmover wrasse
Kyphosidae (Sea chubs) • Oxycheilinus bimaculatus (native), Two-spot wrasse
• Kyphosus bigibbus (native), Grey sea chub • Oxycheilinus digramma (native), Cheeklined wrasse
• Kyphosus cinerascens (native), Blue seachub • Pseudocheilinus hexataenia (native), Sixline wrasse
• Kyphosus vaigiensis (native), Brassy chub • Pseudodax moluccanus (native), Chiseltooth wrasse
• Pteragogus flagellifer (native), Cocktail wrasse
Labridae (Wrasses) • Stethojulis albovittata (native), Bluelined wrasse
• Anampses caeruleopunctatus (native), • Stethojulis balteata (questionable), Belted wrasse
Bluespotted wrasse • Stethojulis strigiventer (native), Three-ribbon wrasse
• Anampses meleagrides (native), Spotted wrasse • Stethojulis trilineata (native), Three-
• Bodianus neilli (native), Bay of Bengal hogfish lined rainbowfish
• Cheilinus chlorourus (native), Floral wrasse • Thalassoma amblycephalum (native),
• Cheilinus fasciatus (native), Redbreast wrasse Bluntheaded wrasse
• Cheilinus oxycephalus (native), Snooty wrasse • Thalassoma hardwicke (native), Sixbar wrasse
• Cheilinus trilobatus (native), Tripletail wrasse • Thalassoma jansenii (native), Jansen's wrasse
• Cheilinus undulatus (native), Humphead wrasse • Thalassoma lunare (native), Moon wrasse
• Cheilio inermis (native), Cigar wrasse • Thalassoma purpureum (native), Surge wrasse
• Choerodon anchorago (native), Orange- • Thalassoma quinquevittatum (native),
dotted tuskfish Fivestripe wrasse
• Choerodon robustus (native), Robust tuskfish • Choerodon typus (native), Blue-banded wrasse
• Cirrhilabrus exquisitus (native), Exquisite wrasse • Iniistius bimaculatus (native), Two-spot razorfish

53
 • Xyrichtys cyanifrons (native) • Lethrinus microdon (native), Smalltooth emperor
• Iniistius pentadactylus (native), Fivefinger wrasse • Lethrinus miniatus (questionable), Trumpet emperor
• Novaculops rajagopalani (native) • Lethrinus nebulosus (native), Spangled emperor
• Lethrinus obsoletus (native), Orange-
Lactariidae (False trevallies) striped emperor
• Lactarius lactarius (native) Big-jawed jumper • Lethrinus olivaceus (native), Longface emperor
• Lethrinus ornatus (native), Ornate emperor
Latidae (Lates perches) • L e t h r i n u s r u b r i o p e r c u l a t u s   ( n a t i v e ) ,
• Lates calcarifer (native) Barramundi, Barramundi Spotcheek emperor
• Psammoperca waigiensis (native) Waigeu seaperch, • Lethrinus semicinctus (native), Black blotch emperor
Waigieu seaperch • Lethrinus variegatus (native), Slender emperor
• Leiognathidae (Slimys, slipmouths, or ponyfishes) • Lethrinus xanthochilus (native), Yellowlip emperor
• Gazza achlamys (native), Smalltoothed ponyfish • Monotaxis grandoculis (native), Humpnose big-
• Gazza minuta (native) Toothpony, Toothpony eye bream
• Gazza rhombea (native), Rhomboid toothpony • Wattsia mossambica (native), Mozambique large-
• Leiognathus berbis (native), Berber ponyfish eye bream
• Photopectoralis bindus (native) Orangefin ponyfish
• Nuchequula blochii (native) Twoblotch ponyfish Lobotidae (Tripletails)
• Leiognathus brevirostris (native), Shortnose ponyfish • Lobotes surinamensis (native) Tripletail
• Leiognathus daura (native), Goldstripe ponyfish
• Nuchequula decorus (native) Shortnose ponyfish Lutjanidae (Snappers)
• Karalla dussumieri (native), Dussumier's ponyfish • Aphareus furca (native), Small toothed jobfish
• Equuilites elongatus (native), Slender ponyfish • Aphareus rutilans (native), Rusty jobfish
• Leiognathus equula (native) Common ponyfish • Aprion virescens (native), Green jobfish
• Aurigequla fasciatus (native), Striped ponyfish • Apsilus fuscus (questionable), African
• Equuilites leuciscus (native), Whipfin ponyfish forktail snapper
• E. lineolatus (native), Ornate ponyfish • Etelis carbunculus (native), Ruby snapper
• Aurigequula longispinis (questionable) • Etelis coruscans (native), Flame snapper
• Eubleekeria splendens (native), Splendid ponyfish • Etelis radiosus (native), Scarlet snapper
• Leiognathus striatus (native) • Lipocheilus carnolabrum (native), Tang's snapper
• Deveximentum insidiator (native) Pugnose ponyfish • Lutjanus argentimaculatus (native), Mangrove
• Secutor ruconius (native) Deep pugnose ponyfish red snapper
• Lutjanus bengalensis (native), Bengal snapper
Lethrinidae (Emperors or scavengers) • Lutjanus biguttatus (native), Two-spot
• Gnathodentex aureolineatus (native), Striped large- banded snapper
Central Marine Fisheries Research Institute

eye bream • Lutjanus bohar (native), Two-spot red snapper

• Gymnocranius elongatus (native), Forktail large- • Lutjanus carponotatus (native), Spanish
eye bream flag snapper
• Gymnocranius grandoculis (native), Blue-lined • Lutjanus decussatus (native), Checkered snapper
large-eye bream • Lutjanus ehrenbergii (native) Blackspot snapper
• Gymnocranius griseus (native), Grey large-eye bream • Lutjanus erythropterus (native), Crimson snapper
• Lethrinus conchyliatus (native), Redaxil emperor • Lutjanus fulviflamma (native), Dory snapper
• Lethrinus erythracanthus (native), Orange- • Lutjanus fulvus (native), Blacktail snapper
spotted emperor • Lutjanus gibbus (native), Humpback red snapper
• Lethrinus harak (native), Thumbprint emperor • Lutjanus guilcheri (native), Yellowfin red snapper
• Lethrinus lentjan (native) Pig-face bream, Pink • Lutjanus johnii (native) John's snapper
ear emperor • Lutjanus kasmira (native), Common
• Lethrinus mahsena (native), Sky emperor bluestripe snapper

54
• Lutjanus lemniscatus (native), • Planiliza melinoptera (native) Giantscale mullet,
Yellowstreaked snapper Otomebora mullet
• Lutjanus lunulatus (native), Lunartail snapper • Planiliza parsia (native) Goldspot mullet
• Lutjanus lutjanus (native), Bigeye snapper • Planiliza subviridis (native) Greenback mullet
• Lutjanus madras (native), Indian snapper • Planiliza tade (native) Tade mullet, Tade mullet
• Lutjanus malabaricus (native), Malabar • Ellochelon vaigiensis (native) Squaretail mullet
blood snapper • Mugil cephalus (native) Flathead mullet
• Lutjanus monostigma (native), Onespot snapper • Plicomugil labiosus (native), Hornlip mullet
• Lutjanus quinquelineatus (native), Five- • Rhinomugil corsula (native) Corsula mullet
lined snapper • Minimugil cascasia (native) Yellowtail mullet
• Lutjanus rivulatus (native), Blubberlip snapper • Sicamugil hamiltonii (questionable) Burmese mullet
• Lutjanus rufolineatus (questionable), Yellow- • Crenimugil buchanani (native) Bluetail mullet
lined snapper • Osteomugil cunnesius (native) Longarm mullet
• Lutjanus russellii (native), Russell's snapper • Crenimugil seheli (native) Bluespot mullet
• Lutjanus sanguineus (native), Humphead snapper • Osteomugil speigleri (native) Speigler's mullet
• Lutjanus sebae (native), Emperor red snapper
• Lutjanus vitta (native), Brownstripe red snapper Mullidae (Goatfishes)
• Macolor niger (native), Black and white snapper • Mulloidichthys flavolineatus (native),
• Paracaesio sordida (native), Dirty ordure snapper Yellowstripe goatfish
• Paracaesio xanthura (native), Yellowtail blue snapper • Parupeneus barberinus (native), Dash-and-
• Pinjalo lewisi (native), Slender pinjalo dot goatfish
• Pinjalo pinjalo (native), Pinjalo • Parupeneus ciliatus (native), Whitesaddle goatfish
• Pristipomoides filamentosus (native), • Parupeneus cyclostomus (native),

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Crimson jobfish Goldsaddle goatfish
• Pristipomoides multidens (native), • Parupeneus heptacanthus (native),
Goldbanded jobfish Cinnabar goatfish
• Pristipomoides sieboldii (native), Lavender • Parupeneus indicus (native), Indian goatfish
jobfish • Parupeneus macronemus (native),
• Pristipomoides typus, Sharptooth jobfish Longbarbel goatfish
• Pristipomoides zonatus, Oblique-banded snapper • Parupeneus margaritatus (questionable),
Pearly goatfish
Malacanthidae (Tilefishes) • Parupeneus multifasciatus (native),
• Hoplolatilus fronticinctus (native), Pastel tilefish Manybar goatfish
• Parupeneus pleurostigma (native), Sidespot goatfish
Menidae (Moonfish) • Parupeneus trifasciatus (native), Doublebar goatfish
• Mene maculata (native), Moonfish • Upeneus japonicus, Bensasi goatfish
• Upeneus luzonius, Dark-barred goatfish
Monodactylidae (Moonyfishes or • Upeneus moluccensis (native), Goldband goatfish
fingerfishes) • Upeneus sulphureus (native), Sulphur goatfish
• Monodactylus argenteus (native) Silvery moony • Upeneus sundaicus (native), Ochre-banded goatfish
• Monodactylus falciformis (native) Full moony • Upeneus taeniopterus (native), Finstripe goatfish
• Upeneus tragula (native), Freckled goatfish
Mugilidae (Mullets) • Upeneus vittatus (native), Yellowstriped goatfish
• Crenimugil crenilabis (native), Fringelip mullet
• Planiliza carinata (native), Keeled mullet Nemipteridae (Threadfin breams,
• Planiliza klunzingeri (native), Klunzinger's mullet Whiptail breams)
• Planiliza macrolepis (native) Largescale mullet • Nemipterus bipunctatus (native), Delagoa
• Planiliza mandapamensis (native), Indian mullet threadfin bream

55
 • Nemipterus furcosus (native), Fork-tailed • Psenes cyanophrys (native), Freckled driftfish
threadfin bream
• Nemipterus japonicus (native), Japanese Pempheridae (Sweepers)
threadfin bream • Parapriacanthus ransonneti (native), Pigmy sweeper
• Nemipterus marginatus (questionable), Red • Pempheris mangula (native), Black-edged sweeper
filament threadfin bream • Pempheris molucca (questionable)
• Nemipterus mesoprion (questionable), Mauvelip • Pempheris oualensis (native), Silver sweeper
threadfin bream • Pempheris vanicolensis (native), Vanikoro sweeper
• Nemipterus nematophorus (native), Doublewhip
threadfin bream Percophidae (Duckbills)
• Nemipterus nemurus (questionable), Redspine • Bembrops caudimacula (native)
threadfin bream • Bembrops platyrhynchus (native), Natal duckbill
• Nemipterus peronii (native), Notchedfin
threadfin bream Pinguipedidae (Sandperches)
• Nemipterus randalli (native), Randall's • Parapercis alboguttata (native), Whitespot sandsmelt
threadfin bream • P a r a p e r c i s hexophtalma (native),
• Nemipterus zysron (native), Slender Speckled sandperch
threadfin bream • Parapercis maculata (native), Harlequin sandperch
• Parascolopsis aspinosa (native), Smooth dwarf • Parapercis pulchella (native), Harlequin sandsmelt
monocle bream • Parapercis punctata (native)
• Parascolopsis boesemani (endemic), Redfin • Parapercis quadrispinosa (native)
dwarf monocle bream • Parapercis tetracantha (native),
• Parascolopsis eriomma (native), Rosy dwarf Reticulated sandperch
monocle bream
• Parascolopsis inermis (native), Unarmed dwarf Plesiopidae (Roundheads)
monocle bream • Acanthoplesiops indicus (native), Scottie
• Scolopsis bilineata (native), Two-lined • P l e s i o p s c o e r u l e o l i n e a t u s   ( n a t i v e ) ,
monocle bream Crimsontip longfin
• Scolopsis bimaculata (native), Thumbprint • Plesiops corallicola (native), Bluegill longfin
monocle bream
• Scolopsis ciliata (native), Saw-jawed Polynemidae (Threadfins)
monocle bream • Eleutheronema tetradactylum (native) White
• Scolopsis frenata (native), Bridled monocle bream salmon, Fourfinger threadfin
• Scolopsis ghanam (native), Arabian monocle bream • Filimanus heptadactyla (native) Sevenfinger threadfin
• Scolopsis lineata (native), Striped monocle bream • Filimanus similis (native)
Central Marine Fisheries Research Institute

• Scolopsis margaritifera (questionable), Pearly • Filimanus xanthonema (native),

monocle bream Yellowthread threadfin
• Scolopsis taeniata (native), Black-streaked • Leptomelanosoma indicum (native) Indian
monocle bream threadfin, Indian threadfin
• Scolopsis taenioptera (questionable), Lattice • Polydactylus macrochir (native), King threadfin
monocle bream • Polydactylus microstoma (native), Small-
• Scolopsis vosmeri (native), Whitecheek mouthed threadfin
monocle bream • Polydactylus mullani (native)
• Scolopsis xenochrous (native), Oblique-barred • Polydactylus plebeius (native), Striped threadfin
monocle bream • Polydactylus sexfilis (native), Sixfinger threadfin
• Polydactylus sextarius (native) Blackspot threadfin
Nomeidae (Driftfishes) • Polynemus dubius (questionable) Borneo threadfin,
• Cubiceps whiteleggu (native), Indian driftfish Eastern paradise fish

56
• Polynemus paradiseus (native) Paradise threadfin, • Neopomacentrus taeniurus (native),
Paradise threadfin Freshwater demoiselle
• Plectroglyphidodon dickii (native), Blackbar devil
Pomacanthidae (Angelfishes) • Plectroglyphidodon lacrymatus (native),
• Apolemichthys xanthurus (native), Whitespotted devil
Yellowtail angelfish • Plectroglyphidodon leucozonus (native), Singlebar devil
• Centropyge bicolor (native), Bicolor angelfish • Pomacentrus pavo (native), Sapphire damsel
• Centropyge multispinis (native), Dusky angelfish • Premnas biaculeatus (native),
• Chaetodontoplus melanosoma (native), Black- Spinecheek anemonefish
velvet angelfish • Pristotis obtusirostris (native), Gulf damselfish
• Pomacanthus annularis (native) Ringed angle fish • Stegastes albifasciatus (native), Whitebar gregory
• Pomacanthus imperator (native), Emperor angelfish • Stegastes lividus (native), Blunt snout gregory
• Pomacanthus semicirculatus (native), • Stegastes nigricans (native), Dusky farmerfish
Semicircle angelfish • Stegastes obreptus (native), Western gregory

Pomacentridae (Damselfishes) Pomatomidae (Bluefishes)
• Abudefduf bengalensis (native), Bengal sergeant • Pomatomus saltatrix (native), Bluefish
• Abudefduf septemfasciatus (native),
Banded sergeant Priacanthidae (Bigeyes or catalufas)
• Abudefduf sexfasciatus (native), Scissortail sergeant • Heteropriacanthus cruentatus (native), Glasseye
• Abudefduf sordidus (native), Blackspot sergeant • Priacanthus hamrur (native), Moontail bullseye
• Abudefduf vaigiensis (native), Indo-Pacific sergeant • Priacanthus macracanthus (native), Red bigeye
• Amphiprion bicinctus, Twoband anemonefish • Priacanthus prolixus (native), Elongate bulleye

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

• Amphiprion chrysogaster, Mauritian anemonefish • Priacanthus tayenus (native), Purple-spotted bigeye
• Amphiprion ephippium (native), Saddle anemonefish • Pristigenys niphonia (native), Japanese bigeye
• Amphiprion frenatus, Tomato clownfish
• Amphiprion nigripes, Maldive anemonefish Pseudochromidae (Dottybacks)
• Amphiprion ocellaris (native), Clown anemonefish • Congrogadus subducens (native), Carpet eel-blenny
• Amphiprion percula, Orange clownfish • Halidesmus thomaseni (native), Thomasen's snakelet
• Amphiprion sebae (native), Sebae anemonefish • Pseudochromis caudalis (native)
• Cheiloprion labiatus (native), Big-lip damsel • Pseudochromis tapeinosoma (native),
• Chromis dimidiata (native), Chocolatedip chromis Blackmargin dottyback
• Chromis opercularis (native), Doublebar chromis
• Chromis ternatensis (native), Ternate chromis Ptereleotridae
• Chromis viridis (native), Blue green damselfish • Ptereleotris evides (native), Blackfin dartfish
• Chromis weberi (native), Weber's chromis • Ptereleotris microlepis (native), Blue gudgeon
• Chrysiptera biocellata (native), Twinspot damselfish
• Chrysiptera brownriggii (native), Surge damselfish Rachycentridae (Cobia)
• Chrysiptera cyanea (native), Sapphire devil • Rachycentron canadum (native), Cobia
• Chrysiptera glauca (native), Grey demoiselle
• Chrysiptera unimaculata (native), Scaridae (Parrotfishes)
Onespot demoiselle • Calotomus spinidens (native), Spinytooth parrotfish
• Dascyllus aruanus (native), Whitetail dascyllus • Calotomus viridescens, Viridescent parrotfish
• Dascyllus trimaculatus (native), Threespot dascyllus • Chlorurus enneacanthus (native), Captain parrotfish
• Dischistodus perspicillatus (native), White damsel • Chlorurus gibbus (native), Heavybeak parrotfish
• Dischistodus prosopotaenia (native), Honey-head damsel • Chlorurus oedema (native), Knothead parrotfish
• L e p i d o z y g u s t a p e i n o s o m a   ( n a t i v e ) , • Chlorurus sordidus (native), Daisy parrotfish
Fusilier damselfish • Hipposcarus harid (native), Candelamoa parrotfish

57
 • Leptoscarus vaigiensis (native), Marbled parrotfish • Johnius plagiostoma (native), Large-eye croaker
• Scarus ghobban (native), Blue-barred parrotfish • Kathala axillaris (native), Kathala croaker
• Scarus globiceps (native), Globehead parrotfish • Nibea maculata (native), Blotched croaker
• Scarus niger (native), Dusky parrotfish • Nibea soldado (native), Soldier croaker
• Scarus prasiognathos (native), Singapore parrotfish • Otolithes cuvieri (native), Lesser tigertooth croaker
• Scarus psittacus (native), Common parrotfish • Otolithes ruber (native), Tiger-toothed croaker
• Scarus quoyi (native), Quoy's parrotfish • Otolithoides biauritus (native) Bronze croaker
• Scarus rubroviolaceus (native), Ember parrotfish • Otolithoides pama (native), Pama croaker
• Scarus russelii (native), Eclipse parrotfish • Panna heterolepis (native) Hooghly croaker
• Scarus scaber (native), Fivesaddle parrotfish • Panna microdon (misidentification) Panna croaker
• Scarus tricolor (native), Tricolour parrotfish • Paranibea semiluctuosa (native), Half-
mourning croaker
Scatophagidae (Scats) • Pennahia anea (native), Greyfin croaker
• Scatophagus argus (native) Spotted scat • Pennahia macrocephalus (questionable), Big-head
Pennah croaker
Schindleriidae • Pennahia ovata (native)
• Schindleria pietschmanni (questionable) • Protonibea diacanthus (native) Spotted croaker
• Schindleria praematura (questionable), Schindler's fish • Pterotolithus maculatus (native) Blotch
• Umbrina canariensis (native), Canary drum
Sciaenidae (Drums or croakers)
• Argyrosomus amoyensis (native), Amoy croaker Scombridae (Mackerels, tunas, bonitos)
• Argyrosomus hololepidotus (misidentification), • Acanthocybium solandri (native), Wahoo
Madagascar meagre • Auxis rochei (native), Bullet tuna
• Argyrosomus japonicus (native), Japanese meagre • Auxis thazard (native), Frigate tuna
• Atrobucca alcocki (native) • Euthynnus affinis (native) Mackerel tuna, Kawakawa
• Atrobucca antonbruun (native) • Grammatorcynus bicarinatus (questionable),
• Atrobucca nibe (native), Longfin kob Shark mackerel
• Atrobucca trewavasae (native) • Gymnosarda unicolor (native) Dogtooth tuna,
• Bahaba chaptis (native) Chaptis bahaba Dogtooth tuna
• Chrysochir aureus (native), Reeve's croaker • Katsuwonus pelamis (native), Skipjack tuna
• Daysciaena albida (native) Two-bearded croaker, • Rastrelliger brachysoma (native), Short mackerel
Bengal corvina • Rastrelliger faughni (native), Island mackerel
• Dendrophysa russelii (native) Goatee croaker • Rastrelliger kanagurta (native), Indian mackerel
• Johnius amblycephalus (native), Bearded croaker • Sarda orientalis (native) Oriental bonito,
• Johnius belangerii (native) Belanger's croaker Striped bonito
Central Marine Fisheries Research Institute

• Johnius borneensis (native), Sharpnose • Scomber japonicus (native), Chub mackerel

hammer croaker • Scomberomorus commerson (native) King seer
• Johnius carouna (native) Caroun croaker • Scomberomorus guttatus (native) Spotted Spanish
• Johnius carutta (native) Karut croaker mackerel, Indo-Pacific king mackerel
• Johnius coitor (native) Coitor croaker • Scomberomorus lineolatus (native) Streaked seer
• Johnius dussumieri (native) Sharptooth • Thunnus albacares (native) Yellow fin tuna
hammer croaker • Thunnus obesus (native), Bigeye tuna
• Johnius elongatus (native), Spindle croaker • Thunnus orientalis (native), Pacific bluefin tuna
• Johnius gangeticus (endemic) Gangetic bola • Thunnus tonggol (native) Blue fin tuna, Longtail tuna
• Johnius glaucus (native), Pale spotfin croaker
• Johnius macropterus (native), Largefin croaker Serranidae (Sea basses: groupers and
• Johnius macrorhynus (native), Big-snout croaker fairy basslets)
• Johnius mannarensis (native), Mannar croaker • Aethaloperca rogaa (native), Redmouth grouper

58
• Anyperodon leucogrammicus (native), • Epinephelus merra (native), Honeycomb grouper
Slender grouper • Epinephelus morrhua (native), Comet grouper
• Cephalopholis argus (native) Balufana, Peacock hind • Epinephelus octofasciatus (native), Eightbar grouper
• Cephalopholis aurantia (native), Golden hind • Epinephelus poecilonotus (native), Dot-
• Cephalopholis boenak (native), Chocolate hind dash grouper
• Cephalopholis formosa (native), Bluelined hind • Epinephelus polylepis (native), Smallscaled grouper
• Cephalopholis leopardus (native), Leopard hind • Epinephelus polyphekadion (native),
• Cephalopholis miniata (native), Coral hind Camouflage grouper
• Cephalopholis sexmaculata (native), Sixblotch hind • Epinephelus quoyanus (native), Longfin grouper
• Cephalopholis sonnerati (native), Tomato hind • Epinephelus radiatus (native), Oblique-
• Cephalopholis urodeta (native), Darkfin hind banded grouper
• Chelidoperca investigatoris (native) • Epinephelus rivulatus (native), Halfmoon grouper
• Cromileptes altivelis (native), Humpback grouper • Epinephelus spilotoceps (native), Foursaddle grouper
• Diploprion bifasciatum (native), Barred soapfish • Epinephelus tauvina (native) Greasy rockcod,
• Epinephelus areolatus (native), Areolate grouper Greasy grouper
• Epinephelus bleekeri (native), Duskytail grouper • Epinephelus undulosus (native), Wavy-lined grouper
• Epinephelus chabaudi (native), Moustache grouper • Plectropomus areolatus (native), Squaretail
• E p i n e p h e l u s c h l o r o s t i g m a   ( n a t i v e ) , coral grouper
Brownspotted grouper • Plectropomus maculatus (questionable),
• Epinephelus coeruleopunctatus (native), Spotted coralgrouper
Whitespotted grouper • Pogonoperca sp. (native), Soapfish
• Epinephelus coioides (native), Orange- • Pseudanthias conspicuus (native)
spotted grouper • Pseudanthias squamipinnis (native), Sea goldie

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

• Epinephelus corallicola (questionable), • Variola louti (native), Yellow-edged lyretail
Coral grouper • Variola albimarginata 
• Epinephelus diacanthus (native), Spinycheek grouper
• Epinephelus epistictus (native), Dotted grouper Siganidae (Rabbitfishes)
• Epinephelus erythrurus (native), Cloudy grouper • Siganus argenteus (native), Streamlined spinefoot
• Epinephelus fasciatus (native), Blacktip grouper • Siganus canaliculatus (native), White-
• Epinephelus faveatus (native), Barred-chest grouper spotted spinefoot
• Epinephelus flavocaeruleus (native), Blue and • Siganus corallinus (native), Blue-spotted spinefoot
yellow grouper • Siganus fuscescens (native), Mottled spinefoot
• Epinephelus fuscoguttatus (native), Brown- • Siganus guttatus (native), Orange-spotted spinefoot
marbled grouper • Siganus javus (native), Streaked spinefoot
• Epinephelus hexagonatus (native), • Siganus lineatus (native), Golden-lined spinefoot
Starspotted grouper • Siganus punctatus (questionable),
• Epinephelus lanceolatus (native) Bridlebass, Goldspotted spinefoot
Giant grouper • Siganus spinus (native), Little spinefoot
• Epinephelus latifasciatus (native), Striped grouper • Siganus stellatus (native), Brownspotted spinefoot
• Epinephelus longispinis (native), Longspine grouper • Siganus vermiculatus (native),
• Epinephelus macrospilos (native), Snubnose grouper Vermiculated spinefoot
• Epinephelus maculatus (native), Highfin grouper • Siganus virgatus (native), Barhead spinefoot
• Epinephelus malabaricus (native) Malabar rockcod,
Malabar grouper Sillaginidae (Smelt-whitings)
• Epinephelus marginatus (questionable), • Sillago aeolus (native), Oriental sillago
Dusky grouper • Sillago chondropus (native), Clubfoot sillago
• Epinephelus melanostigma (native), One- • Sillago indica (endemic), Indian sillago
blotch grouper • Sillago ingenuua (native), Bay sillago

59
 • Sillago intermedius (native), Intermediate sillago • Benthodesmus oligoradiatus (native), Sparse-
• Sillago maculata (questionable), Trumpeter sillago rayed frostfish
• Sillago parvisquamis (questionable), Small- • Eupleurogrammus glossodon (native), Longtooth hairtail
scale sillago • E u p l e u r o g r a m m u s m u t i c u s   ( n a t i v e ) ,
• Sillago sihama (native) Silver sillago Smallhead hairtail
• Sillago soringa (endemic), Soringa sillago • Lepidopus caudatus (questionable),
• Sillago vincenti (endemic) Estuarine whiting Silver scabbardfish
• Lepturacanthus pantului (native)
Sparidae (Porgies) Coromandal ribbonfish
• Acanthopagrus berda (native) Riverbream, • Lepturacanthus savala (native) Small headed
Picnic seabream ribbon fish
• Acanthopagrus bifasciatus (native), • Trichiurus auriga (native), Pearly hairtail
Twobar seabream • Trichiurus gangeticus (native) Gangetic ribbonfish
• Acanthopagrus latus (native) Yellow seabream • Trichiurus lepturus (native), Largehead hairtail
• Argyrops spinifer (native), King soldierbream • Trichiurus russelli (native), Short-tailed hairtail
• Cheimerius nufar (native), Santer seabream
• Crenidens crenidens (native), Karenteen seabream Trichonotidae (Sand divers)
• Rhabdosargus sarba (native) Natal stumpnose • Trichonotus cyclograptus (native)
• Sparidentex hasta (native), Sobaity seabream • Trichonotus setiger (native), Spotted sand-diver

Sphyraenidae (Barracudas) Tripterygiidae (Threefin blennies)

• Sphyraena acutipinnis (native), Sharpfin barracuda • Enneapterygius elegans (native), Hourglass triplefin
• Sphyraena barracuda (native), Great barracuda • Enneapterygius fasciatus (native)
• Sphyraena flavicauda (native), Yellowtail barracuda • Enneapterygius pusillus (native), Highcrest triplefin
• Sphyraena forsteri (native), Bigeye barracuda • Helcogramma ellioti (native)
• Sphyraena jello (native), Pickhandle barracuda
• Sphyraena obtusata (native), Obtuse barracuda Uranoscopidae (Stargazers)
• Sphyraena putnamae (native) Sawtooth barracuda • Ichthyscopus lebeck (native), Longnosed stargazer
• Sphyraena qenie (native), Blackfin barracuda • Uranoscopus crassiceps (native)
• Pampus argenteus (native) Silver pomfret • Uranoscopus guttatus (native)
• Pampus chinensis (native) Chinese pomfret
Xenisthmidae
Symphysanodontidae • Gobiopterus smithi (endemic)
• Symphysanodon andersoni (native)
Central Marine Fisheries Research Institute

Terapontidae (Grunters or tigerperches) Xiphiidae (Swordfish)

• Pelates quadrilineatus (native) Fourlined terapon • Xiphias gladius (native), Swordfish
• Helotes sexlineatus (questionable), Six-
lined trumpeter Zanclidae (Moorish idol)
• Terapon jarbua (native) Jarbua terapon • Zanclus cornutus (native), Moorish idol
• Terapon puta (native) Smallscale terapon
• Terapon theraps (native) Banded grunter Pleuronectiformes
Bothidae (Lefteye flounders)
Toxotidae (Archerfishes) • Arnoglossus aspilos (native), Spotless
• Toxotes chatareus (native) Spotted archerfish lefteye flounder
• Toxotes jaculatrix (native) Banded archerfish • Arnoglossus tapeinosoma (native)
• Bothus leopardinus (questionable), Pacific
Trichiuridae (Cutlassfishes) leopard flounder

60
• Bothus myriaster (native), Indo-Pacific oval flounder Psettodidae (Psettodids)
• Bothus pantherinus (native), Leopard flounder • Psettodes erumei (native) Indian halibut
• Chascanopsetta lugubris (native), Pelican flounder
• Engyprosopon grandisquama (native), Samaridae (Flounders)
Largescale flounder • Samaris cristatus (native), Cockatoo
• Grammatobothus polyophthalmus (native), righteye flounder
Threespot flounder • Samariscus longimanus (native),
Longfinned flounder
Cynoglossidae (Tonguefishes)
• Cynoglossus arel (native), Largescale tonguesole Soleidae (Soles)
• Cynoglossus bilineatus (native), • Aesopia cornuta (native), Unicorn sole
Fourlined tonguesole • Brachirus orientalis (native) Oriental-sole
• Cynoglossus carpenteri (native), • Brachirus pan (native) Pan sole
Hooked tonguesole • Heteromycteris oculus (native), Eyed sole
• Cynoglossus cynoglossus (native), Bengal • Liachirus melanospilos (native)
tongue sole • Pardachirus marmoratus (native), Finless sole
• Cynoglossus dubius (native), Carrot tonguesole • Pardachirus pavoninus (native), Peacock sole
• Cynoglossus lida (native) Shoulder spot tongue, • Solea elongata (native) Elongate sole, Elongate sole
Roughscale tonguesole • Solea ovata (native), Ovate sole
• Cynoglossus lingua (native) Long tonguesole • Soleichthys heterorhinos (native)
• Cynoglossus macrostomus (native) Malabar-sole • Dagetichthys albomaculatus (native) Kaup's sole
• Cynoglossus puncticeps (native) • Dagetichthys commersonnii (native),
Speckled toungesole Commerson's sole

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

• Cynoglossus semifasciatus (native) Malabar sole • Zebrias altipinnis (native)
• Paraplagusia bilineata (native) Fingerlip tonguesole, • Zebrias annandalei (native)
Doublelined tonguesole • Zebrias keralensis (native)
• Paraplagusia blochii (native), Bloch's tonguesole • Zebrias quagga (native), Fringefin zebra sole
• Zebrias synapturoides (native), Indian zebra sole
Paralichthyidae (Large-tooth flounders) • Zebrias zebra (questionable), Zebra sole
• Cephalopsetta ventrocellatus (native)
• Pseudorhombus arsius (native) Largetooth flounder Polymixiiformes
• Pseudorhombus dupliciocellatus (native), Polymixiidae (Beardfishes)
Ocellated flounder • Polymixia fusca (native)
• Pseudorhombus elevatus (native), Deep flounder • Polymixia japonica (questionable), Silver eye
• Pseudorhombus javanicus (native),
Javan flounder Pristiformes
• Pseudorhombus malayanus (native), Pristidae (Sawfishes)
Malayan flounder • Anoxypristis cuspidata (native), Knifetooth sawfish
• Pseudorhombus natalensis (native), • Pristis microdon (native) Smalltooth sawfish,
Natal flounders Largetooth sawfish
• Pseudorhombus triocellatus (native) Three • Pristis zijsron (native), Longcomb sawfish
sopt flounder
Rajiformes
Pleuronectidae (Righteye flounders) Dasyatidae (Stingrays)
• Poecilopsetta colorata (native), Coloured • Hemitrygon bennetti (native), Bennett's stingray
righteye flounder • Negatrygon kuhlii (native), Bluespotted stingray
• Poecilopsetta praelonga (native), Alcock's narrow- • Megatrygon microps (native), Smalleye stingray
body righteye flounder • Dasyatis pastinaca (questionable), Common stingray

61
 • Telatrygon zugei (native) Pale-edged stingray Rhinobatidae (Guitarfishes)
• Pateobatis bleekeri (native) Whiptail stingray • Rhina ancylostoma (native), Bowmouth guitarfish
• Himantura chaophraya (questionable), • Rhinobatos annandalei (native) Annandale's guitarfish
Freshwater whipray • Glaucostegus granulatus (native),
• Himantura fai (native), Pink whipray Sharpnose guitarfish
• Maculabatis gerrardi (native), Sharpnose stingray • Glaucostegus halavi (native), Halavi's guitarfish
• Brevitrygon imbricata (native) Scaly stingray • G. obtusus (native), Widenose guitarfish
• Pateobatis jenkinsii (native), Pointed-nose stingray • G. thouin (native), Clubnose guitarfish
• Himantura marginatus (native) Blackedged stingray • G. typus (questionable), Giant shovelnose ray
• Himantura uarnak (native) Leopard stingray, • Acroteriobatus variegatus (native),
Honeycomb stingray Stripenose guitarfish
• Himantura undulata (native), Leopard whipray • Rhynchobatus djiddensis (native),
• Brevitrygon walga, Dwarf whipray Giant guitarfish
• Pastinachus sephen (native) Feathertail stingray,
Cowtail stingray Salmoniformes
• Taeniura lymma (native), Bluespotted ribbontail ray Salmonidae (Salmonids)
• Taeniura meyeni (native), Blotched fantail ray • Oncorhynchus mykiss (introduced) Rainbow trout
• Urogymnus asperrimus (native), Porcupine ray • Salmo trutta fario (introduced) Brown trout
• Salmo trutta (introduced), Sea trout
Gymnuridae (Butterfly rays) • Salvelinus fontinalis (introduced) Brook trout
• Gymnura tentaculata (native), Tentacled butterfly ray
• Aetoplatea zonura (native), Zonetail butterfly ray Scorpaeniformes
• Gymnura japonica, Japanese butterflyray Apistidae
• Gymnura micrura, Smooth butterfly ray • Apistus carinatus (native), Ocellated waspfish
• Gymnura poecilura (native), Longtail butterfly ray
Aploactinidae (Velvetfishes)
Myliobatidae (Eagle and manta rays) • Acanthosphex leurynnis (native)
• Aetobatus flagellum (native) Plain eagleray • Cocotropus roseus (native)
• Aetobatus narinari (native) Spotted eagleray
• Aetobatus ocellatus (native) Caracanthidae (Orbicular velvetfishes)
• Aetomylaeus maculatus (native), Mottled eagle ray • Caracanthus maculatus (native), Spotted
• Aetomylaeus milvus (native) coral croucher
• Aetomylaeus nichofii (native) Nieuhof's eagle ray, • Caracanthus unipinna (native), Pygmy
Banded eagle ray coral croucher
• Mobula birostris (native), Giant manta
Central Marine Fisheries Research Institute

• Mobula eregoodootenkee (native), Pygmy devilray Dactylopteridae (Flying gurnards)

• Mobula japanica (native), Spinetail mobula • Dactyloptena gilberti (native)
• Mobula kuhlii (native), Shortfin devil ray • Dactyloptena macracantha (native), Spotwing
• Mobula mobular (questionable), Devil fish flying gurnard
• Rhinoptera javanica (native), Javanese cownose ray • Dactyloptena orientalis (native), Oriental
flying gurnard
Plesiobatidae (Deepwater stingray) • Dactyloptena peterseni (native), Starry flying gurnard
• Plesiobatis daviesi (native), Deepwater stingray
Peristediidae (Armored searobins or
Rajidae (Skates) armored gurnards)
• Dipturus johannisdavisi (native), Travancore skate • Scalicus investigatoris (native)
• Fenestraja mamillidens (native), Prickly skate • Satyrichthys laticeps (native)
• Orbiraja powelli (native), Indian ringed skate

62
Platycephalidae (Flatheads) • Minous monodactylus (native) Grey goblin fish
• Cociella crocodilus (native) Crocodile flathead • Synanceia verrucosa (native), Stonefish
• Sunagocia carbunculus (native), Papillose flathead • Trachicephalus uranoscopus (native),
• Grammoplites scaber (native) Rough flathead Stargazing stonefish
• Grammoplites suppositus (native), Spotfin flathead
• Inegocia japonica (native), Japanese flathead Tetrarogidae (Wasp fishes)
• Kumococius rodericensis (native), Spiny flathead • Ocosia ramaraoi (native)
• Platycephalus indicus (native) Bartail flathead • Paracentropogon longispinis (native),
• Rogadius asper (native), Olive-tailed flathead Wispy waspfish
• Rogadius serratus (native), Serrated flathead • Pseudovespicula dracaena (native), Draco waspfish
• Rogadius tuberculata (native), Tuberculated flathead • Richardsonichthys leucogaster (native),
• Suggrundus macracanthus (native), Large- Whiteface waspfish
spined flathead • Snyderina guentheri (native), Günther's waspfish
• Sunagocia otaitensis (native), Fringelip flathead • Tetraroge niger (native)
• Thysanophrys chiltonae (native), Longsnout flathead
Triglidae (Searobins)
Scorpaenidae (Scorpionfishes or • Lepidotrigla bispinosa (native), Bullhorn gurnard
rockfishes) • Lepidotrigla faurei (native), Scalybreast gurnard
• Brachypterois serrulata (native) • Lepidotrigla longipinnis (native)
• Dendrochirus brachypterus (native), • Lepidotrigla omanensis (native), Oman gurnard
Shortfin turkeyfish • Pterygotrigla hemisticta (native), Blackspotted gurnard
• Ebosia falcata (native)
• Parascorpaena picta, Northern scorpionfish Siluriformes

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

• Pteroidichthys amboinensis (native) Ariidae (Sea catfishes)
• Pterois antennata (native), Broadbarred firefish • Arius arius (native) Threadfin sea catfish
• Pterois mombasae (native), Frillfin turkeyfish • Netuma bilineatus (native), Bronze catfish
• Pterois radiata (native), Radial firefish • Plicofollis tonggol (native), Roughback sea catfish
• Pterois russelii (native) Russell's fire fish, • Plicofollis dussumieri (native), Blacktip sea catfish
Plaintail turkeyfish • Arius gagora (native) Gagora catfish
• Pterois volitans (native), Red lionfish • Arius maculatus (native) Spotted catfish
• Scorpaenodes parvipinnis (native), • Nemapteryx nenga (native)
Lowfin scorpionfish • Arius parvipinnis (native)
• Scorpaenopsis gibbosa (native), • Plicofollis platystomus (native), Flatmouth
Humpback scorpionfish sea catfish
• Taenianotus triacanthus (native), • Hexanematichthys sagor (native) Sagor catfish
Leaf scorpionfish • Hemiarius sona (native) Sona sea-catfish
• Arius subrostratus (native), Shovelnose sea catfish
Setarchidae • Hemiarius sumatranus (native), Goat catfish
• Setarches guentheri (native), Deepwater scorpionfish • Plicofolis layardi (native) Thinspine sea catfish
• Netuma thalassina (native), Giant seacatfish
Synanceiidae (Stonefishes) • Arius venosus (native), Veined catfish
• C h o r i d a c t y l u s m u l t i b a r b u s   ( n a t i v e ) , • Batrachocephalus mino (native) Frog-headed catfish
Orangebanded stingfish
• Inimicus caledonicus (native), Chinese ghoul Dalatiidae (Sleeper sharks)
• Inimicus sinensis (native), Spotted ghoul • Centroscyllium ornatum (native), Ornate dogfish
• Minous dempsterae (native),
Obliquebanded stingfish Echinorhinidae (Bramble sharks)
• Minous inermis (native), Alcock's scorpionfish • Echinorhinus brucus (native), Bramble shark

63
 Stomiiformes • Hippichthys cyanospilos (native), Blue-
Gonostomatidae (Bristlemouths) spotted pipefish
• C y c l o t h o n e s i g n a t a   ( q u e s t i o n a b l e ) , • Hippichthys penicillus (native), Beady pipefish
Showy bristlemouth • Hippichthys spicifer (native), Bellybarred pipefish
• Gonostoma elongatum (native), Elongated • Hippocampus fuscus (questionable) Chilka
bristlemouth fish seahorse, Sea pony
• Hippocampus histrix (native), Thorny seahorse
Sternoptychidae • Hippocampus kelloggi (native), Great seahorse
• Polyipnus spinosus (questionable) • Hippocampus kuda (native), Spotted seahorse
• Stomiidae (Barbeled dragonfishes) • Hippocampus trimaculatus (native),
• Chauliodus pammelas (native) Longnose seahorse
• Chauliodus sloani (native), Sloane's viperfish • Microphis brachyurus (native) Short-tailed pipefish
• Stomias affinis (native), Günther's boafish • Microphis cuncalus (native) Crocodile-tooth pipefish
• Microphis insularis (native) Andaman pipefish
Synbranchiformes • Phoxocampus belcheri (native), Rock pipefish
Chaudhuriidae • Syngnathoides biaculeatus (native),
Alligator pipefish
Synbranchidae (Swamp-eels) • Trachyrhamphus serratus (native)
• Monopterus albus (native) Rice swampeel
• Monopterus cuchia (native) Cuchia Tetraodontiformes
• Monopterus digressus (native) Balistidae (Triggerfishes)
• Monopterus eapeni (endemic) • Abalistes stellaris (native), Starry triggerfish
• Monopterus fossorius (endemic) Malabar swampeel • Balistapus undulatus (native), Orange-lined triggerfish
• Monopterus hodgarti (endemic) Indian spaghetti- • Balistes rotundatus (native)
eel • Balistes vetula, Queen triggerfish
• Monopterus indicus (endemic) Bombay swampeel • Balistoides conspicillum (native), Clown triggerfish
• Monopterus roseni (native) • Balistoides viridescens (native), Titan triggerfish
• Ophisternon bengalense (native) Bengal mudeel • Melichthys niger (native), Black triggerfish
• Odonus niger (native), Redtoothed triggerfish
Syngnathiformes • Pseudobalistes flavimarginatus (native),
Aulostomidae (Trumpetfishes) Yellowmargin triggerfish
• Aulostomus chinensis (native), Chinese trumpetfish • Pseudobalistes fuscus (native), Yellow-
spotted triggerfish
Centriscidae (Snipefishes and • Rhinecanthus aculeatus (native), Blackbar triggerfish
shrimpfishes) • Rhinecanthus rectangulus (native), Wedge-
Central Marine Fisheries Research Institute

• Centriscus scutatus (native), Grooved razor-fish tail triggerfish

• Sufflamen chrysopterum (native),
Fistulariidae (Cornetfishes) Halfmoon triggerfish
• Fistularia petimba (native), Red cornetfish • Sufflamen fraenatum (native), Masked triggerfish

Solenostomidae (Ghost pipefishes) Diodontidae (Porcupinefishes)

• Solenostomus cyanopterus (native), Ghost pipefish • Cyclichthys orbicularis (native), Birdbeak burrfish
• Cyclichthys spilostylus (native), Spotbase burrfish
Syngnathidae (Pipefishes and seahorses) • Diodon holocanthus (native) Bloched porcupine
• Choeroichthys brachysoma (native), Short- fish, Long-spine porcupinefish
bodied pipefish • Diodon hystrix (native) Spotted porcupine fish
• Choeroichthys sculptus (native), Sculptured pipefish • Lophodiodon calori (native), Four-bar porcupinefish
• Doryrhamphus excisus (native), Bluestripe pipefish

64
Molidae (Molas or Ocean Sunfishes) backed blowfish
• Mola mola (native), Ocean sunfish • Lagocephalus lagocephalus (native), Oceanic puffer
• Lagocephalus lunaris (native) Moontail blaasop,
Monacanthidae (Filefishes) Green rough-backed puffer
• Aluterus monoceros (native), Unicorn leatherjacket • Lagocephalus spadiceus (native) Chinese blaasop
• Aluterus scriptus (native), Scrawled filefish • Takifugu oblongus (native) Lattice blaasop,
• Anacanthus barbatus (native), Bearded leatherjacket Lattice blaasop
• Lalmohania velutina (native) • Leiodon cutcutia (native) Ocellated pufferfish,
• Oxymonacanthus longirostris (native), Harlequin filefish Ocellated pufferfish
• Paraluteres prionurus (native), Blacksaddle filefish • Dichotomyctere fluviatilis (native) Green pufferfish
• Paramonacanthus choirocephalus (questionable) • Dichotomyctere nigroviridis (native)
Pig faced leather jacket Burmese pufferfish
• Paramonacanthus japonicus (native), • Torquigener hypselogeneion (native), Orange-
Hairfinned leatherjacket spotted toadfish
• Paramonacanthus oblongus (native), Hair- • Tylerius spinosissimus (native), Spiny blaasop
finned filefish
Paramonacanthus tricuspis (native) Triacanthidae (Triplespines)
Thamnaconus modestoides (native), Modest filefish • Pseudotriacanthus strigilifer (native) Long spined
tripod fish
Ostraciidae (Boxfishes) • Triacanthus biaculeatus (native) Short-nosed
• Lactoria cornuta (native), Longhorn cowfish tripodfish, Short-nosed tripodfish
• Ostracion cubicus (native), Yellow boxfish • Triacanthus nieuhofii (native), Silver tripodfish
• Ostracion meleagris (native), Whitespotted • Tripodichthys oxycephalus (native), Short-

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

boxfish tail tripodfish
• Ostracion nasus (native), Shortnose boxfish
• Tetrosomus gibbosus (native), Humpback turretfish Triacanthodidae (Spikefishes)
• Macrorhamphosodes platycheilus (native),
Tetraodontidae (Puffers) Trumpetsnout spikefish
• Arothron hispidus (native) White spotted blow fish, • Triacanthodes ethiops (native), Shortsnout spikefish
White-spotted puffer
• Arothron immaculatus (native) Immaculate blow fish Order Torpediniformes
• Arothron meleagris (native), Guineafowl puffer
• Arothron nigropunctatus (native) Black spotted Narcinidae (Numbfishes)
blow fish • Benthobatis moresbyi (native), Dark blind ray
• Arothron reticularis (native) Reticulated blowfish • Narcine brunnea (native), Brown numbfish
• Arothron stellatus (native) Star blaasop • Narcine lingula (native), Chinese numbfish
• Canthigaster amboinensis (native), Spider- • Narcine prodorsalis (questionable), Tonkin numbfish
eye puffer • Narcine timlei (native), Blackspotted numbfish
• Canthigaster bennetti (native), Bennett's • Narke dipterygia (native), Spottail sleeper ray
sharpnose puffer
• Canthigaster coronata (native), Crowned puffer Torpedinidae (Electric rays)
• Canthigaster margaritata (native) • Torpedo fuscomaculata Black-spotted torpedo
• Carinotetraodon imitator (endemic) • Torpedo panthera (native), Panther electric ray
• Carinotetraodon travancoricus (endemic) • Torpedo sinuspersici (native), Marbled electric ray
Malabar pufferfish
• Lagocephalus guentheri (native), Zeiformes
Diamondback puffer Caproidae (Boarfishes)
• Lagocephalus inermis (native) Smooth- • Antigonia indica (native)

65
 (Cypriniformes: Balitoridae) from Manipur, India. J. Bombay
• Antigonia rubescens (questionable), Indo- Nat. Hist. Soc. 102(1):79-82
Pacific boarfish Vishwanath, W. and K. S. Devi (2005) A new fish species of the
genus Garra Hamilton-Buchanan (Cypriniformes:Cyprinidae)
from Manipur, India. J. Bombay Nat. Hist. Soc. 102(1):86-88
Parazenidae (Parazen) Beevi, K.S.J. and A. Ramachandran (2005) A new species of Puntius
• Cyttopsis rosea (native), Rosy dory (Cyprinidae, Cyprininae) from Kerala, India. J. Bombay Nat.
Hist. Soc. 102(1):83-85
Kurup, B. M., T. G. Manojkumar and K. V. Radhakrishnan.
Zeidae (Dories) 2005. Salarias reticulatus, a new freshwater blenny from
Chalakudy river, Kerala, South India. J. Bombay Nat. Hist.
• Zenopsis conchifer (native), Silvery John dory Soc. 101(2):195-197
• Zenopsis nebulosa (native), Mirror dory  Fishbase
 Ralf Britz, Krishna Kumar & Fibin Baby, 2012. Pristolepis
rubripinnis, a new species of fish from southern India (Teleostei:
Suggested readings Percomorpha: Pristolepididae). Zootaxa 3345: 59-68
Ng, Heok Hee (2005) Amblyceps carinatum, a new species of hill
Froese, R. and D. Pauly. Editors. 2006. FishBase. World Wide Web stream catfish from Myanmar (Teleostei: Amblycipitidae). The
electronic publication. [1], version (05/2006) Raffles Bull. Zool 53(2): 243-249
Raghavan, R., J. Tharian, A. Ali, S. Jadhav & N. Dahanukar (2013). Kosygin, L. and W. Vishwanath (2005) Validity and redescription
Balitora jalpalli, a new species of stone loach (Teleostei: of  Glyptothorax manipurensis Menon and record of G.
Cypriniformes: Balitoridae) from Silent Valley, southern sinense (Regan) from India. J. Bombay Nat. Hist. Soc. 102(1):61-
Western Ghats, India. J. Threatened Taxa 5(5): 3921-3934; 65
doi:10.11609/JoTT. o3277.3921-34. Vishwanath, W and Linthoingambi I. 2005. A new Sisorid catfish of
Vishwanath, W. & K. Nebeshwar Sharma (2005) A new the genus Glyptothorax Blyth from Manipur, India. J. Bombay
Nemacheiline fish of the genus Schistura McClelland Nat. Hist. Soc. 102(2):201-203
Central Marine Fisheries Research Institute

66
Crustacean Diversity
S. Lakshmi Pillai* and G. Maheswarudu
Principal Scientist
Crustacean Fisheries Division, CMFRI, Kochi
e-mail: [email protected]

The Ocean has always fascinated mankind, the both from the east and west coasts of the country
quest for unravelling its mystery leading to several by several workers. They are fed upon by fishes and
exploratory surveys. The most notable as far as marine other crustaceans.
crustaceans are concerned was the exploratory
surveys by the Indian Royal Marine Survey Ship Isopods
‘Investigator’ (1888–1892) which brought to the fore
several marine crustacean species till then unknown. They are a morphologically diverse group of
Majority of the fauna collected came from depths crustaceans that live in sea, in freshwater and on
between 100-1900 fathoms. Later, mechanization
of crafts and gears and extension of fishing into
deeper waters enhanced our knowledge of the
biodiversity of crustaceans. Crustacean biodiversity
is as vast and deep as the oceans itself, difficult

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

to comprehend. Crustaceans first appeared in the
fossil records half a billion years ago. They have been
important components of marine biodiversity and
compose virtually the entire fossil arthropod fauna
through the Mesozoic and Cenozoic Eras. The most
abundant crustaceans in the ocean are copepods
and they have no fossil record.
land. They have segmented and rigid exoskeleton and
Amphipods vary in shape. Smaller ones are parasitic on fishes and
some larger species inhabit the deep sea. Some live
They have a laterally compressed body without in coastal and shelf waters. The largest isopod is in
carapace. They are distributed in marine, brackish the genus Bathynomus found in deep sea.

Barnacles
They are highly specialized crustaceans belonging
to the group known as Cirripedia and are exclusively
marine forms. Most of the species live attached to
inanimate objects, some to living objects like sponge
and bury into the flesh of whales and turtles. They
attach to substratum with their cement glands. They
are mostly found at depths less than 100 m and
also in the intertidal zone. Common barnacles are
water, freshwater and terrestrial environments. In the goose barnacle (Lepas anserifera), Balanus spp.,
India, there is lot of information on their diversity Sacculina spp. Sacculina is parasitic on crabs. It lives

67
 Lepas sp A female opossum shrimp carrying eggs

Ostracods
They range from 0.1 to 32 mm in size. Body is
flattened from side to side and protected by a bivalve

Balanus sp

like calcareous valve or shell. They are also called seed

shrimps. Besides marine, there are freshwater and
terrestrial species also.

Sacculina in crab
Copepods
inside crabs and produce hormones that lead to They are found in sea and in freshwater. Their
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feminization of crabs (changes the host from male body is cylindrical or round, the head is fused
to female) which causes the crabs to nurture the with the first or two thoracic segments. They may
barnacle egg mass as its own.

Mysids / Opossum shrimp

They are commonly called the opossum shrimps as they
have a pouch or marsupium in females. Their larvae
are reared in this pouch and are not free swimming.
They may be pelagic or benthic and are filter feeders
feeding on algae, detritus and zooplankton. They are
found in both marine and freshwater environments.
They are used as feed in aquaculture.

68
be free living, symbiotic or internal or external and Axiidea. The majority of the shrimps and prawns
parasite. They mainly feed on algae. Among the belong to the superfamily Penaeoidea. Around 120
zooplankton community they are usually the
dominant members and are the major food of
fishes, seabirds and Krills.

Brine shrimp Fenneropenaeus indicus Melicertus latisulcatus

Metapenaeopsis stridulans

species of penaeoid shrimps are recorded from the

Indian coast.
They inhabit saltwater lakes and belong to the genus
Artemia. They can produce dormant cysts which can Penaeid shrimps
be stored and hatched when required. The young
ones from hatched cysts are used as live feed in The commercially important genera in Indian waters
aquaculture industry. belonging to family penaeidae are represented by
Fenneropenaeus, Penaeus, Melicertus, Marsupenaeus,

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Euphasiids Parapenaeus, Metapenaeopsis, Penaeopsis,
Metapenaeus, Trachypenaeopsis, Atypopenaeus,
Parapenaeopsis, Megokris and Trachysalambria.
Genera Solenocera represent the family Solenoceridae
and Aristeidae by Aristaeomorpha, and Aristeus.
Under the family Penaeidae there are 71 species
recorded from Indian waters.

Carideans
a. Alpheid shrimps

They are marine crustaceans occupying surface to at

least 4000 m depth and are distributed from Arctic
to Antarctic waters. Euphasia species are fished
commercially in Japan, Canadian and Russian waters
for aquarium feed, pharamceutical purpose and as food
for humans. They are the main prey of baleen whales.

Shrimps/prawns
The species of shrimps and prawns are placed under
four infra orders- Penaeidea, Stenopodidea, Caridea

69
 They are commonly known as the pistol shrimps or
snapping shrimps having asymmetrical claws, the
larger of the claw produces snapping sound. They
dig burrows and inhabit coral reefs. Maximum species
are under the genera Alpheus and Synalpheus. They
are caught as bycatch in trawls.

b. Crangonid shrimps Plesionika quasigrandis

d. Pandalid shrimps
The genera Aegaeon, Pontocaris and Parapontocaris
are known from Indian waters like Aegaeon lacazei A number of pandalid shrimp species are fished on
and Pontocaris pennata. The genera Aegaeon a commercial scale for human consumption from
the southern coasts of India. Notable are Plesionika
quasigrandis, Plesionika martia, Heterocarpus

Acanthephyra armata Acanthephyra sanguinea

Pontocaris pennata

are found from shallow waters to up to 800 m, Oplophorus typus

Parapontocaris at and beyond 200 m depth and
Pontocaris from shallow to deeper waters. gibbosus and Heterocarpus woodmasoni landed by
deep sea trawlers.

e. Oplophorids

The species that have commercial value are

Acanthephyra sanguinea, Acanthephyra armata,
Oplphorus typus and Oplophorus gracillirostris. They
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form a minor fishery. They are found at depths of 300

m and beyond.

Glyphocrangon investigatoris

c. Glyphocrangonid shrimps

They are a single genus–Glyphocrangon under

the family Glyphocrangonidae. Around 10 species
have been recorded from Indian waters. Body is
hard, thick and with spines or tubercles. They are
deep sea inhabitants found at depths of 300 m
and beyond. Nematopalaemon tenuipes

70
f. Palaemonid shrimps spend most part of their life in burrows. The largest
They are found in freshwater and also in marine individuals may reach 10 cm (excluding appendages).
habitats. The species that are usually encountered They have an exceedingly large claw which may be
in the fishery are Nematopalaemon tenuipes and on the right or left side.

Podopthalmus vigil Calappa calappa

Expalaemon styliferus.

g.Stenopid shrimps
Calappa japonica Galene bispinosa
They are usually associated with reefs. They have
enlarged third pair of walking legs. Members of the
genus Stenopus are commonly known as cleaner

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shrimps. Stenopus hispidus is a common aquarium
pet under the family stenopodidae. This banded
cleaner shrimp or banded coral shrimp uses its
three pairs of claws to remove parasites, fungi and
damaged tissue from fish. Liagore rubromaculata Jonas indica

Cryptopodia fornicate Parthenope longimanus

Mud shrimp species

h. Mud shrimps (Thalassinideans)

Dorippe quadridens
They construct burrows in intertidal and subtidal
soft sediments. Their burrows are considered to be
the deepest and complex among the crustaceans. Brachyuran crabs
They use the burrows for shelter, reproduction and
feeding. They have a brief pelagic larval stage but The brachyuran crabs or true crabs belonging to the

71
 infraorder Brachyura are the most diverse groups
among the decapod crustaceans. They lack tail and
their abdomen are reduced and curved or tucked under
their body. Around 991 species of brachyuran crabs
have been recorded from Indian waters. Commercially
important crabs belong to the family Portunidae. The
important commercial brachyuran crabs are Portunus Albunea
pelagicus, Portunus sanguinolentus, Charbydis feriata,
Charybdis lucifera and Scylla spp. Lesser important
crabs belonging to family Portunidae are Portunus
gladiator, Portunus argentatus, Podopthalmus vigil,
Chraybdis natator, and Charybdis smithii. There are
several species of crabs caught as bycatch in trawlers
in India belonging to different families – Calappidae,
Matutidae, Dorippidae, Dromiidae, Leucosiidae,
Emerita
Xanthidae, Corystidae, Galenidae, Majidae, Grapsidae,
Geryonidae, Ocypodidae etc.
very small and positioned under the abdomen hence
Anomurans not visible externally.

The anomurans include the hermit crabs, porcelain Mole crab/sand crab
crabs, mole crabs or sand crabs, hairy stone crab, king
crab and squat lobsters. They have telson and uropods They are placed under the family Hippidae. They have
which the brachyuran crabs lack. Majority of anomurans an oval shaped body and are filter feeders burrowing in
live in tropical and temperate marine habitats sand in the intertidal zone. Albunea spp., Hippa spp.,
Emerita spp. are common and are fed upon by seabirds.

Hermit crabs Eumunida funambulus Munidopsis scobina

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These organisms are also consumed by humans.

Hermit crabs
Squat lobsters
They have a soft body and carry a shell to protect
their-self. Their entire body can be retracted into the In countries like Argentina, Mexico and New Zealand
shell. The shells used by them are mostly that of snails the squat lobsters are considered as potential fishery
and also bivalve shells. As they grow they change their resource. They are found in great abundance in several
shell to accommodate themselves. They range in size countries as bycatch and in India too several species have
from a few millimeters to the size of a coconut. The been recorded- Munidopdis scobina, Munida squamosa,
hermit crabs belong to the superfamily Paguroidea Munida japonica, Munida heteracantha) etc.
of the infraorder Anomura. They inhabit both aquatic
and terrestrial habitats. Generally they have only three Genus Eumunida is mostly represented by deep water
pair of walking legs with the last pair of legs being squat lobsters. In India, Eumunida funambulus was

72
 Thenus unimaculatus Petractus rugosus

or spots. They are mostly coastal and species of the

genera Linuparus, Peurulus are found beyond 300 m
depth. They are usually caught in traps and also form
reported from off Arabian Sea, coast of Tamil Nadu bycatch in trawls. The genera Panulirus in Indian waters
and Lakshadweep. Their first pair of appendage is (including Andaman & Lakshadweep) is represented by
much longer than their body length. Panulirus homarus, P. versicolor, P. ornatus, P. longipes,
P. pencillatus and P. polyphagus, all being commercially
Porcelain crabs exploited. The deep water lobster Peurulus sewelli is
fished along the southern coasts of India. Linuparus
The porcelain crabs are small in size usually less than somniosus is distributed in Andaman waters.
15 mm in carapace width. They are fragile and have
the habit of shedding their limbs when attacked by Scyllarid lobsters
predators, hence the name Porcelain crabs. These
shed appendages often grow back after moulting. They have flattened firm body, lacking rostrum or with
They use their fifth pair of pereiopods for cleaning rudimentary rostrum. They also are members of the

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

and chelate appendages for territorial struggle. infra order Achelata. They range in total length from
a few to 40 cm. Antennae is short and broad, plate
Lobsters like or flattened lacking flagella, hence they are also
called shovel nosed lobsters. Body colour is usually
in various shades of brown. Thenus unimaculatus is
Palinurid lobsters fished commercially in Indian waters (Kerala, Tamil
The Palinurid lobsters are moderate to large sized, Nadu & Maharashtra). Certain other species recorded
lacking rostrum and having spines or granules on the are Petractus rugosus, Scyllarides elisabethae and
carapace and are placed under the infra order Achelata. Scyllarides tridacnophaga. Petractus rugosus have
Most of the species are brightly coloured have bands ornamental potential and Scyllarides tridacnophaga
grow to large size and are edible.

Polychelids
They possess extremely long and chelate first

Palinustus waguensis Palinustus waguensis

Peurulus sewelli Panulirus versicolor Enoplometopus macrodontus

73
 pereiopod and are inhabitants of very deep water,
hence are blind eg Stereomastis nana from Indian
waters. All five pairs of pereiopods may be clawed
from which they get the name polycheles which
means many clawed.

Astacids
The reef lobsters comprise single family and one genus
Enoplometopus. Only two species are recorded from Squilloides leptosquilla
Indian waters – Enoplometopus macrodontus and

Oratosquilla woodmasoni

Nephropsis stewartii

Enoplometopus occidentalis. They usually inhabit

reefs and can be distinguished from the Nephropsid
lobsters by the presence of fully developed claws on
Lysiosquilla tredecimdentata
the first pair of pereiopods, the second and third pair
of pereiopods being only subchelate. They are caught
from a depth of 90 to 200 m. found to inhabit sandy or muddy bottom in the
littoral region up to a depth of 100 m. One species
Members of the family Nephropidae have tubular Squilloides leptosquilla was recorded from a depth of
body with well–developed rostrum, first pair of legs 150 to 300 m. Some species belonging to the family
larger than the other pairs. The first three pairs of legs Gonodactylidae inhabit coral reefs. Several species
are with pincers. Nephropsis stewarti and Nephropsis form commercial fishery in the Mediterranean Sea.
carpenteri are recorded from Indian waters and have In East Asia, Oratosquilla oratoria and in Indo-Pacific
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commercial value in some countries. various species of squillids and lysiosquillids are fished.

Stomatopods Suggested readings

Stomatopods are commonly known as praying mantis Radhakrishnan E.V., Josileen Jose and Lakshmi Pillai, S. 2011.
shrimp. They are predatory, the second maxilliped Handbook of Prawns. Central Marine Fisheries Research
Institute, Kochi
modified as large raptorial appendages. The last three Kathirvel, M. 2008. Biodiversity of Indian Stomatopods. Glimpses of
segments of the second maxilliped fold against each Aquatic Biodiversity, Rajiv Gandhi Chair Spl. Pub., 7:93 – 102.
Galil, B.S. 2000. Crustacea Decapoda: Review of the genera and
other forming the raptorial claw similar to the praying species of the family Polychelidae, Wood-Mason, 1874. In
mantis insect. There are 65 species of stomatopods A.Crosnier (ed.) Résultats des Campagnes MUSORSTOM,
recorded from Indian waters. Maximum numbers Volume 21. Mémoires du Muséum national d’Histoire naturelle,
184: 285-387.
(38) have been recorded from Tamil Nadu, 22 from Ng Peter, K.L., Guinot, D & Davie, P.J.F. 2008. Systema Brachyurorum:
west Bengal and 17 from Maharashtra. They are Part I. An annotated checklist of extant brachyuran crabs of the

74
 world. The Raffles Bulletin of Zoology, 17 : 1-286. Komai, T. 2004. A review of the Indo West Pacific species of the
Fischer, W & Bianchi, G. 1984. FAO species identification sheets genus Glyphocrangon A Milne Edwards, 1881 (excluding
for fishery purposes, Western Indian Ocean, Fishing area 51. G. caeca species group) (Crustacea Decapoda:Caridea:
FAO, Rome. Glyphocrangonidae) in Marshall B and Richer D Forges B. (eds),
Baba, K. 2005. Deep sea Chirostylid and Galatheid crustaceans Tropical Deep sea benthos, Volume 23, Mem.Mus.Natl.Hist.
(Decapoda Anomura) from the Indo Pacific with a list of Nat., 191: 375-610.
species. In Torben Wolff (ed). Galathea Report-Scientific results
of the Danish deep sea expedition round the world, 1950-
52, 20:1-317.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

75
 Molluscan Diversity
Geetha Sasikumar*, V. Venkatesan and K. S. Mohamed
Principal Scientist
Molluscan Fisheries Division, Mangalore RC of CMFRI
e-mail: [email protected]

Introduction Classification
Phylum Mollusca is the second most diverse group In terms of taxonomic rank, phyllum Mollusca consist
under the animal Kingdom. They are soft bodied of two subphyla, namely Aculifera and Conchifera.
invertebrates often enclosed in a hard exoskeleton Aculifera includes all molluscs that either primitively
or shell. Therefore the word mollusc is derived from lack a shell (aplacophora) or have a series of plates
the Latin word mollis with the meaning soft. The instead of a single shell (polyplacophora). The
branch of zoology that deals with the study of subphylum Aculifera includes the class Polyplacophora
mollusc, known as malacology, originates from the (chitons) as well as the classes Caudofoveata and
Greek word for soft ‘malacos’. Solenogastres (or Solenogasters), along with their
fossil relatives. The subphylum Conchifera (shell-
Molluscs are among the most successful of the bearers) comprises of five classes, which includes the
animal phyla in terms of numbers of species and
also in terms of the wide range of habitats to
which they have become adapted. They occupy a
vast range of habitats, both aquatic and terrestrial,
from the arctic seas to small tropical streams, from
valleys to mountainsides and few are adapted to
live in deserts while some are parasitic. In the sea
they occur from the deepest ocean trenches to
the intertidal zone.
Fig. 1. Molluscan phylogeny based on morphological and larval features
(Adapted from von Salvini-Plawen (1990a, b)
Molluscs also exhibit an enormous range in size,
from species which are almost microscopic to dominant molluscan classes the Gastropoda, Bivalvia
the largest of all invertebrates, the giant squid and Cephalopoda.
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which can weighs 270kg and measures up to 12

m in the body length, with tentacles as much as There are various estimates of the number of species
another 50 m in length. The diversity of specific of molluscs from different parts of the world.
shapes within the phylum makes it difficult to Appeltan et al., (2011) places the number of marine
define mollusc in terms of any distinctive trait. molluscs between 135,887 and 164,107 (Table. 1) in
Structures that are prominent in some groups may a recent compilation. Their estimates of the number
be completely absent in others. One of the most of extant described marine molluscs vary from 43,689
characteristic features of molluscs is the possession to 51,689. The number of marine species described
of a hard calcareous shell by the majority of per year continues to rise for Bivalvia and Gastropoda
species offering protection, shape and rigidity to among several other marine taxonomic groups.
the soft visceral mass. These shells are long lasting
and have been collected by human beings for In India, major studies have been carried out mainly
thousands of years. on the commercially important molluscan groups.

76
Nearly, 5,070 species of molluscan species are and 133 species of Caudofoveata are described so
recorded under the phylum Mollusca. Among the far (Table 1).
molluscan biodiversity, India contributes 7.21% of
the world’s biodiversity (UNEP-GBA, 1995). Within Solenogastres are worm-like marine animals without
the number of molluscs recorded from India, 3,370 shells, having a narrow, ciliated, gliding sole located
species of molluscs are recorded from marine habitat in a ventral groove (possibly related to the foot of
(Venkataraman and Wafar, 2005). other mollusc) on which they crawl on hard or soft
substrates, or on the cnidarian colonies on which they
Solenogastres, feed. The solenogasters range in size from less than
a millimeter (0.8 mm) in body length (Meiomenia
Caudofoveata swedmarki) to more than 30 cm long (Epimenia
and Polyplacophora babai) and often colorful. Caudofoveata are worm-like
lacking a ventral groove and foot. They are covered
Two groups of aplacophorans, Caudofoveata by cuticle and aragonitic scales. They are infaunal,
(Chaetodermomorpha) and Solenogastres, are feeding on detritus or selectively on foraminiferans.
universally recognized but their current classification Hence they are adapted to burrowing in the mud
is still contentious with two different systems in use. with their oral shield. Caudofoveates range in length
One system treats Caudofoveata and Solenogastres from 2 mm (Prochaetoderma raduliferum) to 14 cm
as subclasses of a class Aplacophora, which is (Chaetoderma Productum).
accepted to be monophyletic while the other system
consider Solenogastres and Caudofoveata to be The representatives of Polyplacophora are commonly
paraphyletic. The latter view is largely corroborated referred to as the Chitons. They are dorsoventrally
by studies comparing morphological characteristics flattened, exclusively marine molluscs characterized

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and molecular analyses, but the interrelationship by the presence of eight dorsal aragonitic shell plates
between the two remains ambiguous with recent (valves) and a broad ventral ciliated foot. There is
studies supporting either of the classification systems. thick marginal girdle surrounding the dorsal shell
The classification recognizing Caudofoveata and plates covered by a chitinous cuticle. About 930 living
Solenogastres as separate classes is considered here. species are reported from the marine intertidal or
The two classes are however still known collectively sublittoral, including deep-sea species ranging from
as aplacophorans. Though they are not reported 3mm to 43 cm in size. Nearly 21 species are reported
from Indian Sea, about 263 species of Solenogastres from the Indian Seas.

Table 1. Estimates of Known and Unknown global marine molluscan species diversity (Adapted from Appeltans et al., 2012)
Total known % Undescribed Undiscovered Total Total Total % New spp.
(Described) Synonyms collected (Morphospecies) unknown unknown estimated Known (1999-2008)
(Experts) (Model)
Bivalvia 9000 55 2000 3000 5000 ** 135,887- 64  
164,107
Caudofoveata 133 8 Not 500 500 No data 21  
estimated
Cephalopoda 761 No data 150 500 650 No data 54  
Gastropoda 32000- 69-75 35000- 50000-60000 85000- ** 23-32  
40000 45000 105000
Monoplacophora 30 No data 3 50 53 No data 36  
Polyplacophora 930 52 50 50-100 100-150 No data 86-90  
Scaphopoda 572 33 55 500 555 No data 51  
Solenogastres 263 21 20-30 320-480 340-510 No data 34-44  
Total Mollusca 43,689- 37,278- 54,920-65,130 92,198-   28-36 4022
51,689 47,288 112,418
** rate of discovery still rising

77
 Monoplacophora Class Bivalvia
(Lamellibranchia
Extant Monoplacophora live in deep cold waters
and resembles chitons in several characters. They
or Pelecypoda)
are molluscs with a cap-shaped shell. They are not
reported from Indian Seas. Thirty species are described
under the class Monoplacophora. They are smaller in
size ranging from 1.5 to 37 mm.

Scaphopoda
Scaphopods are inhabitants of the sea floor. The shell
is tubiform to barrel-shaped and is open at both
ends. The foot is pointed and cylindrical. The foot
extends from the anterior (front) opening, with which
scaphopods dig into the sandy sea floor. Tusk shells
are exclusively marine and 18 species are reported
from India. They range in size from 2-150 mm, eg.
Dentalium (Dentalium) aprinum, Dentalium elp.
(Adapted from Poutiers, 1998a)

Marine bivalve families reported from India

Solemyidae Pectinidae Semelidae Lyonsiidae
Nuculidae Spondylidae Dreissenidae Myochamidae
Nuculanidae Anomiidae Trapezidae Pandoridae
Yoldiidae Placunidae Glossidae Penicillidae
Malletiidae Lucinidae Vesicomyidae Poromyoidea
Arcidae Galeommatidae Corbiculidae Ungulinidae
Noetiidae Kelliidae Veneridae Unionidae
Cucullaeidae Carditidae Petricolidae Verticordiidae
Limopsidae Chamidae Turtoniidae
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Glycymerididae Crassatellidae Corbulidae

Mytilidae Cardiidae Myidae
Pteriidae Tridacnidae Gastrochaenidae
Malleidae Mactridae Pholadidae
Isognomonidae Mesodesmatidae Teredinidae
Pinnidae Solenidae Astartidae
Limidae Pharidae Cuspidariidae
Gryphaeidae Tellinidae Cyrenidae
Ostreidae Donacidae Euciroidae
Plicatulidae Psammobiidae Glauconomidae
Propeamussiidae Solecurtidae Laternulidae

78
Characteristics of important share a basic morphology. The bivalves are bilaterally
bivalve families (marine) symmetrical, laterally compressed molluscs, with
extensive mantle lobes which secrete a single shell
(Source: Poutiers, 1998a) composed of two valves. The bivalves are mainly
The Bivalvia is the second largest class of the marine, but a few species are found in freshwater
molluscs. They show much variation in body form yet habitats, although none have invaded the land.

Pteriidae (Pearl oysters, wing oysters)

Pteriidae is a bivalve family of great economic importance. The dorsal shell margin is
often produced at each end into a wing-like ear, sometimes very long behind. Shell
slightly inequivalve. Right valve is with a byssal notch anteriorly. Hinge toothless or
with denticles. Interior brilliantly nacreous. Only one adductor muscle scar. Pallial line
without a sinus. In Indian Seas 12 species are reported under this family including
the six species of pearl oysters, eg. Pinctada fucata, P. margaritifera, P. chemnitzii, P.
sugillata, Pteria spp.

Pectinidae (Scallops)
Pectinidae have circular shells with radiating ribs, shell more or less inequivalve,
ovate to subcircular with a straight dorsal margin forming wing-like ears. A byssal
notch and a ctenolium at right valve. Ligament internal, in a small trigonal pit
pointing under the umbones. Hinge without teeth. A single adductor muscle scar.
Pallial line without a sinus. Thirty one species are reported from Indian waters, eg.
Chlamys tranquebaricus

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Anomiidae (Jingle shell)
The shells are inequivalve, often irregular, adhering to substrate by means of a
calcified byssus passing through a hole-like embayment of right valve. Ligament
internal. Hinge without teeth. Central area of the interior thickened, with 1 or 2
retractor muscle scars in left valve, in addition to the single adductor scar. No pallial
sinus. Four species are reported from India, eg. Anomia ephippium

Placunidae (Windowpane oysters)

Placunidae includes only a single genus and single species. Shell thin, rounded to
saddle-shaped, very compressed laterally, slightly inequivalve. Ligament internal,
forming an inverted V-shaped structure. Hinge without teeth. A single adductor
muscle scar. Pallial line without a sinus, eg. Placuna placenta

Ostreidae (Oysters)
Ostreidae and Gryphaeidae are known as the true oysters. Shell inequivalve,
cemented to substrate by the left valve, right valve quite flat. Ligamental area with
a shallow median groove and 2 lateral thickenings. Hinge without teeth. A single
adductor muscle scar, median in position or nearer to the ventral margin. Internal
margins smooth or with simple short marginal crenulations. About 11 species
are recorded from India, eg. Crassostrea madarsensis, C. gryphoides, C. rivularis,
Saccostrea cucullata.

Gryphaeidae (Honeycomb oyster)

Gryphaeidae is having only a single species reported from India, the giant honeycomb
oyster (Hyotissa hyotis). Shell more or less inequivalve, cemented to substrate by the left
valve, with a microscopic vesicular structure. Ligamental area with a shallow median
groove. Hinge without teeth. A single adductor muscle scar, closer to the hinge.

79
 Pholadidae (Angelwings, paddocks)
Pholadidae have uniquely evolved shells. They burrow a cavity into wood, rock and
other materials for protection. Shell subequivalve, gaping. Dorsal margin forming
an umbonal reflection. A number of accessory calcareous plates about the main
shell. Ligament reduced. Hinge without teeth. A finger-like internal apophysis. Three
adductor muscle scars. Pallial line deeply sinuated, eg. Martesia striata

Teredinidae (Shipworms)
Teredinidae are known for boring into wood structures that are immersed into
seawater. Shell reduced, equivalve, widely gaping. Anteroventral margin with a
deep, right-angled notch. Dorsal margin forming an umbonal reflection. Ligament
reduced. Hinge without teeth. A finger-like internal apophysis. An internal
umbonoventral ridge, with a knob at both ends. Three adductor muscle scars.
Accessory calcareous tube lining burrow long, closed by a pair of pallets. eg.
Uperotus panamensis. Teredora malleolus.

Mytilidae (Mussels)
Mytilidae include the green mussel Perna viridis, and P. indica, important mariculture
species which also contribute to the fishery. Thirty two species are recorded from India,
of which 5 species are endemic to India. Shell equivalve and very inequilateral, with
a byssal gape. Umbones at or near anterior end. Periostracum prominent. Ligament
external, deep-set, supported by a whitish ridge. Hinge teeth absent or reduced.
Adductor muscle scars unequal, the anterior one small to absent. Pallial line without
a sinus. Inner side with an extensive nacreous layer, eg., Perna spp., Modiolus spp.

Pinnidae (Pen shells)

The pen shells are large, brittle, equivalve, subtrigonal, ventrally and posteriorly
gaping; very inequilateral, pointed in front. Anterior end eroded and internally
closed by small transverse partitions. Ligament linear. Hinge without teeth. Interior
with a thin nacreous layer, restricted to the anterior half. Two unequal adductor
muscle scars, eg. Pinna spp. Seven species are recorded from India.

Tridacnidae (Giant clams)

The family includes the largest living bivalve species, the giant clams, Tridacna spp.
Four species are reported from India. Shell equivalve, thick, heavy and often very
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large, with strongly scalloped free margins. Umbones ventral, free margins of the
valves dorsal-most in position. Byssal gape, when developed, internally plicate.
Outer surface with strong radial folds. Ligament external. Hinge with ridge-like
cardinal and lateral teeth. A single adductor muscle scar, associated with a pedal
retractor scar, submedian in position. Pallial line without a sinus.

Arcidae (Ark shells)

About 37 species are recorded from Indian Seas under the family Arcidae. This
includes the commercially important blood clam, Anadara granosa. Shell equivalve
or slightly inequivalve, mostly longer than high, more or less inequilateral.Umbones
prosogyrate, on top of a wide cardinal area. Ligament external, often with V-shaped
grooves. Hinge elongate, almost straight, with numerous small transverse teeth.
Two subequal adductor muscle scars. Pallial line without a sinus.

80
Mactridae (Trough shells)
The shell is shaped like a rounded-cornered equilateral triangle and there is a slight
gape at the posterior, shell equivalve. Umbones prosogyrate. Internal ligament well
developed, in a trigonal pit of hinge plate. Hinge characteristic, with 2 cardinal
teeth and lateral teeth; cardinal teeth of the left valve forming an inverted V-shaped
process. Two adductor muscle scars. Pallial line with a well-developed sinus. The
superfamily Mactroidea, which includes Mactridae and Mesodesmatidae contains
32 species, of which 3 species are endemic to India. eg. Mactra violacea.

Mesodesmatidae (Wedge clams)

Shell equivalve, inequilateral, subtrigonal to wedge-shaped. Umbones opisthogyrate.
Internal ligament in a deep pit of hinge plate. One or 2 cardinal teeth and lateral
teeth. Two adductor muscle scars. Pallial line with a short sinus. eg. Mesodesma
glabaratum.

Solenidae (Razor shell)

Members of the genus Solen are with a narrowly elongate shape, gaping at both
ends. Umbones more or less near the anterior end. Ligament external. Hinge feeble.
Two adductor muscle scars, the anterior one larger. Pallial sinus relatively shallow,
eg., Solen spp.

Corbiculidae (Basket clams)

Shell equivalve, solid, umbones prosogyrate. No lunule or escutcheon. Periostracum

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conspicuous. Ligament external. Three diverging cardinal teeth in each valve, and
strong anterior and posterior lateral teeth. Two adductor muscle scars. Pallial sinus
reduced to absent. The family includes the commercially important black clam,
Villorita cyprinoides, the mangrove clam, Polymesoda erosa.

Veneridae (Venus clams)

The superfamily Veneroidea having three families (Petricolidae, Turtonidae,
Veneridae) is the second largest after the Tellinids. Ninety species are reported
under this superfamily from India. Shell mostly solid, equivalve, inequilateral, with
prosogyrate umbones. Lunule and/or escutcheon usually present. Ligament external.
Three cardinal teeth in each valve, anterior lateral teeth sometimes present. Two
adductor muscle scars. Pallial sinus usually present. eg. Meretrix spp., Paphia spp.

Cardiidae (Cockles)
Twenty five species are recorded from Indian Seas. One species is endemic to
Andaman and Nicobar. Shell equivalve, inflated, oval to subquadrate, sometimes
heart-shaped. Umbones prominent. External sculpture mostly radial. Ligament
external. Hinge characteristic, with teeth curving outwards; 2 cardinal teeth and
lateral teeth in each valve; cardinal teeth cruciform in arrangement. Two adductor
muscle scars. Pallial line without a sinus. eg. Cardium flavum, Cardium asiaticum.

Carditidae (Carditas)
Carditidae family is represented by 7 species in Indian Seas. Shell equivalve, stout
and inflated, inequilateral. Exterior mostly with radial ribs. Ligament external. Two
cardinal teeth, unequal and with fine transverse striations; lateral teeth frequently
reduced to absent. Two adductor muscle scars. Pallial line without a sinus. eg.
Cardita antiquata

81
 One of the most widely accepted classification features of the animals. In the Bouchet and Rocroi
systems for the class Bivalvia was that which taxonomy, clades are unranked and used between the
employed a grouping system based on the shell rank of class and the rank of superfamily. They use
shape, microstructures and hinge configuration by six main clades: Patellogastropoda, Vetigastropoda,
Newell (1965, 1969). Because features such as hinge Cocculiniformia, Neritimorpha, Caenogastropoda,
morphology, dentition, mineralogy, shell morphology and Heterobranchia. The first three of these major
and shell composition change slowly over time, clades have no nesting clades within them: the
therefore these characteristics are used to define taxonomy goes immediately to the superfamily level.
major taxonomic groups. Within the Caenogastropoda there is one extra clade.
Cladogram showing gastropod clads, groups and informal
Among the 652 species of marine bivalves reported groups (Bouchet and Rocroi, 2005)
from India, 88 species are endemic to Indian waters 1. Patellogastropoda
(Tripathy and Mukhopadhyay, 2014). 2. Vetigastropoda
3. Cocculiniformia
4. Neritimorpha
Class Gastropoda «« Paleozoic Neritimorpha of uncertain systematic
position (fossil)
Gastropods are the most successful group of molluscs «« Cyrtoneritimorpha (fossil)
not only in terms of the number of species, but also «« Cycloneritimorpha
in the wide range of habitat in which they may 5. Caenogastropoda
«« Caenogastropoda of uncertain systematic position
be found. Marine gastropod species have become «« Architaenioglossa
adapted to living on all types of substratum and some «« Sorbeoconcha
have even adopted a pelagic existence. Generally a «« Hypsogastropoda
univalve spirally coiled shell is present in the majority ªª Littorinimorpha
ªª Ptenoglossa
of gastropods, although the shell may be poorly
ªª Neogastropoda
developed or lacking in the opisthobranchs. Their soft 6. Heterobranchia
body is divided into 4 main regions: the head, which «« Lower Heterobranchia
normally protrudes anteriorly from the shell; the foot, «« Opisthobranchia
ªª Cephalaspidea
a muscular ventral organ with a flattened base used
ªª Thecosomata
for locomotion (creeping or burrowing); the visceral ªª Gymnosomata
mass, which fills dorsally the spire of the shell, and ªª Aplysiomorpha
contains most organ systems; the mantle, a collar- ªª Acochlidiacea
like tegument which lines and secretes the shell, ªª Sacoglossa
ªª Cylindrobullida
and forms a mantle cavity normally provided with ªª Umbraculida
respiratory gills in aquatic species. The noteworthy ªª Nudipleura
asymmetry of the internal anatomy of gastropods ©© Pleurobranchomorpha
Central Marine Fisheries Research Institute

results from a twisting through 180º called the ©© Nudibranchia

»» Euctenidiacea
“torsion”, which occurs in the first few hours of larval
»» Dexiarchia
development. Part of the paired organs of the visceral • Pseudoeuctenidiacea
mass cease developing, and the animal begins to be • Cladobranchia
asymmetrical. This internal asymmetry persists in the ◊◊ Euarminida
◊◊ Dendronotida
adult, even when a subsequent detorsion occurs.
◊◊ Aeolidida
«« Pulmonata
The taxonomy of the Gastropoda was revised by ªª Basommatophora
Philippe Bouchet and Jean-Pierre Rocroi in 2005. They ªª Eupulmonata
have grouped both living and extinct gastropods, as ©© Systellommatophora
©© Stylommatophora
well as some fossils as clades, derived from research
»» Elasmognatha
on molecular phylogenetics. This is in contrast to »» Orthurethra
the taxonomic schemes relying on morphological »» Sigmurethra

82
In contrast, within the Heterobranchia, for some of and freshwater snails and some deepwater
the nudibranch groups there are six separate clades limpets (Neritidae).
above the level of superfamily, and in the case of most
of the land snails, there are four clades above the level Caenogastropoda is a large diverse group of
of superfamily. Since the publication of this taxonomic sea snails, land snails and freshwater snails, which
system in 2005, various proposals for changes have includes 60% of the extant gastropods. They include
been published by other authors. the shelled marine gastropods, the periwinkles,
cowries, moon snails, murexes, cone snails, turrids
Patellogastropoda, are the true limpets, historically and other families.
called Docoglossa. They are found attached to hard
surfaces in the intertidal zone, and are capable of Heterobranchia includes families which were
locomotion (Patellidae, Nacellidae, Pectinodontidae). historically placed in many different class of
gastropods. This includes three informal groups; the
Vetigastropoda are considered among the most lower Heterobranchia (shelled marine and freshwater
primitive living gastropods, and are widely distributed species), the Ophisthobranchia (mostly marine
from the intertidal areas to the deep-sea in all oceans species) and the Pulmonata (land snails and slugs;
of the world. They include the keyhole limpets, many freshwater snails and few marine species).
abalones, top shells, turban shells and other families
(Trochidae, Turbinidae). Marine gastropod diversity

Cocculiniformia includes the deep-sea Nearly 32,000-40,000 marine gastropods are described
limpets (Cocculinidae). globally and an additional 35,000-45,000 more are
collected but undescribed. In India, there are 1487 listed

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Neritimorpha includes the terrestrial, marine marine gastropod species along the east coast of India,

Characteristics of important gastropod families (marine)

(Source: Poutiers, 1998b)

Haliotidae (Ear shells)

Shell ear-shaped, not permanently cemented to a substrate depressed and loosely
coiled. Spire eccentric. A spiral row of holes on body whorl. Aperture occupying
most of the underside. Interior nacreous. No operculum. Haliotis varia (variable
abalone)

Trochidae (Top shells)

Shell conical to globose, often with a flattened base. Aperture without a siphonal
canal, nacreous within. Operculum corneous, nearly circular. Trochus radiates, T.
niloticus (Commercial top), Umbonium vestiarium, (Common button top)

Turritellidae (Screw shells)

Shell elongate, sharply conical, with numerous whorls and a small aperture. Whorls
sculptured with spiral ribs or keels. Siphonal canal absent. Operculum corneous,
rounded. Turritella attenuata, T. acutangula

83
 Buccinidae (Babylon shells)
Shell with a fairly high spire and large body whorl. Outer surface smooth or with
sculpture, without axial varices. Siphonal canal rather short. Operculum corneous.
Babylonia spirata, B. zeylanica, Nassaria sp.

Muricidae (Murex snails)

Shell variably shaped, generally with a raised spire and strong sculpture with axial
varices, spines, tubercles or blade-like processes. Periostracum absent. Aperture
with a well-marked siphonal canal. Operculum corneous. Murex sp. Haustellum
Haustellum, Thais sp., Drupa sp., Rapana rapiformis

Cerithiidae (Horn shells)

Shell sharply conical, with a high, many-whorled spire and rather small aperture.
Sculpture variable. Aperture with a siphonal canal. Outer lip somewhat expanded.
Operculum ovate, corneous, with a few spiral coils. Cerithium spp.

Cassidae (Helmet shells)

Shell thick and solid, with a large body whorl and rather small, conical spire.
Sculpture variable, axial varices sometimes present. Aperture elongate, with a short
siphonal canal, recurved dorsally. Outer lip thickened. Inner lip with a shield-like
callus. Operculum quite small, corneous. Cypraecassis (C.) rufa, Phalium glaucum,
Semicassis (S.) bisulcata

Tonnidae (Tun shells)

Shell thin, globose, with a short spire and very inflated body whorl. Sculpture
only spiral. Siphonal canal short. Operculum absent. Tonna dolium, T. cumingii, T.
tessellata
Central Marine Fisheries Research Institute

Conidae (Cones)
Shell cone-shaped, with a low spire and a well-developed body whorl tapering
towards the narrow anterior end. Aperture very long, with a short siphonal canal.
Operculum corneous, quite small. Conus sp.

Olividae (Olive shells)

Shell elongate-ovate, with a short spire, a large body whorl and channeled sutures.
Surface smooth, highly polished. Aperture elongate, with a short siphonal canal.
Inner lip calloused, with oblique grooves anteriorly. Operculum absent. Oliva sp.

84
Ranellidae
Shell ovate-fusiform, with a strong sculpture and axial varices. Periostracum
frequently well developed and hairy. Aperture with a siphonal canal. Operculum
corneous. Cymatium (L.) lotorium, C. (M.) pileare.

Ficidae (Fig shells)

Shell thin, pear-shaped, drawn out anteriorly into a long, tapered and gracefully
curved siphonal canal. Operculum absent. F. gracilis, F. investigatoris, F. Ficus.

Volutidae (Volutes)
Shell variable in shape, often glossy and brightly coloured. Aperture long, with a
short siphonal canal. Inner lip with strong folds, weaker posteriorly. Operculum
horny, often absent. Voluta lapponica. Melo melo, Voluta sp.

Turbinellidae (Chank shells)

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Shell thick and heavy, biconical to fusiform, often nodulose to spinose on shoulder.
Periostracum conspicuous. Siphonal canal present. Inner lip with strong folds.
Operculum corneous. Turbinella pyrum

Bursidae (Frog shells)

Shell ovate, often slightly dorsoventrally compressed, with 2 strong axial varices
per whorl. Periostracum obsolete. Aperture with a short siphonal canal and a
distinct posterior canal. Operculum corneous. Bufonaria crumena, B. echinata, B.
margaritula

Naticidae (Moon snails)

Shell globular to ovate-conical. Outer surface smooth or with reduced sculpture.
Aperture large, semicircular. Siphonal canal absent. Umbilicus open or closed,
sometimes with an internal rib. Operculum corneous or calcified. Natica sp.

Turbinidae (Turban shells)

Shell thick, turbinate to conical. Outer sculpture often spiral to nodular. Aperture
rounded, without a siphonal canal, nacreous within. Operculum strongly calcified.
Turbo marmoratus (Great green turban) T. (M.) radiates, T. intercostalis, T. (M.)
brunneus (Brown Pacific turban)

85
 Strombidae (True conches)
Shell thick and solid, with a relatively large body whorl. Aperture with a well-
marked siphonal canal. A distinct notch along the anterior margin of the outer lip.
Operculum corneous, claw-like. Tibia curta, T. deliculata, Lambis (Lambis) lambis
(common spider conch), Strombus (Laevistrombus) canarium (dog conch), S.
(Dolomena) marginatus (marginate conch), Terebellum terebellum

Cypraeidae (Cowries)
Shell ovate or oblong, spire concealed under body whorl. Surface highly polished,
smooth. Aperture long and narrow, channeled at both ends. Both lips with teeth.
No operculum. Cypraea sp.

Ovulidae
Shell globular to spindle-shaped, with more or less expanded extremities. Spire
concealed under body whorl. Surface often smooth, porcellaneous. Aperture very
long, channeled at both ends. Inner lip smooth. No operculum. Volva volva

Mitridae (Mitre shells)

Shell fusiform-ovate, with a predominantly spiral sculpture. Aperture notched by a
short siphonal canal. Outer lip not lirate inside. Columella with strong folds, larger
posteriorly. No operculum. Mitra sp.

Potamididae (Horn shells)

Shell high-conical, with many spire whorls. Sculpture generally coarse. Aperture
relatively small, with a short siphonal canal. Outer lip often flaring. Operculum
rounded, corneous, with many spiral coils. Telescopium telescopium, Cerithidea
(Cerithideopsilla) cingulate, Terebralia sp.
Central Marine Fisheries Research Institute

of which 222 are repeated and 7 species are freshwater and Classification of the recent Cephalopoda is
gastropods (Tripathy and Mukhopadhyay, 2014). under considerable discussion. The families of living
cephalopods are, for the most part, well resolved
Class Cephalopoda and relatively well accepted. Species-level taxa usually
can be placed in well-defined families. The higher
The class Cephalopoda is the most complex in the classification, however, still is not resolved. Jereb and
phylum Mollusca, and indeed, in all of the invertebrate Roper (2005) have used an ‘operational breakdown’ for
phyla. It includes exclusively marine animals represented classification. For practical purposes they have separated
by the squids, octopuses and cuttlefishes. At the present the cephalopods into several groups, without assigning
time the status and understanding of the Systematics or implying taxonomic relationships.

86
The living cephalopods are at present not the most in squids (the gladius or pen) and to a cartilaginous
successful of the molluscan groups, although there structure in finned octopods. In some sepiolids no
is fossil evidence to suggest that they were once a vestige of shell is found. A true external shell occurs
much more important group. There are only about only in the nautiluses, although a shell-like egg case
761 living species (Appeltans et al., 2012) described is produced and carried by female argonauts (pelagic
compared to 7500 fossil species so far discovered. octopods often misnamed ‘paper nautilus’). The loss
of the external shell allowed the development of a
Cephalopods are soft-bodied animals with a well- powerful muscular mantle that became the main
developed head, bearing an anterior circumoral locomotory organ for fast swimming, via water jetting
(surrounding the mouth) crown of appendages (arms, from the funnel. The funnel (siphon) is a unique,
tentacles) (Jereb and Roper, 2005). This characteristic multifunctional, muscular structure that aids in
feature reflects the origin of the name Cephalopoda, respiration and expulsion of materials in addition
which derives from the union of the two Greek words: to locomotion. Oxygenated water is drawn through
‘kefale’, head, and ‘pous’, feet. Arms and tentacles the mantle opening around the head (neck) into the
bear suckers and/or hooks (except in Nautilus), which mantle cavity, where it bathes the gills for respiration.
are powerful tools to seize prey. The mouth has a pair Mantle muscular contraction expels the deoxygenated
of chitinous jaws (the beaks) and, as in other molluscs, water from the mantle cavity through the ventrally
a chitinous tongue-like radula (band of teeth) occurs located funnel. The discharge jet serves to eliminate
in most cephalopod species. The ancestral mollusc nephridial and digestive wastes, as well as to complete
shell is variously modified, reduced, or absent in the respiratory cycle and for locomotion. Female
living coleoids. It is a calcium carbonate structure reproductive products (eggs, egg masses) also are
in cuttlefishes, reduced to a rigid chitinous structure discharged through the funnel. Most coleoids produce

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Cephalopod classification (Modified from Jereb and Roper, 2005)

87
 ink, a dark, viscous fluid to decoy potential predators, communicates with the exterior via 3 openings.
or a cloud to obscure the escaping cephalopod. Chitinous shell present.

Cephalopod Groups Family: Loliginidae

Internal shell straight, feather or rod-shaped,
Squid (Source: Jereb et al., 2010) chitinous; tentacles contractile, not retractile, no
Suckers (and/or hooks) present; no external shell. pockets; fins usually joined posteriorly; mantle
Suckers stalked with chitinous rings; 10 circumoral edge near mantle cartilages with small projections
appendages, 8 arms and 2 ventrolateral tentacles; or ‘angles’. Eye covered by transparent membrane
contractile not retractile, no pockets. Mantle cavity (cornea); Four longitudinal rows (series) of suckers on

Uroteuthis (Photololigo) duvauceli

Uroteuthis (Photololigo) chinensis

Central Marine Fisheries Research Institute

Uroteuthis (Photololigo) edulis

Uroteuthis (Photololigo) sinhalensis

88
manus of tentacular clubs; fins united at posterior end Family: Thysanoteuthidae
of mantle; medial posterior border of fins concave.
Internal shell straight, feather-or rod-shaped,
Family: Ommastrephidae chitinous; tentacles contractile, not retractile, no

Internal shell straight, feather or rod-shaped,

chitinous; tentacles contractile, not retractile, no
pockets; fins usually joined posteriorly; mantle edge
near mantle cartilages with small projections or
‘angles’. Eye without cornea; lens in open contact

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Thysanoteuthis rhombus

pockets; fins usually joined posteriorly; mantle edge

near mantle cartilages with small projections or
‘angles’. Eye without cornea; lens in open contact
with seawater. Funnel free from mantle; funnel-
mantle locking apparatus present; Funnel-locking
cartilage with a longitudinal groove from which a
shorter groove branches medially, ┤shaped; fins more
than 80% of mantle length

Cuttlefish (Source: Reid et al., 2005)

Suckers (and/or hooks) present; no external shell,
suckers stalked with chitinous rings; 10 circumoral
appendages, 8 arms and 2 ventrolateral tentacles.
Mantle cavity communicates with the exterior through
3 openings. Internal shell straight, laminate, calcified;
Stenoteuthis oualaniensis
tentacles contractile and retractile into pockets
between arms III and IV; fins not joined posteriorly;
with seawater. Funnel free from mantle; funnel- mantle edge near mantle cartilages straight.
mantle locking apparatus present; Funnel-locking
apparatus not a simple, straight groove and ridge; Family Sepiidae:
Funnel-locking cartilage with a longitudinal and a
transverse groove shaped. Sepia: Cuttlebone outline elliptical to lanceolate;

89
 cuttlebone length approximately equal to mantle
length; dorsal anterior edge of mantle usually with
tongue-like projection. Gland and gland pore absent;
mantle-locking apparatus semicircular, without
triangular projection, cuttlebone inner cone with
relatively long limbs; outer cone usually calcareous,
not obviously spatulate posteriorly. Species: Sepiella
inermis, Sepia elliptica, S. prashadi, S. trygonina, S.
aculeata, S. brevimana, S. arabica.

Sepiella: Cuttlebone outline elliptical to lanceolate;

cuttlebone length approximately equal to mantle
length; dorsal anterior edge of mantle usually with
tongue-like projection. A gland and gland pore located
on the ventral side of the posterior end of the mantle;
mantle-locking apparatus with triangular projection;

Sepiola: Paired, kidney-shaped light organs on

anterior surface of ink sac; tentacular club suckers
usually in 4 to 8 transverse rows.

Octopus
Sepiella
Suckers without stalks, bases sometimes constricted
in finned (cirrate) octopods, without chitinous rings;
8 arms, no ventrolateral tentacles. Mantle cavity
communicates with the exterior via one opening,
rarely 2. Octopods have short, sac like body, eight
circum oral arms connected at the base by a numerous
web, no tentacles. Octopods are divided into two
suborders. Cirrata, mostly deep sea forms possess cirri
along the arms and paddle shaped fins dorsal lateral
to the mantle. Incirrata, mostly shallow living forms
instead of cirri have one or two series of non-stalked
suckers along the arms; and no fins.
Central Marine Fisheries Research Institute

Sepiella inermis

Family: Octopodidae
cuttlebone inner cone with very short limbs; outer Funnel-locking apparatus absent; water pores on head
cone a wide, spatulate, chitinized border around absent; males not very much smaller than females,
posterior end of cuttlebone. Species: Sepiella inermis with left or right ventrolateral arm hectocotylized
(never in pocket), with spoon-shaped, non-
Family: Sepiolidae filamentous tip; females without dorsal arm flaps or
permanent reticulate sculpturing of ventral mantle
Internal shell straight and chitinous; tentacles
contractile and retractile into pockets between arms Species: Octopus vulgaris, Amphioctopus marginatus,
III and IV; fins not joined posteriorly; mantle edge Amphioctopus aegina, Amphioctopus neglectus,
near mantle cartilages straight. Amphioctopus rex, Cistopus indicus

90
Nautilus (Source: Jereb, (2005)) Catalogue for Fishery Purposes. No. 4, Vol. 1. Rome, FAO.
pp. 51–55.
Jereb P. and C.F.E. Roper 2005. Cephalopods of the world, An
annotated and illustrated catalogue of species known to date.
Family Nautilidae FAO Species Catalogue for Fishery Purposes No.4, Vol.1.
Jereb, P., Vecchione, M. & Roper, C.F.E. 2010. Family Loliginidae.
Nautilus possess more than ten (63-94) circumoral In P. Jereb & C.F.E. Roper, eds. Cephalopods of the world.
An annotated and illustrated catalogue of species known to
appendages, without suckers. The shell is external date. Volume 2. Myopsid and Oegopsid Squids. FAO Species
and coiled with chambers. The living nautiluses are Catalogue for Fishery Purposes. No. 4, Vol. 2. Rome, FAO.
pp. 38–117.
limited belonging to 1 family and 2 genera. Newell, ND. 1965. Classification of the Bivalvia. Amer. Museum
Novit. 2206: 1-25.
Newell, ND. 1969. Classification of the Bivalvia. In: Treatise on
Invertebrate Paleontology, Part N, Mollusca 6, Vol. 1. Bivalvia.
Moore R, ed., pp. N205-N244. Geological Society of America
and University of Kansas, Boulder-Lawrence.
Poutiers, J.M. 1998a. Bivalves. In Carpenter, K. E. and V. H. Niem
(Eds). In FAO species identification guide for fishery purposes.
The living marine resources of the Western Central Pacific,
Volume 1. Seaweeds, corals, bivalves, and gastropods. edited
by K.E. Carpenter and V.H. Niem. Rome, FAO. 123-362.
Poutiers, J.M. 1998b. Gastropods. In Carpenter, K. E. and V. H. Niem
(Eds). In FAO species identification guide for fishery purposes.
The living marine resources of the Western Central Pacific,
Volume 1. Seaweeds, corals, bivalves, and gastropods. edited
by K.E. Carpenter and V.H. Niem. Rome, FAO. 363-648.
Reid, A., Jereb, P. & Roper, C.F.E. 2005. Family Sepiidae. In P. Jereb
& C.F.E. Roper, eds. Cephalopods of the world. An annotated
and illustrated catalogue of species known to date. Volume
1. Chambered nautiluses and sepioids (Nautilidae, Sepiidae,
Nautilus pompilius Sepiolidae, Sepiadariidae, Idiosepiidae and Spirulidae). FAO

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Species Catalogue for Fishery Purposes. No. 4, Vol. 1. Rome,
FAO. pp. 57–152.
Tripathy B., Mukhopadhyay A. K., 2014 Marine molluscan diversity
Suggested readings in India. In: Marine faunal diversity in India: taxonomy, ecology
and conservation. Elsevier Inc., pp. 39-74.
UNEP [United Nations Environment Programme]. 1995. UNEP
Appeltans W., P. Bouchet, G.A. Boxshall, K. Fauchald, D.P. Gordon, releases first global biodiversity assessment report. UNEP Press
B.W. Hoeksema, G.C.B. Poore, R.W.M. van Soest, S. Stöhr, Release HE/916 of 14 November 1995.
T.C. Walter and M.J. Costello. (Eds). (2012). World Register Venkataraman, K. and M. Wafar. 2005. Coastal and marine
of Marine Species. Accessed at http://www.marinespecies. biodiversity of India. Indian J. Mar. Sci., 34(1): 57-75.
org on 2012-01-23. Von Salvini-Plawen L. 1990a. Origin, phylogeny and classification
Bouchet, P. and Rocroi, J.P. 2005. Classification and nomenclature of the phylum Mollusca. Iberus 9: 1-33.
of gastropod families. Malacologia, 47(1-2): 1-397. Von Salvini-Plawen L. 1990b. The status of the Caudofoveata and
Jereb, P. 2005. Family Nautilidae. In P. Jereb & C.F.E. Roper, eds. the Solenogastres in the Mediterranean Sea. Lavori S.I.M. 23:5-
Cephalopods of the world. An annotated and illustrated 30.
catalogue of species known to date. Volume 1. Chambered
nautiluses and sepioids (Nautilidae, Sepiidae, Sepiolidae,
Sepiadariidae, Idiosepiidae and Spirulidae). FAO Species

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 Bryozoa – Taxonomy and Diversity
N. Nandini Menon
Scientist
Nansen Environmental Research Centre India,
6A, Oxford Business Centre, Sreekandath Road, Cochin – 16
e-mail: [email protected]

Introduction gave the name POLYZOA (Many animals) as the

colonies are composed of numerous connected
Bryozoans are an ancient, aberrant phylum of units called zooids. Ehrenberg named them
microscopic coelomate and often beautiful animals as BRYOZOA–“moss animals” (Greek Bryo –
that build intricate colonies. Bryozoan phylum moss and Zoion – animal). Hyman renamed it
contains around 3,500 living species and 15,000 as ECTOPROCTA as the animals had their anus
extinct species. They are found both in marine and outside the crown of tentacles (Greek ecto –
freshwater environments, living at all latitudes and outside, proktos – anus). The controversy over
at depths ranging downward to 8,500 meters. which name to be selected still continues, and
They have a well recorded fossil history due to their today, the phylum has three names–PHYLUM
zooecial nature and have been around since the BRYOZOA or ECTOPROCTA or POLYZOA.
Ordovician era.  The bryozoans are almost always
sedentary and sessile animals that live exclusively Habitat
in colonies, normally adhered to a substratum.
The only exception to this colonial existence Bryozoans are benthic sessile forms found from
is  Monobryozoon, which contains species that continental shelf to bottom depths. In 1995,
may be solitary, depending upon conditions.  the first free-swimming bryozoan colonies were
recorded from Weddell Sea, Antarctica  - 400m
History deep and some distance from the normal bryozoan
habitats.  They resembled floating brown golf balls
Early naturalists classified Bryozoans as members in an area free of sea ice for most of the year. It is
of the plant kingdom and Linnaeus invented suspected that the colonies may have been released
the name ‘zoophytes’. In early 16 th century, from under the sea ice because of ice break-up.
Imperato asserted the animal nature of the Another possibility is that the colonies may feed
Central Marine Fisheries Research Institute

zoophytes and noted the pores on the colonies. on the microscopic algae that grow on the under-
The ‘pores’ that he noted as the unique features layer of sea-ice in this area.  A further advantage is
of these organisms, are still retained in many that mobile species are able to exploit patchy food
generic names of the phylum like Retepora, resources which are some distance apart. This is
Membranipora etc. Despite this, until the latter important because the plankton levels in the Weddell
part of 18 th century, naturalists like Linnaeus, Sea are very seasonal. Scientists currently speculate
Cuvier etc. continued to use the term Zoophyta to that the new bryozoan may be a juvenile bryozoan
describe Bryozoans. By 19 th century, they began of the genus Alcyonidium. 
to be considered as the polyps of cnidarians. In
the second quarter of 19th century, Thompson and Bryozoans are well distributed in the Indian waters.
Ehrenberg independently found out that they are They show exceptional levels of species diversity than
not ordinary polyps and that they have a separate any other organisms known today (Menon, 1967;
digestive tract with two openings. Thompson Menon and Menon, 2006).

92
 zooids. The colony is composed of individuals (zooids),
each of which is typically enclosed in a secreted
exoskeletal case. The case is termed zooecium; the
zooecia of a colony has an opening to the exterior
called orifice, provided with a closing apparatus called
the operculum. The ectoproct individual or zooid
consists of two main parts, the tentacular crown or
lophophore, protrusible through the orifice, and the
trunk, permanently fastened in the zooecium. In the
marine forms or gymnolaemates, the lophophore
is circular but has the shape of a horseshoe in the
fresh-water or phylactolaemate forms. lophophore
always embraces the mouth but never the anus.
Lophophore is raised above the zooid on a slender
extension of the body wall (the tentacle sheath, or
introvert). When not spread for feeding, the tentacles
are withdrawn into the coelom by the action of paired
retractor muscles. Eversion of the tentacle sheath and
tentacles is effected by raising the hydrostatic pressure
of the body fluid. 

The colony is composed of polymorphic zooids.

• Autozooid: zooids responsible for feeding

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

and excretion.
• Heterozooid: specialized non-feeding
zooids which get nutrients from autozooids
through channels.
(Adapted from Poutiers, 1998a)
• Avicularia: small heterozooids in which the
zooecium and operculum form a beak-like,
Basic Morphology snapping structure–defensive in function.
of Bryozoa • Vibracula: zooids that bear long setae, or bristles–
The phylum name, Bryozoa, literally means ‘moss clean the bryozoan colony and supposed to aid
animals’ and refers to the bushy, moss-like colonies in chemoreception.
of some species. Flat encrusting forms are sometimes • Kenozooids: small heterozooids that strengthen
called sea mats. Erect, lacy forms are often called lace and support the colony as well as fill space.
corals, a name that could also be applied to the thin, • Ooecia / ovicells: zooids specialised for
lace-like sheets that encrust kelp fronds. reproductive activities, they act as brood chambers,
producing and holding safe the eggs until they are
Bryozoans are defined as microscopic, sessile, ready to hatch.
colonial coelomates that are permanently fastened
in exoskeletal cases or gelatinous material of their Also there are individuals that serve as stolons,
own secretion, that are provided with a circular or holdfasts and attachment discs.
crescentic lophophore and a recurved digestive tract
bringing the anus near the mouth, and that lack Bryozoans are filter feeders. They have a U-shaped
nephridia and a circulatory system. The colony can gut and a lophophore of ciliated tentacles. Retraction
be arborescent or frondose or can very often form and protrusion of the lophophore aids in feeding.
flat spreading incrustations on objects, or sometimes Tentacles generate a water current and trap small
become adherent or erect by stolons bearing the particles of food on a constantly moving stream

93
 of mucous. This mucous stream moves down the colony breaks off, the piece can continue to grow and
tentacles and into the digestive tract, taking anything will form a new colony. In old and senile zooids, the
it has caught with it.  The digestive tract consists of whole body of the zooid retracts to form a small ball,
a pharynx, oesophagus, a stomach and an intestine which then degenerates to form a mass of non-living
which terminates in an anus.  The re-curved digestive debris. This mass is often brownish and is called a
tract hangs freely in the coelom with few attachments “brown body.” A short time after this process, either
to the body wall. In some forms, proximal part of the organism regenerates around the brown body,
stomach is altered into a gizzard that helps in grinding which is often incorporated into it or the brown body
the siliceous and calcareous exoskeleton of diatoms is incorporated into the gut and expelled from the
and dinoflagellates. new individual.  

The nervous system consists of a main ganglionic mass In fresh water forms, during winter, the colonies
situated between the mouth and the anus encircles become dormant or inactive. At this point colonies
the pharynx. The nerves ascend into the tentacles form statoblasts which are modified unopened
and descend along the digestive tract and other parts zooid buds. They are cold resistant and survive the
of the trunk. Most ectoprocts are hermaphroditic. freezing waters when the rest of the zooid colony
Circulatory, respiratory and excretory systems as dies off. When the water warms up, each statoblast
organic assemblages are wanting. germinates into a functional zooid with the ability to
form between one and five newer buds.
Bryozoans can reproduce both sexually and asexually.
Most of the zooids are hermaphrodite, but the testes Peculiarities in Reproduction
and ovary usually do not mature at the same time.
Some species shed both eggs and sperm directly
into the water where they fuse. But the majority
Selfing vs Outcrossing
of species brood their eggs within the zooecium or Studies have shown that selfing (sexual reproduction
in special chambers known as ovicells, and capture involving egg and sperm from the same zooid) is
free-swimming sperm with their tentacles to fertilize not routinely occurring in Bryozoans as the fitness of
the eggs. selfed offspring is significantly reduced compared to
outcrossed offsprings. However, in certain situations
The fertilized eggs divide and develop into free- like the following, selfing in Bryozoans could lead to
swimming ciliated larvae called Cyphonautes. These the production of viable, self-compatible offspring
animals exist as colonies that are typically derived (Johnson, 2010).
by asexual reproduction from a single progenitor • If the population within a given locale were
(ancestrula), originating by the metamorphosis of founded by few individuals, the selection for
a sexually produced larva. In those forms where selfing would be greater, where solitary colonies
Central Marine Fisheries Research Institute

embryo develops in the ovicell, larvae escape from were indeed able to reach reproductive maturity,
the brood chamber and swim away. The escape self, and release offspring and result in the
of larva appears to be a response to illumination establishment and subsequent propagation of
(positive phototaxis). These larvae eventually settle self-compatible individuals.
on a suitable substrate and metamorphose into a • Results from investigations with Celleporella
new zooid. The first formed zooid or the parent hyalina showed a differential ability to self among
zooid is called ancestrula. Ancestrula initiates colony geographically distinct populations (Hughes et
formation by asexual reproduction. al., 2009).

Asexual reproduction occurs by budding and is Cloning

the main way by which a colony expands in size.
Proliferation proceeds according to a pattern Prolific polyembryony (the splitting of a single sexually
characteristic of the species. If a piece of a bryozoan produced embryo into many clonal copies), is reported

94
in marine bryozoans of the order Cyclostomata. sequential order of the morphological complexity.
Microsatellite genotyping of brooded embryos
and maternal colonies conclusively demonstrated • The ctenostomes are the gymnolaemates with a
polyembryony. The characteristically voluminous simple, flexible, uncalcified zooecium composed
brood chamber of cyclostomes is judged to be an of chitinous cuticle. Usually represented by
adaptation linked to larval cloning and hence an stoloniferous vase like zooids, which are greatly
indicator of polyembryony. Embryos are always misunderstood as the coelenterates. The orifice
genetically identical within broods but genetically lacks a closing apparatus called the operculum
distinct among broods and from their mother. Each but the diaphragm usually bears a pleated collar
brood, therefore, result from vegetative budding of a which when folded blocks the vestibule.
primary embryo, itself derived from a zygote resulting
from outcrossed mating via water-borne sperm.  The • The Cheilostomata are the calcified group and the
reasons of such cloning is not sure, but it is speculated most dominant group in a bryozoan assemblage. The
that either colony usually consists of box-like contiguous zooids
• Low sperm output from colonies (Pemberton et arranged as branches, continuous incrustations or
al., 2011)or lamellate expansions. They are unique in having
• Unpredictable environmental conditions or an operculum, the closing apparatus of the orifice.
• Phylogenetic constraint (Hughes et al., 2005) may Polymorphism, a phenomenon of variation in zooidal
be responsible. structure and function within a species reaches its
paramount in this group. Based on the extent of
Classification calcification of the exoskeleton and the presence
or absence of ascopore, they are subdivided into
The marine bryozoans, the Gymnolaemata, may or Anasca and Ascophora.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

may not be calcified, at least to some extent, this helps
in classifying this group, which is subdivided into two »» Anasca having a less calcified membraneous
living orders, based on the complexity involved by front which itself regulates the in and
the calcification of the exoskeleton. The two living out movements of their feeding organ–
orders are the ctenostomes and cheilostomes in the the lophophore.

95
 »» Ascophora has a distinctly calcified front due in marine creatures at the end of the Palaeozoic
to which the in and out movements of the greatly affected bryozoans. During the following
lophophore is facilitated by the possession of Mesozoic Era (251-65 mya) they recovered and new
a compensation sac–the ascus. major groups appeared, including those groups that
are most common today. During the Caenozoic Era
The family Cribrilinidae needs to be mentioned, which (less than 65 mya) bryozoans continued to increase
is the evolutionary link between the Anascans and in variety. Cyclostomes, a stenolaemate order of
Ascophorans, is featured by its calcified costulate minor importance in the Palaeozoic, radiated in the
armour with partially exposed front and possesses Jurassic and are extant today. Another extant order–
characters of both the classes. cheilostomes–first appeared in the Late Jurassic and by
the end of the Cretaceous had surpassed cyclostomes
Class Stenolaemata has one extant order named in both diversity and abundance in fossil assemblages.
Cyclostomata and two extinct orders. Cheilostomes belong to the Class Gymnolaemata
and represent an independent origin of a calcareous
• The cyclostomes are more complex group skeleton from a non-mineralized ancestor belonging
possessing fully calcified tubular zooids. to the paraphyletic Order Ctenostomata. Bryozoan
Lophophore protrusion is by the action of annular diversity today may be greater than at any time
muscles and is devoid of avicularia and vibracula in the geological past, except perhaps during the
but heterozooids occur in the form of gonozooids, Pliocene before the cooling and other changes that
nannozooids and kenozooids accompanied Pleistocene glaciation.

Palaentology Economic Importance

The Bryozoa have a long and eventful fossil history. Fresh water Bryozoans (Plumatella species) are one of
However, records do not appear in Cambrian (when the major foulers in pipelines. They disrupt the public
most metazoans appeared) or late Pre-Cambrian water service in many countries. Many techniques like
rocks. Bryozoa might have existed in the Cambrian but Sodium hypochlorite (commercial bleach) washing,
were soft-bodied or not preserved for some reason. painting the pipelines with Cu containing paints and
A poorly preserved fossil called Archaeotrypa from spraying with water of increased velocity (>1m/sec)
the upper Cambrian is perhaps the oldest Bryozoan. have been tried, but the following three factors hinder
The oldest known fossil bryozoans appeared in the control efforts:
Early Ordovician (diversity of marine invertebrates),
470 million years ago. Freshwater bryozoans are 1. statoblasts that tolerate harsh physical and
virtually unknown as fossils, presumably because chemical treatments;
they did not have mineralized skeletons. From the
Central Marine Fisheries Research Institute

time of their appearance in Ordovician, they evolved 2. regeneration of bryozoan colonies from pockets
rapidly into many diverse forms. During Ordovician, of living tissue;
the rate of appearance of genera was practically
equalled by the rate of extinction. For the remainder 3. easy dispersal of bryozoans through air and water.
of the Palaeozoic Era (until 251 mya) they were
abundant in shallow marine environments and were Marine Bryozoa are serious foulers on the hulls of
an important component of coral reefs formed ships. Many fouling bryozoans are even resistant to
during this time. Dominant Palaeozoic orders all Cu containing anti-fouling paints. Through ships and
belonged to the Class Stenolaemata and included through ballast water, they reach new environments
trepostomes, cystoporates, cryptostomes and and colonise there as invasive alien species. Tufted
fenestrates. The last of these orders became extinct forms like Bugula and Zoobotryon foul the intake
at the end of the Permian and the other three did not pipes of power station and ships’ cooling systems.
survive beyond the Triassic. The massive extinctions Bugula neretina, an inhabitant of tropical waters have

96
 Millions of
· YearsAgo
GEOLOGIC TIME SCALE Millions of
YearsAgo

ERA PERIOD
0 Diversification and colonizationof estuaries
0 2
Quarternary
Cenozioc Tertiary
65 End of the Dinosaurs
100 Cretaceous Recovery and Diversity
Mesozoic 144
Jurassic Radiation-Cyclostomes.Entry- Cheilostomes
200
213
Triassic First Dinosaurs, Mammals, Birds
248
Permian Stenolaemate extinction
286
300 Pennsylvanian
Carboniferous 320
Mississippian First Reptiles
360
Devonian First Amphibians
Paleozoic
400 408
Silurian
438 First Bryozoan Fossil
Ordovician First Land Plants
500 505 First Fishes
Cambrian

590 Archaeotrypa and other soft bodied bryozoans ?

600

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

700 700 First Invertebrates

4,600 4,600

reached the British seas and has invaded the area and reached the pharmaceutical markets (September,
established itself as a dominant species. 2005). Over 20 different bryostatins have been
isolated from this particular species (Faulkner,
Species like Alcyonidium is common in N. Sea and gets 1990, 1991, 1992, 1993, 1994).
trapped in piles in trawling nets. Repeated handling
of this can cause allergic dermatitis with painful rash According to scientists at the Blanchette Rockefeller
and weeping blisters. Neurosciences Institute (BRNI) and the Marine
Biological Laboratory, a cancer drug may stimulate
Bryozoans are biochemically important and the production of proteins needed for long-term
have been proved to be a rich source of novel memory, suggesting the compound may be a possible
compounds or bioactive agents. Bryostatin-1, a treatment for Alzheimer’s disease. Both these drugs
macrocyclic lactone compound is isolated from are in the stage of Phase II clinical trials.
the bryozoan Bugula neritina. It has antineoplastic
activity. Bryostatin-1 binds to and inhibits the Scientists at BRNI have discovered that Bryostatin
cell-signaling enzyme protein kinase C, resulting and a related class of drugs discovered at BRNI
in the inhibition of tumor cell proliferation, the administered 24 hours after stroke can rescue and
promotion of tumor cell differentiation, and the repair brain tissue. These findings are markedly
induction of tumor cell apoptosis. This agent may advanced compared to current stroke treatments
act synergistically with other chemotherapeutic that must be administered within three hours and
agents. A draft developed from this has already are unable to repair damaged brain tissue.

97
 B. dentata was shown to contain an anti-microbial Faulkner, D.J. 1993. Marine natural products. Natural Product
Reports, pp: 497-539.
blue pigment (Matsunaga et al.1986). Faulkner, D.J. 1994. Marine natural products. Natural Product
Reports, pp: 355-394.
A series of brominated alkaloids have been isolated Hughes, R. N.,  M. Eugenia D’Amato, John D.D Bishop, Gary R
Carvalho, Sean F Craig, Lars J Hansson, Margaret A Harley,
from Flustra foliacea (Wright, 1984) of which and Andrew J Pemberton. 2005 Paradoxical polyembryony?
Flustramines A and B having muscle relaxant Embryonic cloning in an ancient order of marine bryozoans.
Biol. Lett.; 1, 178–180.
activity and Dihydroflustramine exhibiting strong Hughes, R. N., P. J. Wright, G. R. Carvalho, and W. F.
antimicrobial activity. Hutchinson. 2009. Patterns of self-compatibility, inbreeding
depression, outcrossing, and sex allocation in a marine
bryozoan suggest the predominating influence of sperm
Chartella papyracea and Cribricellina cribraria are competition.  Biol. J. Linn. Soc. 98:519–531.
biochemically important for its biological activities  Hyman, L.H. 1959. The Invertebrates. McGraw Hill Book Company,
Inc., Vol. 5, pp: 275–515.
including anti-tumor and antifungal activities (Prinsep Johnson, C. H. 2010. Effects of Selfing on Offspring Survival and
et al., 1991). The calcium carbonate of these animals Reproduction in a Colonial Simultaneous Hermaphrodite
is in a highly pure form for the utilisation in dentistry. (Bugula stolonifera, Bryozoa). Biol. Bull., vol. 219 (1): 27-37.
Matsunaga, S., N. Fusetani and K. Hasimoto. 1986. Bioactive
Chitin extraction from bryozoan is another field that marine metabolites VII. Isolation of an antimicrobial blue
is developing. These chemicals open up an important pigment from the bryozoan Bugula dentata. Experientia, Vol.
42, pp: 84.
field in biotechnology research of pharmaceutical Menon, N.R. 1967. Studies on the Polyzoa of the south west coast
importance. The experimental studies to understand of India. Ph.D Thesis, University of Kerala, 548 p.
cloning and mapping of genes is a recent field of Menon, N.R. and N.N. Menon. 2006. A Monograph on the
taxonomy of bryozoans from the Indian EEZ, 325p.
research in which bryozoans are being used extensively Ryland, J.S. 1970. Bryozoans. Cain. AJ. (Ed.), Huchinson University
by genetic engineers. Library, London. 175p.
Pemberton A. J, Lars J. Hansson, John D. D. Bishop. 2011.
Does sperm supply limit the number of broods produced
Suggested readings by a polyembryonous bryozoan? Mar. Ecol. Prog. Ser., Vol.
430: 113–119.
Prinsep, M.R., J.W. Blunt and M.H.G. Munro. 1991. New cytotoxic
Faulkner, D.J. 1990. Marine natural products. Natural Product [3-carboline alkaloids from the marine bryozoan, Cribricellina
Reports, pp: 269-309. cribraria. J. Nat. Prod., Vol. 54, pp: 1068-1076.
Faulkner, D.J. 1991. Marine natural products. Natural Product Wright, J.L.C. 1984. A new antibiotic from the marine Bryozoan
Reports, pp: 97-147. Flustra foliacea. J. Nat.Prod., Vol. 47, pp: 893-895.
Faulkner, D.J. 1992. Marine natural products. Natural Product
Reports, pp: 323-364.
Central Marine Fisheries Research Institute

98
Seaweeds and Marine Biotechnology
P. Kaladharan
Principal Scientist
FEM Division, CMFRI, Kochi
e-mail: [email protected]

Introduction Tamilnadu, Gujarat, Lakshadweep and Anadaman-

Seaweeds or marine macroalgae consist of Nicobar Archipelagos. Rich seaweed beds occur at
taxonomically distinguished groups of Chlorophyta Dwaraka, Okha, Mumbai, Ratnagiri, Goa, Karwar,
(green seaweeds), Phaeophyta (brown seaweeds) and Thikodi, Varkala, Vizhinjam, Rameswaram and
Rhodophyta (red seaweeds). They are generally found Chilka Lakes. There are about 40 seaweed industries
attached to rocks, pebbles or other aquatic plants in functioning in India producing algin and agar,
the intertidal or subtidal regions of the sea. Seaweeds depending only on natural resources.
are valued for the natural source of phycocolloids
such as agar-agar, algin and carrageenan. A number In a checklist of marine algae (Oza and Zaidi, 2001)
of tropical seaweeds including green algae (Ulva, 844 species of marine algae have been reported from
Enteromorpha, Monostroma, Caulerpa) brown India, comprising 216 species of Chlorophyta, 191
seaweeds (Dictyota, Laminaria, Cladosiphon, Padina) species of Phaeophyta, 434 species of Rhodophyta

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

and red seaweed (Gracilaria, Porphyra, Eucheuma) and 3 species of Xanthophyta, which is again revised
are eaten directly (sea vegetables) for their minerals, to 896 species from 250 genera (Umamaheswara
vitamins, proteins, essential aminoacids and low fat Rao, 2011) indicating a considerable increase in the
content. The major economic significance of seaweeds species of seaweeds of India.
is the polysachharides (agar, algin, carrageenan,
agarose etc) certain red and brown seaweed species Seaweed utilization
contain. Mariculture of seaweeds is essential for the
steady supply of raw materials to seaweed industries Agar is the major constituent of the cell wall of
and to reduce the exploitation pressure being faced by certain red algae(Rhodophycae), especially the
the seaweed beds along the coast. Already countries members of families Gelidiaceae, Gelidiellaceae and
like China, Japan, Philippines, Korea are widely Gracilariaceae. Agar-agar is the Malay word a gelling
cultivating seaweeds and wild harvests are regulated. substance extracted from Eucheuma, but now known
to be carrageenan. The term agar is now generally
Seaweed Resources applied to those algal galactans which have agarose,
the disachharide agarobiose as their repeating unit.
Economically important seaweed resources of the Raw materials for the production of agar are red
world, as per the harvests made during 1971-1973 is algae such as Gelidiella acerosa, Gracilaria edulis,
estimated to 2.105 million tones wet weight (about G. verrucosa and species of Gelidium, Pterocladia
1460 million tones of brown algae; 261 million and Ahenfeltia.
tones of red algae) dominated by brown seaweeds
(Michanek, 1975). The south east and north west Agar is an important colloid used extensively if
coasts of India and the Andaman- Nicobar and biomedical laboratories and in R& D labs as a basal
Laccadive archipelagoes harbour a variety of seaweeds medium for the culture of microbes, cells and tissue.
with rich biomass and species diversity. Luxuriant In food sector, agar is used for gelling and thickening
growth of seaweeds is found in southern coast of in the confectionary and bakery purposes and as

99
 a stabilizer for the preparation of cheese. In fish pyrotechniques. Mannitol finds its use as plasticizers
and meat processing industries, agar is applied for for the production of resins
canned products as a protective coating against the
effect of metal containers. In brewery agar is used Liquid seaweed fertilizer Seaweed extract is made
as a clarifying agent for wines, beer and liquors. In into mineral rich liquid seaweed fertilizer (LSF) and
pharmaceutical industry agar is used as a laxative marketed under various trade names. Studies have
for chronic constipation, as a drug vehicle. Agar is proved that extracts of Sargassum wightii, Ulva lactuca,
an ion exchanger and is used in the manufacture of and Spathoglossum asperum at 1% strength show
ion exchange resins. In cosmetic industry agar serves favourable response on the germination, seedling
as a constituent of skin creams and ointments. Agar vigour, fruit setting and on the weight of the fruit
is also employed in paper and textile industries as in crops such as groundnut, maize, gingelly, tomato
finishing and sizing agents. and ber. Liquid seaweed extract was first patented
in the year 1912. Another patent was offered in
Algin or alginic acid is a membrane mucilage and a 1962 by Maxicrop Ltd and marketed as “Maxicrop”
major constituent of all alginates. The various salts and “Bioextract”. When foliar feeding became an
of alginic acid are termed “alginates” (eg., sodium orthodox method of plant nutrition in the 1950s
alginate, calcium alginate etc). In pharmaceutical ‘Marinure’, ‘SM-3’ and ‘Trident’ brands were made
industry alginic acid is used as emulsifiers in watery in the UK in 1966 and ‘Algifert’ in Norway in 1970.
emulsions with fats, oils and waxes as filters in In India SPIC manufactures and markets LSF in the
the manufacture of tablets, pills and as base name of ‘Cytozyme’.
of any ointments. An alginate gauze is used as
a blood stopping plaster. As a slimming agent, Cattle feed from seaweeds Shrinkage of cultivable
the alginate forms a jelly in the stomach which land due to urbanization and shortage of water limit
produces the feeling of satiation. Ammonium the possibility of producing more feed and fodder
alginate wool is used as a filter for microorganisms to livestock from land. Sea remains untapped and
for laminar flowhood. the seaweed resources has got immense potential
to fill the gap in India. Seaweeds are the marine
Carrageenan is a sulphated galactan polymer macrophytic thallophytes and as animal feed had
obtained from various red seaweeds belonging been in use as early as first century BC by the Greeks.
to families such as Gigartinaceae, Soliriaceae and Seaweed has been used by farmers living near the sea
Hypneaceae.In food industry carrageenan finds its use in Europe. In Norway Ascophyllum is used as pigmeal.
in bakery, confectionery and for the culinary purposes Rhodymenia palmata a red seaweed is called cow
especially in the preparation of condiments, syrups, weed in Brittany and horse weed in Norway. Dried
whipped creams, ice disserts, cheese etc. Carrageenan and processed seaweeds have been used as animal
is used for clarifying fruit juices and other beverages. feed in Europe and North America.
Central Marine Fisheries Research Institute

Quality of wheat flour is improved for making

spaghetti and parotta by adding carrageenan. The Seaweeds are rich in protein (20- 25%), carbohydrate
food sector accounts for nearly 70% of the world (50-70%), vitamins, minerals and certain drugs. When
market for carrageenan. used in animal feed, cows produced more milk, chicken
eggs became better pigmented and horses and pets
Mannitol is an important sugar alcohol of the hexite became healthier (White and Keleshian, 1994). Feed
series found in the cell sap of brown algae. Mannitol supplemented with Gracilaria and /or Spirulina to layer
also occurs as mannitan. The chief raw material for chicks (white leghorn) increased the number of eggs,
the extraction of mannitol are Fucus vesiculosus, size and colour of yolk(Chaturvedi et al. 1985). Dave
Bifurcaria brassiformis, Sargassum spp, Turbinaria spp et al. (1977) assessed the possibility of seaweeds being
etc. In pharmacy mannitol is used for the preparation used as supplementary animal feed and they reviewed
of tablets, for making diabetic diet, chewing gum the feeding trials of farm animals with seaweeds
etc. Mannitol is also used in explosives and other conducted in Japan, Germany, the UK and Norway

100
Seaweed farming in ponds at Porbander by attaching small plants of
brown alga Sargassum to coir nets (Thivy, 1964). The
Seaweed farming is encouraged in developing plants of Sargassum grew to a height of 15-52 cm
countries as it provides employment for poor in 40 days from the initial height of 5-10 cm. This
fishermen in growing the seaweed as well as in experiment revealed good possibilities for cultivation
processing industries. One of the biggest exporters of of Sargassum and other seaweeds in India. Agar
cultured seaweed is China where the industry employs yielding seaweed Gracilaria edulis was first cultured
between 100,000-120,000 people. Seaweeds are by long line rope method in a sandy lagoon on the
cultivated for their commercial importance of eastern side of the Kurusadi Island (Rameswaram).
phycocolloids such as agar, algin and carrageenan
besides, their use as food, source of enzymes, dyes, Since 1972 the Central Marine Fisheries Research
drugs, antibiotics etc. Institute is engaged in the cultivation of several
economically important seaweeds such as Gracilaria
Cultivation of seaweeds edulis, Gelidiella acerosa, Sargassum wightii,
Acanthophora spicifera and Ulva lactuca. There are
in India two methods for cultivation of seaweeds; one by
Seaweed mariculture in India mostly dealt with means of vegetative propagation using fragments
cultivation of Gracilaria edulis due to its high from mother plants and the other by different kinds
regenerative capacity. Very recently the cultivation of of spores such as zoospores, monospores, tetraspores
Kappaphycus, an exotic carrageenophyte introduced and carpospores. In the vegetative propagation
in Indian waters found to be very encouraging and method, the fragments are inserted in the twists of
shall help overcome the shortage for raw materials ropes, tied to nylon twine or polypropylene straw and
for extraction of carrageenan and will offer livelihood cultured in the inshore areas of the sea. The fragments

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

security to the coastal fishers. In India mariculture are also cultured by broadcasting them in outdoor
of seaweed was attempted by Central marine ponds and tanks.
Fisheries Research Institute, Central Salt and Marine
Chemicals Research Institute and National Institute Vegetative propagation
of Oceanography.
Through the vegetative propagation method, where
In 1964 seaweed culture experiments were conducted seaweed thallus (fronds) is the seed material and
for the first time in ponds at Porbander by attaching these can be collected from the natural bed from
small plants of brown alga Sargassum to coir nets the intertidal area during the low tide. Seaweeds are
(Thivy, 1964). It was during the Second World War, cultivated on substrata such as 2 x 2 m nets (20
due to the shortage of agar, that the Board of Scientific cm mesh) made of either nylon or coir (obtained
and Industrial Research started manufacture of agar in from coconut husks) and on 10 m long ropes.
India at the Research Department of Kerala University Approximately 5 g of fronds is inserted or sandwiched
(the erstwhile Travancore University). Since then, much between the twists of the rope at a distance of 10 cm
stride has been made in these lines on the economic in the long line or at each mesh in the 2 sq m nets.
utilization of algae and the Central Marine Fisheries These “seeded” ropes or nets attached to floating rafts
Research Institute developed a cottage industry or bottom set fixed structures in the sea, especially
method for the manufacture of agar from Gracilaria protected bays, lagoons or shallow coast. The seeded
spp. and Gelidium micropterum (Thivy,1960). ropes/nets are kept afloat in water either at surface or
at the subsurface, suitable to the species cultivated
In India mariculture of seaweed was attempted by by series of floats and sinkers.
Central marine Fisheries Research Institute, Central
Salt and Marine Chemicals Research Institute and There are many techniques of seaweed farming
National Institute of Oceanography. In 1964 seaweed through vegetative propagation method to suit the
culture experiments were conducted for the first time location, season of farming and the species cultivated.

101
 • Inserting the fragments to the twist of the nylon that its cultivation would be ecologically safe. The
or coir long line rope or net and allow them to species is a source of carrageenan, a gel-forming
grow in natural environment. They can be placed agent widely used in the pharmaceutical and food
in sea either by stationary structure or by floating industries. This algae is commercially cultivated in
raft (Gracilaria). the open sea in a large area by the Pepsi Foods
• Placing the fragments in the nylon net bag and Limited (PFL) with the help of fishermen with
tied to 10mm HDP rope at a fixed interval of whom the company has a “buy back” agreement.
distance (Eucheuma) Kappaphycus also gives a liquid fertilizer that the
• Broadcasting the fragments in ponds, raceways company intends to market.
and tanks.
• Tying the fragments in sand filled polyethylene Seaweed farming and
tube (Gracilaria).
• Fronds can be fixed to small coral stones or pebbles
carbon sequestration
and these ‘seeded’ stones are broadcast on the There has been a 35% increase in CO2 emission
shallow sea bottom. worldwide since 1990 (IPCC, 2007). Carbon fixation
by photoautotrophic algae has the potential to
Reproductive method diminish the release of CO2 into the atmosphere.
Phytoplankton, seaweeds and seagrasses are excellent
The propagation of seaweed through reproductive carbon sequestering agents than their terrestrial
method can be carried out by using the reproductive counterparts (Zou, 2005). It was estimated that
units like zoospores, carpospore, tetraspore, the seaweed biomass occurring along the Indian
conchospore etc. In India, reproductive propagation coasts is capable of utilizing 9052 t of CO2 day-1
of seaweed was successfully done in Gracilaria edulis against emission of 365 t CO2 day-1 indicating strong
liberating the carpospores on different substrata like sequestration of 8687 t of CO2 day-1 by seaweeds
nylon twine, cement blocks, HDP rope and old fishing (Kaladharan et al., 2009). Large scale mariculture
net (Reeta and Ramoorthy, 1997). The spores liberated of seaweeds along the Indian continental shelf is
on the substrata were allowed to grow to germling recommended as one of the positive anthropogenic
stage in a nursery and then transplanted to natural activities to sequester CO2 that can check global
environment during favourable period of growth. The warming to a larger extend and in turn can check
spores reached to germling stage within 13-17 days the ocean acidification.
of liberation and attachment to the substrata. Three
consequitive harvest can be made from the same Total estimated CO2 absorbed (t/day) and emitted
seed after 105 days till 135 days of culture period. (t/day) by seaweed biomass along the Indian coasts
It was observed that the growth is very encouraging
after first and successive harvest by hand pruning. Seaweed industry in India
Central Marine Fisheries Research Institute

The favourable period of growth for cultivation of

Gracilaria in southeast and south west coast was The seaweed industry in India is mainly a cottage
found to be from November to March. industry and is based only on the natural stock of
agar-yielding red seaweeds, such as Gelidiella acerosa
Mariculture of Kappaphycus and Gracilaria edulis, and algin yielding brown
seaweeds species such as Sargassum and Tubineria.
The Central Salt and Marine Chemical Research The production of total seaweeds in India in 2000
Institute (CSMCRI) introduced this fast growing was approximately 600,000 tons (wet weight). India
species of seaweed in the Diu coast (Gujarat) in produces 110-132 tons of dry agar annually utilizing
1995 for experiments in confined waters from about 880-1100 tons of dry agarophytes. Annual
the Philippines. After successful introduction and algin production is 360 to 540 tons from 3,600 to
acclimatization it transferred the material and the 5,400 tons dry alginophytes. Perhaps, the first large
technology to PepsiCo only after convincing itself scale commercial cultivation of seaweeds in India

102
Type of seaweeds Standing crop (t) Efficiency to absorb CO2 absorbed (t/day) Efficiency to emit CO2 emited (t/day)
(mg/g/h) (mg/g/h)
Red 36523 1.60 584 1.0 365
Brown 41740 2.35 981 0 0
Green 182613 4.10 7487 0 0
Total 260876 8.05 9052 1.0 365
Tech. Papers No.138.127 p.
Kaladharan, P., N.Kaliaperumal and J.R. Ramalingam, 1998.
has been embarked upon by Pepsi Foods Ltd. (PFL)
Seaweeds- Products, Processing and Utilization. Mar. Fish
along a 10 km stretch of the Palk Bay side towards Infor. Serv., T & E Ser., No. 157: 1-9.
Mandapam (Ramanathapuram Dist.) in Tamil Nadu, FAO., 1989. Culture of Kelp in China, Training Manual 89/6
(RAS/86/024) 204 p.
with technical support from Marine Algal Research Kaladharan, P. and N. Sridhar, 1999. Cytokinin production
Center, CSMCRI, Mandapam. Furthermore, many agar, from green seaweed, Caulerpa racemosa. Fish. Technol.,
36(2): 87-89.
algin and carrageenan extracting industries have been
Kaladharan, P. and N. Kaliaperumal, 1999. Seaweed Industry in
established in different places along the maritime India. NAGA the ICLARM Qtrly., 22(1): 11-14.
states of Tamil Nadu, Andhra Pradesh, Kerala, Kaladharan, P., S.V.Alavandi and V.K.Pillai, 1990. Volatilization of
inorganic mercury by Isochrysis galbana Parke. from aquatic
Karnataka and Gujarat. The seaweed industry is systems. Indian J. Fish., 37(2): 163-65
certainly on its way marching towards socio economic Kaladharan, P. and K.Seetha, 2000. Agarolytic activity in the enzyme
extracts of Oscillatoria sp. J. Mar. Biol. Assn. India,42(1&2): 151-
development of our nation.
152.
Kaladharan, P., R.Gireesh and K.S.Smitha. 2003. Cost effective

Biotechnological medium for the laboratory culture of live feed microalgae. J.

Seaweed Res. Utiln., 24(1): 35-40.
interventions in seaweed Kunda, S.K. and P.Kaladharan, 2003. Agar factory discharge as
fuel and manure, J. Seaweed Res. Utiln., 25(1 & 2): 165-168.
Kaladharan, P., S.Veena and E.Vivekanandan, 2009. Carbon
• Barcoding seaweeds for eliminating ambiguity in
sequestration by a few marine algae: Observation and
species identification

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

projection. J. Mar. Biol. Assn. India,51(1): 107-110.
• Improvement of species for better yield of Kaladharan, P., K.Vijayakumaran and V.S.K. Chennubhotla, 1996.
Optimization of certain physical parameters for the mariculture
phycocolloids and for fast growth of Gracilaria edulis in Minicoy lagoon of Laccadive archipelago.
• Isolation of growth promoters, pigments, enzymes, Aquaculture, 139: 265-270.
Kaladharan, P. 1998. Protoplasts- a powerful tool in Genetic
drugs and nutraceuticals
manipulation of commercial seaweeds . Proc. First Natl.
• Isolation and culture of protoplasts for propogation Sem.Mar. Biotechnol. Institute for Coastal Area Studies,
and strain improvement Nagercoil 81-88.
Kaladharan, P. and T.S. Velayudhan. 2005. GABA from Hypnea
• Production of biofertilizers and animal feed valentiae and its effect on larval settlement of Perna viridis.
• Bioremediation and water quality management and Seaweed Res. & Utiln., 27: 8-14
Vinoj Kumar, V. and P. Kaladharan. 2006. B i o s o r p t i o n of
• Production of biofuel and bioenergy from seaweeds
metals from contaminated water using seaweed. Current
Science, 90(9):1263-1267.

Suggested readings
Michanek, G. 1975. Seaweed Resources of the Ocean. FAO Fish.

103
 Seagrass Diversity
M. P. Prabhakaran
Assistant Professor
Kerala University of Fisheries and Ocean Studies
e-mail: [email protected]

Seagrasses are the marine monocots, which constitute characteristics (Fig. 1). Many meadows are not
about 0.01% of flowering plants and have adapted uniform in appearance, due to biological and physical
to the submerged marine habitat. They occur in disturbances. Seagrasses grow in soft sediments, from
most shallow, soft-bottomed marine coastlines, the low water mark to the depths of about 3-5 m
estuaries and lagoons. They are submerged marine and are inhabited by a rich associated biota. At the
angiosperms and mainly distributed in Southeast deeper end of the seagrass meadows, light becomes a
Asian countries, Australian and Caribbean coasts. limiting factor, strongly affecting photosynthesis and
Seagrass ecosystem is associated with several faunal the lower limit is usually related to light irradiance.
and floral assemblages such as algae, sponges, As the rhizome system grows and extends laterally,
corals, crustaceans, molluscs and fishes. The growth shoots may be sent up. A well-developed seagrass bed
and distribution of seagrasses are controlled by a
number of physical parameters such as temperature,
salinity, light regime, sediment type and availability of
nutrients. Seagrasses form the nursery and feeding
ground for a number of marine organisms. They are
highly involved in the detritus food web and play an
important role in the recycling of nutrients.

Seagrass ecosystem forms one of the important

coastal ecosystems of tropical and temperate
regions. This ecosystem is conspicuous and often
dominant habitats in shallow water coastal areas Fig. 1. Key morphological features of seagrass (adapted from
Hemminga and Duarte, 2000)
(den Hartog, 1970). This ecosystem is well known
for its high primary and secondary productivity, ability may extend laterally into bare sediments by means
to stabilize sediments, production of vast quantities of the rhizome system. Dissolved nutrients are taken
Central Marine Fisheries Research Institute

of detritus and support of diverse faunal and floral up by the rhizomes and roots mainly from the pore
communities (Phillips and Mc Roy, 1980). Seagrasses water present in sediments.
stabilize and hold sediments, thus preventing erosion.
Due to the shallow water existence, seagrasses are Seagrasses are flowering plants and the pollens
generally subjected to anthropogenic activities, such are transported through the water currents. They
as water sports, dredging, sewage disposal, etc. produce seeds and are borne by water currents.
When considering all these, seagrass bed monitoring Seagrasses appear to reproduce more by asexual
demands prime importance in the integrated coastal method, through the rhizome system. Colonization
zone management. of new areas by seedlings is difficult unless the
sediment is already physically stable and rich in
Seagrass meadows may include mono-specific dissolved nutrients. This can be accomplished by
or multi-species communities. They exhibit a the presence of other plants such as seaweeds,
variety of leaf shapes, shoot densities and rhizome which stabilize the sediments and add nutrients.

104
Thus, through succession a patch of bare sand may proposal of a gradual transition of hydrophytic
change to a bed of seagrass. species into saline habitats was the prevailing view
until den Hartog (1970) suggested that fossils from
The complex ecology and multiple roles (Fig. 2) that cretaceous deposits in Japan (Archeozostera) and
seagrass communities carry out thrust the need for the Netherlands (Thalassocharis) represent primitive
maintaining and improving these communities. Like seagrasses. According to him, seagrasses evolved
mangrove and salt marsh communities, seagrasses from xerophytic plants that tolerated salt, they then
are important primary producers. The abundance and would have to become tolerant of a hydrophytic
diversity of ichthyo-fauna in seagrass meadows is well habitat. Seagrasses probably arose in the mid- to late
known. The roles of benthic algae are less understood, Cretaceous (65 to 40 million years) after angiosperms

Fig. 2. Summary of seagrass ecosystem (adapted from Fortes, 1990)

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

although drift species are known to serve as habitats began to evolve and spread on land, in the earlier
and food source for gammaridean amphipods. portion of this period (120 million years).
Further, these submerged flowering plants can be
used to monitor the health of coastal ecosystems. Seagrasses belong to the families Hydrocharitaceae
So the need for conservation and management of and Cymodoceaceae. den Hartog (1970) recorded
seagrass meadows is evident when their extensive 49 species and 12 genera in 2 families of the class
ecological roles are considered. Helobiae (Monocotyldonae). Kuo and Mc Comb
(1989) recorded 58 species and 12 genera, which
They have originated from freshwater and estuarine are placed in 4 families, 2 orders (Hydrocharitales
hydrophytic relatives (Arber, 1920) or from xerophytic and Potamogetonales) and 1 class Liliopsida. They
salt marsh like plants (den Hartog, 1970). The include 3 genera in the family Hydrocharitaceae, 5

105
 genera in the family Cymodoceaceae, 1 genus in the features, which obviously form the constraints in
family Posidoniaceae and 3 genera in Zosteraceae, their geographic distribution and speciation. They
Ruppiaceae also included in seagrass community. The possess a well-developed creeping rhizome and an
genera Enhaulus, Halophila, and Thalassia belong to erect shoot bearing several foliage leaves. A well-
the family Hydrocharitaceae; Syringodium, Halodule developed seagrass system may develop laterally into
Cymodocea, Amphibolis and Thalassodendron bare sediment by means of rhizome system. Thus
belong to the family Cymodoceaceae. Family bare sand may change by succession to a seagrass
Zosteraceae include Zostera, Heterozostera and bed and appear to reproduce more by vegetative
Phyllospadix, and Posidonia included in the family method i.e., through the rhizome system. Distribution
Posidoneaceae. Lee Long et al., (2000) described of seagrass in the deeper areas of the water body is
60 species worldwide within 12 genera, 4 families limited by intensity of light, since they depend on
and 2 orders. According to him, Halophila and light for photosynthesis.
Thalassia belong to the family Hydrocharitaceae and
order Hydrocharitales; Cymodocea, Halodule and For the protection and management of seagrass
Syringodium belong to the family Cymodoceaceae meadows, information about their ecosystem services is
and the order Potamogetonales. essential. It is important to document floral and faunal
species diversity, distribution and abundance in seagrass
Five characteristics have contributed to making meadow to identity the areas requiring conservation
seagrasses the most successful tropical shallow measures. In such a point of view, this study forms the
marine community. These characteristics (den Hartog, baseline information about the seagrass meadow of
1970) are: Minicoy lagoon. Responsive management based on
1. The ability to live in a saline medium. Seagrasses adequate information will help to prevent any further
are actually killed in low salinities. significant areas or species being lost.
2. The ability to function physiologically while fully
submerged, unlike mangroves, which rely on air Seagrass ecology has evolved as a major part of aquatic
exposure and pneumatophores for gas exchange. ecology, from a descriptive stage, focused on the
3. A well developed anchoring system and the ability distribution and biology of the plants, to a quantitative,
to slow near bottom currents that aids in the process-oriented stage. Research efforts over the past
accumulation of sediments. four decades have generated widespread awareness
4. The ability to reproduce while submerged. of the importance of seagrass meadows as marine
5. The ability to compete with other marine organisms ecosystems, thereby placing seagrass ecosystems as
for space and resources. primary targets for marine conservation and restoration
programmes. These achievements have resulted from
The importance of seagrasses in coastal and near the efforts of a growing community of seagrass
shore environments and their contribution to the ecologists. The scientific studies on seagrass ecology
Central Marine Fisheries Research Institute

productivity of the world’s oceans has become are still limited compared to many marine ecosystems.
increasingly recognized over the last four decades. Moreover, understanding the ecology of seagrass
They play a significant role in the processes and meadows would enable a better basis to sustainably
resources of near shore coastal ecosystems. manage these ecosystems, because seagrass meadows
They having adapted to the submerged aquatic are still being lost from the world’s coastal ocean at
environment, show morphological and anatomical alarming rates.

106
Molecular Biology
Standard Operating Procedure (SOP)
M. P. Paulton
Senior Technical Officer (Training)
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction be given to avoid the access of the same in the hands

The primary aim and idea behind formulating a of unwanted social elements. Further proper inventory
code of conduct classified as standard operating register and a record of the usage of such materials is
procedure in a biotechnology laboratory is to to be maintained. Unauthorised handling or removal of
ensure the safety and security of personals involved hazardous materials is to be restricted in the laboratory.
in research in different capacities. Such a protocol Students who are new to the system are to be trained in
would certainly ensure the judicious use of sensitive the safety protocols to be followed to avoid the theft of
and expensive chemicals and enzymes in a completely such hazardous materials as the same is important from
secured manner. One can easily assess the level of the angle of National security. The protocols related to
understanding and to the extent of practising SOP security are to be designed and practiced based on the
through a self-valuation procedure. The questions to situations in each laboratory with the due approval of
be answered in this process are the following. certifying agencies. Laboratory group is to be trained and

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

responsibilities are to be entrusted to ensure the error
• Are you aware about the security and safety rules free practice of the protocols related to security. Each
to be followed in a biological laboratory using personal working in laboratory should be aware about
sensitive and hazardous chemicals? an emergency plan and security protocols for reporting
• Do you know the procedure for storing and immediately in the event of any kind of incidents leads
handling hazardous materials? to causality. Proper training should be given to operate
• Are you practising scientific protocols during fire extinguishers and other emergency handling devices.
handling and disposal of biological and
chemical materials? Inventory of equipment,
• What is your level of understanding about the
health hazards which could cause by the chemicals
Operational knowledge and
in regular use? Periodical servicing
• What sort of personal protective equipment is
required to protect you from hazardous chemicals? The laboratory manager or the personal concerned
• How do you respond in the event of causality? should be aware of the instruments and other minor
• Are you well aware about the medical attention equipment available. The new recruits should be trained
to be followed during emergency? properly on the operational protocols which should
be displayed for speedy reference. A log book is to be
If you are successful in responding properly to the above maintained to record the use of the instruments with
queries, you could asses yourself as proficient in SOP. adequate instructions to note remarks. Periodic service
of the instruments should be monitored to ensure the
Laboratory Security break down free service. All documents related such
correspondences should be made available in the
As biotechnology Institutes and Industries use variety of laboratory as the same may be reviewed in social auditing.
hazardous materials, the proper care and security is to Adequate power supply depending on the power

109
 requirements of the instruments should be made available drinking inside the laboratory and also avoid leaving
in the laboratory. the lab with gloves and without washing hands.
• Inhalation–Use masks and fume hoods.
Storage & handling of • Injection- Ensure the Proper disposal of all broken
glasses and needles.
chemicals, enzymes • Always try to remove gloves while using phones
and reagents and touching light switches etc…

There is no universal formula or procedures for handling List of common

sensitive materials like chemical and enzymes used in a
biotechnology laboratory. Some common methodologies
hazardous chemicals in a
to be followed by one and all include proper labelling of biotechnology Lab
all chemicals so that even a person who is new to the
system would find it easy. There should be a Material • Formaldehyde (carcinogen).
Safety Data Sheets (MSDS) in file for each chemicals and • Acrylamide (Neurotoxins).
enzymes available in the laboratory. In many countries • Acetonitrile (Nephrotoxins).
MSDS is a legally required technical document which is • Ethidium Bromide (mutagens).
normally being provided by the suppliers. • Formamide (Teratogens).
• Chloroform (Hepatotoxins).
Specific parameters to be included in the MSDS sheet • Phenol, strong acids and bases (Corrosives).
are as follows
Handling Biological hazards
• Identity of the chemical along with contact
information’s of manufacturer. As all well aware, biotechnology laboratories are
• The identity of hazard ingredients routinely doing experiments with many biological
• Physical and Chemical characteristic of the product. organisms and few of them are harmful to the user as
• Nature of reactivity –This is vital information to well as to the environment. Bacteria, fungi and viruses
determine the place of storage isolated in a biotechnological laboratory include both
• Examples- The acids and bases reacts each other harmless and harmful microorganisms and many of its
and should not be stored in adjacent places). toxicological and pathogenic properties are unknown
Certain other chemicals with chances to react with to us. Hence as a general rule, extreme precautions
water should be stored with desiccants. are to be followed while handling microorganisms.
• Health related details- include the toxicological Stringent protocols have to be designed especially in
properties like carcinogenicity, lethal dose (LD50). Indian Scenario as we do not have generalised pattern
• Precautions and how to handle the chemical safely. in connection with the microbial safety to be followed
Central Marine Fisheries Research Institute

• Details regarding the personal protective equipment by all laboratories. The recombinant DNA molecules
(PPE) suggested wearing. produced in biotechnology laboratories are to be
considered as a biohazard as it could bring about
Safety precautions to dangerous changes in the host. Same is the case with
animal and tissue cultured organisms as they can make
handle hazardous chemicals an impact in the environment. General precautions
One should be well aware about the general route such as wearing PPE, washing hands, wearing gloves
through which a toxic chemical can enter into the etc. described above for chemicals are to be followed
body. There exist primarily four routes. for handling live organisms as well. One has to be
more careful and must avoid touching face wearing
• Skin and eye –avoid with the use of lab coats, gloves and keep away all personal belongings in the
gloves and goggles. designated area. Following are the essential precautions
• Ingestion–keep away the habit of eating and to be followed while dealing with live organisms.

110
• Avoid mouth pipetting and use mechanical devices. Chemicals wastes have to be neutralised before
• Splashes and aerosol generation is to be kept to disposal through proper treatment. Buffers, acids and
minimum as majority of the incidents of infection reagents have to be properly diluted before disposal.
is through inhalation. The real challenge in a biotechnology laboratory is the
• Always try to ensure the shaking or mixing in closed management of the disposal of biological wastes. Wastes
containers to avoid the release of aerosols. from microbial cultures and other biological samples have
• Disinfect the glassware and workbench after each to be collected in autoclave bags made of high melting
work session. point plastic so that they can be killed at high temperature
• Specific microbial disinfectants like formalin are to and pressure before disposal. Students have to brief about
be used along with common bleaching powder the proper waste management and disposal before
or ethanol. starting each session which involve lengthy experiments
• Use laminar flow hood safety cabinet. like tissue culture and cell line maintenance. Radioactive
waste materials including the laboratory wares and filter
Handling of papers used have to be collected in separate containers
following the guidelines. Usually the radioactive wastes
Radioactive materials are disposed by separate department like health physics
It is quite common nowadays to use radioactive isotopes department upon receiving the same from laboratories.
in biotechnology laboratories working with protocols in
genomics and proteomics though non isotopic labelling Summary of Good
methods are coming up for labelling experiments.
Radioactive safety measures are generally followed
Laboratory Practices
by all the laboratories owing to awareness of health In order to solve the problems faced by a researcher,
hazard associated. Laboratory must have the services he/she should be aware about the role of the reagent

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

of a trained person in radioactive safety as a mandatory in isolating macromolecules having different physical
requirement. Specific precautions to be practiced while and chemical properties. You should be able to
dealing with radioactive materials are as follows. anticipate the molecular interaction between different
buffers while using simultaneously.
• Earmark certain specific area displayed with
radioactive safety symbols to carry out radioactive Quality of the Reagents
labelling experiments.
• Always stick to the guidelines prescribed by the Always try to use good quality enzymes and reagents
suppliers (BARC) in handling the material. in molecular biology and even if substitute go for a
• Work behind radioactive shields. higher grade than a lower grade.
• Radioactive detectors are to be used to ensure the
area is free from radiations after each work session. Quality of the Water
• The wastes including the tissue papers are to be
stored separately for disposal. Try to ensure that you are using good quality
water suiting to the requirements in molecular
Disposal of biological, biology. Water prepared through reverse osmosis
is the best quality preferred nowadays over
chemical and distilled and deionized.
radioactive waste
Storage life
One of the most sensitive and complex area with
lots of challenges involved is the perfect and speedy Try to dispose buffers and other reagents beyond
disposal of wastes generated from the biotechnology storage life and always check the microbial
laboratory. Methodologies to be followed depend on contamination within the stored reagents and buffers.
the characteristic feature of the waste to be disposed. Media and other buffers should be chemically as well

111
 as microbiologically sterile to avoid contamination
and precipitation.

Suggested readings
Molecular Biology Problem Solver, edited by Alan S. Gerstein
ISBN 0-471-37972-7.
Central Marine Fisheries Research Institute

112
Principles of Isolation, Purification and
Analysis of Nucleic Acids
M. P. Paulton
Senior Technical Officer (Training)
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
conditions. Even the rapid stirring of solution can
Advanced Biotechnological research is largely depended break high molecular weight DNA into shorter
on the genome analysis and recombinant DNA technology. fragments. Vigorous shaking will cause nicks
Good quality nucleic acid is an essential prerequisite for and even cut open the covalently closed circular
consistent results in most of the downstream applications structures of plasmid and mitochondrial DNA. If
in the genome analysis and recombinant DNA technology. physical disruption is necessary as is the case with
The general principle underlying the isolation of nucleic certain types of tissues, it should be kept to the
acids is common with few modifications depending on minimum, and should involve cutting or squashing
the type of nucleic acids being isolated. The type of the of cells, rather than the use of shear forces. Ultra
nucleic acid intending to isolate is to be made free from sonic sounds are used to disrupt the tough cell wall

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

the other biological macromolecules and cell debris .This is of certain bacteria. Care has to be taken to prevent
achieved by properly lysing the cell wall or cell membrane degradation of DNA by deoxyribonucleases. These
as the case may be and by selectively denaturing the other enzymes are found in most cells, and may also be
macromolecules like proteins. Nucleic acids thus recovered present in dust which could contaminate laboratory
in its native form is to be purified by removing the very glass wares. Hence all the glass wares, plastic wares
closely associated molecules. The finely purified molecule and the homogenizing buffer are to made sterile by
is precipitated by alcohol and suspended in sterile buffer autoclaving. This enzyme activity can be inhibited by
or distilled water. Finally, the qualitative integrity of the using EDTA in buffers which will chelate the Mg++
isolated nucleic acids is to be checked by agarose gel ions needed for DNase activity. Cell disruption and
electrophoresis and ethidium bromide staining before most of the subsequent steps should be performed
proceeding with the further downstream applications. at 4˚C. The cell wall could be lysed enzymatically
Quantitative estimation of nucleic acids are carried out as well. The bacterial cell wall is usually lysed by
by spectrophotometric and fluorimetric methods. the enzyme Lysozyme. The cell membranes on the
other hand are solubilised by including suitable
The types of nucleic acids usually isolated on a routine detergent in the homogenizing buffer. Upon lysis
basis are: the nucleic acids will be released into the cytoplasm
• Total genomic DNA and now the target molecule, DNA, is to be made
• Total RNA free from RNA and other associated proteins. The
• Plasmid DNA & Mitochondrial DNA RNA molecules can be selectively denatured by
enzymatic treatment with RNase. Prior to its use, the
Total Genomic DNA RNase is to be heat treated to inactivate any DNase
contaminants. RNase is relatively stable to heat as
Breaking of the bacterial and plant cell walls as a result of its disulphide bonds, which ensure rapid
well as solubilizing the cell membrane of animal renaturation of the molecule on cooling. The other
tissue are to be carefully carried out under optimum major contaminant, protein, is removed by enzymatic

113
 treatment with proteinase K followed by shaking with ethidium bromide. Because of this density difference,
water saturated phenol or with phenol-chloroform plasmid and mt.DNA can be separated from other
mixture, either of which will denature proteins but DNA by ultra-centrifugation.
not nucleic acids. Centrifugation of the emulsion
formed by this mixing produces a lower, organic Another method which is relatively fast is based
phase, separated from the upper, aqueous phase on alkaline lysis. In this method the property of
by an interface of denatured protein. It is advisable super coiled DNA to remain intact at pH between
to use cut micro tips while proceeding through 12 and 12.5 is exploited for the isolation. At this
these steps. The aqueous solution is recovered pH selective denaturation of linear DNA will occur
and deproteinised repeatedly until no material whereas the super coiled DNA will remain intact.
is seen at the interface. Finally the deproteinised Further reduction of the pH to acidic condition will
DNA preparation is mixed with two volumes of enhance the formation of a complex network of
absolute ethanol, and allowed to precipitate out of proteins and linear DNA and the resultant supernatant
solution in a freezer. After centrifugation, the DNA after centrifugation will contain the intact plasmid
pellet is redissolved in a buffer containing EDTA for or mt.DNA .This can be purified and precipitated
protection against DNases, and this solution can be as in DNA isolation procedures. For mitochondrial
stored at 4˚C for at least a month. DNA solutions can DNA, this method works well with fresh tissues with
be stored frozen, but repeated freezing and thawing minimum nicks.
tends to damage long molecules by shearing and
hence the DNA preparations in frequent use are Mitochondrial DNA can also be isolated by differential
normally stored at 4˚C. centrifugation technique. This involves the selective
isolation of the mitochondria which is further lysed
Plasmid & with suitable detergents to release the mt.DNA.
This will be further purified and precipitated by
Mitochondrial DNA conventional means.
The principle of isolation of plasmid and mitochondrial
DNA is based on the structural characteristics. Plasmids RNA
are double stranded, Covalently Closed Circular
(CCC) or super coiled structures. Similarly mt.DNA RNA molecules are relatively short, and therefore less
is also having the same structural characteristics affected by shearing. RNA is, however very vulnerable
and hence almost the same isolation procedure can to digestion by RNases which are present abundantly
be adapted. Bacterial cell wall is to be broken by even on fingers. These enzymes are stable and
enzymatic treatment (lysozyme) in a suitable buffer generally require no co-factors. Hence gloves should
with a suitable metallic chelator like EDTA before be worn, and a strong detergent should be included
initiating the isolation process. The tissue for the in the isolation medium to denature any RNases
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mt.DNA isolation is to be thoroughly homogenized immediately. The solutions used are to be treated
under ice cold conditions. with nuclease inhibitors like Diethyl pyrocarbonate
(DEPC).Care should be taken while using DEPC as
The classical method is to isolate the plasmid and it is a suspected carcinogen. Glass wares should be
mitochondrial DNA by Caesium chloride density baked at 300˚C for 4 to 5 hours as autoclaving alone
gradient ultra-centrifugation in the presence of may not be sufficient to fully inactivate RNases. The
ethidium bromide. Ethidium bromide causes plastic ware can be rinsed with chloroform. Tissue
unwinding of DNA as it binds to it, simultaneously homogenization is to be carried out under ice cold
producing a decrease in its buoyant density .Since conditions with all the precautions detailed above. As
the super coiled plasmid and mt.DNA can unwind in the case of DNA, RNA is to be made free from DNA
to only a very limited extent, it will not bind as much and proteins. Proteins are denatured by proteinase K
ethidium bromide as with the linear and open circle treatment followed by phenol chloroform extraction.
forms of DNA in the presence of saturating levels of This is followed by the ethanol precipitation of

114
RNA in the presence of sodium acetate or sodium liquid connective tissues like blood, haemolypmph
chloride. The overnight precipitated pellet is washed etc. Meanwhile, the commercial kits are effectively
with 70% ethanol to remove the salts and finally used for the isolation of total RNA and mRNA as the
dissolved in DEPC treated water. Contaminating manual isolation is a sensitive process with increased
DNA can be removed by treatment with RNases free chances of degradation.
DNase. The RNase can be inactivated by RNAsin or
vanadylribonucleoside complex. Quantitative Estimation of
nucleic acids
Commercially available kits
DNA and RNA can be spectrophotometrically
Several readymade kits are available commercially and estimated by taking optical density (OD) at 260nm,
many laboratories are depending on such products 1 O.D corresponds to 50 micro gram of DNA and
for the isolation of nucleic acids. In most of these 40 micro gram of RNA. Purity of the DNA can also
kits, the nucleic acids are either trapped by ultra- checked spectrophotometrically by taking O.D at 260
filtration membranes or allowed to bind with certain &280nms. The ratio of 260 and 280 will result a value
resins which have affinity towards nucleic acids. The of 1.8 with pure nucleic acid preparations.
advantage with these kits is that the process is very
fast and devoid of using corrosive organic chemicals Suggested readings
like phenol. The main disadvantage is that they are
quite expensive and hence unaffordable to many Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982). Molecular
laboratories. Hence it is advisable to use alternative cloning. Cold Spring Harbor, New York.
Old, R.W. and Primrose, S.B. (1985). Principles of Gene manipulation,
non organic protocols, for DNA isolation, based on 3rd edition. Blackwell Scientific Publications, Oxford.
the use of high concentration of salts for removing Walker, J. M. and Gaastra, W. (Eds) (1983). Techniques in Molecular

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

biology. Croom Helm, London.
proteins in place of phenol, which are easy to perform
in the laboratories especially while isolating from

115
 Polymerase Chain Reaction and its
various modifications
P. C. Thomas
Principal Scientist (Rtd.)
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
secured tissues such as fin clips and scales. Even
Polymerase Chain Reaction or PCR is a molecular partially degraded DNA from poorly preserved sources
technique which allows in vitro synthesis of billions can be analyzed if sufficiently small PCR products
of copies of a target DNA fragment within hours using are identified.
a simple enzymatic reaction. This is achieved by using
a pair oligonucleotide primers that hybridize (anneal) Discovery of PCR
to the complementary sequences on the opposite
strands of the target DNA, at positions flanking the The concept of PCR was first conceived by Dr. Karry
region to be amplified. New strands are made through Mullis in 1983, first reported in1985, while working
the simultaneous extension of both the primers by at the Cetus Corporation in Emeryville, CA, along with
addition of nucleotides to the primers by the enzyme other researchers at Cetus Corporation (Molecular
DNA polymerase. A repetitive series of cycles involving Station, 2006). Karry Mullis discovered that by
template denaturation, primer annealing and harnessing one component of molecular reproduction
extension of the annealed primers by the enzyme DNA technology, ie a basic principle of replicating a piece of
polymerase results in the exponential accumulation DNA using two primers, a target DNA of interest could
of the DNA whose termini are defined by the 5’ ends be amplified exponentially. This DNA amplification
of the primers. Since the primer extension products procedure was an in vitro process (meaning in a test-
synthesized in one cycle can serve a template for the tube). The first ever PCR product was the 110 base
next, the number of target DNA copies approximately pair DNA fragment of a cloned segment of the human
doubles at every cycle. Thus 20 cycles of PCR can beta-globulin gene at the company labs, being the
yield about a million-fold amplification. The method beginning of PCR as a basic technique in molecular
is simple, as the PCR can be performed in a single biology (Mullis et al.,1986, Mullis and Faloona 1987).
Central Marine Fisheries Research Institute

tube. It can be performed on relatively crude DNA Dr. Mullis was awarded the Nobel Prize in Chemistry
containing samples. These factors have made the in 1993 for his development of the Polymerase Chain
PCR an attractive method for amplification of specific Reaction (PCR), a central technique in biochemistry
sequences. This method is extremely rapid; it takes and molecular biology. Dr. Mullis subsequently was
only 3 hours to amplify a known sequence of interest. awarded the Japan Prize that same year.
PCR generates sufficient copy numbers of target DNA
sequences for their routine visualization through Materials and reagents
standard procedures such as electrophoresis followed
by staining with ethidium bromide. The PCR products
for PCR
may be sequenced to determine the exact sequence The components required for the PCR are the
of the nucleotides within the amplified product. As template (the DNA to be amplified), a pair of
a result, the PCR permits routine analysis of DNA primers, thermostable polymerase, the four types
from single egg and larvae, and from non-invasively of de-oxynucleotide triphosphates (dATP, dCTP,

116
dGTP and dTTP) and appropriate reaction buffer extended toward one another with DNA polymerase,
containing magnesium ions (KCl, Tris-HCl (pH 8.4), forming new DNA molecules. The most important
MgCl2 and gelatin). They are assembled in a tube property of a primer is its sequence specificity, which
and the amplification reaction is carried out by determines what nucleic acid sequence it can bind
manipulating the temperature within the reaction to, how well it will bind, and how well it will serve
tube, in cyclic manner, using a thermal cycler. For any as a site for extension of new nucleic acid molecules.
given pair of primers, the optimal concentrations of Generally, a “specific” primer is designed to target a
all the above ingredients and parameters have to be DNA sequence in a closely related group of organisms,
standardized. Even though there is no single set of while not matching organisms outside that group.
conditions and concentrations that will be optimal for “Universal” primers are designed to target DNA
all reactions, the parameters outlined below defines sequences shared by any species that contains the
a common starting point from where modifications sequence of interest. Thus, care should be taken while
can be attempted. designing the primers for a particular experiment.
Oligonucleotide primers in the range of 18 to 30 bases
Target DNA (Template): An advantage of PCR is are generally used for the PCR. Though there are no
that it can amplify relatively impure DNA or DNA set rules that will ensure the synthesis of an effective
from blood spots, archival material and ancient primer pair, the following guidelines are useful.
DNA. Concentration of template DNA also affects • Wherever possible, select primers with a random
the degree of amplification. The sample DNA base distribution and with a GC content similar to
generally contains 102 to 105 copies of template. that of the fragment being amplified. Avoid primers
Too high or too low concentration will result in poor with stretches of polypurines, polypyrimidines or
amplification. Therefore, it is useful to optimize the other unusual sequences.
template concentration in a PCR reaction to obtain • Check the primers against each other for

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

maximum product. While typically DNA quantity is sequence complementarity. Use primers with low
measured in ng, the relevant unit is actually moles, complementarity to each other. Avoid primers
i.e., how many copies of the sequence that will anneal with 3’ end overlaps in particular. This will reduce
with the primers are present. Thus, the amount of incidence of “primer dimers”. Most primers are
DNA in ng that is needed to add is a function of generally 18 to 30 bases in length and the optimal
its complexity. In theory, a single molecule of DNA length to be used in an amplification will vary.
can be used in PCR but normally between 1000 and Longer primer may be synthesized but are seldom
100,000 molecules for eukaryotic nuclear DNA are necessary. If shorter primers or degenerate primers
used. The nucleotide composition of target DNA are used, the thermal profile should be modified
also affects the PCR amplification. Extremely GC rich considering the lower stability of the primed target.
DNA strands are difficult to separate. Addition of However, the 3’ end of the primer should match
denaturing agents like formamide or DMSO can help the template exactly. Generally, concentrations
to overcome the problem. ranging from 10 to 50 p moles of each primer
should be used.
Primers: Primers are the most important components
of PCR, and the success of a PCR largely depends on The optimum length of a primer depends upon its
the primers. Primers are short, single stranded DNA (A+T) content, and the Tm of its partner. A prime
molecules which will bind (anneal) to either ends of consideration is that the primers should be complex
region to be amplified (one on each strand) and serve enough so that the likelihood of annealing to
as the starting point for attaching nucleotides on its 3’ sequences other than the chosen target is very low.
end, leading to the building of a new complementary For example, there is a ¼ chance (4-1) of finding
nucleic acid strand. Primers are generally made in an A, G, C or T in any given DNA sequence; there
pairs, called “forward” and “reverse”. These primers is a 1/16 chance (4-2) of finding any dinucleotide
are complimentary to the regions flanking the DNA sequence (eg. AG); a 1/256 chance of finding a given
segment to be amplified such that they can be 4-base sequence. Thus, a given sixteen base sequence

117
 will statistically be present only once in every 416 lead to mis-incorporations. Low dNTP concentration
bases (= 4 294 967 296, or 4 billion): this is about reduces mispriming at non-target sites. The lowest
the size of the human or maize genome, and 1000x dNTP concentration appropriate for the length and
greater than the genome size of E. coli. Thus, the composition of the target must be standardized.
association of a greater than 17-base oligonucleotide As a thumb rule, 20mM of each dNTP in a 100 ml
with its target sequence is an extremely sequence- reaction is sufficient to synthesize 10 p Mol of a
specific process. Generally, 17-mer or longer primers 400 bp sequence. In the standard reaction, all four
are routinely used for amplification from genomic triphosphates are added to a final concentration of
DNA of animals and plants. Long primers will result 0.8mM; this leaves 0.7 mM of the original 1.5mM
in mismatch pairing and non-specific priming even MgCl2 not complexed with dNTP. Therefore, if dNTP
at high annealing temperatures concentration is changed significantly, a compensatory
change in MgCl2 may be necessary.
Melting temperature (Tm) of primers: The
annealing temperature is dependent on the Tm of Taq DNA polymerase: The discovery of
primer. Annealing temperature can be the Tm value thermostable DNA polymerase has revolutionized the
calculated using the following formulae: PCR technology. They are obtained from organisms
that thrive in extreme temperatures and have an
(1) Tm = [(number of A+T residues) x 2ºC] + optimum activity at 72º C. It is able to withstand
[(number of G+C residues) x 4ºC] the denaturing conditions (over 90ºC) required
during PCR cycling. Thus, unlike thermo-labile
This formula was determined originally from polymerase, with Taq polymerase there is no need
oligonucleotide hybridization assays, which were for extra addition of enzymes during cycling process
performed in 1 M NaCl, and appears to be accurate where strands separation required heating to over
in lower salt conditions only for primers less than or 90ºC. There are now a plethora of commercially
about 20 nucleotides in length. available enzymes to choose from that differ in
their thermal stability, processivity, and fidelity. The
(2) Tm p = 22 + 1.46 ([2 x (G+C)] + (A+T)) choice of the DNA polymerase employed by PCR
is determined by the goals of the experiment. The
This formula is reportedly useful for primers of 20-35 most commonly used thermostable polymerase is
bases in length. The calculated annealing temperature Taq DNA polymerase isolated from the bacterium
is only a reference temperature from which to initiate Thermus aquaticus which inhabit the hot springs
experiments. The actual annealing temperature with extremely high temperatures. Isolation of the
may be 3-12ºC higher than the calculated Tm. The DNA polymerase from this bacterium yielded a PCR
actual annealing temperature condition should be polymerase that was not rapidly inactivated at high
determined empirically. The optimum annealing temperatures. In 1986, Dr. David Gelfand and Ms.
Central Marine Fisheries Research Institute

temperature which gives the best PCR product should Susanne Stoffel of Cetus Corporation purified such
be used. a thermostable DNA polymerase, referred to as
native Taq (Thermophilus aquaticus in short), from
Deoxynucleotide triphosphate: The dNTPs are the the organism Thermus aquaticus. Taq polymerase
building blocks of DNA. Once the primer binds to its was shown to work successfully in PCR, enabling
target site, synthesis of the complementary strand of the process to be performed much more easily.
DNA takes place through primer extension by linking Today, almost all PCR is done using recombinant
of nucleotide to its 3’end with the help of Taq DNA Taq, a cloned version of the enzyme, as it is less
polymerase. Precursor dNTPs can be obtained as a expensive to manufacture than the native form of
neutralized solution, which are stable at –20ºC for the enzyme (Roche Diagnostics, 2007). Taq was
months. The doxynucleotide triphosphate (dATP, dCTP, the first polymerase that was able to withstand
dGTP and dTTP) is generally used at concentrations of the denaturing conditions (over 90ºC) required
200 mM (0.2mM) each. Higher concentrations may during PCR cycling. Taq has an enzymatic half life

118
at 95ºC of about 40 min. Taq DNA polymerase is of PCR buffer, particularly the concentration of MgCl2
unique in that it produces PCR products with A have a profound effect on the specificity and yield of
(Adenine) overhangs. This was found to be quite an amplification product. Success of PCR is dependent
useful, and was exploited to produce TA Cloning on MgCl2 concentration in the reaction to a great
and TOPO cloning. One of Taq polymerases’ major extent. Mg2+ ions form a soluble complex with
disadvantages is its low replication fidelity. As Taq dNTPs which is essential for dNTP incorporation,
does not have 3’ to 5’ exonuclease proofreading stimulate polymerase activity and increase the Tm
mechanism to replace an accidental mismatch in (melting temperature) of primer / template interaction
the newly synthesized DNA strand, Taq produces (i.e. it serves to stabilize the duplex interaction).
more errors than proofreading polymerases, such Concentration of about 1.0 to1.5 mM is usually
as Pfu (Roche Diagnostics, 2007) Pyrococcus optimal (when 200uM each of dNTPs are used).
furiosus, where it functions in vivo to replicate the Excess of Mg 2+ will result in the accumulation of
organism’s DNA. Pfu’s have superior thermostability non-specific amplification products and insufficient
and ‘proofreading’ properties compared to other Mg2+ will reduce the yield. Optimization by titration
thermostable polymerases. Unlike Taq DNA of MgCl2 concentration is recommended to establish
polymerase, Pfu DNA polymerase possesses 3’ to an optimum concentration for a particular reaction.
5’ exonuclease proofreading activity and corrects
nucleotide-misincorporation errors. Thus Pfu DNA Several buffer formulations have been published & a
polymerase-generated PCR fragments will have fewer consensus is emerging. The recommended PCR buffer
errors than Taq-generated PCR inserts. It also results should contain 10mM Tris-HCI (pH 8.4) also. KCl up
in blunt-ended PCR products. to 50mM can be included in the reaction mixture
to facilitate primer annealing. Excess KCl inhibits
The required concentration of Taq DNA polymerase is Taq polymerase activity. Gelatin or bovine serum

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

between 1 and 2.5 units per 100 ml reaction when albumin (100 mg/ml) and nonionic detergents such
other parameters are optimum. When optimizing as Tween- 20 and NP40 (0.05–0.1%) are included to
a PCR, enzyme concentration ranging from 0.5 to help stabilize the enzyme. The nonionic detergents
5 units/ 100ml are tried and resultant products can be replaced by 0.1% Triton X-100, but some
are visualized by agarose gel electrophoresis. If detergent is essential.
the enzyme concentration is too high, non-specific
background products may accumulate and if too low, Thermal Cycles for PCR
an insufficient amount of desired product is made.
Amplification of a target DNA is achieved by
The Reaction Buffer: The PCR buffer contains KCl, repeated cycles of denaturation, primer annealing
Tris HCl (pH 8.4), MgCl2 and gelatin. The components and extension. These events are controlled by
manipulation of temperature. The above three major
Table 1. Thermostable DNA polymerases steps in a PCR are repeated for 35 to 40 cycles. This is
DNA Polymerase Source
done using an automated thermal cycler, which can
heat and cool the tubes with the reaction mixture in
Taq Thermus aquaticus
a very short time.
Amplitaq® T. aquaticus
Amplitaq (Stoffel fragment)® T. aquaticus
Denaturation: Double stranded DNA used for the
Hot Tub™ Thermus flavis
PCR is separated into single strands in the initial
Pyrostase™ T. flavis
denaturation step. Typical denaturation temperature
Vent™ Thermococcus litoralis
is 94ºC for 15 to 60 seconds. Higher temperatures
Deep Vent™ Pyrococcus GB-D
e.g. 97ºC may be necessary for G + C rich targets.
Tth Thermus thermophilus
Denaturation steps that are too long or too high lead
Pfu Pyrococcus furiosus
to unnecessary loss of enzyme activity. Denaturation
ULTma™ Thermotoga maritima of nucleic acid (NA) is carried out to make it

119
 single-stranded for the purpose of annealing with amplification pair of primers can be attempted. The
primers. It is done by heating it to a point above annealing temperature chosen for a PCR depends
the “melting temperature” of the double or partially directly on length and composition of the primer(s).
double stranded form, and then flash-cooling it: this Annealing temperature in the range of 55 to 65ºC
ensures the “denatured” or separated strands do not generally yield the best results. At the optimal
re-anneal. Additionally, if the NA is heated in buffers of primer concentration annealing will require only a
ionic strength lower than 150 mM NaCl, the melting few seconds. Increasing the annealing temperature
temperature is generally less than 100ºC–which is why enhances discrimination against incorrectly annealed
PCR works with denaturing temperatures of 91-97ºC. primers and reduces mis-extension of incorrect
The main reason of importance of denaturing nucleotides at the 3’ end of the primers. Therefore,
temperature and time in relation to number of cycles stringent annealing temperature, especially during
is because Taq polymerase has a half-life of 30 min at initial few cycles will help to increase specificity.
95ºC. This half-life supports not more than about 30
amplification cycles. However, it is possible to reduce Primer Extension: The DNA polymerase works
the denaturation temperature after about 10 rounds ideally at temperature 72ºC. The annealed primers, to
of amplification, as the mean length of target DNA is which a few bases have been added, have a stronger
decreased. “Time at temperature” is the main reason attraction to the template, created by hydrogen
for denaturation / loss of activity of Taq. Thus, with bonds, than the forces breaking these attractions.
reduction of time, increases the number of cycles are Primers that are on positions with no exact match
possible, whether the temperature is reduced or not. get loose again (because of the higher temperature)
It is possible, for short template sequences, to reduce and do not give an extension of the fragment. The
this to 30 sec or less. nucleotides (complementary to the template) are
linked to the primer on the 3’side by the polymerase,
Primer annealing: At temperatures ranging from from 5’ to 3’, reading the template from 3’ to 5’
47ºC to 62ºC, the primers anneal to its complimentary side and bases are added complimentary to the
region on the template. The complimentary template. Extension time depends on the length and
sequences will form hydrogen bonds between their concentration of the target sequence and upon the
complimentary bases (G to C, and A to T or U) and temperature. Primer extensions are usually performed
form a stable double stranded, anti-parallel molecule. at 72ºC. The rate of nucleotide incorporation at
During PCR, the primers are moving around, caused 72ºC varies from 35 to 100 nucleotides per second
by the Brownian motion in the reaction mix. Hydrogen depending upon the buffer, pH, salt concentration
bonds are constantly formed and broken between and the nature of the DNA template. The length of
the single stranded primer and the single stranded the elongation step (30 seconds to three minutes)
template. The more stable bond lasts a little bit longer is determined by the speed of the enzyme, its ability
(primer that fit exactly) and on that little piece of to continue moving down the template DNA and
Central Marine Fisheries Research Institute

doubling stranded DNA (template and primer), the length of the DNA segment to be amplified.
the polymerase can attach and starts copying the At around 70ºC, the polymerase activity is optimal,
template. Once there are a few bases built in, the and primer extension occurs at upto 100 bases/
hydrogen bond is so strong between the template sec. A general guideline is 1 minute / kb of product
and the primer that it does not break any more. This length. An extension time of one minute at 72ºC is
is usually performed at temperatures between 47ºC considered sufficient for products up to 2 Kb size.
and 65ºC for 30 to 60 seconds. Longer products require longer times: approximately
3 min for 3kb and longer products.
The temperature and length of time required for
primer annealing depends upon the base composition, Cycling could include an initial denaturation at 94ºC
length and concentration of the primers. As a rule and a final extension at 72ºC for 5 min. At the end
of the thumb, annealing temperature (Ta) of 5ºC reactions are stopped by chilling at 4ºC or by addition
below the lowest melting temperature (Tm) of the of EDTA at 10mM.

120
Cycle number: The optimum number of is also in practice for conformation.
amplification cycles necessary to produce a band
visible on a gel depends largely on the starting Types / Variants of PCR
concentration of the target DNA when other
parameters are optimal. Because both strands are PCR has been adapted to fit many different
copied during PCR, there is exponential increase of applications and to achieve amplification of other
the number of copies of the gene. For example if molecules of interest like RNA. Hence, there are
the PCR is initiated with one copy of the gene, after many different types and each one is unique to the
one cycle there will be 2 copies, after two cycles application for which it is designed. The common
there will be 4 copies, three cycles will result in 8 types of PCR are: conventional/basic PCR, multiplex
copies and so on. Too many cycles may increase the PCR, reverse transcription (RT)-PCR, nested PCR, real-
amount and complexity of non-specific background time PCR and random primed PCR. And there are
products. Too few cycles give low product yield. many other types for specific purposes.
Innis and Gelfand (1990) recommend from 40–45
cycles to amplify 50 target molecules, and 25–30 to Conventional/ Basic PCR: Conventional PCR uses a
amplify 3x105 molecules to the same concentration. thermostable DNA polymerase to amplify a region of
This non-proportionality is due to a so-called plateau the DNA defined at each end by a specific primer. The
effect (Rybicki, 2001), which is the attenuation in exponential replication of the same target sequence
the exponential rate of product accumulation in produces enough DNA product or amplicons for use
late stages of a PCR, when product reaches 0.3–1.0 in subsequent analyses. PCR typically consists of three
nM. This may be caused by degradation of reactants basic steps, as mentioned earlier.
(dNTPs, enzyme); reactant depletion (primers, dNTPs–
former a problem with short products, latter for long Multiplex PCR: Multiplex PCR is a modification of

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

products); end-product inhibition (pyrophosphate conventional PCR in which two or more different
formation); competition for reactants by non- PCR products are amplified simultaneously within
specific products; competition for primer binding the same reaction. This type of PCR consists of
by re-annealing of concentrated (10nM) product the same steps as conventional PCR, except that
(Innis and Gelfand, 1990). multiple sets of primers are used, each one priming
a PCR product. Annealing temperatures for each of
Detection and analysis of PCR product: The the primer sets must be optimized to work correctly
PCR product will be DNA fragments (amplicons) of within a single reaction, and amplicon sizes, i.e.,
defined length. The simplest way to check the PCR their base pair length, should be different enough
to form distinct bands when visualized by gel
electrophoresis. The advantage is that it requires
less time and effort in amplifying multiple target
templates or regions than individual reactions
and may be a useful screening assay. However,
significant optimization is required to obtain all
of the products with equal efficiency and sensitivity.
By simultaneously amplifying more than one locus
in the same reaction, multiplex PCR is becoming a
rapid and convenient screening assay in both the
product is to load a portion of it into an agarose gel clinical and the research laboratory. Since its first
containing ethidium bromide along with molecular description in 1988 (Chamberlain et al., 1988), this
weight markers and carry out an electrophoresis. method has been successfully applied in many areas
The DNA fragments generated by the PCR should of DNA testing, including analyses of deletions,
be readily visible over an ultraviolet transilluminator. mutations and polymorphisms, or quantitative
Hybridizing the PCR product with suitable DNA probe assays and reverse transcription PCR.

121
 Nested PCR: Nested PCR is a very specific PCR during cycling when amplification of the PCR product
amplification and is a variation of the conventional PCR, of interest is first detected rather than the amount of
in that two pairs (instead of one pair) of PCR primers the PCR product which has accumulated at the end
are used to amplify a fragment. The first PCR utilizes point. Real Time PCR does this by using fluorescent
a pair of primes flanking the gene in question while dyes such as Sybr Green, or fluorophore-containing
the second PCR uses another pair of primers having DNA probes such as Taq Man which get incorporated
complementarity to an internal segment of the gene, into each of the new strand, and monitoring the
which was amplified in the first PCR. The fragment amount of fluorescence emitted during the PCR. This
produced by the first reaction is used as the template for acts as an indicator of the amount of PCR amplification
the second PCR. The second set of primers called nested that occurs during each PCR cycle. Thus, in Real Time
primers (as they lie / are nested within the first fragment) PCR machines, one can visually see the progress of the
is specific to the DNA sequence found within the initial reaction in “real time”. Quantification using real-time
PCR product. The use of a second amplification step with PCR can be ‘relative or absolute.
the “nested” primer set results in a reduced background
due to the nested primers’ additional specificity to the Random /Arbitrary primed PCR: Random primed
region. The amount of product produced, which is PCR conceived by Williams et al. (1990) is unique in
shorter than the first one, is increased as a result of the that only single short primer (usually 10 bases long)
second round of amplification. Carrying out nested PCR is used instead of the primer pair in the conventional
can further enhance the reliability of the PCR. Therefore, PCR. Prior knowledge of the sequence of the target
when information on the sequence of specific genes is DNA is not required and primer with any sequence
available, amplification and visualization of that gene can be employed. The underlying theory in AP-PCR
using a nested PCR could be carried out for confirmation. is that the primer may find complimentary sequence
at different locations on the two DNA strands used
Reverse Transcription Polymerase Chain as template, and amplify the intervening regions at
Reaction (RT-PCR): This is based on the processes of low PCR stringency conditions (36 – 40ºC).
reverse transcription and polymerase chain reaction.
RT-PCR is a two-step process. The first step consists This is used to generate Random amplified
of the formation of complementary or copy DNA polymorphic DNA (RAPD) profile which is increasingly
(cDNA) from RNA (generally mRNA). This is followed being used as a method for the DNA finger printing
by the second step which is a conventional PCR using and genetic characterization where prior knowledge
the cDNA as the template. The first step referred to of the sequence of the target DNA is not required.
as the “first strand reaction” uses enzyme reverse RAPD is used as a marker system, where sequence of
transcriptase for the production of the cDNA from the target DNA is not known. This is a rapid technique
the RNA. In the second step, the cDNA sequence is and can be useful for species/strain identification.
amplified by using primers specific to it. The RT-PCR Genomic variations between and within species could
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forms a high sensitivity detection technique, where be identified as the difference in the molecular size
low copy number or less abundant RNA molecules can and number of DNA fragments amplified. The PCR
be detected. It is also used to clone mRNA sequences products variations shall be resolved by agarose
in the form of complementary DNA, allowing cDNA gel electrophoresis.
libraries to be created which contain all sequences of
all the genes expressed in a cell. It allows the creation Modifications of PCR for
of cDNA constructs for the gene expression studies.
specific purposes
Real Time PCR: Real-time PCR is different from other There are several modifications developed from the
PCR as it quantifies the initial amount of the template basic method to improve performance and specificity.
instead of detecting the amount of final amplified Some of these commonly used in laboratories are:
product (Freeman et al., 1999; Raeymaekers, 2000).
Real Time PCR is characterized by the point in time Degenerate PCR: Degenerate PCR is in most

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respects identical to ordinary PCR, but with one major the population in a fast and cost-effective manner.
difference. It is in a situation where the sequence
of the gene to be amplified is not known, insert Hot Start PCR: This technique performed manually
“wobbles” in the PCR primers are inserted. So, instead by heating the reaction components to the DNA
of using specific PCR primers with a given sequence, melting temperature (i.e. 95 ºC) before adding the
mixed PCR primers are used. For example, when a polymerase. In this way, non-specific amplification
protein motif is back-translated to the corresponding at lower temperatures is prevented. Specialized
nucleotide motif (Protein —> Sequence), there enzyme systems have been developed that inhibit the
will be more than one codon coding for particular polymerase’s activity at ambient temperature, either
amino acid, due to degeneracy of genetic code. Thus by the binding of an antibody or by the presence
there will be more than one nucleotide sequence of covalently bound inhibitors that only dissociate
deciphered for a particular protein sequence. at high-temperature. At the desired elongation
temperature the polymerase is instantly activated.
Asymmetric PCR: Asymmetric PCR is used to Hot-start/cold-finish PCR is achieved with these type
preferentially amplify one strand of the original DNA of hybrid polymerases that are inactive at ambient
more than the other. It finds use in some types of temperature and are instantly activated at elongation
sequencing and hybridization probing where having temperature. Inhibition of the polymerase activity
only one of the two complementary stands is required. of the enzymes at lower temperatures during PCR
PCR is carried out as usual, but with a great excess of reaction is achieved by chemical modifications or wax-
the primers for the chosen strand. Due to the slow barrier methods or by a Taq-directed antibody. By
(arithmetic) amplification later in the reaction after the limiting polymerase activity prior to PCR cycling, non-
limiting primer has been used up, extra cycles of PCR specific amplification during the initial set up stages
are required. A recent modification known as Linear- are reduced and the yield of desired PCR product

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

After-The-Exponential-PCR (LATE-PCR), uses a limiting is increased.
primer with a higher melting temperature than the
excess primer to maintain reaction efficiency as the Touchdown PCR: Non-specific primer binding
limiting primer concentration decreases mid-reaction. obscures polymerase chain reaction results, as the
non-specific sequences to which primers anneal in
Thermal asymmetric interlaced polymerase early steps of amplification will “swamp out” any
chain reaction (TAIL-PCR): Thermal asymmetric specific sequences because of the exponential nature
interlaced polymerase chain reaction (TAIL-PCR) is a of polymerase amplification. Touchdown PCR or
fast and efficient method initially developed to amplify touchdown style polymerase chain reaction is a variant
unknown sequences adjacent to known insertion sites of PCR that aims to reduce non-specific background
in Arabidopsis. Nested, insertion-specific primers by gradually lowering the annealing temperature as
are used together with arbitrary degenerate primers PCR cycling progresses. The annealing temperature at
(AD primers), which are designed to differ in their the initial cycles is usually a few degrees (3-5˚C) above
annealing temperatures. Alternating cycles of high the Tm of the primers used, while at the later cycles,
and low annealing temperature yield specific products it is decreased in increments for every subsequent
bordered by an insertion-specific primer on one side set of cycles (the number of individual cycles and
and an AD primer on the other. Further specificity is increments of temperature decrease is chosen by the
obtained through subsequent rounds of TAIL-PCR, experimenter) a few degrees (3-5˚C) below the primer
using nested insertion-specific primers. The increasing Tm. The higher temperatures give greater specificity
availability of whole genome sequences renders TAIL- for primer binding, and the lower temperatures
PCR an attractive tool to easily identify insertion sites permit more efficient amplification from the specific
in large genome tagging populations through the products formed during the initial cycles.
direct sequencing of TAIL-PCR products. For large-
scale functional genomics approaches, it is desirable Inverse PCR: Inverse PCR (IPCR), variant of PCR, was
to obtain flanking sequences for each individual in first described by Ochman et al. (1988). It is used

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 when only one internal sequence of the target DNA Alu PCR: PCR using a primer that anneals to Alu
is known. It is therefore very useful in identifying repeats to amplify DNA located between two
flanking DNA sequences of genomic inserts. The oppositely oriented Alu sequences. Used as a
inverse PCR method includes a series of digestions method of obtaining a fingerprint of bands from an
(DNA being cut by a restriction endonuclease). This uncharacterized human DNA.
cut results in a known sequence at either end of DNA
of unknown sequence. The restriction fragment is self- Colony PCR: Colony PCR is mostly used after a
ligated upon itself to form a circle and the inverse PCR transformation of bacterial (E. coli) or yeast clones
uses standard polymerase chain reaction, however to screen colonies for the desired (correct) ligation
it has the primers oriented in the reverse direction or plasmid products. Primers which generate a PCR
of the usual orientation. Thus the template for the product of known size are used. The colonies which
reverse primers for the inverse PCR is a restriction give rise to an amplification product of the expected
fragment that has been self- ligated upon itself to size are likely to contain the correct DNA sequence.
form a circle. Applications of Inverse PCR in molecular Selected colonies of bacteria or yeast are picked
biology include the amplification and identification of inserted into the PCR master mix or pre-inserted into
sequences flanking transposable elements, and the autoclaved water. PCR is then conducted to determine
identification of genomic inserts. if the colony contains the DNA fragment or plasmid
of interest.
Long PCR: Long PCR is used when large segments
of DNA (frequently over 10 kb) is to be amplified. In Situ PCR: In Situ PCR (ISH) is a polymerase chain
For the accuracy of PCR, special mixtures of reaction that actually takes place inside the cell fixed
proficient polymerases such as Pfu are often used, on slide. In situ PCR amplification can be performed
which possesses 3’ to 5’ exonuclease activity or on fixed tissue or cells. ISH applies the methodology
proofreading activity. The efficiency drastically declines of the nucleic acid hybridization technique to
when incorrect bases are incorporated. The 3’ to 5’ the cellular level. Combining cytochemistry and
exonuclease activity removes these mis-incorporated immunocytochemistry, it allows the identification of
bases and makes the further reaction proceed cellular markers to be identified and further permits
smoothly. Therefore, the amplification of long DNA the localization of to cell specific sequences within
fragments can be achieved. Long PCR is often used to cell populations, such as tissues and blood samples.
clone larger genes or large segments of DNA which
standard PCR cannot. Single Cell PCR: The advent of the polymerase
chain reaction (PCR) has revolutionized the way in
Gradient PCR: When a set of PCRs are run with which molecular biologists view their task at hand,
different annealing temperatures all in the same for it is now possible to amplify and examine minute
block of the thermal cycler, simultaneously it quantities of rare genetic material: the limit of this
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is referred to as Gradient PCR. This is generally exploration being the single cell. It is especially in
carried out for standardizing PCR conditions for the field of prenatal diagnostics that this ability has
heterologous primers. In many models of thermal been readily seized upon, as it has opened up the
cyclers, blocks are available with gradient annealing prospect of preimplantation genetic analysis and
temperatures. This saves time and multiple blocks the use of fetal cells enriched from the blood of
are not needed. pregnant women for the assessment of single-gene
Mendelian disorders. However, apart from diagnostic
AFLP PCR: Amplified Fragment Length Polymorphism applications, single-cell PCR has proven to be of
PCR, also called AFLP PCR was originally described by enormous use to basic scientists, addressing diverse
Zabeau and Vos, 1993. AFLP is a highly sensitive PCR- immunological, neurological and developmental
based method for detecting polymorphisms in DNA. AFLP questions, where both the genome but also
can be also used for genotyping individuals for a large messenger RNA expression patterns were examined.
number of loci using a minimal number of PCR reactions. Furthermore, recent advances, such as optimized

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whole genome amplification (WGA) procedures, Intersequence-specific (ISSR) PCR: A PCR
single-cell complementary DNA arrays and perhaps method for DNA fingerprinting that amplifies
even single-cell comparative genomic hybridization regions between some simple sequence repeats
will ensure that the genetic analysis of single cells will to produce a unique fingerprint of amplified
become common practice, thereby opening up new fragment lengths.
possibilities for diagnosis and research.
Ligation-mediated PCR: Ligation-mediated
Digital PCR: Digital PCR represents an example PCR uses small DNA oligonucleotide ‘linkers’ (or
of the power of PCR and provides unprecedented adaptors) that are first ligated to fragments of
opportunities for molecular genetic analysis in the target DNA of interest. Multiple primers that
cancer. The technique is to amplify a single DNA anneal to the linker sequences are then used
template from minimally diluted samples, therefore to amplify the target fragments. This method is
generating amplicons that are exclusively derived deployed for DNA sequencing, genome walking
from one template and can be detected with and DNA fingerprinting.
different fluorophores or sequencing to discriminate
different alleles (e.g., wild type vs. mutant or paternal Methylation-specific PCR (MSP): The MSP method
vs. maternal alleles). Thus, digital PCR transforms was developed by Stephen Baylin and Jim Herman at
the exponential, analog signals obtained from the Johns Hopkins School of Medicine, and is used to
conventional PCR to linear, digital signals, allowing detect methylation of CpG islands in genomic DNA.
statistical analysis of the PCR product. Digital PCR has DNA is first treated with sodium bisulfite, which
been applied in quantification of mutant alleles and converts unmethylated cytosine bases to uracil, which
detection of allelic imbalance in clinical specimens, is recognized by PCR primers as thymine. Two PCR
providing a promising molecular diagnostic tool for reactions are then carried out on the modified DNA,

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

cancer detection. using primer sets identical except at any CpG islands
within the primer sequences. At these points, one
Single nucleotide polymorphism PCR (SNP PCR): primer set recognizes DNA with cytosines to amplify
SNP PCR involves real-time PCR using single nucleotide methylated DNA, and one set recognizes DNA with
polymorphisms (SNPs) as markers. It is a very sensitive uracil or thymine to amplify unmethylated DNA.
and accurate method to quantify the percentage of MSP using qPCR can also be performed to obtain
recipient and donor cells to monitor the effect of stem quantitative rather than qualitative information
cell transplantation (SCT) and sequential adoptive about methylation.
immunotherapy by donor lymphocyte infusions (DLI).
Semi-quantitative PCR
Assembly PCR: Assembly PCR is the artificial
synthesis of long DNA sequences by performing This technique allows an approximation to the
PCR on a pool of long oligonucleotides with short relative amount of nucleic acids present in a
overlapping segments. The oligonucleotides alternate sample. cDNA is obtained by RT-PCR when sample
between sense and antisense directions, and the is RNA. They are then amplified along with the
overlapping segments determine the order of the internal controls (that are used as markers).
PCR fragments thereby selectively producing the final The markers commonly used are Apo A1 and
long DNA product. β-actin. Amplification product is separated by
electrophoresis. Agarose gel is photographed after
Helicase-dependent amplification: This technique ethidium bromide staining, and optical density is
is similar to traditional PCR, but uses a constant calculated by a densitometer. The disadvantage
temperature rather than cycling through denaturation of the technique is possibility of nonspecific
and annealing/extension cycles. DNA Helicase, an hybridizations, generating unsatisfactory results.
enzyme that unwinds DNA, is used in place of Control of specificity is performed using highly
thermal denaturation. specific probes for hybridization

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 Designing of primers which can become competitive enough to suppress
product formation. The sequences of the primers
A primer is a short oligonucleotide (preferably 18-22 used for PCR amplification can have a major effect
bases) which is the reverse complement of a region of on the specificity and sensitivity of the reaction.
the target DNA template. It would anneal to the DNA When choosing appropriate primer pair for PCR, the
strand to facilitate the amplification of the targeted following guidelines should be considered:
DNA sequence. There will be two primers namely,
forward and reverse primers, for amplifying both the Specificity
strands of target DNA template. Proper primer design
is important for applications in PCR. Optimal primer Primer specificity is dependent on primer length, GC
sequence with required concentration is essential content, internal stability and unique sequence of
for achieving maximal specificity and efficiency of the primer.
PCR. The manual selection of optimal PCR primer set
is tedious process and not efficient. Thus, various • Uniqueness: The primers primer sequence should
bioinformatics tools are available for selection of be unique in the template DNA which means there
appropriate primer pairs from a template sequence. should be only one target site present in the target
Parameters like Specificity, stability, compatibility are template DNA.
to be considered for good primer designing. • Primer Length: The length of the primers need
to between 15 and 30 base pairs so that they are
Types of Primer long enough for adequate specificity and short
enough for them to anneal to the DNA template.
Based on the application, primers are classified into • Internal stability: Internal stability is calculated
different categories. with entropy values (ΔG) of neighbor nucleotides.
To minimize false priming, it is critical that the
• Universal primers: Primers to amplify specific stability at 5’ end be high and the stability at 3’
DNA sequence/Gene/Gene product which is end be relatively low. The optimal internal stability
conserved in nature. These primers amplify specific is ΔG ~ 8.5 Kcal/mol.
sequence across different species in a group. • GC Content: The GC content of the primer
• Guessmer: Primers are used to amplify the sequence should be relatively high as it has a direct
particular sequence back translated from relationship with the Tm. There should be a base
amino acid. composition of GC of about 50%-60%. The 3’ end
• Oligo d (T): Oligo dT is the classic primer mix of the primer should finish with at least one G or
used to prime synthesis of the first strand of cDNA; C to promote efficiency in annealing due to the
generally used for reverse transcription PCR. stronger bonding.
• Degenerate primers: The mixture of primer used
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to amplify the same gene from different organisms, Stability

which are probably similar but not identical. The
other use for degenerate primers is when primer The stability of primer is mainly dependent on GC
design is based on protein sequence. clamp, Annealing Temperature, melting temperature.
• Specific primer: Primers are designed specifically Stretches of A and T are also to be avoided as these
for a particular gene in a particular species. will open up stretches of the primer- template
complex. G or C is desirable at the 3’ end. This GC
Parameters for consideration clamp reduces spurious secondary bands.

Efficacy and sensitivity of PCR largely depend on • Melting temperature(Tm): Tm is the temperature
the efficiency of primers. A poorly designed primer at which one-half of a particular DNA duplex will
can leads to failure of PCR due to nonspecific dissociate and become single strand DNA. The
amplification and/or primer-dimer formation, optimal melting temperature for primers generally

126
 lies in the range of 52- 58ºC. The stability of a One critical feature is their annealing temperatures,
primer template DNA duplex can be measured by which shall be compatible with each other. The
its Tm. The melting temperature of nucleic acid maximum difference allowed is 3ºC. Closer the Ta
duplex increases as the length and GC content of the primer pairs; better the annealing.
increases. A simple formula for calculation of the • 3’-end Sequence: It is well established that the
(Tm) is: 3’ terminal position in PCR primers is essential for
Tm = 2(A+T) + 4(G+C) the control of miss-priming. Primers should be
This formula will be accurate in lower salt “stickier” on their 5’ end than on their 3’ ends. A
conditions only for primers less than or about 20 highly “sticky” 3’ end, as indicated by a high GC
nucleotides in length. content, could potentially anneal at multiple sites
Formula for primers of 20-35 bases in length is on the template DNA. Optimal GC clamp reduces
as below. spurious secondary bands.
Tm p = 22 + 1.46 ([2 x (G+C)] + (A+T)) • Secondary structure: Presence of secondary
The calculated annealing temperature is only structures of the primer, produced by intermolecular
a reference temperature from which to initiate or intramolecular interactions can greatly reduce
experiments. The actual annealing temperature the availability of primers to the reaction which lead
may be 3-12ºC higher than the calculated Tm. The to poor or no yield of the product. Generally three
actual annealing temperature condition should be different secondary structures are encountered
determined empirically. The optimum annealing in primers.
temperature which gives the best PCR product • Hairpins loop formed by intramolecular interaction
should be used. within the primer and should be avoided.
(Optimally a 3’ end hairpin with a ΔG of -2 kcal/
• Annealing temperature (Ta): The annealing mol and an internal hairpin with a ΔG of -3 kcal/

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

temperature (Ta) chosen for a PCR depends directly mol is tolerated generally.)
on length and composition of the primer(s). • Primer self-dimer is formed by intermolecular
Generally, an annealing temperature about 5ºC interactions between the two (same sense) primers.
below the lowest Tm of the pair of primers is When primers form intermolecular dimers much
used. Too high Ta will produce insufficient primer- more readily than hybridizing to target DNA,
template hybridization resulting in low PCR they reduce the product yield. (Optimally a 3’
product yield. Too low Ta may possibly lead to end self dimer with a ΔG of -5 kcal/mol and an
non-specific products caused by a high number internal self dimer with a ΔG of -6 kcal/mol is
of base pair mismatches. tolerated generally.)
• GC clamp: The presence of G or C bases within • Primer cross dimers are formed by intermolecular
the last five bases from the 3’ end of primers (GC interaction between sense and antisense primers,
clamp) helps promote specific binding at the 3’end where they are homologous. (Optimally a 3’ end
due to the stronger bonding of G and C bases. cross dimer with a ΔG of -5 kcal/mol and an
More than 3 G’s or C’s should be avoided in the internal cross dimer with a ΔG of -6 kcal/mol is
last 5 bases at the 3’ end of the primer. tolerated generally.)

Compatibility Other Recommendation

The compatibility of primer is dependent on The concentration of primer in amplification reaction
following factors. should be between 0.1 and 0.5 µm. The forward
primer and the reverse primer should be between 300
• Primer Pair Matching: Primers work in pairs – and 2,000 base pairs apart. Primers designed for a
forward primer and reverse primer. Since they are sequence must not amplify other genes in the mixture.
used in the same PCR reaction, it shall be ensured If possible, a computer search should be conducted
that the PCR condition is suitable for both of them. against the vector and insert DNA sequences to verify

127
 that the primer and especially the 8-10 bases of its more than one set of primers in a PCR for getting
3’ end are unique. desired results.

Tools for Primer Designing PCR applications

The use of software in biological applications has PCR has transformed the way that most studies
given a new dimension the field of bioinformatics. requiring the manipulation of DNA fragments and
There are a numerous web based resources and DNA cloning may be performed as a result of the
different programs available for primer designing. simplicity and usefulness of PCR. Cell-free DNA
There are number of simple standalone programs as amplification by PCR is able to simplify many of
well as complex integrated networked versions of the the standard procedures for DNA cloning, DNA
commercial software available. Some important tools analysis, and the modification of DNA. Previous
are doPrimer, primer 3, web primer, primo pro 3.4, molecular biology techniques for isolating a specific
GAP, PCR primer design, the primer generator etc, piece of DNA had relied on gene cloning, which is
these all are web- based resources and very essential a tedious and slower procedure. An alternative to
for molecular biologist. There are different software cloning, PCR, can be used to directly amplify rare
freely and commercially available for PC installation specific DNA sequences in a complex mixture when
like PrimerSelect, Primer 3, Primer Premier, NetPrimer, the ends of the sequence are known. This method
GenomePRIDE 1.0, OLIGO 6, Primer Designer 4, of amplifying rare sequences from a mixture has
GPRIME, Primer Designer, Primer Premier, Primer numerous applications in basic research, human
Design, Gene Runner, Primer BLAST etc. genetics testing and forensics. Some of the PCR
applications include site-specific mutagenesis studies,
In conclusion, the heart of PCR lies in the proper amplification and detection of DNA in situ from cells
designing of primers. Several basic parameters for rapid diagnosis, genomic subtraction, analysis
including the length of the primer, %GC content of protein functions and intermolecular assembly,
and the 3’ sequence, Tm value need to be optimized DNA fingerprinting (RAPD/AFLP/VNTR/) for evaluation
for successful PCR. Certain of these parameters can of genetic heterogeneity & relationship, paternity
be easily by hand optimized while others are best verification, forensic application, generation of
done with available primer designing tools. The single chain antibody fragments for immunology,
increasing use of information from the internet and sensitive disease diagnosis, cDNA synthesis from RNA
the sequences held in gene databases are practical for cDNA library construction, production of clones
starting points when designing primers and reaction for sequencing, molecular epidemiology, molecular
conditions for the PCR. It is also possible to include taxonomy and many more.
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128
Quantitative Genetics
V. Srinivasa Raghavan
Scientist
Madras Research Center of CMFRI
e-mail: [email protected]

The study on genetics of quantitative characters in a variation wherein the phenotypic values of different
population is called as quantitative genetics. The aim individuals of a population form a continuous graded
of quantitative genetics is to bring about desirable series of different phenotypes. Body weight, fleece
change in the quantitative traits. The change in any production are few examples for quantitative traits.
character is possible only if variation exists in those
characters that are genetically determined and are Genetic basis of a character
inherited. Any measurable or observable property
of living individual that exhibit inherited differences The development of any trait and its final expression in
among the individuals of a population is called a the form of a phenotype is totally under the control of
character or trait. Genetically determined character genes. However, the genes don’t produce the characters
is called an inherited character and those inherited directly but through some biochemical reactions that
characters with variation among individuals of a in turn lead to the development of a character or trait.
population are called as heritable traits. The phenotype so expressed is the joint product of

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

gene and the environment. The phenotype (P) due to
Classification of characters gene effect is called as the genotypic value (G). The
environment modifies the genotypic value before its
final expression as phenotype. This deviation of the
Qualitative characters P from G due to environmental effects is called as
The characters governed by single or few pairs of genes environmental deviation (E). Thus the two components
(Oligogenes) with major effect and least affected by of phenotypic value are genotypic value, G and
environment are called as qualitative characters. The environmental effects, E and denoted as P = G + E.
qualitative characters follow discontinuous variation Thus any change in genotype or environment results
among individuals of a population. The qualitative in a change of phenotype. This interaction of genes
traits are influenced by few genes called as major with the external environment like climate, nutrition,
genes and express mendelian ratios. Color pattern management etc. or the differential response of
in different species of farm animals and presence or different genotypes in different environments is called
absence of body parts are few examples. as genotype–environment interaction, IGE.

Quantitative characters Gene action and its types

The characters governed by many genes with minor The quantitative traits are under the control of
effect of each gene (poly genes or minor genes) polygenes and shows different types of gene action
and the effects of whom are modified to a greater in modifying the expression of a character. The effect
extent by environment are called as quantitative of a gene in modifying the expression of a trait or
characters or polygenic traits. Since the phenotypic phenotype depends upon the presence or absence
expression of these traits is affected by many genes of another gene in the same or different locus. This
and environment, they are also called as multifactorial forms the basis for variation in gene expression
traits. The quantitative character shows continuous dividing into additive and non-additive gene action.

129
 Additive gene action (AGA) P = G + E + IGE

A gene may have an independent effect irrespective of The genotypic component, G can be further partitioned
the presence or absence of other genes in the same or into additive, dominance and epistasis effect.
different locus in the same or different chromosome.
This type of gene action which has the increasing or G=A+D+I
decreasing effect of a gene on the phenotypic value
is called the additive gene action. When the gene Where
action is additive, there is no dominance among the A – Additive gene action or breeding value
alleles. The complete additive gene action produces D – Dominance effect
a linear phenotype. E – Epistasis effect

The major component of the genotypic value is the

additive effect of genes or the individual effect of
genes. This portion of the genotypic value (additive
A – Contributes one unit to the phenotype. genetic value or the breeding value) is a fixed effect
a – Contributes zero units to the phenotype. and transmitted to the progeny as such without
any change. The breeding value is thus defined as
Non-additive gene action (NAGA) the sum of average effects of the genes carried by
an individual.
In this type of gene action, the genes in addition to its
individual effect, produces a different type of action The components of variance and
when it combines with another gene. This interaction of
its significance
genes is non-additive and is called as non-additive gene
action. The interaction occurs between alleles of the
The population mean does not reveal whether the
same locus (intra allelic interaction) or between alleles
phenotypic values recorded on different individuals
of different locus (inter allelic interaction).Thus there are
of a population are similar or vary from the mean
different types of NAGA namely dominance, incomplete
of the population. The individuals differ in their
dominance, over dominance and epistasis. The increased
phenotypic values for quantitative traits. The
vigour observed when animals of different genetic
differences in the phenotypic values of a character
background are mated as in case of cross breeding is
among individuals of a population are called as
called as heterosis, a non-additive gene effect.
variation. The variation is thus the mean of the
squares of the deviated values from the mean and
The phenotypic value is the joint effect of genotype
denoted as
and the environment. i.e.
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P=G+E
Where
Where Xi is the numerical value of variable X
P = Phenotypic value X is the mean of variable X
G = Genotypic value (gene effect) N is the number of observations
E = Environmental effect
The components of the phenotypic variance are same
But there exists a differential response of different as the components of the phenotypic value. Thus the
genotypes in different environment. This forms the partitioning of the phenotypic variance is analogous
component of genotype-environment interaction, to partitioning of phenotypic value as
IGE. Therefore the phenotypic value is made up of
the following components. P = G + E + IGE and VP = VG + VE + CovGE

130
Where it’s called as heritability in broad sense (H2).
VP is the observed phenotypic variance
VG is the genotypic variance
VE is the environmental variance

CovGE is the interaction between genotypic values

and environmental deviation. Heritability measures the strength of relationship
between the performance (phenotype) and
The genotypic variance is further partitioned into breeding value of an individual for a particular
trait. It is a measure of parent’s superiority that is
VG = VA + VD + VI being transmitted to the progeny. The heritability
values ranges from 0 to 1 depending upon the
Where magnitude of phenotypic variability due to
VA is the additive genetic variance genetic effects. Thus heritability is a measure of
VD is the dominance variance population and not an individual. Heritability, an
VI is the interaction variance important genetic parameter plays a vital role while
formulating genetic improvement programs. Its
The partitioning of variance is done by analysis provides the degree of genetic determination of a
of variance. The splitting of variance helps us to trait, amount of parent’s superiority transmitted to
understand the effect of different factors in causing the progeny, accuracy of selection, relation between
variation in a trait and the percentage contribution the phenotypic value and breeding value and mode
of different factors to the total phenotypic variance. of gene action.
Thus the knowledge on variance helps us to determine

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

the genetic properties of a population and to study If the heritability value is 1, it means that the
the inheritance of quantitative trait. The variation differences in phenotypic values are entirely due
is thus an important component which helps the to effects of genes and thus VG = VP. If heritability
breeder to bring about genetic improvement in value is 0, then the differences in phenotypic values
the population. Any genetic improvement program are completely due to environmental factors and
would be effective only if variation exists among thus VE = VP as in case of identical twins and pure
the individuals of a population. The whole of the lines. If a trait is highly heritable, then individual
variation is not transmitted to the next generation. selection will bring about genetic improvement
It’s the genetic part which is heritable. Even in in a particular trait in progeny generation and
the genetic variance, only the additive genetic indicates the additive gene action. In case of
component responses to the selection process and low heritable traits, non-additive gene action
gets transmitted from parents to the offspring. Thus plays a major role in determining the phenotypic
the partitioning of phenotypic variance throws light differences of a trait.
on the role of heredity ad environment in influencing
a trait. This degree of genetic determination The resemblance among relatives (due to sharing of
in modifying the phenotypic value is called common genes) forms the basis for different methods
as the heritability. of estimation of heritability like

Heritability Regression of offspring

Heritability is defined as the ratio of genetic variance
on parent.
to the total phenotypic variance. When only the • Correlation between offspring and parent.
additive genetic variance is considered in the total • Half sibs and full sib’s intra class correlation method.
genetic part, it is called as heritability in narrow sense • Apparent and realized heritability.
(h2). When the total genetic variance is considered,

131
 Selection of progress achieved is less efficient and takes a long
time. In case of independent culling level, selection
superior animals is made simultaneously for all the characters but
The foremost step in a genetic improvement program independently. Minimum standards are fixed for
is to decide the characters that are to be improved. each trait and animals below the standard are culled
These are called as selection objectives. The characters irrespective for its superiority in other characters. In
that are to be improved vary between different classes the case of selection index method, each trait is
of animal. The final aim in any selection program is weighed by a score and the scores for individual
to maximize the genetic gain and in turn response characters are summed up to get a total score for
to selection. each animal. Based on the total score, animals with
high scores are selected and those with low scores are
Basis of selection culled from the population. Discriminate function is
used for discriminating the desirable genotypes from
Different criteria’s are to be used while estimating the undesirable ones. The overall genetic worth of an
the breeding value of an individual. These criteria’s individual using the selection index method is
are called as basis of selection.

Where
ai is the relative economic value of the trait
Gi is the additive genetic value

Pedigree selection
The list of record of ancestors like parents,
grandparents and great grandparents is called as
pedigree. The estimation of breeding value of an
Individual selection individual and further selection of that animal based
The fundamental unit of any genetic improvement on the performance of its relatives/ancestors is called
program is the animal itself and the animal’s own as pedigree selection. The basis of pedigree selection
phenotypic value of a trait is considered to estimate is transfer of genes from ancestors to the next
the breeding value of that trait in that individual. generation. The pedigree selection helps to select
This is called as individual selection as animal’s own animals at early age and also for sex limited traits.
performance is used to estimate its breeding value. The main disadvantage is that animals of similar
Individual selection minimizes the environmental pedigree would be culled without giving due credit
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deviations as the individuals to be selected are tested to the individual merit.

under similar environmental conditions. This method
can be applied for traits with high heritability and
can’t be used for sex limited traits. When an animal
has to be selected for different traits at a time, then
it is called as multi trait selection.

The different aids used for multi trait selection

are tandem method, independent culling level
and selection indices. In case of tandem selection,
genetic improvement is practiced for one trait at a
time till satisfactory progress is achieved. Then the
second trait is selected and so on. But the genetic

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Collateral relatives or • Line breeding.

family selection
Outbreeding
The selection of an individual based on the
performance of its collateral relatives is called as • Within species like out crossing, top crossing and
family selection. The collateral relatives may be full pure breeding.
sibs, half sibs, cousins etc. The relatives must be • Between species like inbreed crossing, strain
closely related to the individual as in case of sibs. crossing, criss crossing, rotational crossing, grading
If the individuals own performance is included in up and species hybridization.
estimating the family mean, it is called as family
selection. If the individual records are excluded Inbreeding
in estimating the family mean, it is called as sib
selection. The sib selection is practiced for slaughter The mating of very closely related individuals
traits, sex limited traits and low heritable traits. than the average members of the breed is called
as inbreeding and the mates should have one or
Progeny selection more common ancestors in the immediate 4-6
generations. Inbreeding increases the homozygosity
The selection of an individual based on the by decreasing the percentage of heterozygotes
performance of its progeny is called as progeny without disturbing the gene frequency. The
selection. The progeny of an individual is the index inbreeding increases the frequency of recessive
of parent’s genetic worth as the progeny inherits genes in the population. This mating system fixes
half of genes from each parent. The progeny characters in an inbred population which may be
selection method is used for low heritable traits, either favourable or unfavourable. Inbreeding thus

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sex limited traits and proves whether the male is changes the genetic structure of the population
free from any recessive lethal effects. This method by changing the genotypic frequencies. Due to
can also be used for traits that can’t be measured inbreeding depression, the performance of the
during the life time of an individual like carcass individual is greatly affected in terms of its vigour,
traits. As many progenies as possible should growth and reproduction. Inbred animals are more
be considered for selecting an individual as the susceptible to change in environmental conditions.
segregation may cause the progenies to receive Inbreeding is mainly followed to develop inbred
good or bad genes from parents and environment lines, elimination of undesirable genes from the
is not same for all progenies of single parent. The population, production of distinct families and to
accuracy of selection increase with increase in increase the prepotency of an individual.
number of progenies.
Outbreeding
Mating Systems
The mating of unrelated individuals is called as
The genetic relationships between the mates are outbreeding. If the mating occurs between individuals
also important in bringing about genetic progress of the same breed it is called as out crossing, between
using different selection methods. These are called individuals of different breeds it is called as cross
as mating systems. The different types of mating breeding and between species it is called as species
systems present in front of an animal breeder along hybridization. This is more popular in case of highly
with different selection tools are domesticated farm animals and uncommon in case of
aquaculture species. Outbreeding is mainly practiced
Inbreeding to increase the heterozygosity thereby increasing the
performance of the individual called as hybrid vigour
• Close inbreeding like selfing, parent-offspring and or heterosis. This increase in vigour is mainly due to
sib mating non-additive gene action.

133
 Selective breeding Selective breeding program was also carried out in
tilapia, one of the most famed aquaculture species
programme in of the world known as Genetic Improvement of
aquaculture species Farmed Tilapia (GIFT) by world fish centre, ICLARM in
In contrast to livestock, only very few genetic collaboration with NFFTRC, AKVAFORSK and FAC. The
improvement programs have been carried out in main objective of the study was to develop a synthetic
aquaculture species worldwide (Nile Tilapia in Asia, base population and to estimate the phenotypic and
Atlantic salmon in Norway). The genetic improvement genotypic parameters in tilapia. Selective breeding
programs can be applied to variety of aquaculture programs were also carried out in Atlantic salmon
and mariculture species to develop improved breeds by the Norwegians for different traits like weight at
to have a cost effective farming practices. The most slaughter, age of sexual maturation, flesh colour, total
important traits in case of aquaculture species that fat content, and amount of fat tissues.
could be effectively improved through selective
breeding are the growth rate, disease resistance, Conclusion
meat yield and survival rate.
Selection and genetic improvement programs are
In India, for the first time, genetic selection important tools for increasing the aquaculture
program was carried out to improve the growth production in proportion to the growing food demand
performance or body weight of Indian major carps of the world. The scope exists for standardization and
(Labeo rohita) by Central Institute of Freshwater optimization of breeding programs in combination with
Aquaculture, Bhubaneswar in collaboration with the use of advanced molecular tools like implementation
Institute of Aquaculture Research (AKVAFORSK), of marker assisted selection programs. The decision on
Norway. The main objective of this breeding using a selection program depends upon the variation
program was to develop a national breeding existing in the phenotypic performance of different
plan and dissemination of improved variety of characters in a species. With proper investment of
rohu to fish farmers for quality seed production. time, money, labour and long term planning, genetic
The improved variety named as Jayanti Rohu was improvement programs are going to be the future for
produced using a nested design program (2 males improving the performance of different species to meet
nested within a male or vice versa). It was found the demands of the population explosion.
that the maximum genetic gain per generation
in case of rohu selection program after four Suggested readings
generations of selection was 17%. The program
started in 1992 and the first genetically improved Falconer. D.S., 1961. Introduction to Quantitative genetics.
variety Jayanti Rohu was released in 1997. Gjedrem, T. and Robinson, N. 2014. Advances by Selective Breeding for
Aquatic Species: A Review. Agricultural Sciences, 5, 1152-1158.
Central Marine Fisheries Research Institute

Mahapatra, K.D et al. 2007. Genetic improvement and

In CMFRI, bidirectional selection, individual/mass dissemination of rohu (Labeo rohita, Ham.) In India. Proc.
Assoc. Advmt. Anim. Breed. Genet. 17: 37-40.
selection program was carried out for naupliar Mallia, Jyothi V et al., 2009. Performance of triploid edible oyster
length in Artemia fransciscana by taking two sub- Crassostrea madrasensis (Preston): gonad development and
populations namely small naupliar size (SNS) and big biochemical composition. Journal of the Marine Biological
Association of India, 51 (1). pp. 81-86.
naupliar size (BNS) lines with the view of developing Lush.J.L. 1994. The genetics of populations. Iowa State University,
two divergent strains. In case of SNS, reduce naupliar Ames, Iowa, USA.
Sajesh Kumar, N K et al., 2014. Quantitative genetic manipulation
size was the trait of selection and in case of BNS, for nauplii size reduction of Artemia franciscana Kellogg,
bigger naupliar size was the trait of selection. It 1906 from Indian salinas and correlated changes in the
was found that the phenotypic responses in both polyunsaturated fatty acids (PUFA) profile. Indian Journal of
Fisheries, 61 (3). pp. 69-73.
the lines were substantial. CMFRI has also studied Shirdhankar, M M et al., 2006.  Efficacy of selection in
the performance of triploid edible oysters in terms sexually breeding Artemia (Artemia franciscana, Kellogg,
1906). Aquaculture Research, 37. pp. 1276-1281.
of meat yield, quality and growth by chromosomal Tomar,S.S., 1998. Text book of Quantitative genetics.
manipulation techniques. Tomar,S.S., 2004. Text book on Animal breeding.

134
Marker Assisted Selection
V. Srinivasa Raghavan
Scientist
Madras Research Center of CMFRI
e-mail: [email protected]

The majority of economically important traits in The traditional genetic selection programmes involve
livestock is complex and has continuously distributed the selection of animals based on the observable
phenotypes, which are influenced by polygenes phenotypes since the genes contributing to the
dispersed across the genome. The molecular phenotype was unknown. The two stages of selection
architecture of complex quantitative traits gives process are the phenotypic selection (selection based
new insights on understanding of the molecular on phenotypic performance) and genotypic selection
physiology of the phenotypes and yield sophisticated (selection based on breeding value). Thus there exist
selection strategies for an effective marker assisted three types of selection criteria’s namely
breeding The economically important characters of • Individual selection (animals own performance is
central importance such as food production, body considered as a measure of its genetic merit).
weight, and health in terms of disease resistance • Collateral relatives (performance records of
are under the control of multiple genes plus the ancestors and collateral relatives like family
environment and are known as quantitative traits. selection, within family selection, sib selection).

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

The regions within the genomes that contain genes • Progeny selection (selection of an individual based
associated with a quantitative trait are known on its progeny performance).
as quantitative trait loci (QTLs). The benefits of
molecular markers linked to genes transformed the With the advent of genetic markers in 1980’s,
genetic selection program from phenotype based the association of certain genes with our traits of
to genotype based. Since the identification of QTLs importance is established paving way for selection
based on conventional phenotypic methods is not of animals based on animals genotype rather than
possible, the development of molecular markers its phenotype alone. Thus the process of selection of
leads to the characterization of QTLs and in turn animals based on the information available on DNA is
the quantitative traits. called as Marker Assisted Selection (MAS). The MAS
is an indirect selection process wherein the trait of
The selection is a process of giving preference to interest is selected based on a marker linked to the
certain individuals (differential reproduction) in concerned trait.
a population to reproduce and produce the next
generation. Genetic selection therefore changes the Basis of marker
frequency of genes and genotypes without creating
any new genes. Selection is carried out to improve
assisted selection
one or more desirable traits/characters in the Every individual in this world are made up of cells
population of a species. Genetic improvement of any that are in turn programmed by genetic material
population of a species involves periodic evaluation, called DNA. The DNA consists of four different
selection and culling of animals. Selection of animals nitrogenous bases namely, adenine, guanine, thymine
is an important aspect of genetic improvement and cytosine. Genes which are made up of a portion
program, because the selected parents will decide of DNA sequence code for proteins. More than 95%
the genetic makeup of the progeny and hence their of the DNA sequence doesn’t code for proteins and
phenotypic performance. are called as non-coding sequences. The DNA is in

135
 turn packed up into sets of chromosomes and the QTL’s which contribute to variation in growth and
entire set is called the genome. Any gene is made up production performance of animals.
of two alleles, one from each parent (present over
a pair of chromosomes) in a diploid individual. The Efficiency of MAS
genetic information present on a gene is involved
in the synthesis of proteins and polypeptides which • Marker assisted selection is applicable when
interact among themselves and form the phenotype. heritability of the target trait is low because the
production traits of economic importance are
The genetic makeup of an individual is called a identified not by its phenotype but by the genetic
genotype. The genes are made up of a sequence markers. Traditional selection methods fail to
of nucleotides which varies between individuals. significantly improve traits that are having low
The alleles which are end product of nucleotide heritability values. If the additive genetic variance
differences may affect the amino acid sequence component of the total variance explained by
of the proteins there by creating either additions the genetic marker exceeds the heritability of
or deletions and finally altering the phenotype of the character, then selection using the genetic
an individual. The individual animals harboring marker loci is more efficient than selection on the
alleles that codes for quality proteins are selected individual phenotype. But on the other hand, if
to reproduce and propagate their germplasm to the the heritability of a trait is high, it becomes easier
next generation based on genetic markers linked to to select superior animals based on performance
these alleles. MAS necessitate the need for known or phenotypic records.
association between genetic markers and phenotype • Marker assisted selection can also be applied for
considered for selection. MAS have the potential to sex limited traits. In traditional selection methods,
accelerate the genetic potential by identifying animals artificial selection can be applied only in sex where
carrying desirable alleles for a given trait at an earlier the trait is expressed. But when genetic markers
age. MAS could also be practiced at the embryo are used, the molecular score so generated can be
stage in animals, thereby helping the breeder from used for selection of opposite sex also wherein the
maintaining the animals for a longer duration. trait is not expressed.
• Marker assisted selection can be used for selection
Genetic markers of immature animals. Traditional selection methods
based on phenotype can’t be practiced for traits
The genetic markers are varying lengths of DNA like age at sexual maturity, age at first spawning,
sequences which helps to identify genes or short number of eggs spawned etc. until animal attains
regions on a chromosomal DNA having a major sexual maturity. The genetic markers can be applied
effect on trait of a population of animals. The for selection of these traits even before the animal
genetic markers are tightly linked to a gene of expresses the above said traits.
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interest and tags for a particular gene. The genetic • Marker assisted selection can be practiced for
markers are very useful in breeding programmes traits which are very difficult to measure like feed
as it helps the breeders in screening the segment conversion efficiency or disease resistance.
of chromosomes inherited between generations. If • Marker assisted selection can be used for traits
a particular segment contains a gene responsible that cannot be measured during the lifespan of
for variation between animals, then that segment the animal.
could be used for association studies between the • Marker assisted selection can be practiced for species
particular segment received by the animal and its that is not fully domesticated. The situation is very
performance. If a particular genetic marker has no specific to aquaculture/mariculture species that
direct role in effecting the performance of a trait, are very difficult to domesticate. In this situation, if
then it can be used to mark a chromosome segment genetic markers are available for desired traits, MAS
harboring gene(s) which has a major effect on the could be practiced in every generation to decide the
performance trait. These segments are called a parents carrying beneficial alleles.

136
Marker assisted selection strategy and automate them for improving genomic
The MAS strategy of animals depends on the analysis. The high polymorphic genetic markers
following factors. also reveal the genetic variation existing in
• Precise identification of breeding objectives. quantitative traits and help identify the genes
• The status and selection of genetic markers. that contribute to variation. Molecular markers
• Quantum of genetic markers to be used are also widely used in transgenesis and marker
for selection. assisted selection.
• Identification of QTL’s for the traits of interest.
• Genetic marker analysis. Types of Molecular Markers
• Relative efficiency of MAS to traditional selective
breeding programmes. Genetic marker can be defined as any stable
• Economic viability of MAS. and inherited variation that can be measured or
detected by a suitable method and can be used
Precise identification of subsequently to detect the presence of a specific
genotype or phenotype other than itself, which
breeding objectives
otherwise is non-measurable or very difficult to
The identification of breeding objectives depends detect. The markers revealing variations at the
upon the nature of traits, genetic variation present level of DNA are called as molecular markers.
in the traits, number of traits to be chosen for the Molecular markers are usually repetitive sequences
selection process and accuracy of selection process. found in DNA, and the interval of occurrence of
With the help of MAS, single locus affecting a these repetitive sequences varies from individual
quantitative trait could be identified using genetic to individual giving rise to polymorphisms. The
markers so as to increase the genetic progress that macromolecules like proteins, deoxyribonucleic

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

could be achieved. acid and biochemical constituents constitute
different classes of molecular markers.
Status and selection of
Based on their method of detection, they are
genetic markers
classified as hybridization based and PCR based
Number of genetic markers is available for different markers. The analysis of hybridization-based
species of animals. The strategy should be to select markers includes the traditional RFLP analysis and
the suitable marker according to our selection DNA fingerprinting. The PCR based markers include
programmes. The strategy in selecting a marker is Cleaved Amplified Polymorphic Sequence (CAPS),
based on the assumption that a linkage disequilibrium Random Amplified Polymorphic DNA (RAPD) and
exists between the genetic marker and QTL and there Microsatellites. Depending on the size of motif, they
should be a tight linkage between genetic markers can be called as minisatellites comprising of 10 to
and QTL. 60 base pairs or microsatellites composed of 1 to
5 base pairs. Allele Specific Oligonucleotide (ASO)
Molecular Markers and Single nucleotide polymorphisms form a new
group of markers.
The advent of molecular markers over the last
2 decades has revolutionized the research in Markers are of two different kinds, direct and
biological sciences and offered the opportunities indirect. Specific loci or markers may be part of
of using the genomic variation of major genes for the gene and directly influence a quantitative trait.
the genetic improvement of livestock. DNA based The indirect marker loci has no direct effect on
molecular markers are becoming versatile tools the character of interest and could be utilized in
and are of great importance in various fields like selection by means of its associations or linkage
embryology, physiology, genetic engineering and disequilibria between alleles at the marker loci and
are constantly modified to increase their utility quantitative trait loci

137
 Genetic markers used in MAS identifying the QTL depends upon the utilization of F2
and back crossed animals obtained from the parents
Marker Nature of Type of Reference
marker Marker with divergence for the traits of interest. The resources
required for identification of QTL are polymorphic
Restriction Fragment Co-dominant I & II Botstein et al.,
Length Polymorphism 1980 DNA markers, high resolution linkage maps and
(RFLP) mapping populations that are segregating for the
Random Amplified Dominant II Welsh and polymorphic markers. The principles involved in the
Polymorphic DNA McClelland 1990; process of QTL mapping and MAS are as follows.
(RAPD) Williams et al.,
1990
Amplified Fragment Dominant II Vos et al., 1995 Linkage disequilibrium
Length Polymorphism
(AFLP)
Linkage disequilibria between loci are produced by
Microsatellites or Co-dominant II > I Tautz, 1989 three factors, hybridization, random genetic drift
Simple Sequence
Repeats (SSR)
and epistatic selection. In a large population created
by hybridization between genetically differentiated
groups, after T generations of random mating,
substantial linkage disequilibria are likely to be
Quantum of genetic markers maintained between selectively neutral loci with
to be used for selection recombination rates, r < 1/T. In a randomly mating
population of effective size Ne, genetic drift is
The number of genetic markers needed for selection expected to produce substantial associations between
depends upon the length of the marker and length polymorphic loci with recombination rates r < 1/
of the genome (measured in centimorgan cM units). (4Ne). In domesticated populations, except those
A cM is1/100th of a morgan (1cM = 1/100M = 106 of very small size, the number of generations since
bp, basic unit of linkage map) and is equivalent to the last hybridization event usually will be smaller
106 bp. Since there exists variability in the length of than four times the effective population size i.e. T
the genome and marker present on the genome, < 4Ne. Therefore, hybridization is more powerful
a number of evenly spaced markers should be mechanism for generating useful linkage disequilibria
considered for the selection programme. Lande and than random genetic drift.
Thompson (1990) gave a simple formula to arrive at
minimum number of molecular markers (N) required A major limitation with these associations is that the
for the likely detection of associations with important linkage disequilibrium diminishes over generations
QTLs which is calculated as, by recombination during meiosis. Therefore it is
mandatory to identify such indirect markers that are
N = 2TL + C tightly linked to QTL or closely placed along with QTL
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on the chromosome so that the association is not

Where, T = Number of generations since last disturbed by recombination.
hybridization event in the population
Linkage maps
L = Map length in Morgans
C = Haploid chromosome number A linkage map of a species has molecular markers
placed in an order along the length of the
Identification of QTLs for chromosomes in the genome of that species. Linkage
map indicates the position and relative genetic
the traits of interest distances between markers along the chromosomes.
The most important step in framing the strategy for The crossing over is a random event and hence
MAS of animals is identifying as many QTLs as possible has equal probability of occurring at any position
with regard to our trait of interest. The success in along the length of the paired chromosomes during

138
meiosis. Therefore, the markers that are closely A mapping population can be divided into different
placed are less likely to be separated during meiosis genotypic groups on the basis of the genotype at
than distantly placed markers. Further, the frequency the marker locus. A significant difference between
with which the markers are separated during meiosis phenotypic means of the groups for the trait of
(recombination frequency) is a direct estimate of interest indicates that either marker itself is the QTL
the distance between markers. Recombination for the trait of interest or marker is linked to the
frequencies for different pairs of markers could QTL controlling the trait of interest. It should be
be utilized to construct a map showing relative noted that QTLs can only be detected for traits that
distances between markers on the chromosome. segregate between the parents used to construct
Recombination frequencies between any pair the mapping population.
of markers range from 0 % (complete linkage)
to 50 % (no linkage). The three different methods used in identifying QTLs
are single marker analysis, simple interval mapping
The genotype information of polymorphic markers and composite interval mapping. Single marker
in mapping is subjected to linkage analysis for the considers single marker locus at a time. Simple
construction of linkage map. Popular software interval mapping considers a pair of adjacently
packages used for construction of linkage maps in placed linked markers at a time and try to find out
aquaculture species include CARTHAGENE, CRIMAP, whether QTL exists or not in the interval between
JOINMAP, LINKMFEX, MAPCHART, MAPINSECT 1.0 marker pair. Composite interval mapping combines
(http://www.dpw.wau.nl/pv/pub/), MAPMAKER simple interval mapping with linear regression and
and MAPMANAGER. Linkage between markers is considers additional genetic markers along with
calculated as odds ratio i.e. the ratio of linkage adjacent pair of linked markers.
versus no linkage and is expressed as logarithm of

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

odds (LOD) value or LOD score. Generally a pair of Markers identified should be validated for their
markers with a LOD score of >3 would be utilized reliability/repeatability in different environments in
for the construction of linkage map. A LOD score predicting the phenotype in different populations
of 3 between markers indicates that presence of to avoid false positive associations between
linkage between markers is 1000 times more likely markers and the trait of interest. Markers that
than no linkage. could not be validated can’t be used for marker
assisted breeding program.
Large family sizes are required for detecting QTL
of small effects. When the QTL explains 10 percent Genetic marker analysis
of the phenotypic variance in the target trait, the
optimum family size appears to be 50 individuals The following points are to be considered before
per family for a QTL mapping experiment in implementing the MAS such as identification of
outbred populations. When the total population bred specific genetic marker, linkage status of QTL
size (individuals from all families) reaches 1000 with the marker, estimation of gene frequency in
individuals, family sizes of 25/50/100 give similar different generations and number of generation taken
power of detecting a QTL that is explaining 10 % to identify the animals with QTL. Thus 2 strategies
of phenotypic variance. exist for the MAS namely marker linked with QTL and
marker unlinked with QTL.
The basic principle involved in QTL mapping is
detecting an association between phenotype of the Relative efficiency of MAS
trait and genotype of markers. Phenotypic records are
to traditional selective
available for the individuals in mapping population
for the trait of interest. A polymorphic marker locus breeding programmes:
has 2 or more alleles in the population, though The MAS should be evaluated for its efficiency in
only 2 alleles are present in any diploid individual. order to compare its performance with conventional

139
 selective breeding programmes. MAS is highly found to be associated with complex quantitative
effective for traits with low heritability, sex limited traits. The quantitative traits are under the control
traits, immature traits and population size. of several genes (polygenes) before it interacts
with an environment and gets expressed as a
Economic viability of MAS phenotype. Therefore, breeding values estimated
using phenotypic records for the trait should be
The viability of MAS depends upon its cost effectiveness. utilized for making selection decisions as they
Even though the cost involved in genotyping several consider all the genes that contribute to a given trait
locus per animal per generation is enormous, the and marker assisted selection only complements
amount spent on maintaining he animal till later and cannot act as replacement for traditional
age is relatively higher when compared to screening selection methods.
the animals at an early age. This in turn helps the
breeder to select the animals at young age and reduce Conclusions
the amount spent on feeding and maintaining the
animals till it attains maturity and then select the Most of the economically important traits are
animal for its performance. Marker assisted selection quantitative, polygenic and influenced by environment
is advantageous compared to conventional breeding there by making genetic improvement of these traits
methods as phenotypic screening is expensive, difficulty more laborious and time consuming. The traits with
encountered in detecting presence of multiple alleles low heritability such disease resistances which are
related to a single trait based on phenotype and difficult to measure are important potential targets for
selection for alleles of desirable traits at an early age MAS. Marker assisted selection is likely to accelerate
even before the trait is expressed. genetic progress in some traits better than others.
Hence, target traits for MAS should be selected wisely.
Relevance of MAS
Marker assisted breeding should not be considered as
to traditional replacement for traditional selection methods. MAS
breeding programmes only complement the selection process based on
phenotypic records. The traits with high heritability can
Marker assisted selection allows for the accurate be selected based on breeding values or phenotypic
selection of animals with specific DNA markers performance. The decision on selecting an animal

Table 1. List of QTLs identified in aquaculture species.

Species QTL for traits QTL detecting method Reference
Tilapia
Central Marine Fisheries Research Institute

O. mossambicus X O. aureus Cold tolerance & fish size ANOVA, regression interval mapping Cnaani et al., 2003
Arctic charr
Salvelinus alpinus Body weight, condition factor, age Single marker and interval mapping Moghadam et al., 2007
at sexual maturation
Kuruma shrimp
Marsupenaeus japonicus Growth traits Interval and composite interval mapping Li et al., 2006
Rainbow trout
Oncorhynchus mykiss Resistance to IPNV c2 test and interval mapping Ozaki et al., 2001
Oncorhynchus mykiss Meristic traits Composite interval mapping Nichols et al., 2004
European seabass
Dicentrarchus labrax Morphometric traits Half-sib interval mapping Chatziplis et al., 2007
Rainbow trout
Oncorhynchus mykiss Pyloric caeca number Composite interval mapping Zimmerman et al., 2005
Tilapia
O. mossambicus X O. aureus Stress response, body weight & Genome scan using ANOVA Cnaani et al., 2004
sex determination

140
 Breese, E.L. and Mather, K. 1957. The organisation of polygenic
should take into consideration both the breeding activity within a chromosome in Drosophila. I. Hair characters.
value and marker information rather either alone. Heredity, 11: 373-395.
Chatziplis, D., Batargias, C., Tsigenopoulos, C.S., Magoulas, A.,
The weightage to be given to the marker information Kollias, S., Kotoulas, G., Volckaert, F.A.M. and Haley, C.S.
during the process of selection poses a big challenge 2007. Mapping quantitative trait loci in European sea bass
to the breeder. (Dicentrarchus labrax): The BASSMAP pilot study. Aquaculture,
272S1: S172-S182.
Chistiakov, D.A., Hellemans, B., Haley, C.S., Law, A.S., Tsigenopoulos,
In case of aquaculture species, the quantum of C.S., Kotoulas, G., Bertotto, D., Libertini, A. and Volckaert, F.A.
2005. A microsatellite linkage map of the european sea bass
information available on markers and QTLs are very Dicentrarchus labrax L. Genetics, 170: 1821-1826.
less indicating the need for more genetic improvement Chourrout, D. 1984. Pressure-induced retention of second polar
programs. The construction of high resolution linkage body and suppression of first cleavage in rainbow trout:
production of all-triploids, all-tetraploids, and heterozygous
maps, QTL detection programs is the need of hour. and homozygous diploid gynogenetics. Aquaculture, 36: 111-
The whole process of identifying QTLs has become 126.
Cnaani, A., Hallerman, E.M., Ron, M., Weller, J.I., Indelman, M.,
easy due to technological advancements. The breeder Kashi, Y., Gall, G.A.E. and Hulata, G. 2003. Detection of a
should consider the incorporation of QTL information chromosomal region with two quantitative trait loci, affecting
while framing genetic improvement programs. cold tolerance and fish size in an F2 tilapia hybrid. Aquaculture,
223: 117-128.
Cnaani, A., Zilberman, N., Tinman, S., Hulata, G. and Ron, M. 2004.
Even though there is a strong belief on the influence Genome-scan analysis for quantitative trait loci in an F2 tilapia
hybrid. Mol. Gen. Genomics, 272: 162-172.
of QTLs for the phenotypic expression of economically Coimbra, M.R.M., Kabayashi, K., Koretsugu, S., Hasegawa, O.,
important metric traits, its impact on the breeding Ohara, E., Ozaki, S., Sakamoto, T., Naruse, K. and Okamoto,
programs and commercial applications in plants, N. 2003. A genetic map of the Japanese flounder Paralichthys
olivaceus. Aquaculture, 220: 203-218.
livestock, forestry and fishery is yet to be proven. The Collard, B.C.Y., Jahufer, M.Z.Z., Brouwer, J.B. and Pang, E.C.K.
breeder should rationalize the genetic gain made by 2005. An introduction to markers, quantitative trait loci (QTL)
mapping and marker-assisted selection for crop improvement:
MAS and compare it with the conventional breeding

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

The basic concepts. Euphytica, 142: 169-196.
methods with regard to its cost effectiveness and Danzmann R.G and Gharbi, K. 2001. Gene mapping in fishes: a
economic returns. The traditional breeding methods means to an end. Genetica, 111: 3-23.
Darvasi, A., Weinreb, A., Minke, V., Weller, J.I. and Soller, M. 1993.
still holds good for improving several traits. Though Detecting marker-QTL linkage and estimating QTL gene effect
the animals harbor better genes for production traits, and map location using a saturated genetic map. Genetics,
134: 943-951.
the interaction between the environment and the Dekkers, J.C.M. and Hospital, F. 2002. The use of molecular genetics
genotype plays a major role in the final performance in the improvement of agricultural populations. Nature Reviews
of the animal. Therefore, efficient management of Genetics, 3: 22-32.
Dong, S., Kong, J., Meng, X., Zhang, O., Zhang, T. and Wang, R.
animals is must in framing genetic improvement 2008. Microsatellite DNA markers associated with resistance
programs for selection of superior animals. to WSSV in Penaeus (Fenneropenaeus) chinensis. Aquaculture,
282: 138-141.
Du, Z.Q., Ciobanu, D.C., Onteru, S.K., Gorbach, D., Mileham, M.J.,
Suggested readings Jaramillo, G. and Rothschild, M.F. 2009. A gene-based SNP
linkage map for pacific white shrimp, Litopenaeus vannamei.
Animal Genetics, 41: 286-294.
Adams, M.D., Kelley, J.M., Gocayne, J.D., Dubnick, M., Elcio P. Guimarães., John Ruane., Beate D. Scherf., Andrea Sonnino.
Polymeropoulou, M.H., Xiao, H., Nerrid, C.R., Wa, A.B., and James D. Dargie. 2007. Marker Assisted Selection. Current
Moreno, R.F., Kerlavage, A.R., Mc Combie, W.R. and Ventor, status and future perspectives in crops, livestock, forestry and
J.C. 1991. Complimentary DNA sequencing, expressed fish. Food and Agricultural Organization of UN.
sequence tags and human genome project. Science, Garcia, D.K. and Alcivar-warren, A. 2007. Characterization of 35
252: 1651-1656. new microsatellite genetic markers for the Pacific white leg
Alcivar-Warren, A., Song, L., Meehan-meola, D., Xu, Z., Xiang, shrimp, Litopenaeus vannamei: Their usefulness for studying
J. and Warren, W. 2007. Characterization and mapping of genetic diversity of wild and cultured stocks, tracing pedigree
expressed sequence tags isolated from a subtracted Cdna library in breeding programs and linkage mapping. Journal of Shellfish
of Litopenaeus vannamei injected with white spot syndrome Research, 26(4): 1203-1216.
virus. Journal of Shellfish Research, 26(4): 1247-1258. Georges, M., Lesquare, S., Hansel, R. and Vassart, G. 1987. Genetic
Beattie, K.L. 1998. DNA markers, protocols, applications and variation of the bovine thyroglobulin gene studied at DNA
reviews (G. Cetano-Anolles and P.M. Gressoff, eds.). Wiley, level. Animal Genetics, 18: 411-450.
New York, pp.213-224. Givry S.D., Bouchez, M., Chabrier, P., Milan, D. and Schiex, T.
Botstein, D., White, R.L., Skolnick, M. and Davis, R.W. 1980. 2005. CARTHAGENE: multipopulation integrated genetic and
Construction of a genetic linkage map in man using restriction radiation hybrid mapping. Bioinformatics, 21: 1703-1704.
fragment length polymorphisms. American Journal of Human Glenn, K.L., Grapes, L., Suwanasopee, T., Harris, D. L., Li, Y., Wilson,
Genetics, 32: 314-331. K. and Rothschild, M.F. 2005. SNP analysis of AMY2 and CTSL

141
 genes in Litopenaeus vannamei and Penaeus monodon shrimp. Liu, B. 1998. Statistical Genomics: Linkage, Mapping and QTL
Animal Genetics, 36: 235-236. Analysis CRC Press, Boca Raton.
Green P.K., Fall, K. and Crooks, S. 1990. Documentation for CRIMAP, Liu, Z. 2007. Aquaculture genome technologies. Blackwell
version 2.4. publishing Limited, UK.
Haldane, J.B.S. 1919. The combination of linkage values, and the Maneeruttanarungroj, C., Pongsomboon, S.,
calculation of distances between the loci of linked factors. Wuthisuthimethavee, S., Klinbunga, S., Wilson, K.J.,
Journal of Genetics, 8: 299-309. Swan, J., Li, Y., Whan, V., Chu, K.H., Li, C.P., Tong, J.,
Hartl, D. and Jones, E. 2001. Genetics: Analysis of Genes and Glenn, K., Rothschild, M., Jerry, D. and Tassanakajon, A.
Genomes, Jones and Bartlett Publishers, Sudbury, MA. 2006. Development of polymorphic expressed sequence
Hayes, B.J., Gjuvsland, A. and Omholt, S. 2006. Power of QTL tag-derived microsatellites for the extension of the genetic
mapping experiments in commercial Atlantic salmon linkage map of the black tiger shrimp (Penaeus monodon).
populations, exploiting linkage and linkage disequilibrium and Animal Genetics, 37: 363-368.
effect of limited recombination in males. Heredity, 97: 4-18. Manly, K.F. and Olson, J.M. 1999. Overview of QTL mapping
Hemmer-Hansen, J., Eg Nielsen, E., Meldrup, D. and Mittelholzer, software and introduction to Map Manager QT. Mammalian
C. 2011. Identification of single nucleotide polymorphisms in Genome, 10: 327-334.
candidate genes for growth and reproduction in a nonmodel Manly, K.F., Cudmore, R.H. and Meer, J.M. 2001. Map manager
organism; the Atlantic cod, Gadus morhua. Molecular Ecology QTX, cross-platform software for genetic mapping. Mammalian
Resources, 11 (suppl. 1): 71–80. Genome, 12: 930-932.
Hill, W.G. and Robertson, A. 1968. Linkage disequilibrium in finite Martinez, V., Hill, W.G. and Knott, S.A. 2002. On the use of double
populations. Theor. Appl. Genet., 38: 226-231. haploids for detecting QTL in outbred populations. Heredity,
Hubert, S. and Hedgecock, D. 2004. Linkage maps of microsatellite 88: 423-431.
DNA markers for the Pacific oyster Crassostrea gigas. Genetics, Massault, C., Bovenhuis, H., Haley, C. and Koning, D. 2008. QTL
168: 351-362. mapping designs for aquaculture. Aquaculture, 285: 23-29.
Jeffreys, A.J., Wilson, V. and Thein, S.L. 1985. Hypervariable Meehan, D., Xu, Z., Zuniga, G. and Alcivar-Warren, A.
minisatellites regions in human DNA. Nature, 314: 67-73. 2003. High frequency and large number of polymorphic
Jeffreys, A.J., Wilson, V., Thein, S.L., Weatherall, D.J. and Ponder, microsatellites in cultured shrimp, Penaeus (Litopenaeus)
B.A.J. 1986. DNA fingerprints and segregation analysis of vannamei [Crustacea:Decapoda]. Marine Biotechnology,
multiple markers in human pedigrees. American Journal of 5: 311-330.
Human Genetics, 39: 11-24. Melchinger, A.E. 1990. Use of molecular markers in breeding for
Kearsey, M. and Pooni, H. 1996. The genetical analysis of oligogenic disease resistance. Plant Breeding, 104:1-19.
quantitative traits. Chapman & Hall, London. Mitra, A. 1994. Studies on restriction fragment length
Kimura, M. and Ohta, T. 1971. Theoretical aspects of Population polymorphism in cattle and buffaloes. Ph.D. Thesis, National
Genetics. Princeton University Press, Princeton, N.J. Dairy Research Institute (Deemed University), Karnal, India.
Kolbehdari, D., Jansen, G.B., Schaeffer, L.R. and Allen, B.O. 2005. Moen, T., Hoyheim, B., Munck, H. and Gomez-Raya, L. 2004.
Power of QTL detection by either fixed or random models in A linkage map of Atlantic salmon (Salmo salar) reveals an
half-sib designs. Genet. Sel. Evol., 37: 601-614. uncommonly large difference in recombination rate between
Kosambi, D.D. 1944. The estimation of map distances from the sexes. Animal Genetics, 35: 81-92.
recombination values. Annals of Eugenics, 12: 172-175. Moghadam, H.K., Poissant, J., Fotherby, H., Haidle, L., Ferguson,
Kwok, P.Y., Carson, C., Yager, T.D., Ankener, W. and Nickerson, M.M. and Danzmann, R.G. 2007. Quantitative trait loci for
D.A. 1994. Comparative analysis of human DNA variations by body weight, condition factor and age at sexual maturation
fluorescence-based sequencing of PCR products. Genomics, in Arctic charr (Salvelinus alpinus):comparative analysis with
23: 138-144. rainbow trout (Oncorhynchus mykiss) and Atlantic salmon
Lande, R. and Thompson, R. 1990. Efficiency of marker-assisted (Salmo salar). Mol. Genet. Genomics, 277: 647-661.
selection in the improvement of quantitative traits. Genetics, Mukherjee, K. and Mandal, N. 2009. A Microsatellite DNA marker
124: 743-756. developed for identifying disease-resistant population of giant
Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daly, M.J., black tiger shrimp, Penaeus monodon. Journal of the World
Lincoln, S.J. and Newburg, L. 1987. MAPMAKER: an interactive Aquaculture Society, 40(2): 274-280.
computer package for constructing primary genetic linkage Nichols, K.M., Wheeler, P.A. and Thorgaard, G.H. 2004. Quantitative
maps of experimental and natural populations. Genomics, trait loci analyses for meristic traits in Oncorhynchus mykiss.
Central Marine Fisheries Research Institute

1: 174- 181. Environmental Biology of Fishes, 69: 317-331.

Lander, E. and Botstein, D. 1989. Mapping Mendelian factors O’Brien, S.J. 1991. Molecular genome mapping: lessons and
underlying quantitative traits using RFLP linkage maps. prospects. Curr. Opin. Genet. Dev., 1: 105-111.
Genetics, 121: 185-199. Ozaki, A., Sakamoto, T., Khoo, S., Nakamura, K., Coimbra, M.R.M.,
Li, Y., Byrne, K., Miggiano, E., Whan, V., Moore, S., Keys, S., Crocos, Akutsu, T. and Okamoto, N. 2001. Quantitative trait loci (QTLs)
P., Preston, N. and Lehnert, S. 2003. Genetic mapping of associated with resistance/susceptibility to infectious pancreatic
the kuruma prawn Penaeus japonicus using AFLP markers. necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss).
Aquaculture, 219: 143-156. Mol. Genet. Genomics, 265: 23-31.
Li, Y., Dierens, L., Byrne, K., Miggiano, E., Lehnert, S., Preston, N. Paterson, A.H. 1996. Making genetic maps. In: A.H. Paterson (Ed.),
and Lyons, R. 2006. QTL detection of production traits for the Genome Mapping in Plants, pp. 23-39. R. G. Landes Company,
Kuruma prawn Penaeus japonicus (Bate) using AFLP markers. San Diego, California; Academic Press, Austin, Texas.
Aquaculture, 258: 198-210. Perez, F., Erazo, C., Zhinaula, M., Volckaert, F. and Calderon, J.
Li, Z., Li, J., Wang, Q., He, Y. and Liu, P. 2006. AFLP-based genetic 2004. A sex-specific linkage map of the white shrimp Penaeus
linkage map of marine shrimp Penaeus (Fenneropenaeus) (Litopenaeus) vannamei based on AFLP markers. Aquaculture,
chinensis. Aquaculture, 261: 463-472. 242: 105-118.
Litt, M. and Lutty, J.A. 1989. A hypervariable microsatellite revealed Perry, G.M.L., Danzmann, R.G., Fergusson, M.M. and Gibson,
by in vitro amplification of a dinucleotide repeat within the J.P. 2001. Quantitative trait loci for upper thermal tolerance
cardiac muscle actin gene. American Journal of Human in outbred strains of rainbow trout (Oncorhynchus mykiss).
Genetics, 44: 397 – 401 Heredity, 86: 333-341.

142
Prasertlux, S., Khamnamtong, B., Chumtong, P., Klinbunga, S. Van Ooijen, J.W. and Voorrips, R.E. 2001. JoinMap 3.0, Software
and Menasveta, P. 2010. Expression levels of RuvBL2 during for the Calculation of Genetic Linkage Maps. Plant Research
ovarian development and association between its single International, Wageningen, The Netherlands.
nucleotide polymorphism (SNP) and growth of the giant Verhoeven, K.J., Jannink, J.L. and McIntyre, L.M. 2006. Using
tiger shrimp, Penaeus monodon. Aquaculture, 308: S83- mating designs to uncover QTL and the genetic architecture
S90. of complex traits. Heredity, 96: 139-149.
Risch, N. 1992. Genetic linkage: Interpreting LOD scores. Science, Visscher, P., Thompson, R. and Haley, C. 1996. Confidence intervals
255: 803-804. in QTL mapping by bootstrapping. Genetics, 143: 1013-1020.
Smith, C. 1967. Improvement of metric traits through specific Voorrips, R.E. 2001. MapChart Version 2.0: Windows Software
genetic loci. Anim. Prod., 9: 349-358. for the Graphical Presentation of Linkage Maps and QTLs.
Soller, M. 1978. The use of loci associated with quantitative effects Plant Research International, Wageningen, The Netherlands.
in dairy cattle improvement. Anim. Prod., 27: 133-139. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee, T., Hornes,
Sorensen, N. and Robertson, A. 1961. The association between M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. and Zabeay, M.
blood groups and several production characters in three Danish 1995. AFLP: a new technique for DNA fingerprinting. Nucleic
cattle breeds. Acta Agric. Scand., 11: 163-196. Acids Research, 23: 4407-4414.
Spickett, S.G. and Thoday, J.M. 1966. Regular responses to Welsh, J. and McClelland, M. 1990. Fingerprinting genomes using
selection. 3. Interaction between located polygenes. Genet. PCR with arbitrary primers. Nucleic Acids Research, 18: 7213-
Res., 7: 96-121. 7218.
Staelens, J., Rombaut, D., Vercauteren, I., Argue, B., Benzie, J. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.K. and
and Vuylsteke, M. 2008. High-Density Linkage Maps and Sex- Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary
Linked Markers for the Black Tiger Shrimp (Penaeus monodon). primers are useful as genetic markers. Nucleic Acids Research,
Genetics, 179: 917-925. 18: 6531-6535.
Stam, P. 1993. Construction of integrated genetic linkage maps Wilson, K., Li, Y., Whan, V., Lehnert, S., Byrne, K., Moore, S.,
by means of a new computer package: JoinMap. Plant J., Pongsomboon, S., Tassanakajon, Rosenberg, G., Ballment,
3: 739-744. E., Fayazi, Z., Swan, J., Kenway, M. and Benzie, J. 2002.
Stanley, J.G. 1976. Production of hybrid androgenetic and Genetic mapping of the black tiger shrimp Penaeus monodon
gynogenetic grass carp and carp. Transactions of the American with amplified fragment length polymorphism. Aquaculture,
Fisheries Society, 105: 10-16. 204: 297-309.
Sturtevant, A.H. 1913. The linear arrangement of six sex-linked Woram, R.A., McGowan, C., Stout, J.A., Gharbi, K., Ferguson,
factors in Drosophila as shown by mode of association. J. M.M., Hoyheim, B., Sakamoto, J., Davidson, W., Rexroad, C.
Exp. Zool., 14: 39-45. and Danzmann, R.G. 2004. A genetic linkage map for Arctic
Tanksley, S.D. 1993. Mapping polygenes. Annu. Rev. Genet., char (Salvelinus alpinus): evidence for higher recombination

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

27: 205-233. rates and segregation distortion in hybrid versus pure strain
Tassanakajon, A., Klinbunga, S., Paunglarp, N., Rimphanitchayakit, mapping parents. Genome, 47: 304-315.
V., Udomkit, A., Jitrapakdee, S., Sritunyalucksana, K., Wuthisuthimethavee, S., Lumubol, P., Toojinda, T., Tragoonrung, S.
Phongdara, A., Pongsomboon, S., Supungul, P., Tang, S., and Vanavichit, A. 2005. SSLP-based Linkage Map Construction
Kuphanumart, K., Pichyangkura, R. and Lursinsap, C. 2006. in Black Tiger Shrimp (Penaeus monodon Fabricius).
Penaeus monodon gene discovery project: The generation ScienceAsia, 31: 91-97.
of an EST collection and establishment of a database. Gene, Mei, Y., Cheng, Y. and Rothschild, M. F. 2006. SNP analysis of
384: 104-112. molting related genes in Penaeus monodon and Litopenaeus
Tautz, D. 1989. Hypervariability of simple sequences as a general vannamei shrimp (brief report). Arch. Tierz. Dummerstorf,
source for polymorphic DNA markers. Nucl. Acids Res., 49(4): 411-412.
17: 6563-6571. Zhang, L., Yang, C., Zhang, Y., Li, L., Zhang, X., Zhang, Q. and
Tian, Y., Kong, J. and Wang, W.J. 2008. Construction of AFLP-based Xiang, J. 2007. A genetic linkage map of Pacific white shrimp
genetic linkage maps for the Chinese shrimp Fenneropaeneus (Litopenaeus vannamei): sex-linked microsatellite markers and
chinensis. Chinese Science Bulletin, 53(8): 1205-1216. high recombination rates. Genetica, 131: 37-49.
Togashi, K., Lin, C.Y. and Yokouchi, K. 2004. Overview of genetic Zimmerman, A.M., Wheeler, P.A., Ristow, S.S. and Thorgaard, G.H.
evaluation in dairy cattle. Animal Science Journal, 75: 275-284. 2005. Composite interval mapping reveals three QTL associated
Tong, J., Lehnert, S.A., Byrne, K., Kwan, H.S. and Chu, K.H. 2002. with pyloric caeca number in rainbow trout, Oncorhynchus
Development of polymorphic EST markers in Penaeus monodon: mykiss. Aquaculture, 247: 85-95.
applications in penaeid genetics. Aquaculture, 208: 69-79.

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 Cryopreservation of fish spermatozoa
and its Application in Aquaculture and
Conservation
V. S. Basheer* and A. Gopalakrishnan
Principal scientist
PMFGR Centre, NBFGR, CMFRI campus, Kochi-682 018
e-mail: [email protected]

Introduction 1985), but probably much longer. In India, NBFGR

is the primary organization in India carrying out fish
Cryopreservation is a branch of cryobiology which sperm cryopreservation for long term gene banking.
relates to the long-term preservation and storage of
biological material at very low temperature, usually at Cryopreservation
-196ºC, the temperature of liquid nitrogen. It is based
on the principle that very low temperatures immobilize
of spermatozoa
the physiological and biochemical activities of cells, The first success in preserving fish sperm at low
thereby making it possible to keep them viable for very temperature was reported by Blaxter (1953) who
long period in a state known as suspended animation. fertilized herring (Clupea harengus) eggs with frozen
Cryopreservation of fish sperm is one method – thawed semen. Major efforts have been made
successfully adopted from animal husbandry by the during the past 20 years to effectively freeze salmonid
aquaculture industry. Cryopreservation overcomes the sperm (Stoss, 1983) and now sperm from several
problem of males maturing before females, allows species of fish have been frozen. The spermatozoa
selective breeding and stock improvement and enables of several economically important species have been
the conservation of genomes. The basic technique of cryopreserved in the recent past which includes
cryopreservation involves collection of fish gametes in rainbow trout, Atlantic salmon, Tilapias, several
which specific diluent (extender) with cryoprotectant cyprinids including Common, Chinese and Indian
(such as dimethyl sulfoxide, glycerol, ethylene glycol carps. Some economically important marine species
and methanol) is added. After a period of specific of for which spermatozoa have been frozen include
equilibration, it is frozen rapidly and stored in liquid Asian sea bass, Atlantic halibut, milk fish, black
Central Marine Fisheries Research Institute

nitrogen. After thawing, the milt can be activated for porgy, bluefin tuna, various catfishes, Indian marine
use in fertilizing the eggs. In teleosts, spermatozoa fishes. Majority of the works on cryopreservation of
are held within the testis in an immotile state by the fish semen concerns cultivated species or species
chemical composition of seminal plasma. Under natural of commercial interest. Considering the needs to
conditions, motility is initiated when the semen or milt establish gene-banks for endangered fish species and
is diluted with water on release during spawning. to avoid loss of genetic variability – there is still a need
During cryopreservation, the sperms are to be retained for investigation in the field of sperm preservation.
in inactive condition and it is the extender that keeps
the sperm in live but in immotile state so that ideal Extenders
dilution of cryoprotectant can be made to mix with the
milt. Cryopreservation allows virtually indefinite storage Undiluted gametes are not suitable for freezing and they
of biological material without deterioration over a time must be diluted with a suitable extender. An extender is
scale of at least several thousands of years (Mazur, a solution consisting of inorganic and organic chemicals

144
resembling that of blood or seminal plasma in which the only after release into the external medium for a short
viability of spermatozoa can be maintained during in period of motility. The initiation of motility of fish sperm
vitro storage. Extender also helps to reduce the toxicity is essentially a dilution effect upon expulsion of semen
of the cryoprotectant used in cryopreservation. The into the surrounding water. Spermatozoa motility in
efficacy of cryopreservation is greatly enhanced if the fish is usually estimated by an arbitrary scale of intensity
prefrozen milt is diluted with a suitable extender. Various ranging from 0 to 5, by the duration of motility for
extenders, containing KCl, NaCl, glucose, sodium citrate, a given intensity, or by a combination of these two
Ringer’s solution, cow serum and milk fish serum were parameters. Sperm motility is one of the most important
used to preserve fish sperm in liquid Nirtrogen (-196ºC). parameters of sperm quality and is usually expressed in
The sperm of the Indian carp, Labeo rohita were first duration of sperm movement and percentage of motile
preserved in liquid nitrogen using extender comprised sperm immediately after activation.
of NaCl – 730 mg, NaHCO3 – 500 mg, Fructose – 500
mg, Vegetable lecithin – 750 mg, Mannitol – 500 mg, Procedure of cryopreservation
Distilled water – 100 ml.
Milt of the fish has to be collected in a dry and clean
Cryoprotectant box and to be kept on ice till it used. The extender has
to be prepared afresh every time and the cryoprotectant
Cryoprotectants are added to extenders to minimize (@10% of the extender) to be mixed just before filling the
the stress on cells during cooling and freezing. Glycerol, straw. The milt extender ration should be kept 1:3. Just
DMSO and methanol are the most widely used after mixing the milt with the extender, cryoprotectant
cryoprotectants for preserving teleost spermatozoa. to be added and then straws to be filled. and sealed
The optimum concentraºtion used for cryopreservation by poly vinyl alcohol (PVA) powder and keep on ice
may vary species to species. However, the degree of for 10 minutes for bringing down the temperature to

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

success has been achieved with 10-15% glycerol, 0ºC. After that straws should be transferred to liquid
DMSO or methanol for many species. The optimum nitrogen vapour phase (~90ºC) for 10 minutes and
cryoprotectant concentration for a given protocol may then to be immersed in liquid nitrogen for achieving
also depend on the equilibration period that is the time the desired temperature (-196ºC). The filled straw then
allowed for cryoprotectant penetration into cells. There can be stored in liquid nitrogen. The motility of the
are two kinds of cryoprotectants namely permeating cryopreserved milt can be checked after thawing straws
and non-permeating cryoprotectants. The permeating using an activator solution. After confirming the motility
cryoprotectants include DMSO, glycerol, methanol, of the cryopreserved milt, it can be used for fertility
ethylene glycol etc. The non-permeating cryoprotectant experiments. Once fertility trials become successful,
includes egg yolk, milk and some other protein then cryopreservation of milt is complete. From the
(Soyabean protein, Promine D and bovine serum experiments one can find out best combination of
album). A non-permeating cryoprotectant is often extender and cryoprotectant and this can be used for
used in conjunction with a permeating cryoprotectant. long term cryopreservation fish milt.

Dilution ratios Technical problems in fish sperm

cryopreservation
Various milt to diluent ratios have been tested. It is
1:1 to 1:9 in salmonids, 1:3 in rainbow trout and 1:20 The fish sperm cells are small, have no acrosome
in O. niloticus. In Indian cyprinids and catfishes, 1:3 and are available in enormous quantities is semen
and 1:4 ratios were found to be ideal. for experimentation. Hence, recovering motile, fertile
frozen thawed fish spermatozoa is not a problem.
Motility Yet, the published information reveals difficulties
in developing a reliable and reproducible protocol.
The spermatozoa of most teleost fish species are The problem is in the enormous diversity of fish
immotile in testes and the genital tract and are activated spermatozoan physiology. Although all the species

145
 studied are external fertilizers, the behaviour of the Sperm from males in the wild can be cryopreserved
adults at spawning and the conditions in which sperm for the optimal mature season of hatchery stock so
and egg must meet are very different. The two extremes that genes from the fish in the field can be transferred
are represented by salmonids spawning in freshwater, into the hatchery population. Cryopreservation of fish
where sperm is deposited as close as possible to the sperms can be used to guarantee the specific fish
eggs and swim for 30 seconds at most and marine stocks and to ensure against the loss of specific genes
species like herring where spermatozoa are released in caused by natural, over-fishing or pollution causing
a diffuse cloud over the spawning grounds and remain disasters. It can be used in the hatchery management
motile for hours. In species where spermatozoa swim to solve the problem of having a disproportion
for long periods in nature, cryopreservation is generally between the males and the females. Induction of
easier. Tilapias are a good example. They can spawn in gynogenesis becomes highly feasible if the irradiated
fresh to full seawater and their spermatozoa will swim sperms are cryopreserved in advance. Functions of
for hours in a saline solution. The real challenge facing sperms and gene bank can be further facilitated by
cryobiologists interested in freezing fish spermatozoa utilising this technique.
is not recovering enough viable sperm cells to fertilize
small numbers of eggs in the laboratory but in making In this aspect, cryopreservation of gametes, germ
the technique practical in the field. The cryopreservation cells and embryos are useful for genetic resource
strategy and technique used at NBFGR is simple, does conservation and also for genetic improvement in
not need any electrically operated equipment. This aquaculture. Globally, milt cryopreservation has
provides easy adaptability, customization in difficult been successfully accomplished for more than 200
remote locations and successful application has been fish species with production of normal and viable
proved in species of various taxonomic groups. offsprings mainly, salmon, trout, tilapia, sturgeon,
Chinese and Indian major carps. Attempts have
Constraint in milt cryopreservation of fishes is the need also been made to cryopreserve milt in more than
for development of species-specific protocol, unlike 40 marine fish species of aquaculture importance
higher animals. Limited success in cryopreserving fish mainly, grouper, cod, haddock, sea bream, sea bass,
oocyte and embryo is due to their multi-compartmental eel, striped bass, flounder etc. Successful preservation
biological systems, big issues such as chilling sensitivity, of spermatophores in crustaceans was also
low membrane permeability and their large size developed in Macrobrachium rosenbergii, lobsters;
which require extensive screening of cryoprotectants, crabs, and shrimp Penaeus monodon. In India, milt
studies on tolerance to chilling, determination of the cryopreservation protocols have been developed in
appropriate rate of freezing and rate of thawing. Partial more than 30 species, mainly carps and catfishes.
success achieved in cryopreservation of pluripotent
blastomeres and embryonic cells in few fish species, Conclusion
however, cryotoxicity and chilling sensitivity are still
Central Marine Fisheries Research Institute

major problems. Storage of fish spermatozoa, eggs and embryos

without loss of viability is of considerable value
Application of in aquaculture and conservation. The fish sperm
cryopreservation needs development of species-
cryopreservation specific protocols. Such protocols are developed
of spermatozoa through experimental standardization of various
parameters, after the captive breeding protocol
Cryopreservation of fish sperm can be successfully is developed. This becomes a bottleneck due to
used as a fishery management tool. It provides some protracted breeding season and low domestication
means of control relative to mating and reproduction of most of the aquatic species, especially marine
including artificial propagation with individuals of fishes. Nevertheless, in all such cases, time available
same species from the distant locations or of different in a year for conducting experiment is small and
mature season or developing a profitable new hybrid. determined by breeding cycle of the species. In view

146
of the constraint, it is essential that candidate species provide a method of retaining specific genetic lines of
for sperm cryopreservation are prioritized. In artificial fish without the expense of maintaining brood stock
propagation, sperm cryopreservation protocol can populations and would provide a secondary source
be an asset where such milt related problems exist. of a genetic line in case of brood stock loss or would
Cryopreserved sperm was also used to retrieve the allow the preservation of endangered genetic line in
whole species and clones. Sperm retrieved from the wild populations.
fish, stored at -18ºC has been used to produce
androgenetic fish and interspecific androgenetic Fish milt cryopreservation efforts may be prioritized
cloning, as a mode for restoration of species. Though on endangered, commercially important and
milt cryopreservation is successful in many fish species endemic species from various aquatic habitats
fish gamete cryopreservation still faces an important and by establishing cryobanks. For large-scale
challenge in the form of long-term storage of use of cryopreserved fish sperm in aquaculture
finfish eggs and embryos. Owing to large size, large programmes, uninterrupted supply of liquid
amount of yolk and tough chorion or zona radiata nitrogen in areas where aquaculture activities are
with a low permeability coefficient, egg and embryo centred is a pre-requisite. Sensitive and reliable
cryopreservation of teleosts and crustacea have not tools for assessment of frozen-thawed milt quantity,
met with success anywhere in the world so far. The physiological changes during long-time storage, DNA
fundamental problem of sufficient dehydration during damage during cryopreservation and characterizing
cooling due to the relatively large size (1-6 mm) of fish cryoinjury especially in sperm DNA are also essential.
eggs and the presence of membrane of different water Standardization of cryopreservation protocol for
permeability has not been overcome. Cryopreservation pluripotent fish blastomeres needs to be researched
or long term storage of fish eggs or embryos would in detail for providing optimal and uniform results.
be beneficial to the aquaculture industry. It would

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

147
 Cytogenetics and its applications in fishes
Basdeo Kushwaha*, Ravindra Kumar and N. S. Nagpure
Principal Scientist
ICAR-National Bureau of Fish Genetic Resources
Canal Ring Road, PO: Dilkusha, Lucknow-22602
email : [email protected]

In brief, fish cytogenetics may be defined as   a part when the specimen is very large and from freshly dead
of genetic study that concerned with the study of the fish especially under field conditions. the detailed
structure and function of the chromosomes including process of chromosome preparation from fish is as
analysis chromosomes, utilizing the G banding and described below.
or other cytogenetic banding techniques, as well
as fluorescent in situ hybridization (FISH).  Chromosome preparation
The cytogenetic study involved good quality metaphase
chromosomes preparation from any tissue that
A. Detail procedure for
contains good number of dividing cells at metaphase chromosome preparation from
stage by arresting them with colchicine spindle fibber kidney / gill tissues of fishes:
inhibiter at an extremely low concentration. Followed
by hypotonic treatment, fixation, and dropping the cells • Collect healthy fish specimens (preferably weighing
onto microscope slides for chromosome spreading. 20-50 g).
The chromosomal staining is necessary because the • Inject 0.05% colchicine intramuscularly @ 1 ml
colourless chromosomes are difficult to distinguish per 100 g of body weight.
from equally colourless cytoplasm. The chromosome • Allow specimens to swim for 1- 2 h in a bucket
staining is performed with Giemsa. The active after injection.
components of Giemsa are eosin Y and methylene • Anaesthetize fish specimen with ethylene glycol,
blue, which form a magenta compound after binding dissect out the kidney / gill tissues in a Petri dish,
with DNA. Giemsa has no specificity for any particular and cut into small pieces.
base in DNA and in normal circumstances, it stains • Homogenize tissues in 6-8 ml hypotonic solution
chromosomes uniformly. The chromosomes are (0.56% KCl) in glass tissue grinder to make
observed after staining under the microscope. A 10-X cell suspension.
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objective is generally required to screen the slide to find • Pour the cell suspension in 15 ml centrifuge tube
the metaphase plates and for photography, 100-X oil and keep it at room temperature for 20-25 min
immersion objective is used. for cell swelling.
• Stop the hypotonic action KCL by adding 1-2 ml
In case of fishes mitotic chromosomes usually studied freshly prepared chilled Carnoy’s fixative (Methanol:
from the organelles that has rapidly dividing cells (in Acetic acid in 3:1 ratio) gradually. Mix it gently with
vivo) such as kidney and gills which are good source of pasture pipette
dividing cells and the cells are arrested at metaphase • Centrifuge cell suspension at 1200-1500 rpm for
stage using colchicine. The tissues, which do not divide 10 min at room temperature to get cell pellet at
rapidly like peripheral blood cells are cultured in-situ the bottom.
to induce division followed by in-situ colchicinisation. • Remove supernatant with a pipette leaving
Chromosomes can also be prepared by giving approximately 2-3 ml of supernatant. Add 6-8 ml
colchicine treatment to the tissue in-situ especially freshly prepared chilled fixative slowly.

148
• Keep the tube in refrigerator for 1-2 h for
in 8 ml of RPMI 1640 culture medium.
thorough fixation.
• Add 50 µl of 0.05% of colchicine.
• Mix the contents and centrifuge cell suspension at • Incubate the cell suspension in BOD incubator
1200-1500 rpm for 10 min at room temperature. for 30-50 min at 27-32ºC.
• Remove the supernatant without disturbing cell • Centrifuge the cell suspension at 1200 rpm for
pellet at the bottom and add fresh fixative. 10 min. decant the supernatant.
• Repeat steps 12 -13 three times, till clear • Add 8 ml hypotonic solution (0.56% KCl) to the
transparent cell suspension is obtained. cell pellet.
• Take small quantity of cell suspension in a pasture • Incubate the cells in this solution for 20-25 min
pipette and drop it onto grease free, pre-cleaned at room temperature.
glass slide from 1.5-2.0 feet height. • Follow the steps 7-26 described in ‘A’
• Allow the slide to air / flame dry.
• Keep the slide 1-3 days for ageing in dust free place. Analysis of chromosomes
• Stain it with 4-6% Giemsa in phosphate buffer (pH
6.8) for 15-20 min. The chromosomes are observed under microscope
• Wash with DD water thoroughly. after staining with suitable dye under the
• Air-dry and store the slides in a slide box. microscope. A 10-X objective is generally required
• Observe metaphase spreads in bright field to screen the slide to find the metaphase plates,
microscope to ascertain the quality of staining. which are often scarce among a large number of
• Make the slides permanent by mounting with non-dividing nuclei. For photography, 100-X oil
DPX mountant. immersion objective is used and very good quality
• Screen the slides for good spreads and take images/ photographs are required for measurements
photographs of metaphase spreads under oil and further analysis. Ideally, metaphase spread

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

immersion objective (100X). should not contain any overlapping chromosomes.
• For karyotype preparation, cut individual chromosomes In case of overlaps, more images of the metaphase
from the photo prints. Group the chromosomes into will be needed to prepare the Karyotype.
four categories–metacentric (m), sub metacentric (sm),
sub telocentric (st) and telocentric (t). A chromosome in mitotic metaphase has two
• Paste the chromosomes on ivory sheet in decreasing distinguishing features: its length and a transverse
order of size within the group (align centromeres constriction that marks the position of the
of all chromosomes in each row). centromere. From the chromosome length and
• Photograph the karyotype, which can be centromere position, three parameters are calculated:
used as base line data for detection of the centromeric index, the arm ratio and the relative
chromosome aberrations. length. The first two factors, i.e. centromere index
• Karyotype can be also made from digital images of and arm ratio, describe about the chromosome itself
chromosomes acquired from digital camera with and the relative length denotes about the size of
the help of suitable software. the chromosome in relation to other chromosomes
in the complement. Centromere index is defined as
Note: Unused cell suspension can be stored (in 2.5 ml length of shorter arm of the chromosome divided by
eppendorf vials) for further use in refrigerator up to the length of whole chromosome and multiplied by
six months and at -20ºC for years together without 100. Arm ratio is defined as the length of longer arm
marked deterioration in quality. of the chromosome divided by the length of shorter
one. It is, therefore, always ≥1. The Relative length
B. In-vitro of a chromosome is defined as the length of the
particular chromosome divided by the total length of
chromosome preparation:
all the chromosomes in the haploid set including the
• Take kidney tissue from live/ freshly dead fish. one being measured and multiplied by 100.
• Homogenize the tissue to prepare cell suspension

149
 The first step for characterization of a complete (50%) solution and developing solution (2% Gelatin
chromosome set is by karyotyping. The karyotype + 1.0% Formic acid) are prepared. Before staining,
of a chromosome complement is prepared from the the slide is dried at 40ºC for 4-5 h or overnight at
cells exhibiting the complete somatic chromosome room temperature. Place 8 drops of AgNO3 and 4
number and characteristic chromosome morphology. drops of developing solution on a slide and mix the
The homologous chromosomes were paired based on two solutions thoroughly and cover with a cover slip.
their length, morphology and position of centromere. Incubate the slide at 50ºC for 3-5 min, till the solution
The chromosome pairs were arranged in decreasing become golden yellow in color. Rinse the slide with
order of morphology and size in the karyotype. In the double distilled water (DDW) and air-dry the slide.
fishes, the chromosome classification on the basis of Mount the slide with DPX mountant and observe the
arm ratio, as proposed by Levan et al., (1964), has chromosomes under microscope. The pattern of NOR
been widely used, which is described below: for the species is usually determined by studying a
minimum of 20 metaphase spreads per specimen.

Table 1. Chromosome Classification by Arm Ratio Chromosome staining with

Centromeric Arm Ratio Chromosome type/ Chromomycin A3 (CMA3):
position Symbol
Median 1.0-1.7 Metacentric (m) For CMA3 staining with DAPI, the mixture solution
Sub-median 1.71-3.00 Submetacentric (sm)
containing CMA3 (0.5 mg CMA3 in 1 ml DDW) and
buffer (Mcllvaine in DDW in 1:1 ratio, adjust pH at
Sub-terminal 3.01-7.00 Subtelocentric (st)
7, added MgCl2 to make the 2.5 mM conc. of MgCl2)
Terminal > 7.00 Telocentric (t)
in 1:9 ratio is placed properly on the aged slide and
Once the karyotype is prepared, a diagrammatic covered with cover slip. Keep the slide in dark for 1
representation of the chromosome (ideogram) h and then wash in DDW. Place the DAPI mixture (2
can be prepared on the basis of relative length mg/ ml of DAPI in 1 ml of Mcllvaine buffer, adjust pH
of chromosomes. at 7) on the slide and cover with cover slip and keep
the slide in dark for 30 minute. Wash the slide with
Chromosome banding Mcllvaine buffer and add antifade solution followed
by covering with cover slip. The slide is observed under
Karyotyping using conventional ‘Geimsa’ staining of microscope in the dark and bands are determined in
chromosomes does not give greater insight into the each species by studying a minimum of 20 metaphase
structure and organization of chromosomes. Further, spreads per specimen per species.
in many fish species the chromosomal morphology
is more or less similar making it difficult to identify C-banding:
and pair the homologous chromosomes. Unlike
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humans G- banding is not successful due to poor The C-bands are the regions of constitutive
compartmentalization of genome into AT-& CG- heterochromatin, which are predominantly contains
rich isochors, however, NOR staining, Chromomycin transcriptionally inactive, highly repetitive DNA
A3 C-banding has been widely used for genetic sequences. The use of C-banding techniques in fish
characterization of fishes. These techniques have been has made it possible to correctly identify homologous
optimized in our laboratory and the protocol of the chromosomes by revealing the distribution of
same is given below. constitutive heterochromatin and also to determine
its role in karyotype evolution and speciation.
Silver nitrate staining (AgNO3):
Constitutive heterochromatin banding is performed
Staining of Nucleolar Organizer Regions (NORs) is using Barium hydroxide-Saline-Giemsa (BSG)
performed according to the method of Howell and technique of Sumner (1990). The aged air-dried slide
Black (1980) with minor modifications. For this, AgNO3 is treated with 0.2N HCl for 1 h at room temperature

150
followed by rinsing with DDW. The slide is placed in cytogenetic techniques like fluorescence in situ
coplin jar containing 5% aqueous solution of barium hybridization (FISH), Primed in-situ labeling, Fiber-FISH
hydroxide at 50ºC for 35 min followed by rinsing with
DDW. The slide is then incubated for 1 h at 60ºC in 2X Comparative genome
SSC (0.3M Sodium chloride; 0.03M Sodium citrate)
followed by a rinse with DDW. The slide is then stained
hybridization and
with 4-5% Giemsa in Phosphate buffer (pH 6.8) for Spectral karyotyping.
30 min. The slide is then dried and observed under
microscope. C-band pattern is determined in each Fluorescence in-situ
species by studying a minimum of 20 metaphase hybridization (FISH):
spreads per specimen per species.
The FISH technique is based on the discovery that
Molecular cytogenetics: labeled ribosomal DNA hybridizes to acrocentric
chromosomes. It involves a fluorescently labeled DNA
Molecular cytogenetic techniques focus on specific probe being hybridized to genomic DNA sequences,
chromosomes, chromosome regions, and unique DNA and can be used to physically map the specific site
sequences or genes. In general, it involves the use of a on a chromosome. Initially, radioactive isotopes
series of techniques referred to as in situ hybridization were used for this technique, however, the use of
(ISH) that has given researchers a powerful look into fluorochrome is safer that requires a shorter reaction
the cell and its genetic content. The principle behind time and can give rise to different colors.
ISH is the specific annealing of a labeled nucleic acid
probe to complementary sequences in fixed cells/ a. Chromosome preparation:
tissues, followed by visualization of one or more

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

specific regions of the genome. The ISH technique Metaphase chromosome spreads are prepared using
was initially introduced by Gall & Pardue in 1969 to colchicine to arrest the dividing cells during mitosis.
localize nucleic acids in individual cells. During the Hypotonic solution (0.075M potassium chloride) and
time, the in situ hybridization tool was restricted to fixative (methanol and acetic acid in 3:1 ratio) are
highly repetitive DNA sequences using radioactive then applied sequentially for cell preparation. Finally,
labeled probes visualized by autoradiography. The uses the chromosome suspension is dropped onto glass
of radioisotopes had many disadvantages and were slides and chemical aging of slide in 2X SSC is done.
replaced by non-radioactive detection methods. In Before hybridization, metaphase chromosomes and
1982, Langer–Safer group at Yale University published interphase nuclei are pretreated enzymatically (i.e.
a non-radioactive procedure that was convenient and 0.005% pepsin) to enhance their accessibility to the
polynucleotide specific. The most commonly used probe and to reduce the amount of cytoplasm.
reporter molecules are haptens, such as biotin and
digoxigenin, which can be incorporated easily in the b. Probe labeling:
probe DNA. The tagged probes are then detected with
labeled antibodies against the specific tag or with a For probe labeling, pure DNA preparation is required
labeled avidin molecule, as in the case of biotin. The and a fluorochrome must be incorporated into the
biotin and digoxigenin have partly been replaced by DNA probe. DNA probes can be labeled by enzymatic
directly fluorochrome-conjugated nucleotides that procedures such as nick translation, random priming
simplified the laboratory protocol, but the basic or by the polymerase chain reaction (PCR). For nick
principle of FISH has essentially remained the same. translation, 3’-5’exonuclease and 5’-3’ polymerase
activity of DNA Polymerase I is exploited to cause
Fluorescent labeled probes allow researchers to a single strand nick and subsequently, a specific
selectively label a single gene, entire chromosomes or fluorescently labeled nucleotide is incorporated
whole genome and then visualize it using fluorescence into the nicked strand using non-nicked strand as a
microscopy. Here, we are discussing few molecular template. Feinberg and Vogelstein in 1983 introduced

151
 probe labeling by random priming which depends on e. FISH microscopy:
the ability of the Klenow fragment of DNA polymerase
to copy single-stranded DNA templates primed FISH signals are visualized by fluorescence microscopy
with random hexa-nucleotide mixtures. Labeling by using a light source that illuminates the fluorescently
PCR involves a standard PCR reaction with labeled labeled probe. A variety of specific filter sets are
nucleotide in addition to unlabeled nucleotides. used to separate the different fluorochromes and
to visualize the fluorescence signal of the probe.
c. Hybridization and Detection: Digital imaging systems, such as a CCD camera, to
capture the image and quantify fluorescent signals
Following the pre-treatments, hybridization is carried are needed. Resultant images are analyzed on
out under optimal conditions for the annealing of commercially available systems (e.g. from Vysis, Leica
probe to the specific target nucleic acid. The previously Microsystems or Applied Spectral Imaging).
prepared probe and cot-1 or salmon sperm DNA are
mixed, denatured and applied to the preheated slides Types of FISH probe
for hybridization. Hybridization between probe and
target DNA takes place during an incubation period
of ~16–48 h at 37ºC. This incubation time can vary
a. Gene-specific probes:
depending on the probes used. Gene-specific probes target specific nucleic acid
sequences within the chromosome. Examples of such
Detection of the probe permits the visualization of probes include bacterial artificial chromosome (BAC)
target DNA sequences. Detection starts with post- and yeast artificial chromosome (YAC) probes and
hybridization washes of the slide in formamide and cosmids. Gene-specific probes are useful for mapping
SSC (sodium chloride, sodium citrate) salt solutions genes on chromosomes. These probes have proven
to remove any excess probe that is non-specifically useful particularly in the study of micro deletion,
bound. However, a detection step is not required for all syndromes, where the absence of a gene often goes
DNA probes. In a direct-label reaction, a fluorochrome undetected by conventional banding methods.
conjugated nucleotide [e.g. 2’-deoxyuridine
5’- triphosphate (dUTP) conjugated to fluorescein- b. Repetitive-sequence probes:
isothiocyanate (FITC)] is incorporated into the strands.
In an indirect-label reaction, reporter molecules such Repetitive-sequence probes bind to regions that are
as biotin or digoxigenin are incorporated into the rich in repetitive base-pair sequences. Examples of
DNA. Such indirect labels require a detection step such probes include centromeric and telomeric probes.
in which the reporter molecule or hapten is labeled Centromeres frequently contain AT- rich tandem
with an agent such as avidin or antidigoxin that is repeats, whereas telomeres are recognized by the
conjugated to a fluorochrome. The detection methods short repetitive sequence, i.e. TTAGGG. Centromeric
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for biotinylated probes employ avidin–fluorochrome probes have applications in the identification of
conjugates, whereas those for digoxigenin-labeled marker chromosomes and numerical chromosome
probes employ antidigoxin–fluorochrome conjugates. abnormalities in interphase nuclei and when specimens
are sex mismatched. Telomeric and subtelomere
d. Chromosome counterstaining: specific probes are commonly used to identify cryptic
chromosomal translocations such as those occurring
In addition to immunochemistry of signal detection, in cases of unknown mental retardation.
the DNA of the chromosomes or nucleoli must be
counterstained for visualization. Typical fluorochromes c. Chromosome painting probe:
used in conjunction with FISH include Hoescht 33258,
DAPI and Propidium Iodide (PI). It may be necessary Chromosome-painting probes contain sequences
to vary the concentration of counter-stain in order to that are specific to either a single chromosome (i.e.
optimize signal detection. whole-chromosome-painting probe) or an arm of a

152
chromosome (i.e. chromosome arm-painting probe). an invaluable technique for studying chromosomal
After hybridization, one or more chromosomes of aberrations that occur in solid tumors and other
interest are ‘lit up’ in different colors, which are malignancies. A major advantage of the CGH
dependent on type of particular fluorochromes technique is that only DNA from the tumor samples
used. This technique is particularly useful for is needed for analysis, which avoids the often-difficult
identifying chromosome arms that are involved in preparation of tumor metaphase chromosomes that
translocations, as well as for marker chromosomes may have poor morphology and resolution. Instead,
and ring chromosomes. karyotypically normal metaphase chromosomes
are used to detect tumor associated chromosomal
d. Whole-genomic DNA probe: gains and losses. Another advantage of CGH is that
formalin-fixed tissue sections can be used.
Whole-genomic DNA probes are used for the FISH-
based technique CGH. They can be used to detect Spectral Karyotyping
genomic imbalances in tumor genomes by combining
tumor and normal DNA to analyze gains and losses. Spectral karyotyping (SKY) is a FISH-based molecular
cytogenetic technique that allows color karyotyping
Primed in-situ Hybridization of two species chromosomes. Whereas FISH limits
analysis to specific chromosomes or regions of
Primed in-situ Hybridization (PRINS) technique is based chromosomes, and CGH visualizes only those changes
on in-situ synthesis of non-radio isotopic hybridization that result in variations in copy number. The SKY
probe. Unlabeled synthetic oligonucleotide primers permits the visualization of all chromosomes at
anneal to fixed chromosome preparation in sequence one time, ‘painting’ each pair of chromosomes in
specific fashion. This DNA then serve as a primer for different fluorescent color. Before the development

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

chain elongation in-situ, catalyzed by DNA polymerase of this technique, molecular cytogeneticists analyzed
I. Fluorescently labeled nucleotides are used as chromosomes with staining techniques that produced
a substrate for chain elongation. This procedure a black-and-white banding pattern. However, the
has been used for cytogenetic localization of high identification of all chromosomal aberrations in a
copy number, chromosome-specific, tandem repeat complex karyotype was often not possible from such
sequence including centromeric, telocentric and patterns. SKY has been highly effective in deciphering
satellite sequences. many complex karyotypic rearrangements.

Fiber-FISH Application of FISH in

fish genetics:
High- molecular weight genomic DNA or individual
DNA molecules from large-insert DNA clones can be The probes for centromeric repetitive DNAs are
spread on glass slides for FISH analysis. DNA prepared developed and the repeats have been mapped to
from bacterial artificial chromosome (BAC) or tissues chromosomes of some of the fish species, namely
extend approximately 2.5-3.5 kb/ µm on slides. zebrafish, puffer fish, tilapia, rainbow trout, lake
Thus, the fiber-FISH method provides fine-mapping trout and other chars. Most of the fish genomes have
resolution of up to a few kilo-bases. additional families of tandemly repetitive sequences
that do not map to the centromeres but are localized
Comparative in heterochromatic blocks in the genome. In zebrafish,
there is a family of GC-rich repetitive DNA found
genomic hybridization at paracentric location on the long arms of about
Comparative genomic hybridization (CGH) involves one third of the chromosome pairs. In puffer fish,
the differential labeling of test and reference DNA to a 10 bp repeat is found at short arms of most of
measure genetic imbalances in entire genome. It is the subtelocentric chromosomes. In atlantic salmon,
based on quantitative two-colour FISH. It has become three minisatellite sequences have been mapped using

153
 FISH technique. Probes of 18S and/ or 28S rDNA arm specific probes have been made for rainbow
have been used to localize the major ribosomal RNA trout. Another use of paint probes is to identify
cistron in many fish species. Probes of 5S rDNA also intraspecific deletions and translocations. The work
have been localized in zebrafish, puffer fish, atlantic on exploring the possible application of autosome-
salmon, brown trout, chinook, coho, rainbow specific chromosome paint probes is undertaken in
trout and Salvelinus spp. Probes specific to histone zebrafish. Species-specific probes are useful for quick
genes are mapped in Atlantic salmon, brown trout, identification of immature and related species and for
rainbow trout, Coregonus lavertus etc. In mice and detection of chromosomes in inter-specific diploid
humans, FISH experiments have shown that certain and triploid hybrids.
transposable elements accumulate preferentially on
the sex chromosomes, possibly as a result of reduced Application of cytogenetics
crossing over. CiLINE2 sequence is found preferentially
on chromosomes 1 in tilapia, and this has now been
in fishes
shown to be the sex chromosome pair. Cytogenetics is an exciting, dynamic field of study
which analyzes the number and structure of human
Mapping of polymorphic microsatellite markers and animals chromosomes. Changes that affect the
would be especially useful for fish genetics. Since number and/or structure of the chromosomes can
fish chromosomes are smaller than mammalian cause problems with growth, development, and how
chromosome, fewer polymorphic loci would the body functions. Chromosomal abnormalities can
need to be mapped to have informative markers happen when egg and sperm cells are being made,
flanking most genes of interest. Many phenotypic during early fetal development, or after birth in any
traits of importance to aquaculture such as disease cell in the body. Changes to chromosome structure
resistance, rapid growth, fat content of flesh, early can disrupt genes, causing the proteins made from
or delayed maturity etc. may be the result of one or disrupted genes to be missing or faulty. Depending
two major genes. The genes controlling these traits on size, location, and timing, structural changes in
could be mapped using informative microsatellite chromosomes can lead to genetic defects, syndromes,
loci. Once the chromosomal regions are identified, some chromosomal changes may have no effect on
the genes could be isolated by positional cloning individual’s performance/ health. Cytogenetic analyses
or by micro dissection. are commonly performed to determine if an individual
is at risk for common aneuplodies (syndromes caused
To localize single-copy genes on chromosomes, clones by having extra or missing chromosomes), syndromes
from large insert libraries such as cosmids, PACs, caused by structural abnormalities (like unbalanced
BACs or YACs are usually required. In platy-fish and translocations or inversions), or to determine if
medaka, cosmid clones containing sex linked single extra or missing genetic material is present through
copy genes were used as probes in FISH to identify cytogenetic microarray testing. In genetic research,
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the sex chromosomes. Moreover, sex-chromosome chromosome analysis of embryonic stem cells and
specific paint probes have been prepared for lake induced pluripotent stem cells to ensure the genetic
trout and tilapia. Both Yp and Yq probes exist for integrity of these cell lines.
lake trout. Several chromosome or chromosome

154
Molecular Taxonomy
Reynold Peter
Research Associate
Marine Biotechnology Division, CMFRI
e-mail: [email protected]

The field of biology that deals with the theory and transcribed sequences, ESTs, RAPDs are now being
practice of classification of organism is called as used. The choice of these markers for particular
taxonomy. The term Taxonomy was proposed by de applications is not straightforward one and is often
Candolle in 1813 which means law of arrangements based on the prior experience of the investigators. It is
(taxis- arrangement or order; nomos- law). It is the also important to keep in mind that there is no perfect
science of biological classification. It consists of three method and the choice of a particular technique is
separate but interrelated parts i.e. 1) classification, often a compromise that depends on several reasons,
in which we arrange organisms into groups or taxa including: the resources of the laboratory, financial
based on their mutual similarity or evolutionary constraints, available expertise, time limitations and,
relatedness, 2) Naming or nomenclature and 3) more importantly, the research question pursued.
identification which involves process of determining All points should be scrutinized carefully to avoid
a particular isolate belonging to a recognized taxon. an inappropriate choice. It has therefore become
crucial that researchers have a basic understanding

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Taxonomy helps us to organize huge amounts of of molecular tools. The molecular techniques now
knowledge about organisms because all members of have become less time consuming, molecular markers
a particular group share many characteristics. It places combined with new statistical developments enable
them in useful groups with precise names so that finding out differences and similarities between
researchers can work with them and communicate individuals, and the population of origin of single
efficiently. Initially morphological features formed fish, resulting in many new research possibilities
the basis of identification but then other features and applications.
were also taken into consideration for this purpose.
Study of taxonomic features allows the construction “Pre-DNA World” in
of databases that can be used for a further rapid
identification of organisms.
taxonomy research
Conventional method using the morphological
Advances in molecular techniques have helped to and anatomical features of an individual is still the
establish a new area in taxonomy research called most applied process in both identification and
molecular taxonomy, studying genetic relationship of taxonomy. Although useful in several cases, the use
different taxonomic categories using molecular markers. of anatomical characters for species identification
The information encrypted within these molecular data procedures has several disadvantages. First, there is
can dramatically improve the taxonomy studies. This a much morphological plasticity between organisms
made the taxonomists realize significance of molecular of the same species. The use of morphology is also
data and made them understand that other traditional complicated by the existence of sibling species
methods are although important but molecular which can lead to mistaken identifications if a few
evidences could be final or confirmatory evidences. morphological features are considered in the analysis.

Various molecular markers, mitochondrial DNA or Finally, most morphology-based approaches cannot
nuclear DNA markers such as microsatellites, internal be applied in cases where there is just a small amount

155
 of biological material available for examination. For and hybridization developed in the 1970s and DNA
instance, forensic laboratories often have to deal amplification and automated sequencing during 1980s
with low quantities of biological material, most of led to developing various classes of DNA markers. Later,
the times degraded after a prolonged exposition to with the advent of the PCR many different techniques
harsh environmental conditions. Besides a reliable emerged, ranging from sequencing of the DNA of
diagnostic procedure can be time-consuming and need interest to methods analyzing length polymorphisms,
expert taxonomist support. With the convergence of such as microsatellites.
new ideas from genetics and biochemistry and new
technological developments, the field of species DNA based tools used for
identification started to rely on information from the
molecular components of cell. The first molecular
Molecular taxonomy
methods successfully employed were based on the
analysis of proteins: protein electrophoresis, allozyme DNA hybridization:
analysis, immunological reactions, etc. Although it had
its own advantages, several features are known to limit The hybridisation of complementary DNA
the use of proteins: its rapid degradation in samples oligonucleotides is a basic principle of molecular
under stress conditions, the risk of cross-reactions with biology used in various methods with possible
proteins from closely related species, the differential applications in species identification. Some of the
expression of proteins in specific tissues etc. early assays were based on solid-phase hybridizations
conducted on nitrocellulose or nylon membranes
“Post–DNA World” in between whole genomic or synthetic DNA probes
of known origin and DNA extracted from the
taxonomy research target sample. The probe was usually labelled with
A molecular marker is a DNA sequence used to “mark” fluorescent or radioactive molecules. A positive
or track a particular location (locus) on a particular hybridisation signals the presence of biological
chromosome, i.e. marker gene. It is a gene with a material from the species used to make the probe.
known location or clear phenotypic expression that A widely known DNA hybridisation-based approach is
is detected by analytical methods or an identifiable the fluorescence in situ hybridisation (FISH) technique.
DNA sequence that helps the study of inheritance of This technique uses fluorescently labelled probes to
a trait or a gene. detect nucleic acid sequences in whole cells, allowing
the direct detection of organisms
Over the past years, application of molecular tools
has increased dramatically because of the advances Restriction Fragment Length
in PCR, DNA sequencing, data analysis and it has
Polymorphisms (RFLPs)
been possible to apply in several areas such as species
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identification, population genetics, brood stock The RFLP analysis is used for the detection of
development, fish health management, transgenic, interspecies variation at the DNA sequence level.
genetic diversity, conservation and genomics. Today, These restriction enzymes cleave the DNA molecule
there are two general classes of genetic markers at specific recognition sites resulting in a set of
that are routinely used in population genetic and fragments with different lengths that could be
phylogenetic studies: (1) nuclear DNA and (2) extra separated according to their molecular size by
nuclear DNA (mtDNA, cpDNA etc.) markers. conventional gel electrophoresis. The distinctive RFLP
profile of each species is the result of the unique
Major characteristic of the DNA molecule that makes it genomic distribution of recognition sites and the
a useful tool for molecular species identification are that distance between them. RFLP assays usually do
it is stable and can be easily isolated from minimum not require any sophisticated equipment and no
source material and can be stored efficiently for a very prior sequence information about the species. With
long-time. Methods for DNA cloning, sequencing the advent of the polymerase chain reaction (PCR)

156
technique RFLP analysis (known as PCR-RFLP) has Conventional PCR using species
become routinely used for species detection. Most specific primers
PCR-RFLP approaches focus on mtDNA cytochrome
b or ribosomal RNA (rRNA) genes. A major A conventional PCR based method consists of the
disadvantage of the RFLP technique is the possible design of PCR primers that will only produce an
existence of intraspecies mutations at restriction sites amplification product in the presence of DNA from
that can lead to false results due to the gain or loss the target species. The process of designing species-
of restriction fragments. specific primers is now straightforward due to the
vast number of genomic sequences available and
Amplified Fragment Length software programs that assists in primer designing.

Polymorphisms (AFLPs)
The specificity and sensitivity can be enhanced by
The AFLP method combines the reproducibility of performing a nested PCR, in which the target region
restriction fragment analysis with the power of PCR. is first amplified with an outer primer pair followed
It is based on the selective PCR amplification of by a second amplification using an internal primer
restriction fragments from a total digest of genomic pair. The chance of amplifying unspecific genomic
DNA. The method usually works by digesting a regions is reduced with nested PCR as compared
small amount of purified genomic DNA with two to conventional PCR since undesired sequences
or more restriction enzymes. Double-stranded amplified in the first round of PCR are not likely to
oligonucleotide adapters are ligated to the contain a sequence to which the primers for the
sticky ends of DNA fragments. The ligated DNA second amplification reaction will bind, increasing
fragments are then amplified by PCR using primers the specificity dramatically.
complementary to the adapter and restriction

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

site sequence. The AFLP technique allows the Real-time PCR
simultaneous screening of different loci randomly
scattered throughout the genome. However, it is Real-time PCR is used for the detection of a specific
technically demanding, labour consuming and DNA sequence in a sample by measuring the
interpreting results may need automated computer accumulation of amplified products during the PCR
analysis and still the results could produce non- using fluorescent technology. An important benefit
specific outcomes. of this method is the ability to quantify the starting
amount of a specific DNA sequence in the sample
Random Amplified Polymorphic (this approach is also known as quantitative PCR).

DNA (RAPD)
The real-time PCR has the advantage over conventional
RAPD profiles are generated by the random PCR PCR-based identification systems of working without
amplification of DNA segments using short primers of post- PCR handling, with a minimised risk of carryover
arbitrary nucleotide sequence of usually 10 nucleotides contamination in the laboratory. It also offers an
long. These primers hybridize with different genomic increased sensitivity by allowing discriminating
regions at low annealing temperatures. Each species false PCR amplifications from non-target DNAs and
is identified by a specific banding pattern in an is a relatively fast genotyping method, with some
electrophoretic gel or similar technique resulting platforms affording high throughput automation.
from the different genomic location of primer-
binding sites. The RAPD method does not require Sequencing of PCR products
prior sequence information for PCR primer design but
is extremely dependent on variations in laboratorial The DNA sequencing analysis is currently the most
conditions, needing carefully developed laboratory used method for molecular species identification.
protocols to be reproducible and often overlapping The advent of rapid and cost-effective PCR-linked
results leading to confusion. DNA sequence analysis has circumvented the need

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 for screening of genomic libraries and cloning of targeting of only a single DNA region could be
DNA fragments. The identification is achieved by problematic since a failure in the amplification of
comparing the sequence of a genomic region that region due to, for instance, the occurrence of
found in the target sample with a comprehensive a polymorphism in a primer binding region, may
reference database. Ideally, the structure of the DNA originate a false or null result. This problem can be
region to be analysed must consist of a variable overcome by using degenerate primers that pinpoint
sequence (informative enough to discriminate at polymorphic areas in the primer binding sites, in
species) flanked by highly conserved regions (ideal cases where these variants have been previously
to design universal PCR primers that amplify in a identified. Moreover, DNA sequencing methods do
large number of species). not allow the discrimination and identification of
biological material from different species mixed in
In order to attain a correct identification it is crucial a same sample, unless fragments are cloned before
to consult a reliable database, namely one that sequencing to separate each molecule of DNA.
guarantees that (a) the reference specimen was
correctly identified by a taxonomic expert or by other A solution to overcome some limitations of single-
molecular methods, (b) the same sequences were gene approaches is the use of multi gene sequence
obtained in independent studies, preferentially from analysis, a method based on the sequencing of
the full distribution range of the species and that (c) multiple protein-coding genes. A phylogenetic
most related species have distinct DNA profiles. A approach could be used to identify the species of
common way to assign a particular sequence to its origin of an individual by constructing a phylogenetic
species of origin is to perform a BLAST search on the tree with concatenated sequences of multiple genes.
vast GenBank sequence database. However, care must The phylogenetic inference can be established by
be taken when assigning the questioned sequence the clustering pattern of the species of interest with
to the species with the highest similarity, because related species. An obvious limitation to the use of
several gaps and false sequences are known to be this approach is the necessity of sequencing several
present in these databases. Moreover, this approach genomic regions, a fact that can be cumbersome
does not provide any information and can lead to with sophisticated labour, money and expensive labs.
false identifications if the target sample belongs to
a previously uncharacterized species. DNA microarrays or DNA chips
Although different regions have been targeted for DNA microarray consists of small glass microscope
species identification procedures, most studies rely on slides, silicon chips or nylon membranes with a large
sequence information from nuclear ribosomal RNA number of immobilized DNA fragments arranged in a
genes and mtDNA regions. The list of studies using regular pattern. A DNA microarray provides a medium
mtDNA cytochrome b gene for species identification for matching a reporter probe of known sequence
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is extensive. This gene shows a high level of similarity against the DNA extracted from the target sample
with species limits and can be amplified in several of unknown origin.
vertebrate species under standard conditions by using
a single pair of universal primers. A DNA microarray built with species-specific
DNA sequences can be used for identifications
DNA barcoding purposes. For instance, the DNA extracted from
the target sample could be labelled with a specific
A DNA barcoding system for all animal species has fluorescent molecule and hybridized to the
been proposed based on 650 to 750 bp of the mtDNA microarray DNA. A positive hybridization is detected
cytochrome c oxidase (COI) gene. The use of a single with appropriate fluorescence scanning/imaging
gene in delineating and identifying species and the equipment. Advances in printing technology have
extent of separation between intra- and interspecies enabled the production of microarrays containing
variations have been of a concern for many. The hundreds of thousands of probes revealing the

158
potential to achieve sensitive and high-throughput affecting the genetic composition of a population.
species identifications. DNA microarrays need To understand the patterns of natural genetic
specialized robotics and imaging equipment that diversity and basic genetic structure of isolated
are not available in most laboratories. Advanced populations within the species is a basic need for
bioinformatics tools are also necessary to reduce developing scientific management strategies for
the complex data into useful information. conservation. Mitochondrial DNA (mtDNA analysis),
microsatellites, allozymes, RFLP, RAPD and AFLP
Applications of markers are the popular genetic markers employed
in population genetic studies. The data generated
molecular taxonomy in employing these genetic markers are converted into
evolutionary studies numerical data matrix for using in different genetic
data analysis software / bioinformatics tools to
Analyzing and comparing the genetic material of estimate different population genetic parameters.
different species is an important method for studying Based on these analysis and observations, a
species evolution and also for identifying species/sub- conservation policy based on MUs (Management
species/strains/stocks etc. Molecular tools are used to Units) can be framed.
make comparisons between the numbers, locations
and sequences of genes in different organisms Suggested readings
which reveal the evolutionary relationship between
different species and also to identify the taxonomic Identification of species with DNA-based technology: current
position of new or unidentified species. Application progress and challenges. Recent Pat DNA Gene Seq.
2008; 2(3):187-99.
of bioinformatics tools are central and made these Okumus I, Çiftci Y (2003) Fish population genetics and molecular
tasks more simple and accurate. markers: II-molecular markers and their applications in fisheries

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

and aquaculture. Turk J Fish Aquat Sci 3:51–79

Applications of molecular
taxonomy in Population
genetics and conservation
of species
Population genetics is concerned with the
analysis of demographic and evolutionary factors

159
 An overview of the basic concepts and
principles of Population Genetics
N. S. Jeena
Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Population genetics is a branch of the evolutionary sequence, frequency of which is the proportion
biology that tries to determine the level and of chromosomes of that type in the population. A
distribution of genetic polymorphism in natural locus is monomorphic if there is only one allele
populations and also to detect the changes in in the population and polymorphic if there are
genetic composition that results from the operation two or more alleles in the population at appreciable
of evolutionary forces (mutation, migration, frequencies. At any genetic locus the maternal and
selection and drift). It was developed as a theoretical paternal alleles normally have identical or slightly
discipline by R. A. Fisher (a British statistician), J. B. different DNA sequences. If the two alleles are
S. Haldane (a British Geneticist) and Sewall Wright identical, they are homozygotes and if different
(an American geneticist). This discipline focuses termed as heterozygotes.
on the Mendelian population which is a group of
interbreeding individuals who share a common set Every individual has both a phenotype and a
of genes (called the gene pool), to understand the genotype. The genotype is the specific set of genes
genetics of evolutionary processes. An understanding carried by the individual. The phenotype is the set of
of the genetic structure of a population is the key characteristics (e.g., morphological, physiological,
to our understanding of the importance of the behavioral) expressed by the individual. The
genetic resources and the importance of genes for phenotype is produced by the genotype in interaction
the conservation of species and biodiversity. In this with environment. Genotypes are formed due to
chapter, basic concepts of population genetics are pairing of genes (alleles) during union of gametes for
discussed first in order to have a better understanding zygote formation. It is the set of alleles an organism
of the principles and applications that are detailed in carries at one or more loci. If there are `n` alternative
the preceding sessions. alleles, there will be n (n+1)/2 possible genotypes.
The genotypes of the parents are broken down
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Main ideas of and from the genes transmitted in the gametes a

new set of genotypes is constituted in the progeny.
population genetics Consequently, only alleles have continuity over time
Genetic variation describes differences between the while the genotypes do not, and the gene pool
DNA sequences of individual genomes. Each individual evolves through changes in the frequencies of alleles.
has two nuclear genomes (a paternal genome and
a maternal genome). A genetic locus is a DNA The fundamental quantities in population genetics
region having a unique chromosomal location. It is are frequencies of genes (alleles) and genotypes
a sequence of DNA that may or may not code for a by which the genetic structure of populations is
protein. A gene is defined as a sequence of DNA that expressed. A frequency is a proportion or a percent.
codes for a protein. It always ranges between 0 and 1. Genotypic
frequency is the number of individuals with one
Alleles are alternative forms of a particular particular genotype divided by the total number

160
of individuals in the population. This is done for random sampling of gametes from a population is
each of the genotype at the locus of interest. The called genetic drift or simply drift. It is also called
sum of the genotypic frequencies should be 1. The the Sewall Wright Effect in honor of the population
distribution of genes in different individuals of the geneticist who championed its importance in the
population is the allelic (gene) frequencies. The 1930’s. Genetic drift will cause isolated populations
gene pool can be described with fewer parameters to diverge from one another.
when allelic frequencies are used. Allelic frequency is
the ratio of number of copies of a given allele in a Movement of genes takes place only when organisms
population to sum of all alleles in the population. or gametes migrate and contribute their genes to the
Allelic frequencies may be calculated from observed gene pool of the recipient population. This process
numbers of different genotypes at a particular locus, is referred to as gene flow. Gene flow is a source
or from the genotypic frequencies. The frequencies of genetic variation which introduces new alleles to
of two alleles are commonly symbolized as p and the population and spreads unique alleles to other
q where q= 1 – p. A third allele is symbolized as r. populations, long-term effect of which is the opposite
of genetic drift. Through exchange of genes, different
For example, for the two alleles A1 & A2 in the populations remain similar, and thus migration is a
autosomal locus A, allelic frequency p & q respectively homogenizing force that tends to prevent populations
can be calculated as follows: form accumulating genetic differences among them.
p= (frequency of the A1A1 homozygote) + (½ x
frequency of the A1A2 heterozygote calculated as 2pq) Random mating or panmixia means that any
q = (frequency of the A2 A2 homozygote) + (½ x individual has an equal chance of mating with any
frequency of the A1 A2 heterozygote calculated other individual in the population. There should
as 2pq) be no special tendency for mated individuals to be

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

alike in genotype or to be related to each other by
There are several basic evolutionary processes that ancestry. Non-random mating occurs when mating
act on a population and cause genetic variation. The individuals are genetically related to one another or
first and the most fundamental is mutation which is are phenotypically similar to each other than two
defined as any heritable change in the genetic material. individuals chosen at random.
Mutation is the ultimate source of all genetic variation
without which evolution cannot be materialized. Population genetics deals with how the evolutionary
Recombination is the secondary source of genetic forces distribute the DNA polymorphism in biological
variation which can create new combination of alleles, populations. For evolution of a species to occur the
but not new alleles. New recombination of alleles can gene frequencies of that population must undergo
lead to new phenotypes upon which natural selection change and the evolutionary processes described
can act. Natural selection means that individuals with above play a major role in it.
heritable favorable variations survive and reproduce at
a higher rate than other individuals in the population. The Hardy-Weinberg law
It operates through differences in the fertility of parents
that is controlled by genes. Fitness is a concept related Hardy-Weinberg law, formulated in 1908, is a
to selection. It is the number of offspring that an mathematical model and is the fundamental principle
individual leaves during its lifetime or it is the lifetime in population genetics because it offers a simple
reproductive output of the individual. The average explanation for how the Mendelian principles that
fitness of all individuals in the population is called result from meiosis and sexual reproduction influence
population fitness or mean fitness. allelic and genotypic frequencies of a population.
The assumptions or conditions that must be present
Another important evolutionary process is the for the law to apply are (1) an infinitely large (2)
genetic drift. Random change in allelic frequency randomly mating population (3) free from mutation
from one generation to the next due to repeated (4) migration and (5) natural selection.

161
 The law states that “In a large random-mating changes in allelic and genotypic frequencies. Two
population with no selection, mutation or migration, types of processes cause these changes: dispersive
the gene frequencies and the genotype frequencies processes (inbreeding and genetic drift) and
are constant from generation to generation”. If the systematic processes (mutation, migration and
conditions of the Hardy-Weinberg law are met, the selection). Dispersive processes cause changes in allelic
population will be in genetic equilibrium, and two and genotypic frequencies that are random in amount
results are expected. First, the frequencies of the alleles and direction of change. Systematic processes cause
will not change from one generation to the next, changes that are consistent and predictable. The
and therefore the gene pool is not evolving at this allelic and genotypic frequencies of any population
locus. Second, the frequency of any genotype in the are due to the combined effects of inbreeding, genetic
population after one generation of random mating drift, mutation, migration, and selection.
is the product of the parental allelic frequencies.
These two conclusions have been demonstrated Mutation
experimentally to be valid and form the basis upon
which all further population and evolutionary genetics Usually converts one allelic form of a gene to another.
research is based. When the genotypes are in the A1 to A2 is called a forward mutation; A2 to A1 is
proportions, the population is said to be in Hardy- called a reverse mutation. The rate of mutation is
Weinberg equilibrium. Genotypic frequencies after generally low, but varies among loci and among
one generation of random mating are given by terms species. Mutations can change the frequencies of
in the expansion of (p + q) 2 = p2 + 2pq + q2 where alleles. Mutation provides the raw genetic material
p and q represent allele frequencies. An important for evolution. Most mutations will be detrimental and
use of the H-W law is that it provides a mechanism will be eliminated from the population.
for determining the genotypic frequencies from the
allelic frequencies when the population is in genetic Migration
equilibrium. 
Migration has the potential to disrupt H-W equilibrium
Testing for Hardy- and may influence the evolution of allelic frequencies
within populations. Migration among populations
Weinberg proportions tends to increase the effective population size (Ne) of
The H-W law can be used a s null model to which the the populations and divergence among populations.
genetic structure of any particular population can be
compared and it provides a way of evaluating which Natural selection
assumptions are being violated when the expected
theoretical distribution of genotypes does not match Mutation, migration, and genetic drift all influence
an empirically determined distribution. From allelic the pattern and process of adaptation, but adaptation
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frequencies, we can calculate the expected genotypic arises chiefly from natural selection. Natural selection
frequencies and compare these frequencies with the is the dominant force in the evolution of many traits
actual observed frequencies of the genotypes using and has shaped much of the phenotypic variation
a chi-square test. The chi-square test gives us the observed in nature. Selection in natural populations
probability that the difference between what we can be a) Directional selection b) Stabilizing selection
observed and what we expect under H-W law is due or c) Disruptive selection
to chance. (0.05, 0.10, etc.)
Fitness and the coefficient
Factors affecting genetic of selection.
structure of populations We measure natural selection by assessing
In contrast to idealized populations at Hardy-Weinberg reproduction. It is measured in terms of fitness
equilibrium, real stocks and populations experience which is defined as the relative reproductive ability

162
of a genotype or contribution of offspring of an of a large number of individuals, the gene pool of
individual to the next generation. Often symbolized the population is derived from the genes present
as W, and is also called the adaptive value or selective in the original founders (which may have been
value of a genotype.  Selection coefficient, s, is a determined by chance). This has a profound effect
measure of the relative intensity of selection against on the gene pool in subsequent generations.
a genotype and s = 1-W. The coefficient measures • Bottleneck effect–a form of genetic drift that
the selective advantage of the fitter genotype, or the occurs when a population is drastically reduced
intensity of selection against the less fit genotype. in size. Some genes may be lost from the gene
pool as a result of chance. This can be considered
The effect of selection on allelic frequencies depends as a form of founder effect, since the population
not only on the intensity of selection but also on initial is refounded by those few individuals that survive
gene frequency. Mutation can continuously reproduce the reduction. 
the allele lost by selection.
The genetic drift causes a) change in allelic frequencies
Genetic drift of a population over time and cause fixation and
extinction alleles b) Reduction in genetic variation
In small populations, chance deviations from expected within populations c) Differentiation between sub-
ratios can cause changes in allelic frequency (genetic populations and d) Increased homozygosity.
drift). When the sample is small, the sampling error
can be large. All genetic drift arises from such Nonrandom mating
sampling error. Based on population size we can make
predictions about the magnitude of drift, direction In order for the H-W principle to hold, individuals
of which is unpredictable. If the sexes are equal and in a population must mate at random. But many

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

all individuals have an equal probability of producing populations do not mate randomly for some traits,
offspring, the effective population size is the effective and when nonrandom mating occurs, the genotypes
number of breeding individuals. will not exist in H-W equilibrium.

When males and females are not present in equal In the broad sense, there are three kinds of nonrandom
numbers the effective population size (Ne) is mating described as follows. Assortative mating
occurs when two mating individuals are phenotypically
Ne= 4 x Nf x Nm / Nf + Nm more alike than two individuals chosen at random.
Disassortative mating occurs when mates are
Other factors such as differential production phenotypically less alike than two individuals chosen
of offspring, fluctuating population size, and at random. Neither affects the allelic frequencies,
overlapping generations can further reduce the but both may affect the genotypic frequencies if the
effective population size. The amount of variation phenotypes are genetically determined. Inbreeding
among populations resulting from genetic drift is is mating between individuals that are related to
measured by the variance of allelic frequency: sp2= each other by ancestry. Relatively small amounts of
pq/2Ne   inbreeding can cause tremendous damage to the
reproductive potential and productivity of a fish stock.
There are several ways in which genetic drift via
sampling error occurs in natural populations. Inbreeding
• Population size remains continuously small over
many generations Inbreeding is an exception to random mating that
• Founder effect–occurs when a population increases the number of homozygous individuals
is initially established by a small number of above their expectation described by the Hardy-
breeding individuals. Although the population Weinberg principle. It is the mating of consanguineous
may subsequently grow in size and later consist individuals (individuals having a common ancestor)

163
 which may have alleles that are identical by descent. that describe these divisions viz. FIS, FIT & FST which
Two alleles are identical by descent if they have are related by the expression
originated from the replication of one single gene
in the previous generation. Inbreeding is always (1- FIS)(1- FST)=1- FIT
defined relative to some reference population in
which all individuals are assumed to be unrelated. It FIS is a statistic used to estimate the deviations of
is often measured as the coefficient of inbreeding genotype frequencies from the H-W model within
(F) which is the probability that two alleles in subpopulations which make up the total population.
an individual are identical by descent and can be FIT is a statistic used to estimate deviations from the
calculated from pedigrees. It can also be defined H-W model within the total population. FST is an index
as the proportionate reduction in heterozygosity genetic differentiation used to describe the degree
compared to a reference population. to which a total population is genetically divided
into subpopulations and can vary between 0-1. Nei
F = Expected heterozygosity–Observed heterozygosity (1973) developed a similar statistic to FST called the
/ Expected heterozygosity coefficient of genetic differentiation GST which is
based on the heterozygosity values across local and
In a finite population, the average inbreeding total population. FST and GST are valuable measures
coefficient increases each generation, the rate of of population subdivision.
which depends on population size. Self-fertilization
decreases heterozygosity by half each generation and Genetic variation in
in random mating, F=0
natural populations
Inbreeding depression occurs when inbred individuals One of the most significant questions addressed in
have lower fitness than non-inbred individuals because population genetics is how much genetic variation
of the increased homozygosity with harmful alleles in exists within natural populations. Natural populations
double doses and lower frequencies of heterozygotes. show substantial variation for quantitative traits which
Outbreeding is the opposite of inbreeding; i.e. mating are measured on a continuous scale. Phenotypic values
between individuals less related than the average in the of quantitative traits are almost jointly determined by
population. The outbred population can have higher the effects of many genes and environmental effects.
fitness than any of the involved inbred populations
because of “hybrid vigour”, but outbreeding Genetic variation is important for several reasons:
depression can also occur if matings occur between • It determines the potential for evolutionary change
substantially diverged populations. and adaptation.
• The amount of variation also provides us with
Population subdivision important clues about the relative importance
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of various evolutionary processes, since some

If subpopulations are completely isolated and differ processes increase variation and others decrease it.
in allele frequencies, but are sampled without • The manner in which new species arise may depend
distinguishing between them, there will be a upon the amount of genetic variation harbored
perceived deficiency of heterozygotes in the sample, within populations.
even though there is no such deficiency in any • Evolution by natural selection depends on the
subpopulation. This is called Wahlund effect. existence of genetic variation within a population.

In a subdivided population, the overall deviation from Variation that is not subject to natural selection is
H-W expected heterozygosity has two components: the useful in DNA fingerprinting, conservation biology,
deviation due to factors acting within subpopulations, and empirical population genetics. Genetic variation
and the deviation due to subdivision (the Wahlund can be quantified by estimating genotype and allele
effect). Wright (1951) defined three F coefficients frequencies and also by considering the proportion

164
of polymorphic loci in a population (P) or by the (SNPs) are the most common kind of variation at the
observed heterozygosity. The genetic variation can DNA level. Newer techniques like DNA microarrays
be measured both at protein and DNA levels. or gene chips allow rapid automated detection of
SNPs. The variation at DNA level can be quantified
Measuring genetic variation with by calculating the nucleotide polymorphism (the
proportion of nucleotide sites that are polymorphic in
protein electrophoresis
a sample) and nucleotide diversity in the population.
Protein electrophoresis is a biochemical technique that
separates proteins with different molecular structures Indirect estimates of DNA variation are cheaper
which can be used to quickly determine the genotypes and faster than DNA sequencing and are useful for
of many individuals at many loci. Most species possess preliminary screening of a large number of individuals.
large amounts of genetic variation in their proteins. Restriction fragment length polymorphisms (RFLPs),
The technique misses a large portion of the genetic Amplified Fragment Length Polymorphisms (AFLPs),
variation that is present, because only genetic variants Random Amplified Polymorphic DNA (RAPD). Single
that cause a change in the movement of the protein Strand Conformation Polymorphisms (SSCPs) etc.
on a gel will be observed. come under this.

The amount of genetic variation is measured in this Certain kinds of satellite DNA in eukaryotic
via two parameters chromosomes are dispersed more or less randomly
• The proportion of polymorphic loci (P) is calculated throughout the genome. Each cluster of repeats is
by dividing the number of polymorphic loci by the considered a locus and the number of repeats at a
total number of loci examined particular locus is variable. Such loci are called the
• Average observed heterozygosity which is defined VNTR loci for the variable number of tandem repeats.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

as the proportion of heterozygotes, averaged over There are two kinds of VNTR loci depending on the
all loci. length of repeat sequence viz. Minisatellite loci and
microsatellite loci, both of which are useful genetic
Measuring genetic variation via markers. Microsatellite loci are highly variable and
can be analyzed locus by locus, hence more powerful
DNA sequence variation
for detection of genetic variation at population level.
Molecular markers provide useful tools for studying Variation in length of DNA sequences is also common
mutation rates, population sizes, natural selection, and is frequently due to insertion or removal of
population structure, and other important ideas transposable elements.
in evolution and conservation biology. Techniques
in molecular biology provide a means to examine Applications of population
directly nucleotide sequence differences in the DNA.
The genetic diversity between and within populations
genetics in fisheries
displayed by molecular markers receive extensive Fishes are major food source harvested from wild
interest due to the usefulness of this information in populations. Due to increasing levels of exploitation,
breeding and conservation programs. current fishery yields have reached or exceeded
sustainable limits. Management efforts have focused
These techniques fall into two main kinds: Direct on the estimation of sustainable yields and the
methods using DNA sequencing technology, and maintenance of sufficient stocks. Little consideration
indirect methods using restriction enzyme analysis was given to how fishing may affect genetic diversity
and other molecular techniques to infer variation in and selection pressures in the exploited stocks. The
DNA sequence. foundation for understanding of genetic effects caused
by harvesting comes from models in evolutionary
DNA sequencing has revealed high levels of variation biology and population genetics. The high exploitation
in nucleotide sequences. Single nucleotide differences rate may affect fish species in different ways such as a)

165
 extinction of reproductively isolated populations due population size during previous generations. The
to local overfishing, b) irreversible genetic changes in effective size of hatchery populations should be as
exploited populations due to selective removal fishes large as possible in order to minimize loss of genetic
with certain characteristics and c) reduced genetic diversity. It is suggested that Ne of 500-1000 should
variability as well as increased rates of genetic drift be maintained to prevent inbreeding and genetic
and inbreeding as a result of reduction in the effective drift related problems and to maintain the genetic
size of a stock. variance in fish populations that are used for stocking
programmes. Yet, Ne has to be customized at the
An important prerequisite for elaborating management level of farmers as it may not always be possible to
strategies in fisheries is identifying the number of maintain the recommended numbers.
reproductively distinct populations, the presence of
which can be confirmed by genetic assessments. Suggested readings
Significant differences in allele frequencies will be
detected when populations are isolated for a sufficient Allendorf, F. W., G. H. Luikart and S. N. Aitken. 2013. Conservation
number of generations. Measuring genetic diversity and the Genetics of Populations. Second edition. Wiley-
Blackwell, U. K.
in wild fish populations or aquaculture stocks is Beaumont, A., P. Boudry and K. Hoare. 2010. Biotechnology and
essential. Non-interbreeding populations have to be genetics in fisheries and aquaculture. John Wiley and Sons.
pp. 1-176.
identified to assess the gene flow between different Falconer, D. S. and T. F. C. Mackay. 1996. Introduction to
genetic stocks, and to monitor temporal changes in Quantitative Genetics. 4th ed. Longman, London. pp. 1-375
the gene pools. Population size is extremely important Halliburton, R. 2004. Introduction to population genetics. Upper
Saddle River: Pearson/Prentice Hall. pp. 1-587.
in evaluating conservation priorities for a species. Hamilton, B. M. 2009. Population Genetics. Wiley-Blackwell, U.K.
Small populations are at risk of going extinct because Hauser, L., G. J. Adcock and G. Carvalho. Long-term genetic
changes in exploited populations: Effects of fishing on New
of demographic stochasticity and genetic drift. Zealand snapper. courses.washington.edu
Kapuscinski, A. R. and L. M. Miller. 2007. Genetic guidelines for
Population genetics has role in the management fisheries management. Second Edition.University of Minnesota.
pp. 1-113.
of hatchery populations also. Genetic changes to Morrison, B.P. 2012. An annotated bibliography on the impacts
hatchery stocks can occur through selection, drift, of fish hatchery supplementation and enhancement on wild
populations. Ganaraska Region Conservation Authority, Port
or stock transfers. Inbreeding is a major problem Hope, Ontario. pp. 54.
in hatchery stocks. It is influenced by a number of Tave, D. 1999. Inbreeding and brood stock management (No.
factors like the number of breeding individuals in the 392). Food and Agriculture Organization.
Waples, R. S. 1991. Genetic interactions between hatchery and
population, sex ratio, variation in the reproductive wild salmonids: lessons from the Pacific Northwest. Canadian
success of individual spawners, and effective Journal of Fisheries and Aquatic Sciences, 48: 124-133.
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166
Protein Isolation and purification by
different chromatographic techniques.
M. A. Pradeep* and Esha Arshad
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
mixture flows through the column, depends on its
Isolation and purification of proteins from crude affinity for the matrix, and matrices with different
mixture is an imperative step in the identification chemical and physical properties can be used to
and analysis of proteins. Selective methods to separate proteins and peptides according to different
isolate individual proteins from a mixture, usually selective principles. The main LC techniques used in
exploit their very specific properties such as binding protein purification are ion exchange, size exclusion,
specificity or biochemical functions. Proteomic studies affinity, reversed-phase – HPLC, Hydrophobic
have revealed that most effective proteome analysis interaction Chromatography etc.
uses a combination of separation and identification
techniques. The purification methods are chosen Ion

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

depending on the percentage of purity required and
the end use of the protein.
Exchange Chromatography
The basic principle of ion exchange chromatography
Chromatography has been used for centuries as a involves the reversible ionic interaction between the
means of separation and, over time, has developed surface charge of the proteins and the oppositely
into a sophisticated analytical technique. It is a charged groups on the surface of the ion exchange
central technology in many fields of applied science adsorbent. Proteins can be positively or negatively
such as the synthesis of drugs and pharmaceutics, charged depending on its isoelectric point and the
purification of the products of organic synthesis, as pH of the surrounding medium.
well as food science, clinical chemistry and forensic
science. Due to their high resolving power, different Stationary phase: Consist of a column packed
chromatography techniques have become dominant with ion exchange resins which bind to proteins of
for protein purification. Any separation technique that opposite charge. Cation exchangers are negatively
distributes the components of a mixture between two charged and hence bind to positive charges on the
phases, a fixed stationary phase and a free mobile surface of protein. Eg: Sulfopropyl-Sephadex (Strong
phase, is known as chromatography. In proteomics, Cation exchanger), Carboxy Methyl-Cellulose (Weak
liquid chromatography is more often used than other Cation exchanger). Anion exchangers are positively
chromatography formats because of its versatility and charged and bind to the negative surface charges on
its compatibility with MS. In Liquid chromatography, a protein. Eg. Quaternary Amine-Sephadex (Strong
the stationary phase is a porous matrix, usually in anion exchanger), Diethyl amino ethyl-Sephadex
the form of packed beads that are supported on (Weak anion exchanger).Mobile Phase: The mobile
some form of column. The mobile phase, a solvent phase consists of a buffer, the pH and conductivity of
containing dissolved proteins or peptides flows which are of utmost importance. A buffer which gives
through the column under gravity or is forced through low conductivity and with pH that gives an optimal
under high pressure. The rate, at which the protein charge to the protein is chosen.Elution Strategy:

167
 Proteins of the same charge as that of the resin the purified in the chosen buffer, in the purified form.
will pass through the column while the oppositely
charged resins while bind to the column. Stationary phase: The matrices used in SEC are
often composed of natural polymers such as agarose
Change in pH: The elution buffer will have varying or dextran but may also be composed of synthetic
pH which will create a pH gradient in the column polymers such as polyacrylamide. Gels may be formed
and the proteins elute in the order of their isoelectric from these polymers by cross-linking to form a three-
point (at which pH, the net charge on the protein dimensional network. Different pore sizes can be
becomes zero). obtained by slightly differing amounts of cross-linking.
The degree of crosslinking will define the pore size
Change in Ionic strength: The elution buffer will
have varying ionic strength and the relative affinity of Mobile Phase: In contrast to other types of media
the protein to the resin will decrease with increasing the selectivity of a SEC matrix is not adjustable by
ionic strength. Loosely bound molecules will elute at changing the composition of the mobile phase.
lower ionic strength while strongly bound molecules Optimally there is no adsorption involved, and the
require higher ionic strength of the buffer to elute out. mobile phase should be considered as a carrier
phase and not one which has a large effect on the
Size chromatography. However, the sample may require
a buffer solution with a well-defined pH and ionic
Exclusion Chromatography composition chosen to preserve the structure and
Size exclusion chromatography or gel filtration is a biological activity of the substances of interest.
profiling technique which is used to separate proteins
according to their size. The separation process Elution Strategy: Since molecules are not adsorbed
depends on the different ability of various proteins to but only retarded on a SEC column the proteins are
enter all, some or none of the channels in the porous eluted isocratically and will elute in order with the
beads. Molecules running through a SEC column largest first. A single buffer is used, and hence no
have to solve a maze which becomes more complex gradient pumping systems are needed.
the smaller the molecule is, as the small molecules
have more potential channels that they can access. Affinity Chromatography
Larger molecules on the other hand, are for steric
reasons excluded from the channels, and pass quickly Affinity chromatography partitions proteins or
between the beads. The detour through the channels peptides on the basis of their specific, ligand binding
will thus retard smaller molecules in comparison to affinity. It is based on the reversible interaction
larger proteins which elutes first. between a protein or a group of proteins and a specific
ligand attached to the chromatographic matrix. AC
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The pore size of the gel can be adjusted to exclude offers high selectivity, hence high resolution, and
all molecules above a certain size. All molecules usually high capacity for the protein of interest. The
larger than the pore size are completely excluded target protein is specifically and reversibly bound
from the pore channels and thereby unretained and by a complementary binding substance (ligand).
elute together. The size of the pores in the matrix will The sample is applied under conditions that favour
also determine the rate of molecules that can enter specific binding to the ligand. Unbound material
the pores. This technique is ideal for final polishing is washed away, and the bound target protein is
steps in purification when sample volumes have been recovered by changing conditions to those favouring
reduced. Samples are eluted isocratically (single buffer, desorption. Ligand-protein interaction is often based
no gradient). Buffer conditions are varied to suit the on a combination of electrostatic and hydrophobic
sample type or requirements for further purification, interactions and hydrogen bonds. Agents that weaken
analysis or storage step, since buffer composition does the interaction such as a competitive ligand or changes
not directly affect resolution. Proteins are collected in in pH, ionic strength or polarity are used for desorption

168
of the bound protein. Samples are concentrated step wise or decreasing salt gradient. Most commonly
during binding and protein is collected in purified, samples are eluted with decreasing concentration of
concentrated form. The ideal stationary phase consist ammonium sulphate. Target proteins are concentrated
of a gel matrix possessing suitable chemical groups during binding and collected in purified, concentrated
(linkers) that can be covalently coupled to ligand form. The most widely used ligands in HIC are linear
molecules and provide a relatively large surface area chain alkanes, with or without terminal amino groups.
for attachment. The ligands can be extremely selective Aryl groups such as phenyl are also used. The strength
and bind to only a single or very small number of of the interaction between the ligand and the protein
proteins e.g. Antibodies, protein receptors, steroid increases with increase in the number of carbon
hormones, vitamins etc. Some ligands are less selective atoms in the chain and ideally, 4-10 carbon atoms
and bind to a group of closely related compounds. are most suitable for separation.
Immobilized metal affinity chromatography or IMAC
is a type of affinity chromatography where the solid Reverse Phase
phase contains positively charged metal ions such
as Fe2+, Ni2+ etc. This can be used to selectively
Chromatography (Rpc)
isolate phosphoproteins/peptides, proteins with oligo Reverse phase chromatography separates proteins and
histidine tags and other negatively charged proteins. peptides with differing hydrophobicity based on the
Affinity chromatography finds numerous applications reversible interaction with the hydrophobic surface of
such as purification of antibodies (immunoaffinity), a chromatographic medium. The technique is closely
gylco-conjugates, DNA binding proteins, enzymes, related to hydrophobic interaction chromatography.
isolation of nucleic acids etc. In RPC, the stationary phase is more highly substituted
with hydrophobic ligands than in those used for HIC
Hydrophobic Interaction and hence the hydrophobic interaction between the

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

protein and the ligand is much stronger. It is therefore
Chromatography (HIC) possible to adsorb proteins even in pure water.
HIC separates proteins with differences in However, the very strong interaction requires the use
hydrophobicity. The separation is based on the of organic solvents and other additives (ion pairing
reversible interaction between a protein and the agents) for elution. Elution is usually performed
hydrophobic surface of the chromatographic by increase in organic solvent concentration, most
medium. The proteins are separated according to commonly acetonitrile. RPC is often used in final
the differences in the amount of exposed hydrophobic polishing steps and is ideal for analytical separations
amino acids. To facilitate hydrophobic interaction, the such as peptide mapping. RPC is not recommended
protein mixture is loaded onto the column in a buffer for protein purification, if recovery of activity and
with high salt concentration. Samples in high ionic tertiary structure are required.
strength solution (e.g. 1.5 M ammonium sulphate)
bind as they are loaded onto the column. Conditions Suggested readings
are then allowed so that the bound substances are
eluted differentially. Elution is usually performed by Protein Purification Handbook. Amersham Biosciences.
decreases in salt concentration. Changes are made in Twyman.R.M.2004.Principles of Proteomics. Bios
Scientific Publishers.

169
 Genes as Molecular Guardians in
Environment Management and
Aquaculture
M. P. Paulton
Senior Technical Officer (Training)
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction seasonal scale. Among these bivalves the intertidal

molluscs represented by oysters and mussels are
It is a universal phenomenon that all living beings regularly undergoing regimes of immersion and
across the species and taxa have more or less a similar emersion and thereby exposed to abiotic stresses.
system to regulate and protect its own machinery and Stress can be defined as a condition which disturbs
thereby making the species more sustainable. Such the dynamic equilibrium or homeostasis of an
systems play a vital role in the survival of living organism organism by the action of intrinsic or extrinsic forces
amidst the challenging situations which is imposed by usually referred as stressors. The various abiotic
environment in which they live. Environment is prone to stressors encountered by bivalves both in the wild as
routine pressures from various sources such as climatic well as in the aquaculture include thermal variations,
changes, impact of human civilization etc. It is quite oxidative fluctuations and salinity changes. The
obvious that these pressures will be directly transferred bivalves are also prone to biotic stressors like
to living organisms living in the particular environment. microbial infection and other parasitic attack. The
The intensity or depth of these challenges are more increasing contamination of bodies of natural
in aquatic species as the developmental activity like freshwater and marine ecosystem around the world
industrialization leading to and the continuous discharge by anthropogenic substances is another category
of various organic and inorganic waste and other of environmental stressor. The composite threats
industrial sewage into the aquatic medium are on the of all these factors are more relevant in this era of
increase. Further, the phenomenon of global warming global warming causing chronic stress on aquatic
also has got a direct impact in aquatic environment, and organisms. The tolerance to varying temperature
the rising concern associated with global climate change is an indication of population response to climate
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has regained the interest in the physiological mechanism changes which will ultimately determines the
that animals use to tolerate extreme heat and adapt speciation of the region. Living organisms manage
to thermal changes in their natural environment. One to overcome these harsh environmental challenges
could a see a regular interplay between the genes and through the regulating the expression of certain
the changes takes place in the environment. genes playing vital roles in stress management.

Marine habitat as Multigene families linked

a model of gene to stress management and
environment interaction bio monitoring
Marine bivalves are regularly exposed to varying Latest findings based on the transcriptome sequencing
physico-chemical conditions on a day to day or reveal that certain families of genes are readily

170
expressed in response to environmental challenges. like HSPs in promoting immune responses to protect
Among them the prominent ones are heat shock the cytoplasmic components from biotic stressors
family genes, cytochrome P450 multigene families such as bacterial challenge is documented in recent
and anti-oxidant enzyme genes. Among the stress years. The reports on the role of heat shock proteins
related genes, heat shock protein (HSP) genes are in disease control in aquatic organisms are coming up
much studied across different flora and fauna. They fast. The increased expression of heat shock protein
also represent the evolutionarily conserved molecular genes are reported in fishes and shellfishes infected
chaperones with prominent roles in managing all with viruses and bacteria like white spot syndrome
sorts of abiotic and biotic stress. The presence of virus (WSSV), Noda viruses, V. harvei, V. anguiilarum,
both constitutive and induced isoforms indicates the V. parahaemolyticus, V. alginolyticus, Renibacterium
importance of these proteins in bivalve life. Heat shock salmoninarum etc.
protein family members help the inter tidal bivalves
in making themselves ‘prepared for stress’ amidst A new approach to induce the expression of HSPs
the much challenging environment. The role of these in animals to manage disease and other stressors
multigene family members in thermo tolerance is well are gaining attention. Biochemical inducers of
documented among bivalves. Latest report of HSP plant origin are found to be an effective non-
gene sequence divergence and synonymous single stress inducer to enhance the HSP production.
nucleotide polymorphism (SNPs) suggest the potential Administering Feed containing HSP enriched
use of these genes in population genomics as well. bacteria is a new approach of delivering HSPs
The studies co relating the presence of HSP and aging for protection against diseases in aquaculture.
in bivalves has opened up a new way of assessing the HSPs are also used to raise vaccines to prevent
process of aging. diseases in fishes using pathogen derived HSPs
as antigens. Such vaccines are found to be

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

The HSPs play a major role in host-parasite interactions giving long lasting protection and suggested as a
and this has received considerable attention of promising alternative to antibiotics in aquaculture.
researchers as it is relevant from both clinical and The American oyster Crassostrea virginica infected
biological perspectives. HSPs of invading parasites with the parasite Perkinus marinusis found to be
elicit immune response within host and can be used surviving well after a sub lethal thermal shock than
as potential source for generating vaccines. Owing the control group. All these findings suggest that
to the responsiveness to diverse forms of stress, there is a clear case for exploring HSPs further and
heat shock response as detected through HSP gene applying its potentials in management of different
expression provide widespread application in bio kinds of abiotic and biotic stressors in aquaculture.
monitoring and environmental toxicology. HSPs are
believed to play a major role in managing global Aquatic Oxidative stress and
warming. Global warming is a slow process and
HSPs can evolve more efficiently and offer better
role of antioxidant genes
protection to the individuals. Individuals with mutant Oxygen is a vital factor for the existence of life
forms or over expression of HSPs points towards the and it is an important element for all flora and
probable natural selection and open up scope for fauna of terrestrial and aquatic origin. Marine
selective breeding as most characters of economic organisms, especially bivalves like mussels and
importance and evolutionary significance are inherited oysters are sedentary and sessile growing attached
quantitatively (Quantitative trait loci, QTL). on a suitable substratum. They primarily live in the
inter tidal zone within aquatic habitat. Owing to
Role of HSPs in controlling the characteristics of the habitat, these bivalves are
constantly exposed to changing levels of oxygen,
diseases in aquaculture temperature and salinity. Reactive oxygen species
HSPs also play an important role in activating the (ROS) are produced in abundance when the animals
innate system of immunity. The role of stress protein are exposed to oxygen related stress such as hypoxia.

171
 ROS molecules such as super oxide anion radical metal content. Two isoforms are normally present in
(O2-), hydroxyl radical (HO) and hydrogen peroxide most of the organisms. They are the extracellular and
(H2O2) used during oxidation can destroy tissues and intracellular Cu/ Zn SOD present in the extra cellular
cells. Prolonged hypoxia can result in mass mortality matrix and the intra cellular space respectively within
in oysters and a control system is essential to survive the tissues. The intra cellular Cu/ Zn SOD is also
the situation. found in inter membrane space of mitochondria and
nuclei. Recent literature states that the intracellular
Hence suitable physiological mechanisms mediated Cu/ Zn SOD also perform certain key functions
through biochemical pathways are essential to other than dismutation in molluscs. It prevents
maintain the cellular homeostasis. Oxidative reactions the formation of toxic compound peroxynitrite by
are a part of aerobic life and as a consequence results stimulating to produce nitric oxide and superoxide
in the production of reactive oxygen species within molecules. The up regulated expression levels of
the cell. Accumulation of ROS can cause structural SODs as revealed through transriptomic studies
damage to organic macromolecules like protein, in Haliotis discus discus and Crassostrea gigas,
carbohydrate and lipids. Such oxidative damage Crassostrea madrasensis exposed to heavy metals
resulting from reactive oxygen species (ROS) is and thermal stress or hydrocarbon are reported.
called as Oxidative stress. The aquatic organisms Moreover, elevated level of expression of SOD gene
are usually prone to more oxidative stress owing after Vibrio anguillarum challenge point towards
to the chronic exposure to xenobiotic pollutants its involvement in immune function .The potential
resulting in more ROS). Oxidative stress causes benefits of these genes are widely explored in bio
imbalance between exogenous and endogenous monitoring and in selective breeding programs. The
reactive oxygen species which result a decline in SOD, CAT and GPX are being used as a biomarker
cellular defense. The increase in ROS results the to monitor the level of mercury, lead and copper as
slowdown of the cell’s antioxidant defense system the reduction of these enzymes are noticed in the
and consequently free radicals generated are allowed bivalve Chlamys farreri undergoing lipid peroxidation
to disrupt cellular functions, causing damage to on exposure to lead.
DNA with DNA lesions, denaturing the integrity of
cellular membranes by lipid peroxidation, modifying Stress adaptations as
proteins by protein peroxidation. The ROS defense
system comprise of enzymatic antioxidants and non-
revealed through oyster
enzymatic antioxidants like vitamin E, ascorbate, genome sequencing
β-carotene, and urate. Antioxidant enzymes play a
pivotal role in maintenance of homeostasis within The latest report based on the findings of the
the cells and in cell mediated antioxidant defense sequencing of whole oyster genome along with
by removing ROS. Among the antioxidant enzymes, selected transcriptomes have revolutionized the
Central Marine Fisheries Research Institute

Super oxide dismutase (SOD) is the key defense hitherto understanding about the factors behind
molecule which fight ROS followed by others such defense and stress adaptations in Pacific oyster
catalase (CAT), glutathione transferase (GST) and Crassostrea gigas. As reported in the paper
glutathione peroxidase (GPX) playing vital roles in published in the prestigious journal NATURE (SEP
the detoxification pathways. 2012), the Pacific oyster genome contains eighty
eight heat shock protein 70 (HSP70) genes with
Super oxide dismutase (SOD) removes the super oxide potentials roles in protecting cells from thermal
free radicals through the process of dismutation by and other stressors. This huge number of HSP70
converting them into hydrogen peroxide and this genes is a record compared to the seventeen in
in turn is further reduced to water and oxygen by human beings. Further, the report reveals the
the action of CAT and GPX. Among them SOD is expansion of genes like superoxide dismutase,
the leading enzyme readily responding to stress. cytochrome P450 and inhibitor of apoptosis
SODs (EC 1.15.1.1) are categorized based on the proteins (IAPs) in oyster.

172
Conclusion Suggested readings
Guofan Zhang, Xiaodong Fang, XimingGuo, Li Li, RuibangLuo,
From the above descriptions, it is evident that there
FeiXu, Pengcheng Yang, Linlin Zhang, XiaotongWang, Haigang
exists an array of genes regularly in interaction with Qi, ZhiqiangXiong, HuayongQue, YinlongXie, Peter W. H.
the changes in the surrounding habitat. Selection of Holland, JordiPaps, YabingZhu, Fucun Wu, Yuanxin Chen,
Jiafeng Wang, ChunfangPeng, JieMeng, Lan Yang, Jun Liu,
individuals with better presence or expression of these Bo Wen,Na Zhang et al. The oyster genome reveals stress
as brood stock using these genes as biomarkers would adaptation and complexity of shell formation. Nature.490:
49-54, (04 OCT 2012).
be helpful in raising offspring with better survivability
Paulton, M P (2012) Studies on the defence and stress related
and performance in aquaculture. Hence tapping the factors of Oyster and Mussels of mariculture importance. PhD
potentials of this genomic approach would be a thesis, Mangalore University, Mangalore.

better strategy to make the culture practices more

sustainable and profitable.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

173
 Molecular Systematics
Sandhya Sukumaran
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Systematics is the scientific study of the diversity like NCBI, GenBank. The next step is to align sequences
and kinds of organisms and their relationships and identify mutations like insertions and deletions.
based on which biological evolution and diversity is Wherever needed, gaps should be inserted into the
assessed. It comprises of classification of organisms sequences to increase their similarity. There are many
into groups based on phenetic or phylogenetic algorithms to align multiple sequences and these
characters, naming of organisms and final algorithms tries to minimize the total effect of all the
identification organisms and placing them into any possible changes in pair wise sequence comparisons.
of the previously described groups. Systematics When some sequences are ambiguously aligned, they
also provides clues to the evolutionary biology of should be excluded from the data set. A phylogenetic
an organism and it is another way of studying the tree can be constructed using sequence data and the
evolutionary patterns on a temporal scale. Molecules tree could be rooted using outgroup sequences from
carry information regarding the ancestry and history distantly related species so as to get more insights into
of an organism and hence inferring the biological the evolutionary relationships. The choice of outgroup
relationships based on molecular systematics provides is very important as it will affect the topology of the
coherent answers to many biological questions. tree. In addition to the sequence data, a model of
The ease of generating sequence data for many sequence evolution must also be selected as the
independent data sets and relating the phenotyping methods in molecular phylogenetics are based on
variations with sequence divergence is the key to a series of assumptions about substitution process.
taxonomic inferences. The use of molecular data
along with conventional taxonomy has become a Methods for
scientific practice for the past few decades after the
emergence of sequencing technologies and this is
phylogenetic inference
termed as integrative taxonomy. There are four methods that have been widely used
for phylogenetic inference; maximum parsimony,
Inferring neighbor-joining, maximum likelihood and
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Bayesian inference.
Phylogenetic information
Inferring phylogenetic information is the key to Maximum parsimony
molecular systematics. The individuals within and
between populations are genetically related directly It is one of the earliest inference methods used to
or indirectly sharing common ancestors. The degree infer phylogeny. Maximum parsimony method uses
of genetic divergence provides insights into the extent character states as compared to distance methods
of relationships among and between individuals in and it is based on the optimality criterion which is the
populations. The process of phylogenetic estimation rule to decide the best alternative tree; it selects the
begins with the collection of sequence data which are tree or trees which require the fewest character state
homologous. The sequence data could be generated changes and thus attempt to minimize homoplasy.
by collecting the organisms and sequencing a The length of an unrooted tree is directly calculated
particular gene or it could be retrieved from databases using Fitch’s algorithm which moves through the tree

174
assigning one or more character states to each of the the tree and the model of evolution. This also uses
internal nodes. The tree space (theoretically possible an optimality method. The best tree is the one that
tree topologies for a given number of taxa) is usually renders the observed sequences most likely to have
searched using heuristic searches or exact searches. evolved under the expected evolutionary model.
Exact searches examine all possible trees (exhaustive The tree space is usually explored using heuristic
searches) or parts and thus guarantee that the tree searches and the advantage of likelihood is that
found is optimal whereas heuristic searches do not it allows the inference of phylogenetic trees using
search all possible trees. The advantage of this analysis complex models of sequence evolution. Maximum
is that it is very fast for the analysis of large data sets likelihood based methods are the most robust
which contain many sequences and it gives robust way for estimating molecular phylogenies and
estimates when the branches of the tree are short. understanding sequence evolution. But the main
But maximum parsimony performs poorly when the disadvantage is that the results are highly dependent
rates of evolution vary substantially. on the model of sequence evolution used and the
method is computationally demanding when the
Neighbour–joining and number of sequences is large.

minimum evolution
Bayesian inference
It is based on pair-wise distance methods on the
assumption that dissimilarity between any sequences It is the most recent phylogenetic inference method.
is directly related to their phylogenetic relationship. Bayesian statistics is closely related to maximum
This dissimilarity arises from the number of changes likelihood method; the optimal hypothesis is the
along the branches or in other way the evolutionary one which maximize the posterior probability.
distance. Distance methods are clustering methods Bayes’ theorem states that the posterior probability

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

like neighbor-joining or optimality methods like for a hypothesis is proportional to the likelihood
minimum evolution. In neighbor joining methods, multiplied by the prior probability of that hypothesis.
DNA or amino acid sequences are converted into a Bayesian analysis allows complex models of sequence
distance matrix which is then used to construct a evolution to be applied to for the whole sequence
phylogenetic tree. Optimality methods evaluate the data set and for different partitions of it. It involves
score of a tree based on the squared deviation of the specifying a model along with a prior distribution
pair wise observed distances between each pair of and then integrating the product of these quantities
taxa, estimated from the data matrix and the distance over all parameter values that are possible so that
separating those taxa on the tree. In the minimum the posterior probability values of each tree could
evolution, the sum of branch lengths optimized be determined. But the likelihood functions for
according to the least-square criterion is the optimality phylogenetic models are too complex to integrate
criterion. The advantage of distance methods is that analytically and hence Bayesian approaches depend
they are comparatively fast compared to all the other on Markov chain Monte Carlo (MCMC) procedures.
methods and they perform well when divergence This algorithm functions by sampling trees from
between sequences is low. But there occurs loss of the distribution of posterior probabilities. Unlike
information when the sequences are converted to maximum likelihood which looks for the single most
distances and it is difficult to obtain reliable estimates likely tree, Bayesian MCMC searches for the “best set
when the sequences are highly divergent. of trees” in the tree space. Bayesian analysis given a
robust approximation of the evolutionary tree and
Maximum likelihood hence it is based on powerful statistical foundation
as in maximum likelihood method. The disadvantage
Maximum likelihood is one of the standard of Bayesian methods is that prior distributions for the
methods of statistics and it has also been applied parameters must be specified and it is also difficult
to phylogenetics. The likelihood of a phylogenetic to determine if the MCMC approximation has run for
tree is the probability of observing the data given sufficient number of cycles.

175
 Statistical support for detect allozyme variation, the first step is to extract
allozymes from tissues using specific protocols. Then
phylogenetic trees the variation is detected through electrophoresis in
Parametric bootstrapping uses a Monte Carlo an acrylamide or cellulose acetate gel. Individuals
simulation to generate the data. A simulation is that are homozygous show a single band whereas
carried out using replicate data sets of the same size heterozygous individuals show two bands. Allozymes
as original according the null hypothesis being tested. are one of the most studied form of molecular
Likelihoods according both the null and alternative variation due to their simplicity, low cost and the
hypotheses are estimated for each replicate data requirement of little specialized equipment. Any kind
set and the LRT (Likelihood Ration Test) statistic of soluble protein is suitable for allozyme analysis. A
is derived and significance test is implemented large number of loci can be screened at a time. The
based on simulated values. Parametric bootstrap limitations of this technique include requirement of a
is computationally very demanding when the data large amount of tissue and consequently this method
sets are large. Several non-parametric likelihood could not be applied when the organisms are small
based tests are also used which determine whether (for eg; larval forms). The tissue sampling method is
the difference in fit of two or more alternative tree invasive and so the fish needs to be sacrificed and
topologies to the data is significantly higher than the tissue needs to be stored cryogenically. A point
expected under the null hypothesis of random mutation in a nucleotide sequence may not result
sampling error. The most widely used methods are in a change in amino acid at all and thus could not
the Kishino-Hasegawa, the Shimodaira-Hasegawa be detected by protein electrophoresis. In addition
and the unbiased tests. Non-parametric tests appear to that, a change in DNA that results in a change in
to be conservative and parametric tests appear to amino acid will not result in the overall charge of the
be liberal in empirical comparisons. But generally, to protein and therefore is not detected. In spite of their
assess branch support for phylogenetic trees non- limitations, the use of allozyme analysis has been
parametric bootstrapping is used. widespread in fisheries mainly in fish systematics,
population structure, conservation genetics, mixed
Molecular markers used for stock fishery analysis and forensic analysis.

molecular systematic studies

Mitochondrial DNA markers
To infer the information required for phylogenetic
analysis, several molecular markers can be used. Mitochondrial DNA is non- nuclear DNA in the cell
Molecular markers can be characterized as Type I having located in within organelles in the cytoplasm
and Type II markers; Type I markers are associated called mitochondria. Mitochondrial DNA is maternally
with genes of known function and type II markers inherited with a haploid genome. The entire genome
are associated with genes of unknown function. undergoes transcription as one single unit. They are
Central Marine Fisheries Research Institute

Allozyme markers are type I markers as the proteins not subjected to recombination and thus they are
they encode are associated with some functions. homologous markers. They are selectively neutral
Microsatellites and other neutral markers are type occurring in multiple copies in each cell. Mitochondrial
II markers unless they are associated with genes of DNA is physically separate from the rest of cell’s DNA
some known function. and so it is relatively easy to isolate from any tissue
or blood sample. Due to the maternal inheritance of
Allozymes mitochondrial DNA, the effective population size is
smaller than nuclear DNA and so mitochondrial DNA
Allozymes are codominant markers having been variation is more sensitive to population bottlenecks
expressed in a heterozygous individual in a Mendelian and hybridizations. The differences in the nucleotide
way. Thus allozyme analysis provides us with data sequence of DNA molecule in the mitochondria can be
on single locus genetic variation which can answer determined directly or indirectly by several methods.
many questions about fish and fish populations. To Many population genetic studies have employed RFLP

176
(Restriction Fragment Length Polymorphism) analysis slight changes in amplification conditions. Amplified
of mitochondrial DNA for understanding population Fragment Length Polymorphism (AFLP) markers
genetic variation either by digesting the whole purified combine the benefits of both RFLP and RAPD. The
mtDNA with restriction endonucleases or by DNA total genomic DNA is digested using two restriction
sequencing of small segments of mtDNA molecule enzymes. Double–stranded nucleotide adapters are
obtained by PCR amplification. These techniques with ligated to the ends of DNA fragments to serve as
increased resolution and maximum information have primer binding sites for PCR amplification. Primers
made mtDNA analysis very popular. complementary to the adapter and restriction site
sequence, with additional nucleotides at the 3’-end,
The newly emerged sequencing technologies have are used as selective agents to amplify a subset of
enabled direct sequencing of mitochondrial genes ligated fragments. The presence of absence of DNA
and several sets of universal primers have been fragments are detected on polyacrylamide gels and
developed from conserved sequence regions. Slow thus polymorphisms are studied.
evolving gene regions are being used for inter
species comparisons and fast evolving gene regions Specific Nuclear DNA markers
for population comparisons. The only non coding
region of mtDNA is D-loop region and this region Variable Number of Tandem Repeat is a segment
is fast evolving and mostly used for population of DNA that is repeated tens or even hundreds
comparisons. The cytochrome b and ND-1 and to thousands of times in nuclear genome of
ND-5/6 gene regions are also being used widely. eukaryotes. They repeat in tandem; vary in number
Mitochondrial Cytochrome C Oxidase I gene has in different loci and differently in individuals. There
been identified as the universal barcode for species are two main classes of repetitive and highly
level identification due to its conserved nature across polymorphic DNA; minisatellite DNA referring to

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

a wide range of taxa. DNA barcodes are segments of genetic loci with repeats of length 9-65 bp and
approximately 600 base pairs of the mitochondrial microsatellite DNA with repeats of 2-8 bp (1-6)
COI gene which is a fast, efficient and inexpensive long. Microsatellites are much more numerous in
technique helpful in cataloguing the biodiversity. the genome of vertebrates than mini satellites.
During the last two decades, mitochondrial DNA They are widely used in population genetics of
genes have found widespread application fish fishes and aquatic invertebrates. Minisatellites
taxonomy, biology and population genetics. can be classified into multilocus and single-
locus minisatellites. Multilocus minisatellites are
Arbitrary Nuclear DNA markers composed of tandem repeats of 9-65 base pair
and have a total length ranging from 0.1 to 7kb.
Arbitrary markers are used when we target a Minisatellite loci are used mainly in parentage
segment of DNA of unknown function. The widely analysis. They are less useful for population
used methods of amplifying unknown regions are genetic analysis unless we use large sample sizes.
RAPD (Random Amplified Polymorphic DNA) and The complexity of mutation processes undergone
AFLP (Amplified Fragment Length Polymorphism) by minisatellite loci is also a limitation. Due to
DNA. RAPD uses an arbitrary primer which can the difficulties in the interpretation of multilocus
amplify anonymous loci. It is fast, cheap and fingerprints, the research work were concentrated
shows very high amount of polymorphism and this on single locus minisatellite probes and this
marker does not require knowledge of the genetic procedure required reasonable quantities of
makeup of the organism. The major drawback with high-quality DNA. These single locus minisatellite
RAPD markers is the lack of reproducibility and probes have been very useful and successful in
repeatability and the large number of products detecting genetic variations within and between
generated. RAPD is a dominant marker and so populations. It has also been used in fisheries for
homozygous and heterozygous states cannot be forensics, parentage, genetic identity, estimating
differentiated and these patterns are sensitive to mating success and confirming gynogenesis.

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 Microsatellites amounts of stuttering. Microsatellite markers are
used in fisheries and aquaculture for phylogenetics
A microsatellite is a simple DNA sequence which and phylogeography, population genetic structure,
is repeated several times across various points in biodiversity conservation, stocking impacts and
the DNA of an organism. These repeats are highly hybridization. It is also being increasingly used
variable and these loci can be used as markers. for forensic identification of individuals, genome
Microsatellite occur once in every 10 kbp while mapping and determination of kinship and
minisatellite loci occur once in every 1500 kbp in behavioral patterns.
fishes and due this, microsatellites are more useful
in genome mapping and population genetics Single Nucleotide Polymorphisms
studies. They are highly variable, non-coding
and selectively neutral and the basic assumption Single nucleotide polymorphisms arise due to single
while using microsatellite loci is that the predicted nucleotide substitutions (transitions/transversions)
amount of sequence divergence between units of or single nucleotide insertions/deletions. These
interest is directly related to length of time since point mutations give rise to different alleles with
separation. Microsatellites are codominant markers alternative bases at a particular nucleotide position.
which are inherited in a Mendelian fashion and SNPs are the most abundant polymorphisms in the
they are highly evolving with 10-3-10-4 mutation/ genome (coding and non-coding) of any organism.
generation. The high levels of polymorphism shown These single nucleotide variants can be detected
by microsatellites have made them one of the most using PCR, microchip arrays or fluorescence
popular genetic markers. Cross amplification with technology. They are considered as next generation
primers developed in closely related species is also markers in fisheries and can be employed for
possible which minimizes the cost associated with population genetics studies, genomics studies and
detecting microsatellite sequences in a different for detection of diseases.
species. The analysis of microsatellite loci involves
DNA extraction, amplification of the microsatellite DNA microarrays or DNA chips
loci using specific primers in a PCR machine and
examination of the bands using poly acrylamide DNA microarray consists of small glass microscope
gel electrophoresis. The recent introduction of slides, silicon chip or nylon membranes with many
automated genotyping machines has made the immobilized DNA fragments arranged in a standard
analysis of size polymorphisms of microsatellite pattern. A DNA microarray can be utilized as a
loci with automated genotyping using labeled medium for matching a reporter probe of known
primers. The use of large number of samples and sequence against the DNA isolated from the target
loci is now possible due to automated genotyping sample which is of unknown origin. Species-specific
which has increased precision and speed with DNA sequences could be incorporated to a DNA
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microsatellite analysis. The constraints of using microarray and this could be used for identification
microsatellite markers are the presence of null alleles purposes. DNA extracted from a target sample should
and presence of stutter bands. Null alleles are found be labeled with a specific fluorescent molecule
when mutations occur at primer binding sites of and hybridized to the microarray DNA. When the
microsatellite locus. The presence of null alleles hybridization is positive a fluorescent signal is
reduce accuracy especially in parentage or relatedness detected with appropriate fluorescence scanning/
analysis and assignment tests and the best option imaging equipment.
is to discard loci showing null alleles. Stutter bands
occur when a ladder of bands differing between 1-2 Expressed Sequence Tags (ESTs)
bp is seen and these occur due to slipped strands
impairing during PCR or incomplete denaturation ESTs are single-pass sequences which were generated
of amplification products. Tri-nucleotide and tetra from random sequencing of cDNA clones. ESTs can be
nucleotide repeats usually do not show significant used to identify genes and analyze their expression by

178
means of expression analysis. Fast and reliable analysis Davis, G.P. and Hetzel, D.J.S. 2000. Integrating molecular
genetics technology with traditional approaches for genetic
can be made for the genes expressed in particular improvement in aquaculture species. Aquaculture Research,
tissue types under specific physiological conditions 31: 3-10.
Edwards, S.V. 2008. Is a new and general theory of molecular
or developmental stages. Differentially expressed systematics emerging? Evolution 63(1): 1-19.
genes could be identified using cDNA microarrays Hallerman, E.M. 2006. Use of molecular tools for research and
in a systematic way. ESTs are most valuable for improvement of aquaculture stocks. The Israeli Journal of
Aquaculture – Bamidgeh, 58(4): 286-296.
linkage mapping. Hillis, D.M. 1987. Molecular versus morphological approaches
to systematic. Annual Reviews in Ecology and Systematics
18: 23-42.
Future of Liu, Z.J. and Cordes, J.F. 2004. DNA marker technologies and their

Molecular Systematics applications in aquaculture genetics. Aquaculture 1-37.

Mauro, D.S. and Agorreta, A. 2010. Molecular systematic: A
synthesis of the common methods and the state of knowledge.
With the advent of Next generation sequencing, Cellular and Molecular Biology Letters 311-341.
enormous amounts of sequence information are being Okumus, I. and Ciftci, Y. 2003. Fish population genetics and
molecular markers: II- Molecular markers and their applications
generated for a large number of taxa. So the challenge in fisheries and aquaculture. Turkish Journal of Fisheries and
will be in handling the very large data sets and then Aquatic Sciences 3: 51-79.
integrating different levels of genomic information. Presti, R.L., Lisa,C. and Stasio, L.D. 2009. Molecular genetics in
aquaculture. Italian Journal of Animal Science 8: 299-313.
Schwartz, J.H. and Maresca, B. 2006. Do molecular clocks run
Suggested readings at all? A critique of molecular systematics. Biological Theory
1(4): 357-371.
Teletchea, F. 2009. Molecular identification methods of fish species:
Askari, G.H., Shabani, H. and Miandare, H.K. Application of reassessment and possible applications. Reviews in Fish Biology
molecular markers in fisheries and aquaculture. Scientific and Fisheries 19: 265- 293.
Journal of Animal Science, 2(4): 82-88.
Chauhan, T and Rajiv. K. 2010. Molecular markers and their
application is fisheries and aquaculture. Advances in Bioscience

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

and Biotechnology 1: 281-291.

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 The science of ‘omics’ – Genomics,
Proteomics and Metabolomics
M. A. Pradeep*, S. R. Krupesha Sharma and Esha Arshad
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
scientists in the 1950’s that laid the foundation
In Biological science there are more than 1000 for genomics research. Development of techniques
“omics” field. Different views on the origin of the for radiolabelling of biological molecules and
word “ome” exists, the claim that it originated from discovery of the double helical structure of DNA
the Sanskrit word OM is one among them. Just as by Watson and Crick in 1953 propelled more
the Sanskrit word OM stands for completeness and studies on genome. These discoveries led to the
fullness, the different “omics” fields in biology adopts understanding of processes such as DNA replication,
a holistic approach in its study, for eg. genomics is gene expression and protein synthesis. Later,
the study of the complete genome of an organism, technological advances such as the polymerase
whereas proteomics relates to universal detection of chain reaction (PCR) developed in the 1980’s,
all the proteins present in the cell at a particular point allowed DNA amplification from small amounts
of time, and metabolomics studies all the metabolites of starting material. Automated DNA sequencing
present in the cell at a particular point of time. technologies enabled fast genomic sequencing of
DNA. PCR and DNA sequencing can be thought of as
Genomics the prime building stones of the field of genomics.

Genomics is the study of the genome of an organism, The first genome was sequenced in the 1970’s
including the interaction of the genes with each by Fredrick Sanger and was that of a virus and
other and with the environment. Genome broadly mitochondrion. Sanger also developed a technique for
refers to the total amount of DNA that is present in DNA sequencing, known as the “Sangers Sequencing”,
a single cell of an organism, including its genes i.e. for which he received the Nobel Prize in 1980. Since
the whole genetic information encoded in the DNA. then, whole genome of several organisms has been
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Genes are the units of heredity, which encodes the sequenced. Walter Fries in 1972 sequenced the first
information to carry out all cellular processes. The term gene and ultimately the first whole genome sequence
“genomics” was coined by mouse geneticist, Thomas. of bacteriophage MS2, a RNA virus. Haemophilus
H. Roderick in 1986 and has swiftly developed over influenzae is the first free living organism to have
the years into a new discipline. Genomics include the its genome sequenced and Ceanorhabd tiselegans
mapping, sequencing and functional characterization genome is the first multicellular organism genome
of genome using recombinant DNA technology, DNA which has been completely sequenced. In the years
sequencing and bioinformatics tools. that followed, genomes of various organism have
been sequenced including humans at a very rapid
DNA was isolated as early as in 1896; however, pace. Presently, whole genome sequences are
the first genome was isolated only a century available for over 2700 viruses, 1000 bacteria and
later. Early genomics began in the 1970’s, but archae, and 36 eukaryotes, with new sequences
it was the techniques developed by group of added almost every day.

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The field of genomics can be sub classified into (a) can then be visualised. Total strength of the signal
structural genomics involving the construction of emitted from a spot depends upon the amount of
genomic sequence data, genomic maps and gene target sample binding to the probes present on that
discovery (b) Functional Genomics encompassing the spot. Microarray technique uses relative quantitation
study of the function of the genes, its regulation, gene in which the intensity of a feature is under different
products etc. and (c) comparative genomics which is conditions, and the identity of the feature is known
used to compare gene sequences to understand the by its position.
functional or evolutionary relationships.
Microarray analysis is mostly used in functional
The fundamental technologies used in genomics genomics for studying gene expression, in detection
include PCR, microarray analysis, and DNA of single nucleotide polymorphisms and also in
sequencing. PCR revolutionised the study of DNA to comparative genomics where it is used to study the
the extent that it is used every day in identification increase or decrease in chromosomal fragments.
of microbes, disease diagnosis, bar coading, etc. The
basic methodology of PCR involves three important DNA sequencing is the process of determining the
steps: (i)Denaturation : where the template DNA is exact sequence of order of nucleotides within a DNA
denatured into its single strand components as a molecule. The first major sequencing technology to be
result of the destruction of hydrogen bonds which developed was the “dideoxy method” or the “chain
holds the two strands together. It is often carried out termination” method or the “sangers sequencing”
at 94ºC (ii) Annealing: here the primers, which are method. Here, the technique uses DNA polymerases
short stretches of DNA, attach to complementary to synthesis DNA strands and the main feature of
sequences in the template strand. Annealing occurs the technique is the inclusion of dideoxy nucleotides.
at lower temperatures in the range of 50ºC to 60ºC Dideoxy nucleotides lack the 3’ oh group to form the

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

(iii) Extension: DNA synthesis by DNA polymerases phosphodiester group and hence when a dideoxy
now begins at a temperature of 72ºC, which is the nucleotide is added to the growing chain, it gets
optimum temperature for most DNA polymerases. The terminated. Four different dideoxy nucleotides are
cycle of denaturation-Annealing-Extension is repeated used (ddA,C,T,G) so that the synthesis is not always
to obtain multiple copies of the same product. terminated at the same nucleotide and the reactions
are carried in four sets with each set having a different
DNA Microarray is a technique that allows large dideoxynucleotide.This results in a set of fragments
scale genomic analysis. DNA Microarray analysis is of varying lengths which are then analysed by gel
a hybridization based technology which enables electrophoresis and the sequence is determined by
to investigate and address issues which were analysing the distribution of the fragments in the
previously impossible. It is mainly used to study gel. Sanger sequencing methods are used even today
gene expression and a typical a microarray uses for genomic sequencing, however its cost and larger
DNA as the hybridization probes and thousands of time required for sequencing limits in applications.
such probes or spots maybe present in a single chip,
which enables the large scale analysis of multiple Pyrosequencing is another sequencing technology used.
samples. These probes are immobilized on a solid This is a single nucleotide addition mechanism which
support (a microscope glass slides or silicon chips makes use of a DNA polymerase and a chemiluminiscent
or nylon membrane). The mRNA in the tissue is enzyme. The process involves synthesis a complementary
isolated and is converted to cDNA using fluorescently strand to the template, one base at a time and detecting
labelled nucleotides. The cDNA is then incubated which base was added based on the fluorescent signal.
in the microarray and allowed to hybridise.  The After the fluorescent detection, synthesis is resumed.
unbound particles are then washed off. Only those The light emitted is recorded as pyrogram which
target sequences which are attached strongly to the corresponds to the order of the nucleotides added
probes remain in the chip and these fluorescently which in turn gives the sequence of the sequence of
labelled target sequences generate a signal which template DNA. Pyrosequencing have become highly

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 automated and capable of handling large amount of larger capacities. Bio informatics has mainly three
data and in less time. aims:–to organise the existing data in a way which
allows easy access of existing data and submission
Shot gun sequencing techniques are used in the of new entries as they are produced. Secondly, it
sequencing of long DNA strands. The longer aims in the development of tools and resources
sequencing subdivided into smaller groups and for the analysis for data generated. The third aim
the small fragments are sequenced using the is to generate results using tools from these data
chain termination method. Several rounds of in a biologically meaningful manner. The main
such sequencing are carried out using different driving force behind bioinformatics is the search
fragments and the overlapping fragments are then for similarities between different biomolecules.
assembled into one long sequence with the aid of Apart from enabling systematic organisation of
computer programs. As the size of the molecule to data in databases, bio informatics tools helps in
be sequenced increases, shot gun analysis becomes the prediction of secondary structure (Expasy),
more complex and leads to more errors, especially in construction of phylogenetic tree (clustal W), develop
repetitive regions. This is solved by the development antibody, annotating whole genomes, management
of a genome map. A genome map shows the exact of microarray data (Array Track) etc.
position of genes in a chromosome and the relative
distance between genes. Once a genome map has Table 1. List of databases in use
been developed, sequencing can be carried out by
short gun method using the landmarks or distinctive
features on the genome map as reference.

Genomic mapping can be divided intogenetic mapping

and physical mapping. Genetic mapping makes use
of genetic techniques to construct maps showing
the position of genes and other sequence features
on the genome. It makes use of DNA markers such as
Restriction Fragment Length Polymorphisms, Single
Nucleotide Polymorphisms, Simple sequence length
polymorphisms etc. These are DNA markers which
do not contain any genetic information. However,
maps based on genetic methods alone are rarely
sufficient in aiding the sequencing projects because
of their lower resolution and limited accuracy. Thus,
physical maps are developed with techniques such
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as (i) restriction mapping: which locates the relative

position of DNA based on the recognition sequences
for restriction endonucleases (ii) Fluorescent in situ (Source: Luscombe N. M., Greenbaum. D., Gerstein.M.2001. Review
Paper: what is bioinformatics? An introduction and overview. Year book
Hybridization: in which marker locations are identified of medical informatics)
using a fluorescent hybridizing probe containing the
marker of intact chromosomes or (iii) sequenced Applications
tagged site (STS) mapping, in which the position of • Drug development, diagnostics and
short fragments of DNA are mapped by examining prognostic development.
collection of genomic DNA fragments by PCR and or • Genotyping to predict patient susceptibilities
hybridization analysis. to diseases.
• Molecular medicine.
As the scope of genomics grew over the years, the • Microbial Genomics.
field of bio-informatics developed and evolved in • Bioarcehology, Anthropology and Evolution studies.

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• Personalised health care based on an individual’s not contain sufficient information to understand
genomic features. the gene products. There is very little correlation
• DNA identification/Forensics. between gene expression and protein function.
• Agriculture, Plant breeding. The time point of gene expression varies from that
• Livestock breeding. of the protein product, and the activity displayed
• Metagenomics by the protein will become unpredictable by
the gene, Post transcriptional splicing and RNA
Proteomics recombination leads to various protein products
and thus will multiply the number of components
The term proteomics and proteome was introduces and functions. Also, the rate of synthesis and
in 1995 by Maurice Wilkins & colleagues to emulate degradation of nucleotides and proteins vary and
the concept of genomics and genome. It can be their individual stability gives rise to discrepancies
defined as the “large-scale characterization of the in their correlation. In addition, proteins undergo
entire protein complement of a cell line, tissue, various post translational modifications like
or organism”. Proteome refers to the protein addition of functional groups such as sugar or
complement of the genome and proteomics, phosphate. A premature protein is enzymatically
its study. A proteome indicates the quantitative cleaved to become active or intra-or inter molecular
protein expression profile of a cell, an organism interactions give rise to functional oligomeric
or a tissue under any defined condition. However, proteins. All these modifications give rise to the
the concept of proteome is not as literal as it may complexity of the protein products. The different
seem. It essentially deals with understanding the types of biological information cannot be hence
multiprotein systems; the interplay of multiple, correlated or linked quantitatively. The study of
distinct proteins and their functions as part of a genomics and proteomics are different entities,

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

larger complex network or system. thus, becomes imperative.

Essentially, genomics and proteomics are interlinked Proteome Analysis

and synergistic and yet, they are remarkably
different. Genomics has several fundamental Measuring the protein expression or protein analysis
restrictions, since the sequence of the gene does poses many analytical challenges. Analysis of

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 gene expression is made possible by microarrays and can be visualized with naked eye using non
which rely on two essential components: PCR and fluorescent dyes such as silver nitrate or coomassie
hybridization of oligonucleotides to complementary blue or fluorescent dyes. A protein profile map
sequences. Such tools are not available with protein can be drawn by image analysis, displaying and
analysis. Also, since proteins are post translationally matching the spot pattern on computer screen. This
modified, multiple protein products will be present can be then be compared with related proteome.
for a particular gene and differentiating between The spots obtained by 2D-PAGE can also be used for
these products adds to the challenge. However, protein identification through Mass spectrometry.
despite of all these challenges characterizing Although, there are several gel free strategies
the proteome and its components is achievable. available today for protein identification, 2D PAGE
This was made possible with the development of remains a popular method.
techniques which provide sensitive and specific
means to identifying and characterising proteins. Chromatography is any separation technique that
The combination of sequential methods exploiting distributes the components of a mixture between
different properties can provide high resolution two phases, a fixed stationary phase and a free
analysis of very complex mixtures. The present mobile phase. In proteomics, liquid chromatography
analytical strategies can reach different level of is more often used than other chromatography
resolution, depending on platform used. formats because of its versatility and its compatibility
with MS. In Liquid chromatography, the stationary
2D gel electrophoresis and multidimensional liquid phase is a porous matrix, usually in the form of
chromatography are the two methods that are packed beads that are supported on some form of
predominantly used in protein separation. The two column. The mobile phase, a solvent containing
techniques used differ in their sensitivities and high dissolved proteins or peptides, flows through the
throughput possibilities. The 2D PAGE or the two column under gravity or is forced through under
dimensional poly acrylamide gel electrophoresis high pressure. The rate, at which the protein
was developed by Patrick H.O’Farell by combining mixture flows through the column, depends on its
two electrophoretic techniques:SDS PAGE and affinity for the matrix, and matrices with different
Isoelectric focussing. The method uses two non- chemical and physical properties can be used to
related physical properties sequentially. In the first separate proteins and peptides according to different
dimension, proteins are separated owing to their selective principles. The different types of liquid
migration in an immobilized pH gradient. This chromatography used in proteomics are below:
method is also called as iso-eletric focussing. It is an
electrophoretic method, where protein molecules Liquid Chromatography
move under the influence of electric current. Proteins
being amphoteric molecules, presents a negative or Protein subsequent to purification can be used for
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positive charge in their ionised groups depending acquisition of protein structure information and
on the pH of their surrounding medium. When protein identification. One of the earliest methods
the electric current is applied, the proteins migrate used for protein identification was microsequencing
till it reaches the pH where its net charge equals by Edman chemistry (known as Edman Sequencing)
zero (known as pI of Protien). At this point protein to obtain N-terminal amino acid sequences. Although
migration cease, and the proteins get focussed. At the use of Edman sequencing is waning in the field of
this point, the second dimension is applied. The proteomics, it is still a very useful tool particularly so
proteins are now separated according to their mass. because it helps to obtain the N-terminal sequence
The IEF strips are equilibrated with SDS detergent of a protein (if possible) to determine its true start.
which imparts a negative charge to the protein. An It involves labelling the N-terminal amino acid
electric current is applied and the protein moves sequence of a protein or peptide with Phenyl
based on their charge under the influence of the isothiocyanate. Mild acid hydrolysis then results
current. The separated proteins appear as bands in the cleavage of the peptide bond immediately

184
 Liquid Chromatography

Ion Exchange Size Exclusion Affinity Hydrophobic Reverse Phase-

Chromatography Chromatography chromatography Interaction HPLC
chromatography(HIC)
Separates proteins Separation of molecules Affinity chromatography Similar to
based on reversible ionic based on size. The partitions proteins or Separates proteins with HIC, involving
interactions between the stationary media will peptides on the basis differences in hydrophobicity. hydrophobic
proteins and the oppositely have a definite pore of their specific, ligand The separation is based on interactions. The
charged groups on the size and molecules binding affinity. the reversible interaction interactions are
stationary phase. It is of of different sizes pass between a protein and the much stronger, and
2 types: cation exchange through the media at hydrophobic surface of the require harsher
and anion exchange. different rates. chromatographic medium. conditions for
chromatography elution.

adjacent to the modified residue, but leaves rest interaction while MALDI-TOF is more adapted to high
of the residue intact. The terminal amino acid (or throughput approaches and is therefore suited for
rather its phenyl hydrodantoin residue) can then be large scale proteomics. The mass analysers used in
identified by chromatography and the procedure is proteomics are of four type, viz., triple quadruple
repeated on the next residue and next, thus building (TQ), time of flight (TOF), ion trap and Fourier
up a longer sequence. transform ion cyclotron resonance (FT-ICS) analysers.
MS instrumentation can provide mainly three types
Mass spectrometry has become an indispensible tool of highly useful analysis in proteomics:–(i) MS can
in proteomics for acquiring the protein sequence provide accurate molecular mass measurements of

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

information. Mass Spectrometer is instrument that can intact proteins (ii) definitive identification of target
measure the mass/charge ration of ions in vacuum. proteins by digesting the protein with enzymes to
From these data, molecular masses can be determined obtain peptides and comparing the peptide mass
with high accuracy, allowing the composition of against databases and (iii) sequence information of
a given sample or analyte to be determined. In the protein using tandem mass spectrometry.
proteomics two types of analysis can be carried out: (i)
Analysis of intact peptide ions: this allows the mass of Proteins have evolved under selective pressure to
intact peptide ions to be calculated, and these masses carry out different functions which depends on the
can be used to identify proteins in sample by database way in which proteins interact with other molecules.
searching (ii) Analysis of fragmented ions: This enables This interaction ultimately depends on the three
the mass of peptide fragments to be determined dimensional structure of the protein – its overall shape,
which can be used in correlative database searching presence of clefts, cavities complementary to particular
to derive de novo sequences. Mass spectrometers ligand/substrate, distribution of charges internally
have three main components: a source of ions, a and on the surface, positioning of key amino acids
mass analyser and an ion detector. The ionization etc. Thus, structural analysis of a protein is the key to
source converts the analyte into gaseous phase ions understanding its biological function. The two major
in vacuum, ions are then accelerated in an electric techniques that can be used for this purpose are X-ray
field towards the analyser. The analyser separates the crystallography (XRC) and Nuclear magnetic resonance
ions based on their m/z ratios and the ions finally hit (NMR) spectroscopy. X ray crystallography exploits
the detector, which record the impact of individual the fact that X rays are scattered or diffracted in a
ions. MS in proteomics can be categorised into two predictable manner when they pass through a protein
based on the mode of ionisation as: MALDI-TOF MS crystal. Protein molecules, when arranged regularly in
(matrix assisted laser desorption ionisation-time of a crystal, diffract X-rays and the nature of diffraction
flight MS) and ESI-MS (Electrospray Ionisation MS). depends on the nature of electrons that are present
ESI MS is mainly linked to liquid chromatographic in each atom and the organization of atoms in space.

185
 The purified protein sample at high concentration is Tertiary structure of a protein can be predicted with
crystallised and the crystals are then exposed to an x ray reasonable accuracy if the structure of a closely related
beam. The resulting diffraction patterns can then be protein is available and can be used as a template.
processed, initially to generate information about the This approach is known as comparative modelling
crystal packing symmetry and the size of the repeating or homology modelling and generally works well if
unit that forms the crystal. This information is obtained the two sequences show greater than 30 % identity
from the pattern of the diffraction spots. The intensities over 80 or more residues. The first step is to find
of the spots are further analysed to determine the suitable templates, which is achieved by searching for
“structure factors” from which a map of the electron homologous protein sequences and identifying those
density can be calculated. Various additional methods with solved structures. When a suitable template has
are thereafter used to improve the quality of this map been found, the sequence of the template protein(s)
until it is of sufficient clarity to permit the building of and query protein are aligned using an algorithm such
the molecular structure using the protein sequence. as clustal W. Different softwares use slightly different
The resulting structure is further refined to fit the map methods, a common strategy is to identify residues in
more accurately and to adopt a thermodynamically the template that are part of the proteins structural
favoured conformation. core and those forming the surface loops. The main
disadvantage of comparative modelling methods is
Nuclear magnetic resonance is a phenomenon that that only structures with suitable templates can be
occurs because certain atomic nuclei have magnetic modified. In contrast, ab initio methods can predict
properties. These properties are exploited to obtain protein tertiary structures from first principles i.e. in
chemical information in NMR spectroscopy. NMR the absence of any structural information. A typical
spectroscopy is used to determine the structure procedure would involve defining a mathematical
of proteins in solution in size range between representation of polypeptide chain and the
5 and 25 KDa. surrounding solvent, define an energy function that
accurately represents the physicochemical properties
Although the technology for solving protein of proteins and use algorithm to search for the chain
structures has advanced significantly, it still conformation which possesses the minimum free
remains a labour-intensive and expensive process. energy. Ab intio methods are often not used for
An alternative, although somewhat less accurate, polypeptides greater than 200 residues for the fact
method for obtaining structural information is to that larger polypeptide chains can fold into potentially
predict protein structures using bioinformatics tools. infinite number of different structures.
Secondary structure of proteins can be predicted
using three state predictions because residues in a Integration of all the above techniques encompasses
protein can be present in any of the three states, the current technologies used in proteomics. The large
which are alpha helix, beta sheets and coil. Early scale analysis of proteins has generated huge amounts
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methods for three state predictions were based on of data due to the new technologies in proteomics.
the statistical likelihood of particular amino acid Protein sequences database play a vital role as a central
appearing in a given type of structure. For example, resource for storing the data generated. Several large
some amino acids such as glutamate, have helical scale protein databases act as repositories of protein
propensity and hence more likely to occur in alpha information. Some of them are GenPept, NCBI Entrez
helices than anywhere else in the protein. While protein, RefSeq, Swissprot, PIR-PSP (Protein information
other residues such as valine have strand propensity resource-proteins sequence database), TrEMBL etc.
i.e. they are abundant in beta sheets and strands.
Currently, there are sophisticated structural prediction Applications of proteomics
algorithms such as PSI- PRED which uses multiple
alignments and also incorporate evolutionary and Protein Mining: It is the identification of all (or
structural information from databases to increase as many as possible) the proteins present in a
the accuracy of prediction. sample. Mining enables the cataloguing of the

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proteome and reduces the dependence on gene environment”. They are the intermediate and end
expression patterns for identifying composition product of any metabolism and provide information
of a proteome. on the physiology of the cell. Metabolites, unlike
genes and proteins are not subject to regulation and
Protein Expression profiling : This involves the post translational modifications, and hence serve as a
identification of proteins in a particular sample as direct signature of the biochemical activity. Therefore,
a function of the particular state of the organism they are also easier to correlate with the phenotype
or the cell ( eg. Disease state, developmental of an organism. In this context, metabolomics or
state, differentiation state etc.). Protein expression metabolic profiling has become a powerful strategy in
profiles are used in cancer research, for example biochemical profiling of cells for various applications
to identify tumour subtypes and to obtain a more such as disease diagnosis, therapeutic purposes etc.
reliable classification.
“Metabolome” refers to the complete repertoire
Protein Network Mapping: Network mapping of small molecules in cells, tissues, organs, and
is the study of how proteins interact with each biological fluids. The term metabolome was coined
other in a living system. All proteins work in close in synergy with proteome & transcriptome and like
association with each other. Complex network of both; metabolome is dynamic and changing every
protein interactions can be characterised through second. It includes products or metabolites from
methods such as affinity capture techniques. It offers a large network of metabolic reactions where the
the ability to study the status of all the components product of one reaction serves as the input for other
in a biochemical pathway and is one of the most chemical reactions. Hence, it is the combination of
ambitious and powerful potential future applications metabolites, rather than individual metabolites, which
of proteomics. are of biological relevance.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Mapping of protein Modifications: Here, the The concept of metabolic profiling existed as early as
main task includes the identifying the mechanism in the 1940’s. The metabolic profiling of saliva and
of protein modification. A variety of analytical urine were first performed by Roger Williams, who
techniques are available with which it is possible to identified the difference in patterns of metabolic
study and identify modified proteins and also the profiles for patients associated with Schizophrenia.
nature of modifications. The term metabolic profiling was coined in 1971
by Horning, who used Gas chromatography- Mass
Metabolomics spectrometry to measure the components of urine
and tissue extracts quantitatively. By the 1980’s,
Metabolomics is the systematic study of the unique NMR technologies which could identify metabolites
chemical fingerprints that specific cellular processes in unmodified biological samples were developed.
leave behind. In simple terms, it is the overall study NMR based metabolomics took shape in 1984 by the
of metabolic expression at any given point in time. research works done by Jeremy Nicholson who used
Metabolomics is a rapidly emerging field and it HNMR to diagnose Diabetes Mellitus. Metabolomics
combines several strategies to identify and quantify since then has come a long way and in 2005, the first
cellular metabolites using sophisticated analytical metabolomics database called as the METLIN was
technologies with the application of statistical and developed to characterise the human metabolites.
multi-variant methods for information extraction and With over 60,000 deposits, METLIN is currently the
data interpretation. largest repository of data in metabolomics.

Metabolites are the smallest chemical components Metabolome analysis

which are present in any cell at any time and are
known to “act as spoken language, broadcasting Metabolomic assessment can be done using cells,
signals from the genetic architecture and the fluids or tissues. Biofluids are the easiest samples to

187
 work with and include serum, plasma, urine, ascitic tools. Metabolome software such as XCRS enables
fluid, saliva, bronchial washes, prostatic secretions or the users to upload data, perform data processing
fecal water. The Initial step in metabolome analysis and browse results within a web based interface.
involves freezing of all metabolic processes within
the cell using physical agents such as liquid nitrogen. The metabolomics software does not produce any
Following which the metabolites are extracted by metabolite identification. Instead, it searches for
alkali, acid or ethanol extraction. The extracted peaks which vary in groups of sample and the peaks
sample is then analysed for profiling. Subsequent of interest are selected and generate putative matches
to extraction, the sample is then separated using from selected databases by comparing the m/z ratio
chromatographic techniques. Since the physic- of the selected peaks. This must be now confirmed
chemical nature of the different metabolites in a by additional MS/MS and comparing the MS/MS
metabolome is different and metabolites in tissues fragmentation data and retention time with standard
or body fluids are present in a broad range of compounds. Even though metabolite databases
concentration, no single analytical method is capable have increased considerably over the last decade,
of analyzing all the metabolites. Hence, multiplexed a large number of metabolite features detected
methods of separation are used for separation from biological samples do not return any matches.
and extraction. This also increases the number of Identification of these unknown features requires
compounds that are detected. For example, extracting denovo characterization using traditional methods.
the same cell with aqueous and organic solvent Thus, it is important to note that identification of
enhances the number of hydrophilic and hydrophobic all metabolite features detected by LC/MS is not
compound detected. Similarly, using two separation completely possible.
techniques like reverse phase chromatography and
hydrophilic interaction chromatography increases Metabolome study or analysis can be carried out in
the variety of metabolites purified. Reverse phase two approaches:-targeted approach and the Non-
chromatography and hydrophilic interaction targeted approach.
chromatography purifies hydrophobic and hydrophilic
compounds respectively. Targeted approach
Subsequent to purification, the sample is analysed, This approach to metabolomics is when there is a
commonly using mass spectrometry techniques. specific biochemical question or a hypothesis to be
Quadruple TOF MS are often used due to its increased answered or studied like for instance, studying the
sensitivity and reliability, but other TOF and ion trap effect of a drug. This is the most developed approach in
instruments are also used. MS technologies using metabolomics that is used to measure the concentration
MALDI are also used in metabolome analysis but it of a limited number of chemically related metabolites
results in significant background at lower molecular like amino acids or hormones and often used in the
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weights which complicates the analysis. Other surface pharmacokinetic analysis of drug metabolism, measuring
based technologies for MS analysis of metabolome the influence of a drug or genetic modifications of a
include secondary ion mass spectrometry (SIMS), specific enzyme. Triple quadrupole-MS methods are
Desorption electrospray ionisation (DESI) etc. NMR often used in targeted analysis since these methods
techniques are also employed for metabolic profiling are quantitatively reliable and offer the absolute
and are the only detection technique which does not quantification of even low concentration of metabolites.
rely on the separation of analytes. NMR enables the It is a well established approach in the diagnosis of
detection of all types of metabolites and is highly inherited metabolic disorders.
reproducible and simple, but is relatively insensitive
when compared to mass spectrometry. Non-targeted Approach
The metabolite peaks obtained using mass Non targeted approach is global in scope and is used
spectrometry is then analysed using bioinformatics for simultaneous measurement of as many metabolites

188
as possible in a biological sample irrespective of its screening and for following a patient clinically to
chemical class. It can be carried out using either monitor target organ effects.
NMR or MS technologies. Liquid chromatography
coupled with Mass spectrometry is the most common • Therapeutic Metabolomics: Most of the drugs
method of choice for metabolic profiling and can be act at the level of metabolites and metabolomics
used in the detection of almost all analytes. Unlike is a more direct measure of the action of a drug.
targeted approach, non targeted approach generates Thus, measuring the biochemical status using
vast amount of data which are complex and large metabolomics helps in understanding how the
to analyse. Introduction of metabolome software disease manifests, how drugs work and also in
such as MathDAMP, MetAlign, MZmine and XCMS identifying responders and non responders to
has made the analysis of untargeted metabolome treatment. It also involves identifying altered
data easier. Untargeted metabolomics approach has metabolic pathways in diseases that represent
the potential to provide greater insights into many novel drug targets. Example: In cancer cells with
fundamental biological processes. It helps reveal many isocitrate dehydrogenase mutation, an increased
previously uncharacterised metabolites with respect level of metabolite 2-hydroxyglutarate was
to their structure and function. observed. The result suggests the inhibition of
2-hydroxyglutarate as a therapeutic approach to
Applications the cancer.

Metabolomics was originally proposed as a method of • Biomarker discovery for disease identification:
functional genomics, but now its applications extend Here, metabolite profiling is used to generate
well beyond that. It is used for comparing mutants, quantitative data of metabolites from control
assessing responses to environmental stress, studying populations and test population with disease.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

global effects of genetic manipulation, comparing The data is then analysed to and determine which
different growth stages, toxicology, drug discovery, metabolites are discriminatory for the disease
nutrition, cancer, Diabetes and natural product and which of these could be used in predictive
discovery. Some of the important functions are medicine. Characterization of function of unknown
described below: gene and proteins by screening for metabolites
that accumulate after gene metabolism or
• Toxicology: This type of metabolic profiling enzyme inhibition etc. Also in the determination
enables understands the physiological changes in of phenotype as a result of genetic manipulation
response to a toxic insult of a chemical or a mixture such as gene deletion or insertion.
of chemicals. It can be used as a biomarker of
hepatic, renal and lung toxicity by analyzing various • Metabolic foot printing: Metabolic foot
metabolites such as glucose, lactate, lipoproteins printing refers to the extracellular metabolite or
and amino acids which may either increase or exometabolome characterization. The footprint of
decrease thus providing for a recognizable pattern the extracellular medium consists of the medium
associated with an organ dysfunction. Though components (less the substrate uptake) and the
much of the data have not been validated and secreted metabolites. This is applicable only in the
some overlap exists between various toxins, the case of cell culture medium and is useful in studying
pattern, temporal rate of change and extent of the behaviour and responses of the cultured cells.
change in metabolites can still provide toxicity This has potential clinical applications especially in
assessments which can be used for preclinical drug IVF treatments.

189
 Functional Genomics
M. P. Paulton
Senior Technical Officer (Training)
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction Functional
The sequence information comprising of billions of genomic approaches
bases do not communicate the role of the genes
and how they are influencing the formation of The targets in functional genomics is achieved
organism, aging, biological pathways and what through the integration of different branches such
changes in them lead to diseases etc. Functional as transcriptomics, proteomics, metabolomics,
genomics comes into action to answer the above nutrigenomics and epigenetics. Transcriptomics deals
queries and it provides information about the with the quantification gene expression at the transcript
role of different genes in comprising the function level. Proteomics focus on the expression of specific
of cells and organism. The science of functional proteins and their structures. Metabolomics explains
genomics helps the researchers to find out the various metabolites produced in cell during various
gene expression and its regulation and its impact phases in cell cycle. Interactomics is another branch
on complex production traits at the genome level. with applications in agriculture as they focus on the host
The goal of functional genomics could also be pathogen interaction. Another area namely epigenetics
described as to find out the biological factors studies the non-inheritable changes taking place at the
behind a phenotype and its expression in different genome and its impact in phenotype. Nutigenomics
situations or environment. With the advancement deals with the how diets are affecting gene expression.
of sequencing techniques, researchers can directly All these branches are working together to meet the
go into the sequence variant lines and sub types challenges in food security and to ensure sustainable
and thereby to identify the polymorphic forms such production. Further it is anticipated in long term that the
single nucleotide polymorphisms (SNPs) and their results of functional genomics are properly integrated
impact in regulating the expression of certain genes. with quantitative genetics and ultimately expected to
Functional genomics provides a reliable platform contribute improved agricultural productivity.
to understand gene environment interaction at the
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genome level and thereby to design strategies in Single and multiple

breeding and production. Further, the elements
such as non-coding RNAs (ncRNAs) and introns
gene expression
conventionally believed as non-functional are profiling methods
getting into limelight with the advancement in
sequencing technologies. Many such hitherto Analysis of the single gene expression is normally carried
unrevealed entities are coming up and even now out by a variety of techniques which include Northern
the real challenge in functional genomics is not Blotting, Ribonuclease Protection Assay (RPA), Reverse
in obtaining new data but in analysing it. A series transcription PCR and quantitative real time PCR. Expression
of techniques are available and also emerging in profiling of more number of known mRNA is possible by
functional genomics with specific focus on their procedures like Differential Display (DD), Representational
application to enhance production in agricultural, difference analysis (RDA) and suppression subtractive
livestock and aquaculture systems. hybridization (SSH). As the technological advancement

190
increases, following techniques based on sequencing the tag of 10-14 bp in length. In this technique C-DNA is
transcriptome is getting popular. synthesized from mRNA through reverse transcription
and the unique identifying sequence is extracted,
Transcriptomic techniques ligated into a concatemer and the quantification of
the same done through sequencing. This sequencing
Transcriptome constitute the whole population based technique is useful in developing bio markers
of RNA such as m RNA, r RNA, micro RNA and and also enable the identification of differentially
other regulatory RNAs and the study of this expressed genes not revealed through Microarray.
contribute the field of tanscriptomics. The popular
techniques in transcriptome studies are Expressed Massively Parallel signature
sequence Tag(EST), microarray, and serial analysis
gene expression (SAGE). Many candidate genes
sequencing (MPSS)
of commercial importance which could be used in In this process, the cDNA produced from experimental
breeding programmes are being isolated. Further the RNAs are tagged and attached to microbeads for flow
evolution of inflammatory response markers through cell sorting to quantify RNAs. This is useful in studying
transcriptomic studies helped to detect the nutritional gene expression of species with little knowledge on
additives negatively regulating the growth of farmed sequences like pacific oyster.
animals like Atlantic salmon.
Transcriptome
EST and Microarray analysis sequencing (RNA-Seq)
Genomics involve the generation of DNA sequences This is the novel and emerging technology based on
and DNA markers, genetic linkage, physical maps and next-generation sequencing enable the researcher

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

QTL mapping, gene discovery and the genetic control of to simultaneously identify the novel regions of the
complex traits and metabolic pathways .There exist three
levels of genomic branches namely structural genomics,
functional genomics and comparative genomics.
Essential steps or targets of functional genomics involves
the gene expression profiling though monitoring RNA
and protein expressions following proteomic and
transcriptomic tools and also find out mutant forms
of gene. It co relates the gene function and gene trait
relationships through gene expression analysis with the
advanced procedures like DNA microarray. Expressed
Sequence Tags (ESTs) are generated by creating libraries
of mRNAs represented as different clones. The sequences
from ESTs are properly clustered to create EST assemblies
which form the basis to develop microarray probes and
also to generate data for gene identification. National
Center for Biotechnology information (NCBI) keeps
ESTs numbering into millions and serve as a major
database support.

Serial analysis of gene

expression (SAGE)
SAGE works on the principle that each RNA molecule
could be identified through a unique short sequence

191
 sequences generated as well as the quantification of inhibits or negatively regulates muscle growth in
the transcript and thereby measures relative abundance. animals and it is conserved in vertebrates. Interestingly
These advantages make it more suitable than microarray a mutant or polymorphic form of the gene has gained
and the use of this technique is getting popular. attention as the phenotypic growth is enhanced to a
great percentage in muscle growth. Hence the much
Functional genes related focus is given to this gene in farmed animals. Recent
years have witnessed research findings from fishes
growth studied in fishes almost in the same line as in livestock animals. This
Specific genes associated with growth related traits has made the research groups to initiate work in
in fishes involve in regulating the growth of fishes in detecting myostatin polymorphisms from wild to be
wild as well as in culture. Their expression pattern is used in marker assisted selection of the brood stock.
also depending on the environmental parameters.
Hence specific strategies are essential to study the
genes controlling the harvest traits living in diverse
habitats. In many cases, the polymorphism of these
genes negatively or positively correlates the growth of
the fishes. In addition to the growth hormone gene
(GH) genes like myostatin (MSTN), a member of the
transforming growth factor-β superfamily and the
insulin-like growth factor 1(IGF1) are two important
genes which influence the growth.

IGF
IGF1 and 2 along with few binding proteins and Myostatin sequences of Lutjanus argentimaculatus

receptors work together under the stimulation by

growth hormone to development and normal growth. Many results co relate the presence of SNPs within
Polymorphism within this highly conserved IGF gene. the exon of the gene and incidence of higher growth
Genetic variations were detected in the IGF1 gene in many fishes including sea bass, Atlantic salmon,
in Artic charr and sinipercid species where they are common carp etc.
found to be associated with growth traits. Single
nucleotide polymorphisms (SNPs) regulating growth Suggested readings
trait positively were also detected in farmed atlantic
salmon, making them suitable for developing markers Teresia Buza and Fiona M. McCarthy. Functional genomics:
for marker assisted selection in aquaculture industry. applications to production agriculture. CAB Reviews 2013
Central Marine Fisheries Research Institute

8, No. 054.
Carolina Peñaloza, Alastair Hamilton, Derrick R Guy, Stephen C
Myostatin (MSTN) Bishop and Ross D Houston. A SNP in the 5′ flanking region
of the myostatin-1b gene is associated with harvest traits in
Atlantic salmon (Salmo salar). BMC Genetics 2013, 14:112.
Myostatin is a member of TGF -β superfamily that

192
Population Genomics of Fishes
Sandhya Sukumaran
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Fishes are the most successful vertebrates in the estimates could now be evaluated and assessed as
planet with around 28,600 extant species compared continuous distributions across the genome.
to 4,629 species in mammals and 9,946 species in
birds. They live in wide range of habitats from below Population genomic approaches are based on
freezing to more than 40ºC and from freshwater to information from many genome wide markers in
hyper saline waters and exhibit different life history contrast to population genetics approaches where
and reproductive strategies. They have varied only a few markers are employed. In addition,
patterns of biological interactions with respect to neutral and non-neutral markers are employed
prey, predator and parasites. This enormous diversity simultaneously so as to gain information regarding
and divergence makes them excellent models for genic and non-genic regions. When both neutral
genomic investigations on adaptation, selection and and non-neutral markers are used, the effects
consequent evolution. In spite of their importance and due to selection, mutation and recombination
diversity, genome level investigations on fishes are still on populations (locus specific effects) could be

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

in infancy and most of the studies were focusing on differentiated from the effects due to genetic drift,
a few model species. Genomic studies on fishes will inbreeding and gene flow (genome wide effects).
provide vital and revolutionary insights into ecological When neutral markers alone are used, information
speciation and adaptation. about adaptive evolution is missing whilst adaptive
divergence plays a major part in evolution of resident
Population genomics and populations with improved fitness for that particular
environment. Knowledge about locally adapted
population genetics populations also aid in suggesting conservation
Over the last few decades, population genetics has measures aimed conserving genetic diversity
evolved from a theoretical science to a fully developed over ecological time scales. It is also important
empirical science with the extensive use of neutral to understand climate mediated micro-evolution
markers like microsatellites and mitochondrial DNA and information about selection and divergence is
markers to infer genetic stock structure information. crucial to predict climate related habitat shifts and
Thus with the tools of population genetics, inferences movements of marine fishes.
regarding genetic drift, gene flow and inbreeding
could be made with reasonable precision and this has Finding out the footprints of selection on a
improved our understanding regarding evolutionary particular trait at genome level is not an easy task
processes shaping population structure in varied as several genes are involved in determining the
oceanic habitats. With the advent of next generation characteristics of an adaptive trait and consequent
sequencing techniques, population genetics is moving variations in phenotype. In this context, the relevance
towards more inclusive population genomics where of population genomics is more emphasized as
both neutral and non-neutral regions could be studied population genomics aims to gain insights into
simultaneously by whole genome scans with increased genomic variations in several functional genes linked
speed and efficiency. Population genetic statistics like to adaptation and selection within and between
Wright’s F statistics which were considered as point populations in space and time. Expressed Sequence

193
 Tags sequencing (EST), transcriptome sequencing like genes linked to ESTs. Genes linked to ESTs could
and complete genome sequencing are some of be detected through transcriptome sequencing
the methods used to infer information about and genome scan approaches help to detect
functional gene modifications linked to differing loci under selection.
environmental conditions.
Quantitative trait locus, admixture
Approaches in and association mapping
population genomics QTL mapping is based on the information from linkage
maps constructed using a dense set of markers which
Candidate Gene Approach provides information regarding the genetic basis
underlying phenotypic traits through family crosses
Candidate genes are functional genes having a under controlled laboratory conditions. QTL information
known function which has a major influence on could be compared among closely related species which
an adaptive trait. They can be genes linked to helps to minimize cost. QTL studies require controlled
variations in structure of fishes or genes related to experimental conditions and so species amenable to
some physiological processes and they are directly aquaculture will be the best candidates for quantitative
or indirectly linked to phenotypic variation. The trait loci related investigations.
information regarding functional gene variations
can be derived from sequence information or allele Association mapping depends on the information
frequency based tests. Sequence based data is more regarding linkage disequilibrium between large
informative to understand the action of evolutionary number of genetic markers and we try to link linkage
forces. The candidate gene approach is relatively disequilibrium information with phenotypic variations.
faster with the help of comparative genomic Admixture mapping is used to detect natural events
approaches which enable efficient characterization of admixture like intra-specific hybrid zones and the
and comparison of different genes and it does information regarding linkage disequilibrium in wild
not require any controlled experimental set up as populations is utilized for this purpose.
far as a direct linking with phenotypic variation
is not desired. Variations across populations Seascape genomics approaches
could be efficiently detected and the influence of
selective pressures in relation to environmental In Seascape or landscape genomics, information
fluctuations could easily be deduced. The only obtained by the use of genetic markers is combined
challenge with candidate gene approach is to with geographic information so as to derive geographic
identify the correct candidate gene related to the patterns of genetic differentiation. Both neutral and
trait under study and the need for undertaking non-neutral markers are employed in sea scape
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time consuming sequencing. genomics so that information regarding selection

could be deduced whenever there is divergence in
Genome Scan Approach non-neutral markers based on geographic location.
The presence of genetic divergence in non-neutral
Genome scan approach is based on the principle markers based on geographic locations is indicative
of genetic hitchhiking whereby identification of of locally adapted populations.
regions or loci showing high amount of structuring
due to selection is studied. Selective forces acting Transcriptomics approaches
on such loci may either due to balancing selection
or directional selection. This requires large numbers Transcriptomic approaches are based on the data
of genetic markers which are widespread in the regarding differential gene expression patterns and
genome which can be random markers like mass scale RNA sequencing enables identification
microsatellites, SNPs or AFLPs or functional markers of differential gene expression patterns in real

194
time and this method can be applied to all species out substantial divergence in morphological and
due to the ease of transcription profiling and physiological characteristics.
annotation simultaneously. Microarray are used
to measure differential expression of hundreds Genomic resources are available for some of the
or thousands of genes and real time PCR is used model species like zebra fish, Danio rerio; Japanese
to measure differential gene expression of one puffer fish, Takifugu rubripes; medaka, Oryzias latipes;
or a few candidate genes. Real time PCR follows Spotted green puffer fish, Tetraodon nigroviridis and
the principles of normal PCR and the product is three-spined stickleback, Gasterosteus aculeatus.
measured in real time by tagging the reaction There are large scale genome sequencing projects
mixture with fluorescent dyes and measuring for some of the species like flat fish (PLEUROGENE)
the fluorescence after each PCR cycle which and Atlantic cod. In addition to that, linkage maps,
corresponds to expression levels of a gene. But BAC libraries and microarrays are being developed
with real time PCR studies, only a small number worldwide for many species which are relevant to
of genes within a genome could be studied. aquaculture. With the level of genomic information
DNA microarrays are designed to investigate available for species like European Seabass, Japanese
gene expression of thousands of genes at once. flounder and gilt head sea bream, it is now
Microarray are thousands of single stranded DNA possible to link phenotypic variations and genome l
spots attached to surfaces like glass slides and evel alterations.
each spot corresponds to a gene. To measure
the gene expression, fluorescently tagged cDNA EST information is also increasing rapidly for
is washed over the slide, so that complementary many fish species like Three spined stickle back
strands hybridize on to the array and fluoresce. ( Gasterosteus aculeatus ), Atlantic cod ( Gadus
The intensity of each spot is proportional to morhua ), Killifish ( Fundulus heteroclitus ), Gilt

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

the expression of that gene. Gene expression head seabream ( Sparus aurata ), Eurpean sea
studies also require controlled experimental bass ( Dicentrarchus labrax ), Antarctic tooth
conditions so as to relate gene expression data fish ( Dissostichus mawsoni ), Piked dog fish
with adaptive variation. ( Squalus acanthias ), Little skate ( Leucoraja
erinacea), Japanese pufferfish (Takifugu rubripes),
Present status of fish Copper rock fish ( Sebastes caurinus ), Atlantic
halibut ( Hippoglossus hippoglossus ), Turbot
genomics research (Scophthalmus maximus), Grass rock fish (Sebastes
Marine fishes are highly diverse and heterogeneous rastrelliger), Senegalese sole (Solea senegalensis),
possessing large effective population sizes and Pacific electric ray ( Torpedo californica ), Blue
high amounts of gene flow. The high gene flow is fin tuna ( Thunnus thynnus ), Japanese flounder
attributed to pelagic larval phase and consequent ( Paralichthys olivaceus ), Barramundi ( Lates
dispersal of many pelagic and demersal fish calcarifer ), Striped seabream ( Lithognathus
resources. Extensive studies on population genetic mormyrus ), Sheepshead minnow ( Cyprinodon
structure of many fish groups like scombrids (eg: variegates), European flounder (Platycthys flesus),
mackerel, tuna and bonito), clupeids (eg: herring, Long-jawed mudsucker ( Gillichthys mirabilis ),
anchovy and sardine), pleuronectids (eg: plaices, Tongue sole ( Cynoglossus semilaevis ) and Large
soles and flounders) and gadids (eg: cod, hake and yellow croaker (Pseudosciaena crocea). In the
haddock) have been carried out with an aim to find dbEST database of NCBI, over one million teleost
out genetic stock structure and define management EST sequences are currently listed.
units. Most of the studies have concluded weak
genetic structuring and high levels of intra- Several candidate genes have been studied in marine
population diversity values. But, in spite of their large fishes to understand their divergence and selection.
effective population sizes, it is possible to have locally Ectodysplasin (EDA) gene in stickle back, Lactate
adapted populations as many studies have pointed dehydrogenase B (Ldh-B) in killifish and Pantophysin

195
 (Pan I) in Atlantic cod are some of the well studied undertaken to identify variations in gene expression
functional genes in marine fishes. Haemoglobin patterns in killifish distributed across steep thermal
polymorphisms have been studied in Atlantic cod gradients on the east coast of North America. There
which showed clear genetic differentiation across were differing patterns of gene expression in Lactate
populations. Pantophysin gene also showed signals dehydrogenase (Ldh-B) gene for fish kept in controlled
of adaptive differentiation showing diversifying conditions. Similar gene expression studies were also
selection. Heat shock cognate gene Hsc70 has conducted in killi fish to understand differential gene
been studied in European flounder (Platichthys expression patterns in response to chemical pollution.
flesus) and several insertion/deletion polymorphisms In European flounder, a microarray was developed for
have been detected. ecotoxicology studies and to understand differences in
gene expression between populations from saline and
Studies based on genome scans have been conducted brackish water environments.
in three-spined stickle back to derive insights to their
adaptation to freshwater and marine environments. Prospects of population
EST-based microsatellites and EDA gene linked indels
were studied understand divergence in different
genomics of fishes
ecotypes. A mass scale genome scan study was Fishes live in a dynamic environment where they face
conducted in Atlantic Cod identifying many outlier loci many physical, chemical and biological challenges.
potentially subject to selection. The study also proved Adjusting to such varying environmental conditions
that even if geographical differentiation was limited, require altering their phenotypes, moving to more
adaptive population divergence may be possible favorable locations or by adapting genetically to
in fishes. altered conditions. Temporal patterns in changes
in effective population size, migration patterns and
QTL analysis had been carried out in few fishes gene flow are to be studied in detail so as to gain
like three spined stickleback, European seabass, more insights into adapted genotypes and selection
Barramundi and Japanese flounder. QTL analysis in patterns. Population genomic approaches have
three spined stickleback could identify a dominant a great potential to elucidate patterns of natural
gene, Ectodyplasin which determines lateral armour selection in the wild and information from population
plate pattern and number. Another gene, involved genomics could be translated to management
in pelvic skeleton formation, Pitx1 locus was also measures for conservation of marine diversity. The
mapped in stickle backs using QTL analysis. QTLs advent of Next Generation Sequencing technologies
linked with size and growth has been identified in has revolutionized the field of population genomics
both European seabass and barramundi and another expanding the knowledge base with respect to role
QTL linked with disease resistance had been detected and importance of functional genes. More and more
in Japanese flounder. non-model species are to be focused upon so as to
Central Marine Fisheries Research Institute

aid in comparisons and future conservation decisions.

Large – insert libraries (BAC; Bacterial Artificial
Chromosome, YAC; Yeast Artificial Chromosome and Suggested readings
fosmid; A low copy vector which uses Escherichia coli
F-factor origin for replication) construction is another Allendorf, F.W., Hohenlohe, P.A. and Luikart, G. 2010. Genomics
method which is a prelude to full genome sequencing and the future of conservation genetics. Nature Reviews
Genetics 11: 697-709.
and this aims at producing multiple copies of large Cossins, A.R. and Crawford, D.L. 2005. Fish as models for
segments of DNA of the desired organism through environmental genomics. Nature Reviews Genetics 6:324-340.
Crollius, H.R. and Weissenbach, J. 2005. Fish genomics and biology.
cloning. These libraries also serve as tools for genetic Genome Research 15: 1675-1682.
mapping, marker assisted selection and positional Ding, F., Chu, W., Cui, P., Tao, M., Zhou, R., Zhao, F., Hu, S. and
cloning of QTLs for traits that are of interest. Zhang, J. 2011. EST-Based identification of genes expressed
in skeletal muscle of the Mandarin Fish (Siniperca chuatsi).
Genomics, Proteomics and Bioinformatics 9(1-2): 30-36.
Population transcriptomics studies had been Guo, B., DeFaveri, J., Sotelo, G., Nair, A. and Merila, J. 2015.

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 Population genomic evidence for adaptive differentiation in E.A. and Cresko, W.A. 2010. Population genomics of parallel
Baltic Sea three-spined sticklebacks. BMC Biology 13: 19 adaptation in Threespine stickleback using sequenced RAD
Hansen, M.M.,Olivieri, I., Waller, D.M., Nielsen, E.E. and The tags. PLoS Genetics 6(2): 1-23.
GeM working group. 2012. Monitoring adaptive genetic Nielsen, E.E., Hemmer-Hansen, J., Larsen, P.F. and Bekkevold,D.
responses to environmental change. Molecular Ecology 2009. Population genomics of marine fishes: identifying
21: 1311-1329. adaptive variation in space and time. Molecular Ecology
Helyar, S.J., Hemmer-Hansen, J., Bekkevold, D., Taylor, M.I., 18: 3128-3150.
Ogden, R., Limborg, M.T., Cariani, A., Maes, G.E., Diopere, Quere, N., Guinand, B., Kuhl, H., Reinhardt, R., Bonhomme, F.
E., Carvalho, G.R. and Nielsen, E.E. 2011. Application of and Desmarais, E. 2010. Genomic sequences and genetic
SNPs for population genetics of nonmodel organisms: new differentiation at associated tandem repeat markers in growth
opportunities and challenges. Molecular Ecology Resources hormone, somatolactin and insulin-like growth factor-1 genes
11(Suppl.1), 123-136. of the sea bass, Dicentrarchus labrax. Aquatic Living Resources
Hemmer-Hansen, J., Therkildsen, N.O. and Pujolar, J.M. 23: 285-296.
2014. Population genomics of marine fishes: Next – Zhu, M. and Zhao, S. 2007. Candidate gene identification
Generation prospects and challenges. Biological Bulletin approach: progress and challenges. International Journal of
227: 117-132. Biological Sciences 3(7): 420-427.
Hohenlohe, P.A., Bassham, S., Etter, P.D., Stiffler, N., Johnson,

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

197
 Next Generation Sequencing and RAD
sequencing
Sandhya Sukumaran
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

DNA sequence information is fundamental to any copy number plasmid, which is consequently used
research as it helps to decode the mysteries of life to transform Escherichia coli; or in the second
and this knowledge has transformed the way we approach, PCR amplification of the target DNA
perceive natural science for the last two decades. DNA is carried out using primers that flank the target.
sequence analysis has broad range of applications Thus using either of the approaches an amplified
in population genetics, molecular breeding, disease template is produced. Sequencing reactions take
resistance investigations, health care and comparative place in a “cycle sequencing” mode with cycles of
evolutionary studies. DNA sequence development template denaturation, primer annealing and primer
and detection has undergone revolutionary changes extension and the primer used is complementary to
during the past 30 years moving from first generation the sequences flanking the region of interest. Every
to third generation technologies. First draft human step of primer extension is terminated stochastically
genome was completed in 2003 using the Sanger by incorporation of fluorescently labeled dideoxy
sequencing method and from then onwards genomics nucleotides (ddNTPs). The resulting mixture contains
has progressed exponentially with modern techniques end-labelled extension products and the label on the
making it fast, efficient and cheap. The availability terminating ddNTP of any given fragment corresponds
of draft human genome has provided momentum to the nucleotide identity of its terminal position.
to a new era of genomic and personalized medicine. Consequently, the sequence is determined by capillary
based polymer gel by high-resolution electrophoretic
First Generation separation of the fragments which are single stranded
and end-labeled. The fluorescent fragments of discreet
Sequencing Technologies length are laser excited as they exit the capillary and
Sanger sequencing was introduced by Frederick with a four colour detection of emission spectra
Sanger in 1977 and this sequencing technology was which provides the read out that is represented in
Central Marine Fisheries Research Institute

based on chain-termination method. This technique a Sanger sequencing trace. A Software is capable
has undergone improvement over years and Applied of translating these traces into DNA sequence along
Biosystems Company introduced the first automatic with error probabilities for each base call. A limited
sequencing machine (AB370) in 1987 based on level of parallelization is possible with simultaneous
capillary electrophoresis which made sequencing electrophoresis in 96 or 384 independent capillaries.
faster and accurate. AB 370 was capable of detecting
96 bases at a time, and the read length could reach Second Generation/
600 bases. After improvisations, it is now possible
to analyze a read length of up to 900-1000 bases.
Next Generation
In high-throughput sanger sequencing machines, Sequencing Technologies
DNA to be sequenced is prepared by any one of the
two approaches; for shot gun de novo sequencing, Second Generation Sequencing makes use of many
randomly fragmented DNA is cloned into a high- alternative strategies in sequencing and they are

198
1) micro-electrophoretic methods 2) sequencing generate a burst of light and this is detected by the
by hybridization 3) real time observation of single CCD corresponding to the array coordinates of specific
molecules and 4) cyclic array sequencing. 454 Genome wells. When multiple cycles are performed, (eg: A-G-
Sequencers by Roche Applied Science uses cyclic array C-T…….) the incorporation events reveals the sequence
sequencing. It is also called pyrosequencing. Illumina of templates represented by individual beads. Greater
uses reversible terminator sequencing, ABI/SoLiD uses read-length is possible with 454 sequencing. The 454
sequencing by ligation technique and Helicos uses FLX instrument is capable of producing approximately
single-molecule sequencing. 4, 00,000 reads per instrument-run with lengths of 200-
300bp. The presence of homopolymers (consecutive
Sequencing in Roche454 platform/ instances of the same base such as AAA or GGG) may
increase the rate of error. The absence of a terminating
pyrosequencing
moiety which will prevent multiple consecutive
The 454 system was first next generation sequencing incorporations at a given cycle prevents inference
platform which was available as a commercial regarding length of homopolymers and this inference
product. DNA libraries can be constructed by any has to be made from signal intensity. This increases
method which gives rise to a mixture of short, the error rate and the dominant error type in 454
adaptor-flanked fragments. Clonal amplification of platforms is insertion-deletion.
target sequence is achieved by emulsion PCR and the
amplicons are captured to the surface of 28µm beads. Illumina Genome Analyzer
The emulsion is broken to fragments and the beads
are treated with denaturants to remove untethered This also commonly referred to as “Solexa” and four
strands which are then subjected to a hybridization- companies were merged together; Solexa (Essex,
based enrichment for amplicon bearing beads. The UK), Lynx Therapeutics (Hayward, CA, USA), Manteia

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

hybridization reaction with a sequencing primer is Predictive Medicine (Coinsins, Switzerland) and Illumina.
carried out using a universal adapter at the position Any method can be used for the production of libraries
and orientation that is immediately adjacent to the which gives rise to a mixture of adapter-flanked
start of unknown sequence. fragments up to several hundred base pairs in length.
A bridge PCR is used to generate amplified sequencing
Sequencing reactions are carried out using features. In this method, forward and reverse PCR
pyrosequencing method. The amplicon-bearing primers are secured to a solid substrate by a flexible
beads are pre-incubated with the enzyme Bacillus linker and all the amplicons arising from any template
stearothermophilus (Bst) polymerase along with single- molecule during the process of amplification will be
stranded binding protein which is then deposited to immobilized and clustered to a single physical location
a microfabricated array of picolitre scale wells (the on the array. The bridge PCR relies on alternating cycles
dimensions are such that only one bead will fit per of extension with Bst polymerase and denaturation with
well) to make this biochemistry compatible with array- formamide. The clusters that are produced consist of
based sequencing. Smaller beads bearing immobilized approximately 1000 clonal amplicons. Several million
enzymes (ATP sulfurylase and luciferase) required for clusters can be amplified to specific locations within
pyrosequencing are also added. While sequencing, one each of eight independent “lanes” which are on a
side of the array functions as a flow cell for introducing single flow-cell (such that eight independent libraries
and removing sequencing reagents and the other side is can be sequenced in parallel during the same instrument
bonded to a fiber-optic bundle for CCD (charge coupled run). Once the cluster generation is completed, the
device) based signal detection. During the course of amplicons are linearized or single stranded and a
sequencing, a single species of unlabeled nucleotide sequencing primer is hybridized to a universal sequence
is introduced at each of several hundred cycles. flanking the region of interest. Every cycle of sequence
When there is an incorporation event in templates, interrogation is composed of single base extension
pyrophosphate is released. With the help of enzymes with a modified DNA polymerase and a mixture of four
ATP sulfurylase and luciferase, the incorporation events nucleotides. The nucleotides are modified in two ways;

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 reversible terminators in which a chemically cleavable of images tiling the full array is completed, chemical
moiety at the 3’ hydroxyl position allows only a single cleavage and release of fluorescent label permits
base incorporation at each cycle and one out of four subsequent cycle of extension and imaging. Thus
fluorescent labels which are chemically cleavable several hundred cycles of single-base extension (A,
corresponds to the identity of each nucleotide. Read- G, C, T, A, G, C, T) will yield average read-lengths of
lengths of upto 36bp are currently possible and longer 25bp or greater. The dominant error type is deletion.
reads incur higher error rate. The common error type is
substitution than insertions and deletions. Third Generation
Sequencing Technologies
AB SOLiD
Third generation sequencing technologies are
Libraries can be constructed by any method which may characterized by new chemistry, less operation
give rise to a mix of short, adaptor flanked fragments. time, desktop design and lower operation cost.
Clonal sequencing by emulsion PCR and capturing The major third generation sequencers are; Pacific
of amplicons to the surface of 1µm paramagnetic Biosciences real time single molecule sequencing
beads are carried out. The emulsion is broken and the (PacBioRS), Compete Genomics combined pre anchor
beads bearing products of amplification are selectively hybridixation and ligation (cPAL) and Ion Torrent of
recovered and immobilized to a solid planar substrate Life Technologies, Inc.
which generates a dense, disordered array. Sequencing
by synthesis is carried out by a DNA ligase than a PacBioRS is a real time single molecule-single
polymerase. A universal primer which is complementary polymerase sequencing platform which will be able
to adapter sequence is hybridized to the array of to produce 1000bp read. The chip consist of zero-
amplicon bearing beads and each cycle of sequencing mode wave guided (ZMW) nano structures with holes
involves the ligation of a degenerate population of of size 100nm and inside this hole, DNA polymerase
fluorescently labeled octamers. The mixture of octamers performs sequencing by synthesis with the help of
is structured in such a way that the identity of specific phospholinked nucleotides labeled with fluorophores
positions within the octamer (base 5) correlate with the introduced sequentially. With the help of this
identity of the fluorescent label. When octamer ligation instrument, it is also possible to monitor the kinetics
is carried out progressively, every fifth base is sequenced. of nucleotide incorporation and thus epigenetic
Subsequent steps could identify different base positions information may also be extracted in future.
and the sequencing could be completed.
Complete Genomics uses a combined approach
HeliScope of probe-anchor hybridization and ligation (cPAL)
sequencing and they claim the highest throughput
This also depends on cyclic interrogation of a dense among third generation sequencers. The method is
Central Marine Fisheries Research Institute

array of sequencing features. No clonal amplification based on rolling circle amplification of small DNA
is required here. A highly sensitive fluorescence sequences into so-called nanoballs. Nucleotide
detection system is used to directly interrogate single sequence is determined by unchained sequencing by
DNA molecules through sequencing by synthesis. ligation method. In this approach, many DNA nanoballs
Template libraries prepared by random fragmentation could be sequenced per run and with low costs.
and poly A – tailing (without PCR amplification)
are captured by hybridization to surface bound Ion Torrent technology (Life Technologies Inc.) is
poly-T oligomers to produce a disordered array of considered currently as one of the most versatile and
primed single molecular sequencing templates. At low cost method and it is being supplied as a personal
each cycle, DNA polymerase and a single species of genomic machine (PGM) as benchtop instrument
fluorescently labeled nucleotide are added, which to laboratories involved in research and medicine.
results in template dependent extension of surface- The technology is based on proton release during
captured primer-template duplexes. When acquisition incorporation of each nucleotide by DNA polymerase.

200
Applications, Limitations an Illumina platform. Sequence is then generated from
the molecular identifier in the P1 adapter and across
and Future of NGS methods the restriction enzyme site which generates a data set
Next Generation Sequencing methods are fast, of RAD tags which is derived from a reduced part of
efficient and cheap and offer immense possibilities and the original genome.
applications in human and animal medicine, ecological
and evolutionary studies. The major problems with NGS RAD sequencing has many advantages as compared
data is the many number of short reads generated which to whole genome sequencing as regions of interest
has to be assembled and annotated using reference can be selected and sequenced. Since it is one of
sequences. Longer reads may have more error reads a number of reduced representation methods
towards the end. Repetitive sequences may be difficult which is sampling only a shared set of sites across
to read and end up in more errors. In spite of all these the genome in several individuals, population level
problems, NGS technologies find varied applications in sequencing could be carried out at a fraction of the
human medicine mainly in diagnostics, prognostics and cost required for whole genome sequencing. RAD-
therapeutics. It also finds applications in whole genome seq finds many applications for fine scale linkage
sequencing, targeted sequencing of exomes or selected mapping, phylogenetics and phylogeography,
genes related to a specific disorder or category of genome scaffolding and population genetics. It has
disease, epigenetic mapping, transcriptome sequencing recently been applied in many species like salmon,
and microbial population sequencing. cutthroat and rainbow trout for deducing inferences
regarding population genetic structure.
Restriction Site Associated
DNA (RAD) sequencing Suggested readings

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

In RAD sequencing, two techniques in molecular biology Amish, S.J., Hoehenlohe, P.A., Painter, S., Leary, R.F., Muhlfeld, C.,
are used in combination with Illumina sequencing. Allendorf, F.W. and Luikart, G. 2012. RAD sequencing yields a
high success rate for westslope cutthroat and rainbow trout
Restriction enzymes are used to cut DNA into fragments species-diagnostic SNP arrays. Molecular Ecology Resources
and molecular identifiers (MID) are used to relate 12: 653-660.
Catchen, J., Hohenlohe, P.A., Bassham, S., Amores, A. and
sequence reads to specific individuals. DNA from one Cresko, W.A. 2013. Stacks: an analysis tool set for population
individual is fragmented with specific restriction enzyme genomics. Molecular Ecology 22: 3124-3140.
which produces a set of sticky ended fragments. The Davey, J.W. and Blaxter, M.L. 2011. RADSeq: next generation
population genetics. Briefings in Functional Genomics
sticky ended fragments are ligated to a P1 adapter which 9(5): 416-423.
also contains a matching sticky end and a molecular Davey, J.W., Cezard, T., Fuentes-Utrilla, P., Eland, C., Gharbi, K. and
Blaxter, M.L. 2013. Special features of RAD sequencing data:
identifier. The restriction fragments from different implications for genotyping. Molecular Ecology 22: 3151-
number of individuals which are tagged are pooled 3164.
and then randomly sheared to produce fragments Liu, L., Li, Y., Li, S., Hu, N., He, Y., Pong, R., Lin, D., Lu, L. and Law,
M. 2012. Comparison of next-generation sequencing systems.
with an average length of a few hundred base pairs. Journal of Biomedicine and Biotechnology 2:11.
The fragments which are sheared are then ligated to Metzker, M.L. 2010. Sequencing technologies-the next generation.
Nature Reviews Genetics 31-46
a second P2 adapter and amplified using PCR with P1 Pocwierz-Kotus, A., Bernas, R., Kent, M.P., Lien, S., Leliuna, E.,
and P2 primers. The P2 adapter is characterized by a Debowski, P. and Wenne, R. 2015. Restitution and genetic
divergent “Y” structure which will not bind to P2 primer differentiation of salmon populations in the sourthern Baltic
genotyped with the Atlantic salmon 7K SNP array. Genetics
unless it is completely amplified by the P1 adapter. Thus Selection Evolution 47: 1-9.
it is ensured that all the amplified fragments have the Quail, M.A., Smith, M., Coupland, P., Otto, T.D., Harris, S.R.,
Connor, T.R., Bertoni, A., Swerdlow, H.P. and Gu, Y. 2012. A tale
P1 adapter and MID, the partial restriction site, the of three next generation sequencing platforms: comparison of
flanking sequence consisting of a few hundred bases Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.
and a P2 adapter. These fragments which are ready for BMC Genomics 13: 1-13.
Shendure, J. and Ji, H. 2008. Next generation DNA sequencing.
sequencing are selected for size (approximately 200-500 Nature Biotechnology 26: 1135-1145.
base fragments) and the RADSeq library is sequenced on

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 Software Packages used in Population
Genetics
Sandhya Sukumaran*, N. S. Jeena, Reynold Peter and Wilson Sebastian
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Many software packages are freely available for BioEdit (Current version 7.2.5 Last updated
population genetics analysis. Software packages for 12/11/2013) can be downloaded from the following
aligning sequences, construction of phylogenetic Server: http://www.mbio.ncsu.edu/BioEdit/bioedit.
trees using different methods, analysis of genetic html
differentiation, analysis of historical demography,
constructing network diagrams, analyzing genetic Mega (Molecular
population structure and a number other analyses
are available as freely downloadable files. The major
Evolutionary
software packages used for population genetic Genetics Analysis)
analyses are listed below.
The Molecular Evolutionary Genetics Analysis
Bioedit software is a multi platform desktop application
designed for comparative analysis of homologous
Bio Edit is a user-friendly biological sequence gene sequences either from multi-gene families or
alignment editor and analysis program and is an from different species with a special emphasis on
important bioinformatics tool for molecular biologists. inferring evolutionary relationships and patterns of
It was developed initially as a graphical biological DNA and protein evolution. In addition to the tools
sequence alignment editor written for Windows for statistical analysis of data, MEGA provides many
only. The purpose of this program is to provide a convenient facilities for the assembly of sequence
useful molecular biology tool which can be started data sets from files or web-based repositories, and
up and used easily. However, in the last few years it it includes tools for visual presentation of the results
has been improved dramatically to integrate many obtained in the form of interactive phylogenetic trees
other features and functions such as several modes and evolutionary distance matrices.
Central Marine Fisheries Research Institute

of hand alignment, plasmid drawing and annotation,

restriction mapping and much more. The Molecular Evolutionary Genetics Analysis (MEGA)
software aims to serve both of these purposes in
BioEdit can accept a wide variety of formats that is inferring evolutionary relationships of homologous
commonly used with other bioinformatics application. sequences, exploring basic statistical properties
This allows the swapping of Data files between BioEdit of genes and estimating neutral and selective
and other programs. It contains many features for evolutionary divergence among sequences.
sequence alignments like split window view, user
defined color, information based shading and auto MEGA is an integrated workbench for biologists for
integration with other programs such as Clustal W mining data from the web, conducting automatic and
and Blast. Most of sequence alignment programs manual sequence alignment, inferring phylogenetic
do not have many other functions or useful tools for trees, estimating rates of molecular evolution, testing
molecular biologist comparing to BioEdit. evolutionary hypotheses and generating publication

202
quality displays and descriptions. MEGA is used A web-based front-end is available for teaching
by biologists in a large number of laboratories for purposes and for those who, for some reason, cannot
reconstructing the evolutionary histories of species run the Genepop on their local PC or Mac.
and inferring the extent and nature of the selective
forces shaping the evolution of genes and species. GenAlEx
MEGA is distributed in two editions: a graphical user GenAlEx 6.5 is used for a range of population genetic
interface (GUI) edition with visual tools for exploration analysis with a wide range of molecular markers within
of data and analysis results and a command line the Microsoft Excel environment on both PC and
edition (MEGA-CC), which is optimized for iterative Macintosh computers. It has a user friendly interface
and integrated pipeline analyses. Both GUI and with rich graphical outputs for data exploration and
command-line versions of MEGA6 (current version) publication, tools for data manipulation and export
can be downloaded from www.megasoftware.net options to many other software packages. GenAlEx
free of charge. offers analysis of codominant, haploid and binary
genetic loci and DNA sequences. Both frequency-
Genepop based (F-statistics, heterozygosity, HWE, population
assignment, relatedness) and distance-based (AMOVA,
GENEPOP is a population genetics software package. The PCoA, Mantel tests, multivariate spatial autocorrelation)
basic tools of this program are designed for multi-allelic analyses are provided. In GenAlEx 6.5 there are new
markers like microsatellite, short tandem repeat-STR etc. features including calculation of new estimators of
It is used for descriptive statistics, estimates of genetic population structure: G’ST, G’’ST, Jost’s Dest, and
differentiation (traditional & microsat specific estimates F’ST via AMOVA, Shannon Information analysis,
of genetic differentiation), Hardy-Weinberg equilibrium linkage disequilibrium analysis for biallelic data, and

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

(pairwise comparisons of single locus and global tests), heterogeneity tests for spatial autocorrelation analysis.
geographic vs. genetic distance. Direct data export is provided to more than 30 other
software packages, and indirectly via common formats
Three major tasks can be performed in Genepop. 1) It to many more packages.
computes exact tests: for Hardy-Weinberg equilibrium,
for population differentiation and for genotypic DnaSP
disequilibrium among pairs of loci. 2) computes
estimates of classical population parameters, such DNA sequence polymorphism (DnaSP) is an
as Fst and other correlations, allele frequencies, etc. interactive computer program for the analysis of
and 3) converts the input Genepop file to formats DNA polymorphism from nucleotide sequence data.
used by other programs, like Biosys, Fstat and Linkdos. It calculates several measures of DNA sequence
variation within and between populations; linkage
The latest version of Genepop (4.2) is now available disequilibrium, recombination, gene flow and gene
from http://kimura.univ montp2.fr/~rousset/Genepop. conversion parameters. In addition, DnaSP performs
htm. Genepop 4.2 runs under Windows, and can also some neutrality tests. DnaSP is written in Visual Basic
be compiled to run under Unix or Linux. The Genepop v. 6.0 (Microsoft) and DnaSP version 5.10.1 is freely
programs is a simple command-line executable files, downloadable from http://www.ub.edu/dnasp/
where the settings of the computations can often be
specified on a command line. Even though being less DnaSP can automatically read the following types of
user friendly than graphical packages, command-line data file formats: FASTA, MEGA, NBRF/PIR, NEXUS,
programs have the advantage that they can be easily PHYLIP and HapMap3 Phased Haplotypes. In all cases
launched in parallel on computer clusters and can one or more homologous nucleotide sequences
be used to automatically analyze a large number of should be included in just one file (ASCII file). The
input files, like those resulting from large genomic sequences must be aligned (i. e. the sequences must
studies or simulations. have the same length).

203
 Various analysis options are included in the software present in the population by maximum likelihood
such as DNA Polymorphism analysis, GC content methods, linkage disequilibrium which is a test of
at non-coding and coding positions, Haplotype/ non-random association of alleles at different loci.,
Nucleotide Diversity and Divergence. There are options Hardy-Weinberg equilibrium which is the test of
to find out the number of Segregating Sites (S), total non-random association of alleles within diploid
number of mutations (Eta), the number of haplotypes, individuals, Tajima’s neutrality test, Fu’s Fs neutrality
Haplotype (gene) diversity and its sampling variance, test, Ewens-Watterson neutrality test, Chakraborty’s
Nucleotide diversity, Pi (π), The average number of amalgamation test and Minimum Spanning Network
nucleotide differences (k), Nucleotide divergence with (MSN) analyses. Inter-population methods include
Jukes and Cantor, Theta (per gene or per site) from searching for shared haplotypes between populations,
Eta (η) or from S and ZnS statistic, Neutrality tests different hierarchical Analyses of Molecular Variance
such as Tajima’s D with its statistical significance, Fu (AMOVA) to evaluate the amount of population
and Li’s D, Fu’s Fs etc. This software also estimates genetic structure, FST based pairwise genetic distances
the raggedness statistic (r) which quantifies the for short divergence time, exact test of population
smoothness of the observed pairwise differences differentiation, assignment of individual genotypes to
distribution. More powerful statistics for detecting particular populations according to estimated allele
population expansion such as the Fu’s Fs Test and the frequencies, detection of loci under selection from
Ramos-Onsins and Rozas’s R2 can also be carried out F-statistics and mantel test to test for the presence
using the software. of isolation-by-distance.

Arlequin Network
The free population genetic software Arlequin Network is a free phylogenetic network software
provides the user with quite a large set of basic available from fluxusengineering.com. It is used to
methods and statistical tests, in order to extract reconstruct phylogenetic networks and trees, infer
information on genetic and demographic features of ancestral types and potential variants and evolutionary
a collection of population samples. The latest version branching patterns. The Network programs have
Arlequin ver 3.5.2.1 can be downloaded in Windows user-friendly graphical interfaces, allowing users to
XP/Vista/7 based systems with a minimum of 256 MB easily choose the types of analysis and computation
RAM from http://cmpg.unibe.ch/software/arlequin35/ parameters to be performed. The basic algorithms of
Arl35Downloads.html this program are designed for non-recombining bio-
molecules, thus it can be effectively used for mtDNA,
Arlequin can handle several types of data either in Y-STR, amino acid, RNA, viral DNA, bacterial DNA,
haplotypic or genotypic form. The basic data types some non-recombining autosomal DNA, and also for
are DNA sequences, RFLP data, Microsatellite data, non-biomolecule data such as linguistic data.
Central Marine Fisheries Research Institute

Standard data and Allele frequency data. The analyses

Arlequin can perform on the data fall into two main The Network software was developed to reconstruct
categories: intrapopulation and inter-population all possible least complex phylogenetic trees (all
methods. In the first category statistical information maximum parsimony or MP trees) from a given data
is extracted independently from each population, set. The data can be DNA sequences or nucleotide
whereas in the second category, samples are sequences, amino acid sequences or protein
compared to each other. Intra-population methods sequences, STR (short tandem repeat) or microsatellite
include standard diversity indices like the number data, language/linguistic or manuscript data.
of polymorphic sites, gene diversity, molecular
diversity indices like nucleotide diversity, different Two independent network-building options are
estimators of the population parameter θ, mismatch included for arriving at optimal networks; the
distribution of the number of pairwise differences Reduced Median networks or RM network algorithm
between haplotypes, Haplotype frequency estimation for binary data and the Median Joining networks

204
or MJ network algorithm for all types of data. MJ (version 3.2) includes a number of significant new
networks are commonly used to visualize relationships features, such as check-pointing, strict and relaxed
of closely related mitochondrial or nuclear haplotypes, clock models, and functionality for dating. Version 3.2
for which traditional phylogenetic methods yield can be compiled from source code freely available on
multiple possible trees. For interpretation of complex SourceForge (see instructions below).
and large data a neat skeleton phylogeny can be
constructed using Star Contraction pre-processing. MrBayes version 3.1.2 is available for Windows,
Age estimation for ancestral nodes or branching OSX, and Linux. All versions use a command-line
points is also possible in Network software. interface and the program looks virtually the same
on all platforms.
Current Version is Network 4.6.1.3., released on
24 December 2014, and it is available from www. MrBayes 3 is a program for Bayesian inference and
fluxusengineering.com. Network runs on Windows 7, model choice across a large space of phylogenetic and
Vista, XP, 2000, many Linux versions with WINE Windows evolutionary models. The program has a command-
emulation, and Macs with Parallels Desktop Windows, or line interface and should run on a variety of computer
VirtualBox with Windows. Network can run from memory platforms, including large computer clusters and
stick no Windows registry entries and no Windows multicore machines. Depending on the settings,
Administrator required for installation. Network software MrBayes analyses may demand a lot on the machines,
package includes a data editor and a graphics program. both in terms of memory and processor speed. Many
FASTA files can be imported and prepared for Network users therefore run more challenging analyses on
using Fluxus’ DNA Alignment software. Higher-quality dedicated computing machines or clusters. Several
graphics of Network’s results files can be prepared using computing centers around the globe provide web
Fluxus’ Network Publisher software. access to such services.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Mr Bayes MrBayes 3 is distributed without charge by download
from the MrBayes web site, http://mrbayes.net.
MrBayes (Ronquist and Huelsenbeck 2003) is
a program for doing Bayesian phylogenetic WSTRUCTURE is freely downloadable software which
analysis. The posterior probability distribution uses a model based clustering method to infer genetic
is sampled using Markov Chain Monte Carlo population structure. It uses multi-locus genotype
(MCMC) techniques. Metropolis-coupling is used data for investigations on genetic population
to accelerate convergence in MrBayes. In parallel structure with a Bayesian approach (MCMC: Markov
with regular “cold” chain, three heated chains are Chain Monte Carlo). It can be used for knowing the
also run in parallel. The heated chains will sample presence of distinct populations, assigning individuals
from distributions obtained by raising the posterior to populations, studying hybrid zones, identifying
probability with some factor smaller than 1, which migrants and admixed individuals, and estimating
result in flattening (“melting”) of the peaks in the population allele frequencies. Results based on most
landscape defined by the posterior distribution. of the commonly used genetic markers like SNPS,
At specified intervals, the parameter values (the microsatellites, RFLPs and AFLPs could be analyzed
locations in the landscape) are swapped between using this software. This software could also be used
cold and heated chains, making it possible for the to compute the proportion of the genome of an
cold chain to escape local peaks. This comes at the individual originating from each inferred population
cost of increased computational complexity. (quantitative clustering method).

The last official release of the program is version 3.1.2. It can be downloaded from: http://pritch.bsd.
The most recent development version of the program uchicago.edu/software/structure2_1.html).

205
 General Methods of Tissue Culture
Vidya Jayasankar
Senior Scientist
Madras Research Center of CMFRI
e-mail: [email protected]

Introduction experiment, in order to control air-borne bacteria

Tissue culture is a technique that requires a high level in the room. They should not, however, be used for
of expertise, utmost care during the preparatory and long durations when culture work is in progress,
execution phases and a sound knowledge on the because UV light may have a deleterious effect on the
cytology of the culture material being studied. There tissues being cultured.
are certain basic principles and methods that need to
be followed in order to set up a tissue culture facility Basic Equipment
and to establish successful cultures.
• Autoclave: Used to sterilize glassware, stainless
Laboratory Space steel surgical instruments, heat-resistant plastic
lab ware, certain salt solutions etc. Wasted dishes
and Equipment and other disposable material that come in
The space allocated for a tissue culture laboratory contact with pathogens must also be autoclaved
should ideally consist of two rooms, a preparation before they are discarded. The material to be
room opening into a smaller, aseptic culture room autoclaved is usually subjected to high pressure
which should be dedicated exclusively to tissue saturated steam at 121ºC for 15 min. Purified
culture activities. A sliding door should be used to water should always be used in the autoclave
separate the preparation room from the culture to minimize contamination of material being
room, in order to reduce the draft of air that would sterilized due to condensed water.
enter during the opening and closing motions of • Laminar-flow cabinet: Used during media
a regular door. The preparation room is used for preparation and culture handling to reduce
all preliminary activities which do not require a contamination from airborne particles by
sterile environment, including washing/drying and continuous displacement of air that passes
sterilization of glassware, weighing of chemicals, through a HEPA (high efficiency particle air) filter.
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preparation of reagents etc. The culture room should It is more common to use a flow cabinet in which
house a culture table, a closed cupboard for storing air flows from the inside to the outside. However,
sterile supplies and all the necessary cell culture when working with hazardous organisms, it is
equipment, such as the laminar flow cabinet, CO2 preferable to use a vertical flow cabinet (biology
incubator and microscopes. This room should be kept safety cabinet), where the aerosols generated in
sterile, and its walls and floors should be smooth the hood are filtered out before being released
and clean. Entry into this area should be restricted into the surrounding environment. The flow
in order to minimize chances of contamination cabinets are equipped with a short-wave UV
of cultures. If possible, a dressing room through light that must be turned on for 10-20 minutes
which the culture room can be entered can also be before use to sterilize the surfaces of cabinet. The
added. The culture room should be fitted with ultra cabinet should be kept clean and clutter-free. All
violet lamps, which should be turned on for about surfaces need to be wiped with ethanol before
half an hour before commencement of a culture and after each use.

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• CO2 Incubator: Used to provide a temperature Culture System
controlled atmosphere of high humidity and
increased CO2 tension for optimal cell growth. Culture vessels must have a biologically inert,
A mixture of air with 5-10% CO2 and saturated optically transparent surface. Sterile, disposable
water vapor is generally used to keep the pH of polystyrene plastic dishes are preferred over glass
the culture medium constant in mammalian cell because attachment of cells and tissue fragments
culture. Invertebrate cells, on the other hand are is poor on glass. The different types of culture
in general insensitive to changes in pH of culture vessels include petri dishes, multi-well plates (6, 12,
media, and hence a CO2 incubator is not a strict 24, 48 and 96-well configurations) and screw cap
requirement for their culture. flasks (T-25, T-75, T-150). Culture flasks with vented
• Inverted microscope: Used to examine cultured caps containing filters are also available, where gas
cells in flasks and multi-well plates. The inverted exchange can be carried out without having to loosen
microscope should be equipped with a bright the cap. The inner surface of the culture flasks is
field and phase-contrast optic system with 4x, coated with substrates such as collagen, poly L-lysine,
10x, 20x, and 40x objectives, and digital imaging fibronectin etc. to increase adhesiveness, for closed
and processing facilities. If fluorescent microscopy and stationary cultures. For large-scale culture of
is required, an epifluorescent attachment should suspended cells, a spinner bottle and magnetic stirrer,
be included. or an Erlenmeyer flask placed on a rotary shaker are
• Refrigerator and freezer (–20ºC): Used to commonly used. Culture of substrate-dependent cells
preserve culture media, salt solutions, sera, amino usually requires the use of roller bottles.
acid solutions, hormones, vitamins etc.
• Centrifuge: Used to spin down cells in a cell Aseptic Measures
suspension at low speed during washing,

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

harvesting or subculturing of cells from cultures. Aseptic technique and the proper use of laboratory
• Osmometer: Used to measure the osmolality of equipment are very essential when working with cell
culture media prepared in the laboratory. cultures. It is mandatory to use sterile equipment and
• Filtration system: Used to sterilize heat labile reagents, and disinfect hands, reagent bottles and
culture media, enzymes, supplements etc. that work surfaces with 70% alcohol before beginning
cannot be autoclaved. A basic filter system is culture work. Glass pipettes and necks of bottles
comprised of a filter holder set, a membrane filter should be flamed before and after use. Screw caps
having a pore size of 0.22 µm and an aspiration should be placed open side down on the surface of
pump (peristaltic or vacuum). a clean bench. Glass or disposable plastic fast-flow
pipettes should preferably be used to increase speed
Minor equipment of dispensing solutions. Bulbs or electric pipette
controllers should be used to dispense reagents
In addition to these major equipment, a hot air oven since mouth pipetting could be hazardous to the
is required for drying/sterilizing glassware, surgical operator or lead to contamination. Pipettes with
instruments etc., a water purification system for the cotton plugs at the top should be used. Care should
supply of purified water and an electronic balance, pH be taken not to mix liquids by rapidly pipetting up
meter, water bath and magnetic stirrer for preparation and down or releasing the contents of a pipette from
of reagents and culture media. A hemocytometer, a height into the receiving vessel. All liquid waste
electronic cell counter or flow cytometer is required should be treated with bleach or similar detergents
to make accurate counts of cells in cultures. before disposal.

Other requirements include culture dishes, centrifuge Conditions for Culture

tubes, disposable Pasteur pipettes, variable volume
automatic micropipettes, disposable/autoclavable An in vitro culture should recreate the physical,
pipette tips etc. nutritional, and hormonal environment of the cell in

207
 vivo. The physicochemical properties that are required by bicarbonate (NaHCO3). The CO2/HCO3 balance is
include controlling the temperature, pH, osmolality, maintained by gaseous CO2, which requires the use
and gaseous environment. of gassed incubators to maintain an atmosphere of
5-10% CO2 in air.
Temperature
Light
The incubation temperature will depend on the
type of cells being cultured. While mammalian cells Light can have an adverse effect on cells by inducing
require a temperature of 37ºC, invertebrate cells production of toxic compounds in some media. In
require a lower temperature range of 15ºC-30ºC. such cases cells should be cultured in the dark and
Most mammalian cell cultures are grown in exposed to room light as little as possible.
incubators that are set at 37ºC.
Cell Culture Media
pH
Culture medium is the most important factor affecting
Regulation of extracellular and intracellular pH is very the success of a cell culture. The main component of
essential for survival of mammalian cells. The pH is all media being water, it is recommended that the
important for maintaining appropriate ion balance, water being used for media preparation is ultrapure.
as well as for maintaining the optimal function of Culture media are of two types.
cellular enzymes. Minor changes in pH can also
alter cell metabolism and induce the production • Natural media:Media containing natural
of heat-shock proteins, which can ultimately lead sources of nutrients such as biological fluids
to apoptosis. Most media aim to maintain a pH (serum, amniotic fluid, pleural fluid, insect
between 7.0 and 7.4. Different cell types may hemolymph etc.) or tissue extracts (extracts from
however have varying ranges of optimal pH. Cells liver, leukocytes, embryo, bone marrow etc.) as
usually tolerate slow changes in pH as compared to the main components.
rapid changes. Insect cells in culture are insensitive to • Synthetic media:Media which are artificially
wide variations in pH. The pH of media are regulated prepared by the addition of various organic
through a variety of buffering systems. and inorganic nutrients, salts, vitamins, serum,
carbohydrates etc. Synthetic media can further
Osmolality be categorized into two types, serum containing
media and serum-free media.
The osmolality of a culture medium is mainly
determined by the dissolved salts and glucose, Serum containing media are considered advantageous
although amino acids may also contribute for the culture of most types of cells and are
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significantly. Most of the commercial media used supplemented with 5-20% serum. Serum is a complex
for mammalian cell culture are formulated to have mixture of essential growth factors (platelet-derived
a final osmolality of around 300 mOsm. Significant growth factor, epidermal growth factor, fibroblast
alterations in osmolality will affect cell growth and growth factor, endothelial growth factor, etc.),
function. The optimal osmolality for growth of most hormones, attachment and spreading factors
cells has been determined to be around 290 to 310 (fibronectin, serum spreading factor etc.), binding
mOsm. Extreme changes in osmolality will result in and transport proteins, protease inhibitors and trace
loss of membrane integrity leading to explosion or elements. It has the capacity to bind and neutralize
collapse of cells. toxins such as heavy metals and reactive organic
components, and also increases the buffering capacity
CO2, Oxygen, and other Gases of a medium. However, the use of serum in culture
media also has certain disadvantages. Firstly, it is not
The buffering capacity of the medium is increased chemically defined and therefore its composition is

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highly variable. Since serum components are affected Certain media require additional supplements such as
by factors such as age, health and nutrition of the collagen and fibronectin, hormones such as estrogen,
donor animals, the quality of serum varies from batch and growth factors such as epidermal growth factor
to batch, which makes it necessary to test every new and nerve growth factor, to promote cell attachment
batch for the required quality standards. Serum can and proliferation.
also be a source of contamination by mycoplasma,
viruses etc. Fetal calf/bovine serum (FCS/FBS) is the The choice of a cell culture medium depends on
most frequently used serum. the type of cells being cultured and is an extremely
important factor that affects the success of cell
Serum-free media are chemically defined media cultures. Different cell types have highly specific
where the chemical composition and the growth requirements, and the most suitable
concentration of every component are known. medium for each cell type needs to be determined
These media are consistent from batch to batch experimentally. Commonly used basal media include
and are optimized for growth of specific cell types Eagle Minimal Essential Medium (MEM), Dulbecco’s
and product synthesis. Development of serum-free Modified Eagle Medium (DMEM), RPMI 1640, Ham
media for the culture of mammalian cells in vitro F10, M199 and L-15, which contain a mixture of amino
has shown great progress over the last two decades. acids, glucose, salts, vitamins, and other nutrients.
However, they have not yet been able to replace Basal media usually need to be supplemented with
conventional serum based formulations, mainly serum, L glutamine, and antibiotics/fungicides just
due to the high cost involved and the need for prior to use.
maintaining specificity in formulation for each cell
type. Cell lines usually require the use of expensive Use of antibiotics in cultures
serum-free media specific to a particular cell type.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

A number of serum-free medium formulations have Antibiotics are routinely incorporated in cell culture
been described for mammalian and insect cell lines media in order to avoid contamination due to bacteria
as well as for primary cultures. and fungi. However, it is important to keep the
concentration of antibiotics to a minimum amount
Nutritional requirements: The basic components of that is necessary to prevent microbial growth, since
cell culture media are inorganic salts, sugars, amino higher concentrations can have deleterious effects
acids, vitamins, lipids, proteins and peptides, hormones, on cell viability. Their continuous use can lead to
vertebrate sera, invertebrate hemolymph, tissue extracts the development of antibiotic resistant strains,
and growth factors. Basic salts contained in cell culture persistence of low-level contamination and hide the
media include NaCl, KCl, CaCl2, and MgCl2. Inorganic presence of mycoplasma infections and other cryptic
salts are important to adjust the osmotic and ionic
balance of the cells and maintain their membrane Table 1. Commonly used antibiotics and fungicides for
potential. Carbohydrates are an important energy animal cell culture

source, and are mainly used in the form of glucose. Concentration Effective against
Amino acids serve as a source of nitrogen. Media  Penicillin  50–100 U/ml Gram-positive bacteria
also contain lipids, mostly in the form of a mixture of
 Streptomycin  50–100 µg/ml Gram-negative bacteria
fatty acids, or in some cases as more complex lipids
such as cholesterol. Some media formulations (eg.  Kanamycin  100 µg/ml Gram-positive and
gram-negative bacteria;
medium 199) contain detergents such as Tween 80 to mycoplasma
help emulsify the lipids. Vitamins such as niacin, folic
 Gentamycin  5–50 µg/ml Gram-positive and
acid, riboflavin, inositol and thiamine are commonly gram-negative bacteria;
incorporated in many media to augment continued mycoplasma
cell replication. Macromolecules such as thymidine,  Amphotericin B  0.25–2.5 µg/ml Yeasts and molds
adenosine, and hypoxanthine are also included in
 Nystatin  100 U/ml  Yeasts and molds
specific media to improve the growth of some cells.

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 contaminants.  The commonly used antibiotics include morphology, color of the medium, density of cultured
penicillin, streptomycin and gentamycin. cells, dates on which cells were subcultured etc. must
be recorded.
Selection of Animals
Growth Patterns
Successful cell cultures can be obtained by using
animals that are young, actively growing and healthy. Depending on the cell type, seeding density and media
In the case of aquatic invertebrate cell culture, the components used, cells in culture initially go through
animals should be acclimatized to the laboratory a lag phase, followed by an exponential growth
conditions for at least a week before cell culturing is phase where the cells have the highest metabolic
initiated, and starved for a day before they are used activity. The cells finally enter into a stationary phase
for the experiments. where they cease to proliferate and cultures become
confluent (completely covering the surface of the
Types of Cultures culture vessel).

Explant culture: In explant culture, a small fragment Harvesting and Sub-

of tissue is allowed to adhere to an appropriate
substrate, either spontaneously or aided by mechanical
culturing
means, and cultured in a nutrient-rich medium. Cell Cells are usually harvested when they are in a
migration is promoted following attachment to give semi-confluent state and are still in log phase.
rise to an outgrowth of cells. The tissue is usually cut Adherent cell cultures die if they are left in a
with a scalpel into slices of 0.5–1.0 mm thickness. confluent state for too long and therefore need to
One of the main advantages of this method is that be routinely passaged, where a fraction of the cells
some aspects of the tissue’s architecture can be are transferred to a new culture vessel. Suspension
preserved within the explant. cells exhaust their culture medium once cell density
becomes very high, hence these cultures also need
Dissociated Cell culture: Cell culture refers to to be passaged regularly. Most cells are passaged
cultures derived from dissociated cells obtained either (or at least fed) three times a week. Suspension
mechanically or enzymatically from the original tissue. cultures are fed by dilution into fresh medium.
The dispersed cells form a cell suspension which may Adherent cultures that do not need to be divided
then be cultured as a monolayer on a solid substrate, can be fed by just replacing the old medium with
or as a suspension in the culture medium. Tissue fresh medium.
disaggregation is capable of generating larger cultures
more rapidly than explant culture, but explant culture Several methods can be used to remove semi-
may be preferable where only small fragments of confluent cells from the growth surface in adherent
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tissue are available or where the cells are too fragile cultures so that they can be passaged or sub-cultured:
to survive after disaggregation.
Mechanical: A rubber cell scraper can be used to
Organ culture refers to the culture of the whole physically detach the cells from the growth surface.
organ or part of the organ in a way that allows Although this method is quick and easy, it can damage
differentiation and preservation of architecture of cell and may cause cell death. This method can mainly
the tissue. The tissue is usually cultured at the liquid- be used for harvesting cells for extract preparation
gas interface on a grid or gel. etc., where cell viability is not very important.

Maintenance of Cultures Proteolytic enzymes: Trypsin, collagenase,

pronase or dispase are usually used in combination
Cultures should be examined daily and a tissue with EDTA to detach adherent cells from the
culture log should be maintained. Observations on surface of a cell culture vessel. Although this

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method is fast and reliable, it can damage the later stages of contamination, and the culture
cell surface by digesting exposed cell surface becomes turbid. Yeast contamination can also be
proteins. Termination of proteolysis can be quickly visually detected by the presence of turbidity in
achieved by the addition of complete medium cultures. Viruses are microscopic in nature and
containing serum. can be detected only by electron microscopy,
immunostaining, ELISA assays, or PCR.
EDTA: EDTA alone can also be used to detach cells
and seems to be gentler on the cells than trypsin. Mycoplasma contamination: Mycoplasma are
extremely small sized bacteria which are difficult
Cryopreservation to detect until their densities increase to such an
extent that they cause deterioration of cell cultures.
Cell lines can be preserved by freezing to maintain Mycoplasma contamination can be detected only by
stocks without aging and to protect them from periodical testing of cell cultures using fluorescent
contamination. Factors favoring good survival after stains, ELISA, immunostaining, PCR, autoradiography
freezing and thawing are: (i) High cell density at or microbiological assays.
freezing (1 × 106 –1 × 107 cells/ml). (ii) Presence
of a cryopreservatives, such as glycerol or dimethyl Cross-contamination: Although cross-
sulfoxide (DMSO) at 5–10%. (iii) Slow freezing at contamination is not encountered as frequently as
1ºC/min, down to −70ºC and then rapid transfer microbial contamination, it can be a serious problem
to a liquid nitrogen freezer. (iv) Rapid thawing. in the culture of cell lines. Cross-contamination can be
(v) Slow dilution with medium to dilute out the avoided making periodic checks on the characteristics
preservative without causing osmotic damage to cells. of the cell lines and also by practicing good aseptic
(vi) Reseeding at 2- to 5-fold the normal seeding techniques for culture.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

concentration. (vii) Changing medium the following
day to remove preservative. Safety Considerations
Cell Culture Contamination • Appropriate personal protective gear (aprons,
caps, masks, gloves etc.) must be worn always.
Contamination of cultures is one of the most Gloves must be changed when contaminated, and
common problems encountered during cell culture used gloves disposed of with other contaminated
activities. Contaminants can be of two types: chemical laboratory waste.
contaminants which include impurities in media, • Separate footwear must be used in the
sera and water, and biological contaminants such as culture room.
bacteria, molds, yeasts, viruses and mycoplasma, and • Movement of personnel in and out of the culture
cross contamination by other cell lines.  room must be minimized.
• Hands must be washed after working with
Bacterial contamination: Bacterial contamination potentially hazardous materials and before leaving
is a very common occurrence in cell cultures and can the laboratory.
be detected by visual inspection in the initial stages • Talking must be avoided when culture activities
of contamination itself. Cultures appear cloudy/turbid are in progress.
and sometimes have a thin film on the surface. A • Eating, drinking, or storing food for human
sudden lowering of pH of the culture medium is consumption in the laboratory must
also observed. be prohibited.
• Contaminated sharp objects (needles, scalpels,
Mold, yeast and virus contamination: Molds pipettes, broken glassware etc.) must be disposed
have multicellular filaments (hyphae) which form of properly in separate biohazard waste bins.
a connected network called a colony or mycelium. • Care must be taken to minimize the creation of
The pH of the medium increases rapidly in the aerosols and/or splashes.

211
 • All work surfaces must be decontaminated with Suggested readings
an appropriate disinfectant before and after the
experiments, and immediately after any spill or Freshney, R.I. (1993) Culture of Animal Cells: A Manual of Basic
Techniques, 3rd ed., Wiley-Liss, New York.
splash of potentially infectious material. Mather, J.P. and Roberts, P.E. (1998) Introduction to Cell and Tissue
• Laboratory equipment must be cleaned routinely, Culture: Theory and Technique, Genentech Inc., San Francisco.
Plenum Press, New York.
even if it is not contaminated. Culture room must
Doyle, A. and Griffiths, J.B. (1998) Cell and Tissue Culture:
be routinely decontaminated using a UV lamp. Laboratory Procedures in Biotechnology. John Wiley and
• All potentially infectious materials must be Sons, UK.
Spector, D., Goldman, R.R., and Leinwand, L.A. (1998) Cells: a
autoclaved and decontaminated before disposal. Laboratory Manual. Cold Spring Harbor, NY. Cold Spring Harbor
• All liquid waste after each experiment must be Laboratory Press.
Mothersill, C. and Austin, B. (2000) Aquatic Invertebrate cell
treated with bleach before disposal.
Culture. Springer-Praxis Publication, UK.
Mitsuhashi, J. (2002) Invertebrate Tissue Culture Methods. Springer-
Verlag, Tokyo.
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212
In vitro Culture of Finfish Cells – Principle
and its Applications
T. Raja Swaminathan* and V. S. Basheer
Principal Scientist
Peninsular and Marine Fish Genetic Resources Centre of NBFGR
CMFRI Campus, P.B. Number 1603, Ernakulam North, Kochi – 682 018.
e-mail: [email protected]

Introduction increase in the number of laboratories and industries

using cell culture today.
Vertebrate cell cultures are in vitro models. The term in
vitro refers to keeping entities of an organism outside Any interaction of a toxic substance with an organism
the living body in an artificial environment, in contrast is initiated at the cellular level. From cells, alterations
to in vivo, i.e. in the organisms. Primary cultures can translate to changes in tissue, organ function
start from cells, tissues or organs taken directly from and finally impact on whole organism. Based on the
organisms. If a primary culture can be divided into central role of cells in the expression of toxicity, several
new culture vessels and successfully propagated, it cell lines or in vitro models have received regulatory
becomes a cell line. A cell line may be propagated a acceptance by the organization as alternative to whole

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

limited number of times, in which case it is finite, or animal tests in health sciences. Besides their potential
indefinitely, in which case it becomes an immortal or to replace or reduce animals in toxicity tests, cell lines
continuous cell line. have several advantages compared to whole animal
tests. Large numbers of potentially toxic substances
Although animal cell culture was first successfully can be screened rather quickly in multiple-well plates,
undertaken by Ross Harrison in 1907, it was not which can be analysed rapidly. As well, cells can help
until the late 1940’s to early 1950’s that several identify the mechanisms underlying a toxic response.
developments occurred that made cell culture If, for a particular purpose, a suitable continuous
widely available as a tool for scientists. First, there cell line can be used, a donor animal is never again
was the development of antibiotics that made it needed. Based on these reasons, the role of cell lines
easier to avoid many of the contamination problems is expected to significantly increase.
that plagued earlier cell culture attempts. Second
was the development of the techniques, such Fish cell line
as the use of trypsin to remove cells from culture
vessels, necessary to obtain continuously growing Fish cell lines have been useful in many areas of
cell lines (such as HeLa cells). Third, scientists were research. Originally developed to support the growth
able to develop standardized, chemically defined of fish viruses for studies in aquatic animal viral
culture media that made it far easier to grow cells. diseases, fish cell lines have grown tremendously
These three areas combined to allow many more in number covering a wide variety of species and
scientists to use cell, tissue and organ culture in tissues of origin and an array of applications. Fish
their research. During the 1960’s and 1970’s, immunology, physiology, genetics and development,
commercialization of this technology had further toxicology, ecotoxicology, endocrinology, biomedical
impact on cell culture that continues to this day. research, disease control, biotechnology and
The overall result of these and other continuing aquaculture are some of the areas in which fish cell
technological developments has been a widespread lines have made significant contributions.

213
 Fish cell lines being of poikilothermic origin, grow increasingly utilized in research unrelated to disease,
well at room temperatures without the need of and with the recent identification of rickettsial fish
thermo regulated incubators, furthermore, an amino pathogens, the diagnostic role of cultured fish cells
acid-rich nutrient medium such as Leibovitz- 15 that has also expanded. Along with the multiplying uses
does not require CO2 buffering has been successfully of fish cell culture is a concomitant increase in the
used with fish cell lines, thus CO2 incubators are need for guidelines for the health and maintenance
not necessary and cells can be grown conveniently of fish cell lines.
in any undisturbed areas. Additionally, because of
lower metabolic rates than eurythermic cells, fish Fish Cell
cells can be maintained with little care for long
periods of time.
Culture characteristics
The physiology and the blood plasma constituents of
In 1962, the first teleost cell line was reported in the teleost fishes are very much like those of terrestrial
literature. The first continuous fish cell culture (RTG- vertebrates; therefore the methodology for culture of
2) was originated over 20 year ago from rainbow cells and tissues is also similar. Most fish cell lines are
trout gonad tissue. Subsequently, many other cell lines readily propagated in vitro using unmodified media
of poikilothermic origin have been developed. Most and techniques developed for mammalian cells, with
fish cell lines have been established and utilized for appropriate adjustment of incubation temperatures
isolating, identifying, and studying viruses that cause to reflect the temperature range normal for the
economically important diseases. Consequently, the donor fish species. Also, osmolarity of the media
majority of these cell lines originate from species that must be adjusted upward for fishes of marine
are artificially propagated to some extent. Moreover, origin. Most important, fish tissue culture often
most such fish cell lines have been developed in North requires less time for preparation and maintenance.
America or Europe. Nowadays, more fish cell lines Mammalian cell culture techniques need only be
are available from fishes indigenous to Asia, since modified to reflect the lower incubation temperature
aquaculture and fish farming are pursued on a large requirements and the slower replication rates of the
scale in this part of the world. poikilothermic cells.

Wolf & Mann (1962) enumerated 61 cell lines originating Appropriate incubation temperatures for cultured
from 36 fish species, representing 17 families. These cell fish cells correspond to the normal temperature
lines were chiefly used for viral diagnostic purposes, and range of the fish species from which the cell line is
many had not been well characterized or previously derived. For lines from coldwater fishes, incubation
reported. Fryer and Lannan (1993) have compiled a temperatures range from 4-24ºC with an optimal
new listing of the fish cell lines reported in the literature range of 15-21ºC. For lines from warm water fishes,
that have been at least partially characterized. Recently incubation temperatures range from 15-37ºC, and
Central Marine Fisheries Research Institute

Lakra et al. (2010) made a comprehensive review on the the range of optimal incubation temperatures
characterized fish cell lines of both freshwater and salt is 25-35ºC.
water that have been developed after 1993.
Cell Culture Systems
Most fish cell lines originate from normal tissues, and
embryos or normal fins are most frequently listed as Once in culture, cells exhibit a wide range of
the source of the tissue used in the primary culture. behaviors, characteristics and shapes. Some of the
However, few cell lines were initiated from fish tumors, more common ones are described below. Two basic
and in some cases, these cells remained tumorigenic in culture systems are used for growing cells. These are
vivo following repeated in vitro passage. Traditionally, based primarily upon the ability of the cells to either
the chief uses of these cell lines were for detection and grow attached to a glass or treated plastic substrate
study of fish viruses and for diagnosis of the diseases (Monolayer Culture Systems) or floating free in the
caused by these agents. Today, fish cell cultures are culture medium (Suspension Culture Systems).

214
Types of Cells Development of cell line
Cultured cells are usually described based on their Primary Culture
morphology (shape and appearance) or their
functional characteristics. There are several ways with which monolayer
cultures of fish cells may be initiated. This is a quick
There are three basic morphologies: method that employs multiple explants of tissues
of either fresh water or marine fish as the simplest
• Epithelial-like: cells that are attached to a substrate way to produce monolayer cultures. When cells are
and appear flattened and polygonal in shape. surgically removed from an organism and placed
• Lymphoblast-like: cells that do not attach into a suitable culture environment, they will attach,
normally to a substrate but remain in suspension divide and grow. This is called a Primary culture.
with a spherical shape. There are two basic methods for doing this. First, for
• Fibroblast-like: cells that are attached to a Explant Cultures, small pieces of tissue are attached
substrate and appear elongated and bipolar, to a glass or treated plastic culture vessel and bathed
frequently forming swirls in heavy cultures. in culture medium. After a few days, individual
• Endothelial cells: Endothelial cells are very flat, cells will move from the tissue explant out onto the
have a central nucleus, are about 1-2 µm thick culture vessel surface or substrate where they will
and some 10-20 µm in diameter. begin to divide and grow.
• Other types: Macrophages, neuronal cells,
melanocytes, etc. The second, more widely used method, speeds up this
process by adding digesting (proteolytic) enzymes, such
It is important to remember that the culture conditions play as trypsin or collagenase, to the tissue fragments to

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

an important role in determining shape and that many cell dissolve the cement holding the cells together. This creates
cultures are capable of exhibiting multiple morphologies. a suspension of single cells that are then placed into

215
 culture vessels containing culture medium and allowed cells to the substrate. Some cell lines can be harvested
to grow and divide. This trypsinization method describes by gently scraping the cells off the bottom of the
warm (1-2 hrs at 37ºC) and cold (4 - 6ºC overnight (16 culture vessel. Once released, the cell suspension
hr) trypsinization of fish tissues which yields cultivable cells can then be subdivided and placed into new culture
and small aggregates of cells for monolayer cultures. The
disaggregated cells obtained by this procedure generally
yield more uniform monolayer more quickly than do
cultures initiated with minced tissues alone. This method
is called Enzymatic dissociation.

Before starting the preparation of primary cultutre,

food should be withheld from donor fish for a
day or more before use. Healthy specimens free of
external lesions are preferred; otherwise there is risk
of encountering systemically disseminated bacteria.
Cells and tissues should be cultured at a temperature
similar to the environmental temperature preferred
by the donor species. Extended exposure of tissues
from cold-water fishes such as salmon and trout to
30ºC can be lethal. In contrast, many and perhaps
most fish tissues remain viable even if held for a day vessels. Once a surplus of cells is available, they can
or two on ice or at 4ºC to 6ºC. Internal tissues may be treated with suitable cryoprotective agents, such
be safely removed after thorough topical disinfection; as dimethylsulfoxide (DMSO) or glycerol, carefully
this is conveniently done by total immersion of the fish frozen and then stored at cryogenic temperatures
for several minutes. A solution of liquid household (below -130ºC) until they are needed.
bleach having 500 ppm available chlorine, or a 1:1000
dilution of a quartenary ammonium compound may Development of continuous
be used. Excess disinfectant should be rinsed off with
chlorinated tap water or sterile water and the surgical
cell lines
site sponged with 70% isopropanol or ethanol. Some cell lines may give rise to continuous cell
External tissues such as those of fins, gills, corneas and lines. The ability of a cell line to grow continuously
barbels are severely damaged by such disinfection. probably reflects its capacity for genetic variation,
Consequently, such tissues should be excised first allowing subsequent selection. Genetic variation
and decontaminated separately. Immersion for 1 hr often involves the deletion or mutation of the
in a solution containing 500 IU polymyxin B, 500 µg p53 gene, which would normally arrest cell cycle
Central Marine Fisheries Research Institute

neomycin and 40 IU bacitracin is suggested, for these progression, if DNA were to become mutated,
are bactericidal antibiotics. and over expression of the telomerase gene.
Possibly the condition that predisposes most to the
Subculturing development of a continuous cell line is inherent
genetic variation, so it is not surprising to find
When the cells in the primary culture vessel have genetic instability perpetuated in continuous cell
grown and filled up all of the available culture lines. The alteration in a culture that give rise to a
substrate (called monolayer) they must be subcultured continuous cell line is communally called in vitro
to give them room for continued growth. This is transformation and may occur spontaneously or
usually done by removing them as gently as possible be chemically or virally induced. Immortalization
from the substrate with enzymes. These are similar means the acquisition of an infinite life span and
to the enzymes used in obtaining the primary culture transformation implies an alteration in growth
and are used to break the protein bonds attaching the characteristics (anchorage independence, loss

216
of contact inhibition and density limitation of themselves through cell division, sometimes after long
growth) that will often, but not necessarily correlate periods of inactivity. Second, under certain physiologic
with tumorigenicity or experimental conditions, they can be induced to
become tissue- or organ-specific cells with special
Many (if not most) normal cells do not give rise to functions. In some organs, such as the gut and
continuous cell lines. Normal human fibroblasts remain bone marrow, stem cells regularly divide to repair
euploid throughout their life span and at crisis will and replace worn out or damaged tissues. In other
stop dividing (around 50 generations), although may organs, however, such as the pancreas and the heart,
viable for 18 months. Human glia cells and chick stem cells only divide under special conditions.
fibroblasts behave similarly. Epidermal cells, on the
other hand, have shown gradually increasing life span Until recently, scientists primarily worked with two
with improvements in culture techniques and may kinds of stem cells from animals and humans:
yet be shown capable of giving rise to continuous embryonic stem cells and non-embryonic “somatic”
growth. Continuous cell line of lymphoblastoid cells is or “adult” stem cells. Scientists discovered ways to
also possible by transformation with Epstein-Barr virus. derive embryonic stem cells from early mouse embryos
nearly 30 years ago, in 1981. In 2006, researchers
made another breakthrough by identifying conditions
that would allow some specialized adult cells to be
Table 1. Properties of finite and continuous “reprogrammed” genetically to assume a stem cell-
cell line like state. This new type of stem cell, called induced
Properties Finite cell line Continuous cell pluripotent stem cells (IPSCs).
line
Ploidy Euploid, eiploid Aneuploid,
Stem cells are important for living organisms for

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

heteroploidy
Transformation Normal Immortal, growth many reasons. In the 3- to 5-day-old embryo, called
control altered and a blastocyst, the inner cells give rise to the entire body
tumorigeneic
Anchorage Yes No
of the organism, including all of the many specialized
dependence cell types and organs such as the heart, lung, skin,
Contact inhibition Yes No sperm, eggs and other tissues. In some adult tissues,
Density limitation of Yes No such as bone marrow, muscle, and brain, discrete
cell proliferation
Mode of growth Monolayer Monolayer or
populations of adult stem cells generate replacements
suspension for cells that are lost through normal wear and tear,
Maintenance Cyclic Steady phase injury, or disease.
possible
Serum requirement High Low
Given their unique regenerative abilities, stem cells
Cloning efficiency Low High
Markers Tissue specific Chromosomal, offer new potentials for treating diseases such as
enzymic, antigenic diabetes and heart disease. Laboratory studies of
Growth rate Slow Rapid stem cells enable scientists to learn about the cells’
Yield Low High essential properties and what makes them different
Control parameters Generation number, Stain characteristics
tissues specific from specialized cell types. Scientists are already using
markers stem cells in the laboratory to screen new drugs and
to develop model systems to study normal growth
Stem cell cultures and identify the causes of birth defects.

Stem cells have the remarkable potential to develop Stem cell research is one of the most fascinating
into many different cell types in the body during early areas of contemporary biology, but, as with many
life and growth. Stem cells are distinguished from expanding fields of scientific inquiry, research on
other cell types by two important characteristics. stem cells raises scientific questions as rapidly as it
First, they are unspecialized cells capable of renewing generates new discoveries.

217
 Characterization of effect. Their contamination also spreads quickly to
the other cell lines
Cell Lines
In contrast to mammalian cells, are easier to maintain Sources of contamination
and manipulate, and unlike primary cultures, produce
highly reproducible results. This ease of handling Another approach to cell culture contamination is
and simpler growth requirements makes cross- sources of contamination. The sources of microbial
contamination of cell lines a more likely possibility, culture contamination are different and may be
Proper characterization and identification of the cell grouped under four subjects.
lines are hence critical for scientific usage.
Contaminated cells, which are used as the primary
Authentication of a cell line is the sum of the process starting material for cell culture.
by which a line’s identity is verified and shown to
be free of contamination from other cell lines and Glassware or apparatus, including storage bottles
microbes. Tests used to authenticate cell cultures and pipettes
include iso-enzyme analysis, antigenic markers,
karyotyping/ cytogenetic analysis and more recently Culture media (serum, basal cultural media containing
molecular techniques of DNA profiling. Whilst most heat-sensitive essential amino acids and vitamins,
of the techniques above are generalized tests and enzymes like trypsin, pronase and collagenase, and
are applicable to all cell lines additional specific tests basic salt solutions).
may also be required to confirm the presence of a
product or antigen of interest. Airborne modes which can occur anytime the culture
vessel is opened or contact is made with culture
Cell line contamination fluid through a defective culture vessel, stopper, or
poor technique
Cell line contamination is a major drawback of main
cell banks of the world and it has cost of losing Cross-Contamination
important biological products or valuable research.
The causative agents are different chemicals,
and Misidentification
invertebrates, bacteria, fungi, parasites, viral species The problem of intraspecies and interspecies cross-
and even other cell lines. Bacteria, fungi, parasite, contamination among cell lines has been recognized for
viruses, invertebrates and mycoplasmas are main half a century. For those scientists working on cell lines
causative agents of cell line contamination. derived themselves or received from a colleague, basic
authentication tests such as STR profiling, isoenzyme
The bacterial and fungal (including molds analysis, and contamination tests are readily available
Central Marine Fisheries Research Institute

and yeast) contamination of cell lines (except and should be routinely used. Transferring cell lines to
mycoplasmas) can be readily detected, as these colleagues should be avoided, or when it does occur,
organisms cause increased turbidity, shift in media accompanied with comprehensive documentation
pH (change in medium color) and cell destruction. verifying the integrity of the material or tests need to
Some reports have indicated that putative be repeated. Although cross-contamination of fish cells
pathogens such as nanobacteria also will not be with other cell types has not been widely reported,
detected by this method. conveyed the identification of a cell line dubbed Clone
1A believed to be derived from rainbow trout as being
In the case of mycoplasmas their cell line contamination CHSE-214, a cell line derived from Chinook salmon
is always undetected for many passages. They can embryos. Accordingly, awareness of good laboratory
proliferate within the cell, tolerate antibiotics and their practices and careful vigilance with fish cell cultures
growth always does not have any obvious microbial as detailed by Lannan should be followed to avoid
evidence like turbidity and pH changes or cytopathic confusion of cell lines.

218
Applications of cell culture protein of medicinal and commercial value and
replacement of tissues and organs.
Fish cell lines have been useful in many areas of research.
Originally developed to support the growth of fish Genetic Counseling
viruses for studies in aquatic animal viral diseases, fish
cell lines have grown tremendously in number covering Amniocentesis, a diagnostic technique that
a wide variety of species and tissues of origin and an enables doctors to remove and culture fetal cells
array of applications. Fish immunology, physiology, from pregnant women, has given doctors an
genetics and development, toxicology, ecotoxicology, important tool for the early diagnosis of fetal
endocrinology, biomedical research, disease control, disorders. These cells can then be examined for
biotechnology and aquaculture are some of the areas in abnormalities in their chromosomes and genes
which fish cell lines have made significant contributions using karyotyping, chromosome painting and other
molecular techniques.
Toxicity Testing
Genetic Engineering
Cultured cells are widely used alone or in conjunction
with animal tests to study the effects of new drugs, The ability to transfect or reprogram cultured cells
cosmetics and chemicals on survival and growth in with new genetic material (DNA and genes) has
a wide variety of cell types. Especially important are provided a major tool to molecular biologists wishing
liver- and kidney-derived cell cultures. to study the cellular effects of the expression of theses
genes (new proteins).
Cancer Research
Gene Therapy

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Since both normal cells and cancer cells can be grown
in culture, the basic differences between them can be The ability to genetically engineer cells has also led
closely studied. Since, the normal cultured cells could to their use for gene therapy. Cells can be removed
be induced into cancer cells, the mechanisms that from a patient lacking a functional gene and the
cause the change can be studied. Cultured cancer cells missing or damaged gene can then be replaced. The
also serve as a test system to determine suitable drugs cells can be grown for a while in culture and then
and methods for selectively destroying types of cancer. replaced into the patient. An alternative approach
is to place the missing gene into a viral vector and
Virology then “infect’’ the patient with the virus in the hope
that the missing gene will then be expressed in the
One of the earliest and major uses of cell culture is patient’s cells.
the replication of viruses in cell cultures (in place of
animals) for use in vaccine production. Cell cultures Suggested readings
are also widely used in the clinical detection and
isolation of viruses, as well as basic research into how Fryer J L, Lannan C N. 1994. Three decades of fish cell culture: a
they grow and infect organisms. current listing of cell lines derived from fish. Journal of Tissue
Culture Methods 16:87–94.
Freshney R I. 2005. Culture of animal cells: a manual of basic
Cell-Based Manufacturing technique. Wiley, New Jersey.
Lakra W. S., Swaminathan T. R. and K. P. Joy. 2011. Development,
characterization, conservation and storage of fish cell lines: a
Cultured cells can be used to produce many important review. Fish Physiology and Biochemistry. 37(1): 1-20.
products, like viral vaccines, genetically engineered

219
 Methods for examination of Cell Culture
V. Srinivasa Raghavan
Scientist
Madras Research Center of CMFRI
e-mail: [email protected]

The cell cultures have to be evaluated for their associations can be noted down. The images of
growth properties and reproducibility of culture all these changes can be recorded using a camera
conditions. The general health of cell culture attached with the microscope. The different types
depends upon four important characteristics namely of microscope that can be used for evaluating a
morphology, growth rate, plating efficiency and cell culture are.
expression of functions. The morphology of cells
changes with the culture conditions but it is very Phase contrast microscopy
difficult to characterize or quantify the morphology
based on microscopic examination. The growth rate The differences in phases occur by placing a phase
of cells is determined by counting the number of annulus in the objective and mounting a fixed
cells which again vary with the culture conditions. annular phase ring in the condenser system. This
Plating efficiency is done to test the growth rate configuration creates a ring of light in the rear
and survivability of cells by placing few cells on focal plane of the objective, where 75% of the
to media and testing the number and size of central beam is absorbed. The refracted beam
cell colonies formed. The specialized functions of light is brought into focus at the eyepiece,
of cells are evaluated using biochemical and where it produces a bright image. Since the
immunological assays. beam intensity is significantly reduced in the rear
focal plane of the objective, a contrasting dark
Gross examination background is produced for the bright image, called
as “phase contrast.” The inverted phase contrast
Before subjecting the cultures for assays, the culture microscope gives much information when viewing
plates must be looked at morphologically for any unstained living tissue.
observations. The change of color of medium (Purple
coloration/yellow coloration) indicates that the pH of Fluorescence microscopy
the medium has changed either due to problem with
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CO2 supply or acidity of the medium. The plates also The phase contrast microscope is fixed with
should be checked for cloudiness or turbidity, floating epifluorescent objective to form a fluorescent
of debris or any other contamination in the medium. illumination. The cells are labeled with fluorescent
All the above indicate the overall healthiness of the dyes like Fluo-3, Fluo-4, Rhod-2, Calcium Green, and
cell cultures. Calcium Orange for assessing cell viability, function
and proliferation.
Microscopy
Confocal microscopy
The most important step in evaluating a cell
culture is the microscopic examination. The Confocal microscopy is used in the scanning imaging
cultures should be magnified to more than 100 mode for three dimensional imaging of specimens.
folds before making quantitative measurements. This helps in studying the cell’s physiology, cell
The changes in cell shape, cell size and cell to cell motility, and three dimensional structures.

220
Scanning Electron Flow Cytometry: It’s a laser based technology for
Microscopy (SEM) detection of cell concentration by allowing the cell
suspension to pass through an electronic beam.
The SEM helps in looking at the surface of cells at The cells are passed as single file through the flow
higher magnifications. The cells that are coated with cytometer, the laser hits the cells, light is scattered
a reflective metal such as gold are bombarded with and the photomultiplier measures the light intensity
an electron beam and the image thus obtained in the and the sensor measures the scatter pattern. Based on
microscope is created from the reflected electrons. the nature and type of cells, the computer categorizes
The cell surface features like cilia, cell shape and the cells and its quantity. Flow cytometry helps in
cell to cell association can be seen by scanning quantifying the cells, categorizing cells and also
electron microscopy. separate subpopulations of cells.

Transmission Electron MTT assay

Microscopy (TEM)
This assay measures the cell viability and
The TEM visualizes sub-cellular components such as cytotoxicity. The cells are stained with MTT
Mitochondria, Golgi vesicles and other organelles; (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl
even individual macromolecules such as DNA can be tetrazolium bromide). The assay is based on the
observed under TEM. principle of conversion of MTT into formazan
crystals by living cells. This method is easy-to-use,
Cell Counting safe, high reproducible and widely used in both
cell viability and cytotoxicity tests.
The growth of cells can be assessed under a

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

microscope with regard to its stage of culture. But Trypan blue assay
still the cells must be counted during the course
of an experiment for proper analysis which in turn Trypan blue is widely used for staining dead cells.
decides the reproducibility of a culture and also In this method, the cells are mixed with 0.4%
further subcultures. The different methods used for trypan blue dye and centrifuged. The supernatant
counting cultures are is discarded and the cell suspension in pellet form
is loaded on to a hemocytometer chamber and
• Direct methods like Hemocytometer, viewed under a microscope. The live cells have
flow cytometry intact cell membrane and so exclude the dye
• Indirect methods like staining with crystal violet from entering into the cell. In contrast, the dead
and MTT. cells due to the loss of membrane integrity take
up the dye readily and get stained in blue color.
Hemocytometer: In this method, the cells in Thus dead cells are seen as blue in color and the
suspension are placed in an optically flat chamber. viability is determined by counting the unstained
Since the area bounded by the lines and depth of cells with a microscope. However, this method can’t
the chamber is known, it helps us in counting the differentiate the healthy cells from that of live cells
number of cells in a particular volume of suspension losing functions.
and in turn the number or concentration of cells in
overall culture. Neutral red assay
The neutral red assay provides a quantitative
(Number of cells counted) estimation of the number of viable cells in a culture.
Concentration of cells =
(Volume counted) It is one of the most used cytotoxicity tests based on
the ability of viable cells to incorporate and bind the
i.e., Concentration of cells = Number of cells x 104 /ml neutral red dye in the lysosomes.

221
 LDH assay turbidity indicates the presence of bacterial
contamination. The cells may die subsequently.
The assay gives us an indication of the number The fungal/mold contamination is indicated by
of dead cells based on the estimation of lactate the presence of puffy milky white clumps or mat
dehydrogenase released by the dead cells into the like colonies. Under microscopic examination,
medium. Since LDH is a stable enzyme, its release into fungal filaments are seen in the cultures. The
the medium indicates the presence of dead tissues yeast contamination is indicated by budding
or cell damage. ovoid structures in the cell cultures on microscopic
examination. There should be routine examination
Contamination and of the cell culture plates for the presence of bacterial
and fungal contamination. The media routinely
its detection used to test bacterial and fungal contamination are
The detection of contamination and subsequent blood agar, thioglycollate broth, tryptic soy broth,
eradication from the cell culture system poses a major Sabouraud broth, and nutrient broth.
challenge for the researchers dealing with cell cultures.
The contamination may influence all the parameters Insects and parasites also contaminate the cell
that are to be studied during the course of a cell culture culture system. The viral contamination is generally
experiment. Even if one well is contaminated, it is better not a common feature in cell cultures. The use of
to discard the plates safely without retaining other wells infected bovine serum may pose threat in the form
for further experiments. Contaminants like mold can of viral contamination in cell cultures. If present, it
be seen with naked eye while others like bacteria and is very difficult to detect except by immune staining,
yeast are too small to be seen except under high power ELISA and PCR.
microscope. The contamination turns the medium cloudy
and changes the pH of the medium. The contaminated Immunohistochemistry
cultures should be viewed under microscope and if
confirmed, should be discarded immediately in proper Immunohistochemistry is used to visualize the
sealed bags after autoclaving. localization of a specific protein or antigen in cells
by use of a specific primary antibody that binds to
Mycoplasma it. It provides a rapid method for demonstrating
the presence of an antigen in a cell culture
In comparison to bacteria and yeast, mycoplasma system. With advances in antibody labeling and
contaminants grow very slowly. They don’t destroy cell staining techniques, immunohistochemistry
or kill the cells immediately. They alter the function can be a powerful technique for the detection of
and metabolism of the cell culture thereby causing surface and intracellular antigens. Fluorochrome
chromosomal aberrations, cytopathology, slow labeled antibodies is used for detecting sub-cellular
Central Marine Fisheries Research Institute

down growth rate of cells, cell transformation and components at high magnification. The sensitivity
alteration of membrane permeability, interfere with of detecting the antigens is increased by the use of
nucleic acid metabolism and change cell behavior. The enzyme labeled antibodies.
different assays used for the detection of mycoplasma
contamination are fluorescent staining, culture, Karyotyping
DNA probes and co-cultivation. Molecular tools like
hybridization of probes and PCR have been widely used The characterization of a cell line is very important to
for the easy detection of mycoplasma. They can also establish its future value. The analysis of chromosome
be detected using high power microscopy or SEM. known as Karyotyping is one of the conventional
methods utilized for characterization of cell lines.
Bacteria and Fungi Chromosome banding and chromosome painting are
generally used to distinguish individual chromosomes
The change in PH of the medium and subsequent by using specific molecular probes.

222
Isoenzyme analysis Suggested readings
Doyle, A and Griffiths, J.B. 1998. Cell and tissue culture. Laboratory
The analysis of isozymes is one of the qualitative
procedures in biotechnology.
methods to differentiate cell strains based on enzyme Freshney, I.R. 2010. Culture of animal cells (6th Edn).
protein polymorphisms. The enzymes are separated Mather, J.P and Roberts, P.E. 2002. Introduction to cell and tissue
culture, Theory and Techniques.
either by chromatography or electrophoresis and Mitsuhashi, J. 2002. Invertebrate cell culture methods.
the distribution patterns of the enzymes are very Mothersil,C and Austin, B. 2000. Aquatic invertebrate cell culture.
Copyright © Copyright by the World Aquaculture Society 2009
characteristic of a particular tissue or cell line.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

223
 Tissue Culture–Marine Invertebrates
C. P. Suja
Principal Scientist
Tuticorin Research Center of CMFRI
e-mail: [email protected]

Introduction compounds from marine sponges have anti-oxidant,

In biological and medical research, cell cultures are radical scavenging, anti-bacterial anti-inflammatory
valuable tools as it replaces the need for using live properties (Perdicaris, 2013). The cell cultures from
animals in experiments by providing an alternate sponges are getting great importance in producing
method. The culture of animal cells and tissues new medical drugs. Sponge cell culture is still in its
has undergone a major development from being primary stage and facing problems for maintaining
a purely experimental procedure to become an long term cultures. Use of antibiotics for maintaining
accepted technological component of many aspects the axenic condition in sponge cell culture has inhibitive
of biological research and commercial exploitation. effect on cell growth and contamination is the great
Remarkable progress has been achieved in medical problem without antibiotics. Single cells apparently do
and biological sciences in cellular biology, genetics not have the potency to produce secondary metabolites
and pathology by in-vitro investigations. Even though and primorph model is found as a suitable system for
the use of tissue culture in invertebrate physiology and the synthesis of bioactive compounds in vitro. Improved
pathology has been popular in recent years, only few media and improved culture conditions in this group
studies have been reported in marine invertebrates in should prospect the long term cultures (Rinkevich,
countries like Japan, USA, Canada, France and China. 1999, 2005).
Marine invertebrates encompassing 20 different phyla
is the richest source of varied cells and tissue types. Cnidaria
Cell culture works are reported mainly in Porifera,
Cnidaria, Crustacea, Mollusca, Echinodermata and Cnidarians are the more primitive of the animal phyla
Urochordata (Rinkevich, 2005) and no single marine and includes organisms such as jellyfish, sea anemones,
invertebrate cell line has been reported to date. corals and hydras. Primary cultures were also made from
the neurons which are disassociated from the nerve rings
Porifera of the jellyfish, Polyorchis penicillatus (Rinkevich, 1999).
Central Marine Fisheries Research Institute

Symbiotic relationship between many cnidarians and

Cell culture of sponges has been selected as a model unicellular algae was studied through in-vitro cnidarians
system for various cellular aspects. Large scale culture. Tissues of the striated muscle and endoderm
production of novel highly potent pharmacologically of the jellyfish have also been used as the substratum
significant bioactive compounds can be obtained from for primary cell cultures. Anemonia viridis, a temperate
in-vitro culture of sponges. Embryos and larvae from symbiotic sea anemone which belongs to the cnidaria
sponge have been evolved as a new source for cell was used as an experimental tool to study the molecular
culture (continuous cell lines). In comparison with the and cellular events involved in the rupture of symbiosis
adult sponge cells, sponge embryos and larvae are more between animal cells and their microalgae. To study this
adaptable as it has high content of stem cells and also molecular events primary culture of A. viridis from its
shows resistance to infections (De Caralt, 2007). Marine tentacles tissue was carried out. (Verdier, 2013). Corals
sponges are the sources of metabolites including are the most basic cnidarians and culturing their cells
cytotoxic and anticancer compounds. Bioactive in-vitro helps in the production of extra-cellular matrix

224
and precipitation of calcium carbonate (Helman, 2007). and P. japonicas were used widely in cell culture
In vitro crystallization of aragonite in coral cell cultures techniques. The embryonic, ovary and larval tissues
was proved as an innovative approach to investigate were selected for cell culture as these cells are
reef-building coral calcification at the cellular level in mitotically active and resistant to contamination than
the cell cultures of a hard coral, Pocillopora damicornis adult tissues. Cell cultures derived from crustaceans
(Rinkevich, 2005). are widely used in studying pathogens, especially
viral pathogens. Therefore it is necessary that the cell
Echinodermata line should be capable of supporting viral growth in
in-vitro conditions (Rinkevich, 2005). Lymphoid organ
The primary cultures from echinoderms were mainly is mostly selected as a tissue source as it is the target
performed on sea urchin cells, from ovaries of the organ for many viral infections. Tissues from heart, gill,
edible sea urchin Paracentrotus lividus. Primary cell nerve, gut, testis, eye, epidermis have also been used
cultures from starfish, Marthasterias glacialis with as a source for primary cell cultures. The development
tissue samples taken from tube feet and body wall also of cell line from crustaceans will help in understanding
reported (Bavington et al. 2000). Coelomocytes from the viruses that attack them and in developing control
the sea cucumber Holothuria tubulosa and star fish, measures. Primary hemocyte culture from the Penaeus
Asterias rubens were used as a cellular model for toxicity monodon a marine crustacean helps in assessing the
testing and biomonitoring (Ronning, 2006). Primary cell cytotoxicity and genotoxicity of the environmental
cultures of echinoderms were used as an ideal system to pollutants such as cadmium chloride and mercuric
explore the cellular, biochemical, and genomic aspects of chloride (Jose, et al. 2011).
echinoderm regenerative properties (Bello et al., 2015).
Mollusca
Urochordata

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Molluscs are the largest marine phylum which includes
Cell cultures of tunicates, a primitive group of snails, mussels, limpets, oysters, clams etc; Molluscan
phylum Chordata is a powerful tool for studying the cells can be derived from any tissue and can be cultured
phylogeny of different biological characters such as in in-vitro conditions. As for now only one molluscan cell
developmental and cellular biology, immunology and line developed is the Bge cell line (embryonic) which is
genetics. Hemocytes and the pharyngeal explants isolated from a fresh water snail, Biomphalaria glabrata
from the colonial tunicates were the major cell type Say (Hansen, 1976). The early developing embryos and
studied from the tunicate Styela clava. Proliferative the larval stages are the most suitable source of primary
activity of pharyngeal hemocytes and differentiation cell culture. Hemocytes in molluscs exist as different cell
state of circulatory hemocytes was studied by in-vitro types and express different functions like phagocytosis,
culture. Proliferation activity of the pharyngeal cells cell signaling pathways etc., Due to its defensive
of tunicates was enhanced in in-vitro primary cell phagocytic property, it is easier to develop cultures with
culture after stimulating these cells by Interleukin limited contamination than other molluscan tissues.
1 like molecule extracted from the tunicates. Cells Circulatory hemocytes are terminally differentiated and
from the palleal buds of the colonial tunicate Botryllus short lived. However, long term cultures from marine
schlosseri was used as a source in epithelial cell culture clams were reported from neoplastic and cancerous
(Rinkevich, 1999, Rabinowitz and Rinkevich, 2003). hemocytes. Hemocyte cultures play a crucial role in
Kawamura et al., 2006 established in vitro culture investigating pathogens like Perkinsus marinus and
of mesenchymal lineage cells from the colonial immune function of commercially important bivalves.
tunicate Botryllus primigenus. Neuronal cells of molluscs are used as models for
studying the complexity of memory and other
Crustacea behavioural capacities of higher organisms. Primary
cell cultures of neurons were derived from molluscan
Attempts were made to develop cell lines from species of the genus Aplysia, Lymnaea, Helisoma and
crustaceans mainly with penaeid species. P. monodon Helix. Primary cell cultures were also obtained from

225
 tissues derived from organs such as heart, mantle, concavalin A are incorporated to the culture medium
gill, digestive gland and gonads. Cardiomyocyte according to the requirement. Antibiotics such as
cultures of oysters are used as model systems for kanamycin, streptomycin, rifampicin, gentamicin,
electrophysiological studies, molecular genetic studies nystatin, amphotericin, penicillin, ampicillin are added
and environmental toxicological monitoring. Tissues in the culture media to prevent contamination. Sterile
from cardiac muscle of surf clam Spisula solidissimia vessels can be selected according to the requirement
have also been used in growing mono layer cell lines of cell growth. Petri plates, T25 flasks, 6, 12, 24 and
for long periods. As gill and digestive glands have direct 96 well plates are also available and are used for
contact with the natural environment, primary cell different cell culture studies.
cultures of these tissues are considered as a suitable
system for biomonitoring of different chemical Cell cultures are incubated inside a CO2 incubator
contaminants in aquatic systems (Yoshino, 2013). for regulating CO 2 concentration. The marine
In-vitro pearl production was successful through the invertebrates require lower temperature at 20-25ºC
culture of mantle tissue from the abalone Haliotis varia than mammalian cell types at 37ºC. Some media
Linnaeus (Suja and Dharmaraj, 2005). Mechanism of requires no additional CO2, if it is cultured in sealed
cell proliferation, pearl sac formation, crystal secretion vessels. Cryopreservation is mainly used to conserve
and nacre formation was revealed by the mantle tissue various tissues such as embryo, larvae for a long
culture of the pearl oyster, Pinctada fucata, abalone time. Cryopreservation was basically developed for
Haliotis varia and other molluscs (Dharmaraj and Suja vertebrate cells. But few studies reported successful
2006; Awaji and Machi, 2011). Primary cell culture cryopreservation in marine invertebrates also.
derived from molluscs was proved to be a suitable tool Cell cultures obtained from the tissues of marine
for in-vitro studies of bio-mineralization (Bordenave, invertebrates can be frozen upto -196ºC. Dimethyl
2010). In-vitro synthesis of proteoglycans and collagen sulphoxide (DMSO) and trehalose are commonly used
was possible by culturing the mantle cells from as cryoprotectants (Odintsova, 2001).
nacreous mollusc Haliotis tuberculata. Through this
model molluscan extra-cellular matrix was studied well Obstacles in Development
(Rinkevich, 2005). In the last decade in vitro pearl sac
formation and nacre biomineralization created great
of Cell Cultures
interest in the cell culture of molluscs. Organisms in the marine environment are exposed
to natural microbial flora and fauna of the habitat.
Culture Conditions The successful development of cell cultures
depends on axenic conditions. Contamination is
Culture medium is the vital factor to control the the major problem for long term cultures of marine
growth of culture. Basic cell culture media should invertebrates. The control of microbial contaminants
provide an optimal pH value, specific osmolality, with the antibiotics will adversely affect the
Central Marine Fisheries Research Institute

nutrients such as vitamins and other trace elements. growth of primary cells. The common type of
There are several types of media which are enriched contamination in many cultures is the appearance of
in various supplements to support the growth of thraustochytrids. The standardization of optimum
marine invertebrates cell cultures. The basis of cell dose of antibiotics in culture is a vital step for the
culture media is the balanced salt solution and it success of culture. Marine invertebrate organisms
should be sterile. Sea water, Medium 199, L-15, comprise of different cells and tissues and each cell
Ham’s F-12, RPMI 1640, EMEM, and Iscove’s MDM type require different culture conditions. Hence
etc. are the common media available in markets components of culture media should be formulated
which can be procured directly from companies according to the cell types with optimum pH,
or can be prepared as per compositions. Growth and osmotic conditions, and this is detrimental
supplements like tissue extracts, FCS (Foetal calf to cell proliferation. Limited knowledge of cell
Serum), Lactalbumin hydrolysate, growth factors (EGF) adherence preferences, optimum concentration of
vitamins, carbohydrates, lipids, amino acids, salts, inorganic and organic components of media, toxic

226
 studies. J Sci Hal Aquat, 2, 42-47.
compounds released during explant preparation Barnay-Verdier, S., Dall’Osso, D., Joli, N., Olivré, J., Priouzeau, F.,
and culture are some of the factors for the failures Zamoum, T & Furla, P. (2013). Establishment of primary cell
culture from the temperate symbiotic cnidarian, Anemonia
of cell growth in marine invertebrates. Use of trypsin viridis. Cytotechnology, 65(5), 697-704.
and other enzymes for harvesting and subculturing Bavington, C. D., Moss, C., & McKenzie, J. D. (2001). The use
of cells will also affect the viability of cells. Better of lectins, particularly concanavalin -The development of
primary cell cultures from echinoderms. In Echinoderms 2000:
understanding of secondary metabolites during cell Proceedings of the 10th International Conference, Dunedin,
growth can answer the problems in cell viability for 31 January-4 February 2000 (p. 149).
Bello, S. A., Abreu-Irizarry, R. J., & García-Arrarás, J. E. (2015).
long term maintenanace. Primary Cell Cultures of Regenerating Holothurian Tissues.
In Tissue Morphogenesis (pp. 283-297). Springer New York.
De Caralt, S., Uriz, M. J., & Wijffels, R. H. (2007). Cell culture
Conclusion from sponges: pluripotency and immortality. Trends in
biotechnology, 25(10), 467-471.
Marine invertebrate tissue culture has its Dharmaraj S & Suja C. P. (2006). Pearl production techniques
through tissue culture in the Indian pearl oyster (Pinctada
application in various fields such as molecular fucata) and in the abalone (Haliotis varia) and other pearl
biology, medicine, pharmaceutical industries, producing molluscs, International patent,2006 No.PCT/
cosmetics production, detection of marine IB 2006/003299
Helman, Y., Natale, F., Sherrell, R. M., LaVigne, M., Starovoytov,
pollutants, pearl production mechanisms etc. V., Gorbunov, M. Y., & Falkowski, P. G. (2008). Extracellular
Numerous studies on marine invertebrates matrix production and calcium carbonate precipitation by
coral cells in vitro. Proceedings of the National Academy of
primary cell culture indicate that we still need to Sciences, 105(1), 54-58.
establish standard protocols for the production of Jose, S., Jayesh, P., Mohandas, A., Philip, R., & Singh, I. B. (2011).
secondary cell metabolites and cell immortalization Application of primary haemocyte culture of Penaeus
monodon in the assessment of cytotoxicity and genotoxicity
(Rinkevich, 2005). The increasing demand of of heavy metals and pesticides.Marine environmental
biomaterials from marine invertebrates fascinated research, 71(3), 169-177.
Kaneko, H., Kawahara, Y., & Dan-Sohkawa, M. (1995). Primary
new researchers into this challenging field. The

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

culture of mesodermal and endodermal cells of the starfish
development of cell cultures from economically embryo. Zoological Science, 12(5), 551-558.
important species will be a powerful tool for Kawamura, K., Takeoka, S., Takahashi, S., & Sunanaga, T. (2006).
In vitro culture of mesenchymal lineage cells established
disease diagnosis, biomonitoring, drugs and novel from the colonial tunicate Botryllus primigenus. Zoological
biomaterials. Development of enhanced culture science, 23(3), 245-254.
Odintsova, N., Kiselev, K., Sanina, N., & Kostetsky, E. (2001).
media with growth additives and factors will lead Cryopreservation of primary cell cultures of marine
to a better insight in the development of cell invertebrates. Cryo-letters, 22(5), 299-310.
cultures from marine invertebrates. Introduction Perdicaris, S., Vlachogianni, T., & Valavanidis, A. (2013). Bioactive
natural substances from marine sponges: new developments
of novel genomic and proteomic tools will help and prospects for future pharmaceuticals. Nat Prod Chem
in developing continuous cell lines using standard Res, 1(3), 1-8.
Rinkevich, B. (1999). Cell cultures from marine invertebrates:
procedures. Control of contamination through obstacles, new approaches and recent improvements. Journal
advanced laboratory protocols and combination of of Biotechnology, 70(1), 133-153.
new antibiotics will definitely give better growth Rinkevich, B. (2005). Marine invertebrate cell cultures: new
millennium trends. Marine biotechnology, 7(5), 429-439.
and survival of cultures. Modifications from the Rønning, I. (2006). Echinoderm coelomocytes as a cellular model in
existing protocols with modern techniques will toxicity testing and biomonitoring. Master Thesis. Programme
for Toxicology and Ecophysiology, Department of Biology,
certainly give promising results in this field. University of Oslo.
Suja, C. P. & Dharmaraj, S. (2005). In vitro culture of mantle tissue of
the abalone Haliotis varia Linnaeus. Tissue and Cell, 37(1), 1-10.
Suggested readings Yoshino, T. P., Bickham, U., & Bayne, C. J. (2013). Molluscan cells in
culture: primary cell cultures and cell lines 1. Canadian Journal
Auzoux-Bordenave, S., & Domart-Coulon, I. (2010). Marine of Zoology, 91(6), 391-404.
invertebrate cell cultures as tools for biomineralization

227
 Molecular markers in Population Genetics
K. A. Sajeela
Technical Assistant
Marine Biotechnology Division CMFRI
e-mail: [email protected]

Genetic variation describes naturally occurring genetic development of DNA-based genetic markers has
differences among individuals of the same species. had a revolutionary impact on animal genetics. A
Natural populations show variation at all levels, from number of different techniques have emerged,
gross morphology to DNA sequences. They differ in ranging from sequencing of the DNA of interest to
their morphology, their microscopic structure, their methods analyzing length polymorphisms, such as
chromosomes, the amino acid sequences of their microsatellites. It is theoretically possible to observe
proteins, and in their DNA sequences. Population and exploit genetic variation in the entire genome of
genetics is defined as the science of how genetic organisms with DNA markers.
variation is distributed among species, populations and
individuals. Population is a sub-species category, formed Genetic markers can be categorized based on their
by a group of conspecific individuals as a breeding unit mode of transmission and evolutionary dynamics
sharing a particular habitat at a certain time. The stock into (1) protein markers such as allozymes and (2)
concept subdivided species to closed populations or DNA markers such as (2.1) mitochondrial DNA and
‘stocks’ that maintain themselves in a given area and (2.2) nuclear DNA markers like randomly amplified
are reproductively isolated from other such populations. polymorphic DNA (RAPDs) and variable number of
tandem repeats (VNTRs) loci such as minisatellites
The logical and practical reasons for identifying and microsatellites.
population units are that such units may have their
own characteristics of recruitment, growth, natural Principles and
mortality, migration, behaviour etc. Assessment
using ecological parameters, tagging, parasite
categorization of
distribution, physiological, behavioral, morphometric, molecular markers
meristic, calcareous, biochemical and cytogenetic
characters identify stock structure and genetic All organisms are subject to mutations as a result
variation in a population. Different methodologies of normal cellular operations or interactions with
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evaluating genetic variation begin with the very the environment, leading to genetic variation
traditional comparative examination of morphology (polymorphism). In conjunction with selection and
to the most innovative DNA-based genetic markers. genetic drift, there arises genetic variation within
The inefficiency of conventional morphometric and among individuals, species, and higher order
measurements led to the development of ‘truss taxonomic groups. For this variation to be useful to
network analysis’, where the shape of the body forms geneticists, it must be (1) heritable and (2) discernable
of fish or shellfish is also taken into account along with to the researcher, whether as a recognizable
the size. In the mid-fifties, protein electrophoresis and phenotypic variation or as a genetic mutation
histochemical staining methods gained advantage distinguishable through molecular techniques. At
over morphological studies. Analysis of allozyme loci the DNA level, types of genetic variation include:
remained one of the most popular approaches in base substitutions, commonly referred to as single
examining population genetics and stock structure nucleotide polymorphisms (SNPs), insertions or
questions in fishes till the advent of the PCR. The deletions of nucleotide sequences (indels) within a

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locus, inversion of a segment of DNA within a locus, genome evolution, candidate gene identification
and rearrangement of DNA segments around a locus etc. Type II markers are considered to be non-coding
of interest. DNA marker technology can be applied to and therefore selectively neutral. Such markers have
reveal these mutations. Large deletions and insertions found widespread use in population genetic studies
(indels) cause shifts in the sizes of DNA fragments whose characterizations of genetic diversity and
produced upon digestion by restriction enzymes, and divergence within and among populations are based
are among the easiest type of mutations to detect, on assumptions of Hardy–Weinberg equilibrium and
mainly by electrophoresis of the fragments on an selective neutrality of the markers employed.
agarose gel; smaller indels require DNA sequencing
or more elaborate electrophoretic techniques to Protein Markers
determine smaller changes in size. Inversions and
rearrangements that involve restriction sites can be
easy to detect because they disrupt the ability of a
Allozymes
restriction enzyme to cut DNA at a given site and Isozymes are functionally similar and separable forms
thus can produce relatively large changes in DNA of enzymes encoded by one or more loci. Isozyme
fragment sizes. Point mutations are more difficult to products of different alleles at the same locus are
detect because they do not cause changes in fragment termed as allozymes. They inherit co-dominantly
sizes. Several marker types are highly popular in so that genetic interpretation (genotype) of the
genetics. In the past, allozyme and mtDNA (restriction phenotype is facilitated because all products are
fragment length polymorphism (RFLP)) markers have normally visible and not masked by dominance of one
been popular genetics research. More recent genetic over another. Allozyme electrophoresis has been used
markers employed in fisheries and aquaculture are in defining genetic markers for stock identification on
mitochondrial DNA sequence information, randomly the basis of differences in allelic frequencies between

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

amplified polymorphic DNA (RAPD), amplified stocks in many species. Using allozyme markers, it
fragment length polymorphism (AFLP), microsatellite, is possible to determine whether a population is
single nucleotide polymorphism (SNP), and expressed a random mating one with equilibrium genotypes
sequence tag (EST) markers. frequencies or sample comprise of an assembly of
genetically distinct units. Their allele frequencies
Molecular markers are classified into two categories: primarily respond to mutation, gene flow and drift.
type I are markers associated with genes of known One of the limitations of enzyme variants as genetic
function, while type II markers are associated with markers is the low level polymorphism observed in
anonymous genomic segments. RFLP, allozyme and some species and populations. The extensive allozymes
EST markers are type I markers because they were studies undertaken on fish stocks have not only
associated with known genes or transcripts of genes. proven valuable for estimating population divergence,
RAPD, AFLP and Microsatellite markers are type II but also have focussed attention on the underlying
markers because these are amplified from anonymous evolutionary forces that promote differentiation.
genomic regions via the polymerase chain reaction
(PCR). SNP markers are mostly type II markers unless DNA markers
they are developed from expressed sequences (eSNP
or cSNP). Indels are becoming more widely used Based on the source of DNA, markers can be classified
as markers since they often are discovered during into (1) mitochondrial (2) nuclear and (3) chloroplast
genomic or transcriptomic sequencing projects; they DNA markers.
can be either type I or type II markers depending on
whether they are located in genes. In addition to Mitochondrial DNA (mt DNA)
their functions as markers in population studies, type
I markers are becoming very important in studies of The mitochondrial genome is a small and double
genetic linkage and QTL mapping. Type I markers stranded circular DNA molecule. It is haploid,
have utility in studies of comparative genomics, maternally transmitted, non-recombinant and has

229
 high mutation rate compared to nuclear DNA. These other. Considering this, PCR will amplify many random
factors in combination reduce the effective population fragments that can vary in sizes when different species,
size for mt DNA to one fourth in comparison to single subspecies, populations or individuals are analysed and
copy nuclear DNA and making it a powerful tool this will constitute the basis of identification.
for elucidating population structures and estimating
phylogenetic relationships. RAPD analysis has several advantages. These
include relatively shorter time (1-2 days) required
The vertebrate mitochondrial genome is composed to complete analysis after standardization; no need
of about 15 to 20 kb in different organisms, of prior information on the genome of an organism;
coding for 40 genes responsible for 2 ribosomal availability of series of primers for analysis; minimal
RNAs, 22 transfer RNAs, and 13 proteins essential operational cost requirement; relatively smaller
in respiration. It also has a non-coding region amount (»20 ng) of high molecular weight DNA;
(+ 1000bp) responsible for replication, known simpler protocol and involvement of non-invasive
as the “control region” or “d-loop”, that evolves sampling for tissue analysis. However, the application
4–5 times faster than the entire mtDNA molecule and interpretation of RAPD – PCR in population
which itself evolves 5 to 10 times faster than nuclear genetics is not without technical problems and
DNA mainly because the mitochondria do not have practical limitations. The main negative aspect
repair enzymes for errors in the replication, nor of this technique in is the necessity of extensive
for the damages of the DNA. Partial sequences standardization to obtain reproducible results. In
of mitochondrial DNA genes especially 16S rRNA addition, most of the RAPD polymorphisms segregate
and Cytochrome c Oxidase I and Cytochrome b as dominant markers and individuals carrying two
has proved suitable to resolve the phylogenetic copies of an allele cannot be distinguished from
relationships. There are some disadvantages also individuals carrying one copy of an allele. The limited
with this marker like; maternal inheritance does not sample size in each population and the specific RAPD
provide information about males in populations, primers utilized can also have an influence over
which may display different dispersal behavior to the results. Even then RAPD technique is used in
females and its haploid nature is also an issue as microbes, plants and animals for resolving taxonomic
no inferences about the neutrality and equilibrium ambiguities and stock identification.
of populations, as well as other aspects based on
allelic frequencies can be addressed. RAPD-PCR technique can also generate species-
specific, sex-specific and population specific
Beyond discriminating closely related species, mt fragments. These fragments are useful in developing
DNA has been used to explore intra-specific genetic specific “Sequence Characterized Amplified Region
polymorphism. Fast evolving ATPase and COI gene (SCAR) Markers”. For this, SCAR primers need to be
sequence information is useful in assessing population synthesized from specific RAPD fragments. Usually,
Central Marine Fisheries Research Institute

structure of the same species. fragments above 1000 bp and less than 300 bp are
not considered to develop SCAR markers owing to
Nuclear DNA Markers difficulties arising from co-migration and the lesser
possibility of designing suitable primers from smaller
Random Amplified Polymorphic DNA (RAPD): The fragments. The identified fragments are excised from
principle behind Randomly amplified polymorphic DNA the gel, purified and sequenced; and based on the
(RAPD) analysis is that at low annealing temperatures sequence information, suitable SCAR primers are
or high magnesium concentrations, a primer is likely to synthesized. These primers will amplify only specific
find many sequences within the template DNA to which fragments that are useful in settling taxonomic
it can anneal. Depending on the length and complexity disputes and identifying sex or distinct populations.
of genome of an organism, there can be numerous However, to identify specific RAPD fragments,
pairs of these sequences and they will be arranged screening of large number of samples and RAPD
inversely to and within about two kilobases of each primers are required.

230
Microsatellites: Microsatellites–also referred to assays); homoplasy; and need for very high sample size
as short tandem repeats (STRs) or simple sequence if more alleles are noticed. Also, microsatellite flanking
repeats (SSRs), discovered in the early 1980s. regions (MFRs) sometimes contain length mutations
Microsatellites are repeated DNA sequences having which may produce identical length variants that could
a unit length of 1-6 base pairs tandemly repeated compromise microsatellite population level studies.
minimum 6 times usually; maximum several times at
each locus. Depending on the purity and complexity EST-SSR: Expressed sequence tags (ESTs) are single-
of the motifs, SSRs can be perfect (single motif in pass sequences generated from random sequencing
an uninterrupted array) or imperfect (single motif of cDNA clones. ESTs are type I molecular markers
interrupted) or compound (two or more motifs in with known functions which provide gene candidates
interrupted or uninterrupted arrays). Based on the for production traits and are important components
number of base pairs in a repeat unit, microsatellites in genome mapping projects. The vast EST data
can be again classified into mono (e.g. C or A), di collections available from genomic libraries and
(e.g. CA), tri (e.g. CCA), tetra (e.g. GATA) repeat random genomic sequences are a valuable source
unit microsatellites. The most common ones are of gene-based SSR markers for population genetic
dinucleotide repeats. analyses. EST-SSRs have been found to be significantly
more transferable across taxonomic boundaries and
Microsatellites represent ideal molecular markers less polymorphic than traditional ‘anonymous’ SSRs.
because they have multiple alleles that with highly
polymorphic among individuals and are highly Single nucleotide polymorphism (SNP): Single
abundant (once every 10 kbp in fish species) and nucleotide polymorphisms or SNPs (pronounced
dispersed evenly throughout eukaryotic genomes. “snips”) are DNA sequence variations that occur when
Microsatellites are neutral and inherited in Mendelian a single nucleotide (A, T, C, or G) in the genome

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

fashion. High variability, ease and accuracy of assay make sequence is altered between individuals. SNPs are
microsatellites the best for high-resolution population again becoming a focal point in molecular marker
analysis. While genes offer functional sequences, development since they are the most abundant
microsatellites offer highly polymorphic sequences. polymorphism which can be used for population
Length and point mutations are the mutational genetic studies. SNPs can occur in both coding (gene)
events proposed to occur in microsatellites. Length and noncoding regions of the genome. Theoretically, a
mutations, i.e. copy (or repeat) number mutations lead SNP within a locus can produce as many as four alleles,
to new allelic variants. Compared to other genomic each containing one of four bases at the SNP site: A,
markers, microsatellites are highly reproducible and T, C, and G. Practically, however, most SNPs are usually
easily transferable between laboratories. The main restricted to one of two alleles (most often either the
limitation of SSR markers is primer design for each two pyrimidines C/ T or the two purines A/G) and
new species demands considerable time, effort and have been regarded as bi-allelic. Obviously, their PIC
cost. The approaches available to develop microsatellite is not as high as multi-allele microsatellites, but this
markers are: 1) Survey of GenBank and EMBL databases shortcoming is balanced by their great abundance.
2) screening of genomic or cDNA libraries 3) use of SNP markers are inherited as co-dominant markers.
primers already developed in related species 4) next- If SNPs change either the function of a gene or its
generation sequencing (NGS). expression, and the change provides greater fitness
for a population (i.e., a higher capacity to survive and/
Microsatellites are also becoming increasingly or reproduce in a given environment), the change
popular in forensic identification of individuals, and will be favored by natural selection. Therefore, SNPs
determination of parentage and relatedness, genome can be the basis of evolutionary change. The physical
mapping, gene flow and effective population size location of SNPs will be determined in a similar way
analysis. Microsatellites too have some negatives like to micro-satellite markers. SNPs will, at least initially,
appearance of stutter bands, presence of null alleles be selected based on how informative they might be
(existing alleles that are not observed using standard as genetic markers. SNPs in low proportions (<10 %)

231
 Gopalakrishnan, A (2009). Molecular Markers. Training Manual on
will be less informative than SNPs at higher frequency Advances in DNA Marker Techniques. pp. 32-45.
(30-50 %) in a given population. Sajeela, K. A., Gopalakrishnan, A., Basheer, V. S., Bineesh, K. K.,
& Jena, J. K. (2015). Development and characterization of
eighty-one microsatellite markers in Indian white shrimp,
Suggested readings Fenneropenaeus indicus, through cross-amplification. Journal
of genetics, 94(2), 43-50.
Norrgard, K. & Schultz, J. (2008) Using SNP data to examine human
Ellis, J. R., & Burke, J. M. (2007). EST-SSRs as a resource for phenotypic differences. Nature Education 1(1):85
population genetic analyses. Heredity, 99(2), 125.
Central Marine Fisheries Research Institute

232
Recombinant DNA technology and
Molecular Cloning
Reynold Peter
Research Associate
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
Finally, the cloned DNA segments can be recovered
The basis of most molecular biology technologies is from the host cell, purified, and utilized for
the gene and to facilitate the study of genes, they various applications.
have to be isolated and amplified. One method of
isolation and amplification of a gene of interest is Sources of DNA for cloning
to clone the gene by inserting it into another DNA
molecule that serves as a vehicle or vector that can The cloning will work for any random piece of DNA.
be replicated in living cells. When these two DNAs But since the goal of many cloning experiments is to
of different origin are combined, the result is a obtain a sequence of DNA that directs the production
recombinant DNA molecule. The recombinant DNA of a specific protein, we need to first consider where

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

molecule is placed in a host cell, either prokaryotic to obtain such DNA. Sources of DNA for cloning into
or eukaryotic and the host cells are then replicated vectors may be DNA fragments representing a specific
(producing a clone), and the vector with its foreign gene or portion of a gene, or may be sequences
piece of DNA also replicates. The foreign DNA thus of the entire genome of an organism, depending
becomes amplified in number, and following its on the end goal of the researcher. Typical “inserts”
amplification can be purified for future applications. include genomic DNA, cDNA, polymerase chain
reaction (PCR) products, and chemically synthesized
Molecular cloning oligonucleotides. When previously isolated clones
are transferred into a different vector for other
The basic procedure of molecular cloning involves applications, this is called “subcloning.”
a series of steps. First, the DNA fragments to be
cloned are generated by using PCR and restriction Vector DNA
digestion. Second, the fragments produced by
digestion with restriction enzymes are ligated to Cloning vectors are carrier DNA molecules. Four
other DNA molecules that serve as vectors. Vectors important features of all cloning vectors are that
can replicate autonomously (independent of host they: (i) can independently replicate themselves and
genome replication) in host cells and facilitate the the foreign DNA segments they carry; (ii) contain a
manipulation of the newly created recombinant DNA number of unique restriction endonuclease cleavage
molecule. Third, the recombinant DNA molecule sites that are present only once in the vector; (iii) carry
is transferred to a host cell. Within this cell, the a selectable marker (usually in the form of antibiotic
recombinant DNA molecule replicates, producing resistance genes or genes for enzymes missing in the
dozens of identical copies known as clones. As the host cell) to distinguish host cells that carry vectors
host cells replicate, the recombinant DNA is passed from host cells that do not contain a vector; and (iv)
on to all progeny cells, creating a population of are relatively easy to recover from the host cell. There
identical cells, all carrying the cloned sequence. are many possible choices of vector depending on the

233
 purpose of cloning. The greatest variety of cloning vector are not 100% productive, because the two
vectors has been developed for use in the bacterial ends of a plasmid vector can be readily ligated
host E. coli. Thus, the first practical skill generally together, which is called self-ligation. The degree
required by a molecular biologist is the ability to grow of self-ligation can be reduced by treatment of
pure cultures of bacteria. the vector with the enzyme phosphatase, which
removes the terminal 5`-phosphate. When the
Choice of vector is 5`-phosphate is removed from the plasmid it
cannot be recircularized by ligase, since there is
dependent on insert size nothing with which to make a phosphodiester
and application bond. But, if the vector is joined with a foreign
insert, the 5`-phosphate is provided by the
The classic cloning vectors are plasmids, phages, and foreign DNA. Another strategy involves using two
cosmids, which are limited to the size insert they different restriction endonuclease cutting sites with
can accommodate, taking up to 10, 20, and 45 kb, noncomplementary sticky ends. This inhibits self-
respectively. In some cases genes are often greater ligation and promotes annealing of the foreign
than 100 kb in size, so there were limitations in DNA in the desired orientation within the vector.
cloning complete gene sequences. To circumvent this
new generation of artificial chromosome vectors like Cutting and joining of DNA
bacterial artificial chromosomes (BACs), yeast artificial
chromosomes (YACs), and mammalian artificial Two major categories of enzymes are important
chromosomes (MACs) where engineered more tools in the isolation of DNA and the preparation
recently have problem by mimicking the properties of recombinant DNA: restriction endonucleases and
of host cell chromosomes. DNA ligases. Restriction endonucleases recognize
a specific, rather short, nucleotide sequence on a
Plasmid DNA as a vector double-stranded DNA molecule, called a restriction
site, and cleave the DNA at this recognition site
Plasmids are naturally occurring extrachromosomal or elsewhere, depending on the type of enzyme.
double-stranded circular DNA molecules that carry DNA ligase joins two pieces of DNA by forming
an origin of replication and replicate autonomously phosphodiester bonds.
within bacterial cells. The plasmid vector pBR322,
constructed in 1974, was one of the first genetically Transformation: transfer of
engineered plasmids to be used in recombinant DNA.
These early vectors were often of low copy number,
recombinant plasmid DNA
meaning that they replicate to yield only one or two to a bacterial host
copies in each cell. pUC18 is a derivative of pBR322.
Central Marine Fisheries Research Institute

This is a “high copy number” plasmid (> 500 copies The ligation reaction mixture with ‘vector’ and
per bacterial cell). ‘insert’ DNA is introduced into bacterial cells in a
process called transformation. The traditional method
Plasmid vectors are modified to contain a specific is to incubate the cells in a concentrated calcium
antibiotic resistance gene and a multiple cloning salt solution to make their membranes leaky. The
site (also called the polylinker region) which has permeable “competent” cells are then mixed with
a number of unique target sites for restriction DNA to allow entry of the DNA into the bacterial
endonucleases. Cutting the circular plasmid vector cell. Alternatively, a process called electroporation
with one of these enzymes results in a single is also used that drives DNA into cells by a strong
cut, creating a linear plasmid. A foreign DNA electric current.
molecule, referred to as the “insert,” cut with the
same enzyme, can then be joined to the vector Since bacterial species use a restriction-modification
in a ligation reaction. Ligations of the insert to system to degrade foreign DNA lacking the

234
appropriate methylation pattern, including plasmids, nonrecombinant (selfligated vector only) or
the molecular biologists have circumvented this recombinant (vector containing foreign DNA insert).
defense system by using mutant strains of bacteria, Both types of transformed bacterial cells will be
deficient for both restriction and modification, such antibiotic resistant. To distinguish non-recombinant
as the common lab strain E. coli DH5α. from recombinant transformants various screening
techniques like blue-white screening, colony PCR,
Successfully transformed bacteria will carry colony hybridization etc. can be used.
recombinant plasmid DNA. Multiplication of the
plasmid DNA occurs within each transformed Amplification and
bacterium. A single bacterial cell placed on a
solid surface (agar plate) containing nutrients can
purification of recombinant
multiply to form a visible colony made of millions plasmid DNA
of identical cells. As the host cell divides, the
plasmid vectors are passed on to progeny, where When a positive colony containing recombinant
they continue to replicate. Numerous cell divisions plasmid DNA is transferred aseptically to liquid
of a single transfomed bacteria result in a clone growth medium, the cells will continue to multiply
of cells (visible as a bacterial colony) from a single exponentially. Within a day or two, a culture
parental cell. This step is where “cloning” got its containing trillions of identical cells can be harvested.
name. The cloned DNA can then be isolated from The final step in molecular cloning is the recovery of
the clone of bacterial cells. the cloned DNA.

Recombinant selection Plasmid DNA can be isolated by alkaline lysis method

and further purified from crude cell lysates by

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

& Screening chromatography using silica gel or anion exchange
The transformation process generates a mixed resins that preferentially bind nucleic acids under
population of transformed and non-transformed host appropriate conditions and allow for the removal of
cells. As we are interested only in transformed host proteins and polysaccharides. The purified plasmid
cells it becomes necessary to filter them out. This is DNA can then be eluted and recovered by ethanol
exactly what is done in the selection process. There precipitation in the presence of monovalent cations.
are many existing selection strategies and the strategy Ethanol precipitation of plasmid DNA from aqueous
depends on the particular vector. solutions yields a clear pellet that can be easily
dissolved in an appropriate buffered solution. Further
In most cases the vector carries a selectable screening can be done by restriction endonuclease
marker gene for resistance to an antibiotic. If the digest to confirm the presence and orientation of the
plasmid vector is introduced into a plasmid free insert and by DNA sequencing.
antibiotic sensitive bacterial cell, the cell becomes
resistant to antibiotic. Non-transformed host cells Suggested readings
contain no recombinant plasmid, therefore they
will not be antibiotic-resistant, and their growth Allison, Lizabeth A. (2009). Chapter 8 Recombinant DNA
will be inhibited on agar containing antibiotic. technology and molecular cloning. In Fundamental Molecular
Biology (pp202–254). Hoboken, NJ: Wiley-Blackwell.
Transformed bacterial cells may contain either

235
Marine Microbiology
Fish Health Management
N. K. Sanil* and K. K. Vijayan
Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Aquaculture is one of the fastest growing food help groups along the costal belt of Kerala, and the
producing sectors in the world and reduced growth in farming area is growing every year.
agriculture and animal husbandry sectors has shifted
the focus of attention to aquaculture. As aquaculture Diseases
production expands, diversifies and becomes more
intensive, the risk and effects associated with disease Disease is an abnormal condition characterized by
outbreaks and pathogen spread are also enhanced. a gradual degeneration of the animals’ ability to
The growth, economic viability and sustainability maintain normal physiological state due to various
of aquaculture primarily depend on the successful factors adversely affecting its wellbeing. Absence of
prevention/control of diseases. Unlike in land based clinical signs or obvious diseases does not indicate
farming systems, disease problems in aqua farming that pathogens or possibilities of diseases do not exist.
are complicated due to the multi-dimensional nature Domestication and intensive rearing practices always
of culture system where dynamic interactions between carry risks of enhanced incidence of infection and

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hosts, pathogens and environment take place. potential for disease. Creation of Intensive rearing
systems aiming for higher production and profits,
Mariculture – without proper planning and management, always
create stress and pave way for infections and diseases.
Indian scenario
The strength of Indian aquaculture lies in (a) large One of the most important factors in dealing with
water bodies suitable for aquaculture, (b) tropical the disease is INFORMATION. Knowledge is required
Climate, (c) species diversity and (d) availability of on the potential disease causing pathogens, role
cheap labour. While the weakness includes (a) of environmental factors, health status of the host,
unregulated development, (b) lack of regulations (c) diagnostics and therapeutic options. The four K’s essential
disease problems and (d) lack of scientific approaches. for scientific aquaculture health management are:
It is estimated that about 5 million tones of aquatic
animal products can be produced annually through • Knowledge about the disease process
aquaculture, in India. Shrimp farming still dominates • Knowledge about the pathogen
Indian aquaculture scene. The accumulated losses due • Knowledge about the host and
to white spot syndrome virus (WSSV) alone in India, • Knowledge about the environment.
during the past decade is about Rs. 3000 crores. In
this context, finfish and shellfish mariculture as an Information forms the key element in deciding
alternative for shrimp farming has gained importance. upon the best means of dealing with a disease or
The research efforts by CMFRI in the development of formulating a health management strategy. Hence the
maricuture technologies of the candidate species such best approach in collecting the information should be
as bivalves, swimming crabs, sand lobsters, finfishes proactive, rather than waiting for a disease outbreak
and marine ornamentals have shown good success. to happen. One single piece of information, that the
Farming of the green mussel and edible oyster has disease is caused by a viral pathogen and there is
become a popular livelihood activity by the self- no cure, would save the farmer from spending large

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 amount of money for ‘bogus’ cure, and also from can establish on/in the host, proliferate, overcome the
additional losses due to delayed harvesting. non-specific and/or specific defense barriers of the
host, produce the pathogenic factors, cause cellular
Genesis of disease and tissue damage, produce significant pathological
changes, impair the function of the target tissue/
Diseases can be caused by a variety of factors, the most organ and cause mortality. The sequence of events
important being pathogens. Other factors include in an acute infection is as follows.
stress, environmental/water quality, physical agents,
nutritional imbalance, toxins etc. or a combination of • Contact with the pathogen
these. Thus a ‘disease condition’ is actually a complex • Infiltration into the body
situation resulting from the interaction/modification • Development / proliferation–incubation (usually
of the primary disease condition by various biotic short in fishes)
and abiotic factors. • Spreads throughout the body/accumulate in
target organ
Stress and disease development • Symptoms appear
• Mortality
Role of stress in predisposing fish/shrimp to infections is
widely recognized and many of the routine aquaculture In the case of chronic infections the pattern of
practices are known to induce stress. Stress is a non- development is
specific response and involves a series of changes in • Slow
the animal in trying to adapt to the changed situation. • May or may not show pathology / symptoms
Adaptive responses of the animal if extended beyond • Remain in the body and serve as reservoir / carrier
the normal range, disturbs the normal functions, and
these series of changes termed “stress response” tries All infections need not result in disease manifestation.
to help the animal to restore normal homeostasis. The sequence of disease development will depend
This process has both advantages and disadvantages. on the nature of the pathogen, environmental
During stress, in an effort to mobilize additional energy factors, size of the host, pathogen load or intensity
to regain the internal homeostasis, hypothalamus per unit area or unit weight of the host and their
pituitary- inter-renal axis (HPI axis) gets stimulated interactions. The complexity of the situation makes
leading to an increased output of stress hormones health management a difficult proposition.
called corticosteroids. However, these stress hormones
are basically immunosuppressive in nature and reduces Common
the efficiency of both non-specific and specific immune
system increasing the susceptibility to disease. Common
pathogens encountered
husbandry practices like handling, netting, transportation Bacterial Diseases: Fish are susceptible to a wide
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and the normal features associated with intensive culture variety of bacteria (mostly opportunistic) in the
systems like suspended solids, low oxygen, high organic environment and many of these become pathogenic
matter, overcrowding, high ammonia, etc. can elevate when fishes are physiologically unbalanced,
the level of corticosteroids in the blood. Similarly, many nutritionally deficient, or in the presence of other
of the pollutants at very low levels can also stress the fish stressors. Bacteria are known to cause infections /
and make them relatively more susceptible to infection. diseases in shellfish farming also.
Most of the stressors encountered in intensive culture
systems are of chronic nature and can keep the level of Viral Diseases: Viruses are obligatory intracellular
corticosteroids above basal levels for longer duration. parasites requiring a living cell to replicate. Outcome
of diseases due to virus infection is complex and
Disease process depends on several factors including the immune
status of individuals and infectious dose of virus.
A pathogen can cause a clinical disease only when it Mortality rates may vary and in some cases, the

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pathogen remains at a low level of infection serving Disease Diagnosis
as reservoirs/carriers which are difficult to detect. Viral
diseases have been a major cause of diseases and
in Aquaculture
subsequent economic loss. Diagnosis forms the first step in any disease control
programme, which determines the ultimate success
Fungal Diseases: Generally fungal diseases can be or failure of the programme. Once a disease is
external or systemic and are difficult to cure. Except suspected, the next step is to draw a diagnostic
a few, they are generally considered less important procedure, to identify the root cause. The diagnostic
pathogens of fishes. procedure may include a single test or a combination
of tests. In the case of routine pathogen watch or
Parasitic Diseases: Parasitic diseases in fishes range health monitoring, a set of selected diagnostic tests
from extremely pathogenic ones to those, which are are performed to cover the potential pathogens. The
practically harmless. Many of the protozoan parasites are approach generally followed is location specific and
important pathogens of fishes while metazoan parasites problem specific, where the first consideration is the
except a few are generally less pathogenic in fishes. availability of the diagnostic facility and expertise
and there is no hard and fast method, which can
Non-infectious Diseases: Feed-derived wastes be applied for all cases.
also affect the culture environment through direct
Table 1. Diagnostic methods

History History of disease at facility or region, facility design, source of seed, type of feed used,
environmental conditions etc.
Gross clinical signs Lesions visible, behavior, abnormal growth, feeding or food conversion efficiency, etc.

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Direct microscopy Bright-field, phase contrast, or dark field examination of stained or unstained tissue smears, whole-mounts, etc.
of diseased or abnormal specimens.
Histopathology Routine histological or histochemical analysis of tissue sections

Electron microscopy Ultrastructural examination of tissue sections, negatively stained virus preparations, or sample surfaces

Culture and bio chemical Routine culture and isolation of bacterial isolates and identification using biochemical reactions
studies
Enhancement Rearing samples of the appropriate life stages under controlled conditions to enhance expression of latent or low
level infections
Bioassay Exposure to potential pathogens

Serological methods Use of specific antibodies as diagnostic reagents in immunoblot, agglutination, ELISA, IFAT, or other tests.

Tissue culture In vitro culture of pathogens in cell lines

DNA based Diagnostics PCR, nested PCR, Multiplex, real time PCR

pollution, which in turn affects the culture organisms. Once the right diagnostic picture along with the water
Uneaten feeds, faeces and metabolic wastes and soil parameters are available, control measures
contribute to nutrient and particulate loading of with respect to the causative factor(s) can be initiated.
the water and substrate which in turn induce stress,
reduce growth and increase their vulnerability to Treatment
diseases. Improper diets can negatively influence the
health by inducing nutrient deficiencies, imbalances or Treatment or therapy is intended to restore the normal
toxicoses. An impaired nutritional status contributes health of the diseased or infected animal. Drugs can be
to defective host resistance. Malnourished fish may given oral, intramuscular, intraperitoneal, intravenous
harbor latent infections, and certain physiological or topically as baths or dips. Selection of the proper
conditions and environmental stress may predispose route depends on the environmental situation, the
them to infection. species and condition of the animal, and the drug

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 being delivered. Unlike the land-based animal rearing biosecurity, quarantine, screening of broodstock and
systems, where the diseased animals can be identified larvae/fingerlings, Specific Pathogen Free (SPF) animals,
and treated individually, the scope for disease control pond and water quality management etc. are involved.
in aquaculture through detection and treatment is
very limited, mainly due to the co-existence of the Translocation/introduction of aquatic animals has
pathogen and host in the aquatic rearing system. been frequently identified as an event that precedes
In the case of mariculture, where the extent of the major outbreaks of new/emerging diseases in a region
water bodies are without boundaries, the scope of or species. The commercial/ economic reasons for
control over the host, pathogen and environment species introductions in aquaculture include (a)
is all the more difficult than in the case of inshore/ cost-efficient species in terms of production costs
inland aquaculture. to output revenues, (b) high growth potential, (c)
resistance to environmental stressors and pathogens,
Chemotherapy is not advised in culture systems (d) good market opportunities, (e) pre-existing
and should be used only as a last resort since the knowledge of rearing methodologies/ technologies
use of antibiotics can lead to residues in tissues as etc. The potential sources of introduction include live
well as development of antibiotic resistant microbes fish, eggs, larvae, contaminated water, wrappings
in the environment, which in turn can create other or packaging etc. Factors like pathogenicity, host-
public health issues. The fish is constantly bathed in pathogen interactions, vectors, climatic conditions,
potential pathogens, viz., parasites, bacteria, fungi susceptibility and resistance of the hosts etc. play an
and viruses. Separating the infected or diseased important role in deciding/modifying the outcome
animals from the population and subjecting them to of pathogen introductions. Open aquatic farming
individual treatment regimes is impractical. Hence, systems favouring easy dispersal of the pathogens
disease treatment becomes a difficult proposition in along with their ability for long-term survival outside
aquaculture, and prevention remains the only natural the host further complicates the issue.
choice and chemotherapy, if at all required, should
be practiced judiciously and restricted to broodstock WSSV – an example
alone. Though vaccines can play an important role in
controlling/preventing bacterial diseases, in many cases Outbreaks of WSSV, the most virulent virus known to
especially with viral pathogens, protection is found affect cultured shrimps were first reported in Penaeus
to be for short periods with variable results. In the japonicus in Taiwan and China in 1992. In 1993, it
absence of successful drug or vaccine, an integrated has spread to other species of shrimp and resulted in
management approach to tackle the diseases with outbreaks in Japan and Korea. In 1994, it was reported
respect to animal, environment and pathogen using from Thailand, India and Malaysia and by 1996, has
diagnostics as a functional tool is required. spread over the entire Asian continent. In 1995, it was
also reported in the USA, entered the central and South
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Aquaculture Americas in 1998 and Mexico in 1999. Entered Europe

during 1995-2001, Iran in 2002 and Saudi Arabia
Health Management and Mozambique in 2011 (WAHID, 2012). Currently,
The management practices that are designed to WSSV is known to be present in all shrimp-growing
prevent the occurrence of disease in a culture system regions except Australia. The practice of moving grossly
are termed as AQUACULTURE HEALTH MANAGEMENT. normal brood stock and post larvae (PL) freely amongst
This holistic approach using diagnostics along with countries was probably the most rapid and effective
farm management, can avoid the introduction of means of its spread throughout Asia (Flegel, 2006).
the pathogens into the system. The success of this
approach mostly depends on the right choice and use Similarly, Furunculosis in European trout, Whirling
of diagnostics along with other farm management disease in US, Crayfish Plague in Europe, viral nervous
measures, to keep both the animal and its environment necrosis (VNN) in marine fish, and many molluscan
in a healthy condition. Different components viz., diseases are typical examples. Epizootoic Ulcerative

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Syndrome (EUS) epidemic caused by the fungus, pathogens. Fish health management deals with (a)
Aphanomyces invadans in Asian freshwater and proper animal quarantine b) screening of Broodstock
estuarine fishes has spread throughout Asia, Australia and larvae/ fingerlings and (c) crop health monitoring
and has even reached the African continent. and pathogen watch. Effective implementation of all
the above three aspects of fish health management
Emerging diseases depends entirely on the early and accurate diagnosis

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of the disease causing agents.
Emerging disease problems, particularly in developing
countries, are often slow to be recognized. The Applying quarantine and biosecurity principles helps
recent outbreaks of Koi herpes virus (KHV), in the to prevent the entry of disease causing pathogens
neighbouring South-East Asian countries is a cause and thereby avoid serious problems, mainly related
of worry for India. Methods for detecting, reporting to infectious diseases. Bio-security can be defined
and responding much more quickly to such emerging as a set of standard scientific measures, adopted to
diseases should be developed. The design and exclude pathogens from the country (national level
implementation of effective disease surveillance bio-security) and from culture environment and host
programs, early warning and reporting systems and (farm level biosecurity) and, more broadly, to limit
contingency plans for dealing with serious disease pathogen establishment and spread. Biosecurity
outbreaks will help in reducing the social, economic can be more easily implemented in small, intensive,
and biological impacts of disease. and controlled farming systems than in outdoor and
large-scale operations. Biosecurity principles serve as
Health management the cornerstone for implementing the NACA and OIE
guidelines for aquatic animal health management.
Fish health management primarily constitutes two Quarantine helps in the (a) evaluation of the health
aspects, the farm health management and the condition of the new fish (b) reduction of disease
fish health management. Successful integration of transmission risk to pre-existing fish (c) gradual
these two aspects only can deliver a disease free acclimatization of the new fish and (d) convenient
environment. Farm health management constitutes administration of drugs. Avoidance or at least
the maintenance of (a) good soil quality (b) good water minimizing the introduction of known infectious
quality (c) good farm productivity (d) feed management pathogens is also important. Preventative treatments
and (e) maintenance of proper farm quarantine to (“prophylactic” treatments) can be helpful in removing
prevent horizontal transmission of disease causing initial loads of external parasites.

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 Aquatic animal health economic, industrial, environmental, biological and
Management at geographical characteristics, and in this situation a
regionally adopted health management programme
national level is considered a practical approach. An Asia-Pacific
Legislation has an important role in enhancing Regional Strategy better known as “Asia Regional
responses to aquatic animal health emergencies. Technical Guidelines on Health Management for the
It should enable and guide those involved in fish Responsible Movement of Live Aquatic Animals” has
health related activities and should clearly define the been developed through an FAO/NACA initiative
duties of various authorities involved at the national, involving the participation and agreement of 21
provincial and district levels and promote effective regional countries (FAO/NACA. 2001; Subasinghe
coordination, power-sharing and communication and Bondad-Reantaso, 2008).
between all those involved. Each country should
develop a national strategy/plan which includes Conclusion
short, medium and long-term action plans, with the
following components in place The key elements of an ideal health management
system can be summarized as:
• Competent Authority
• Legislative support • Control over the fish/animal stocks at hatchery /
• National advisory committee farm levels
• National list of diseases • Identify excludable disease/pathogens of concern
• National surveillance system • Prophylaxis /Vaccination
• Disease reporting • Diagnostics for the detection of pathogens
• Emergency preparedness and contingency planning of concern
• Quarantine and health certification • Adequate environmental control to prevent the
• Import risk analysis introduction of pathogens of concern (specific
• Zoning pathogen free stock)
• Biosecurity & Quarantine
The plan should be based on the various guidance • Routine management/husbandry practices to
tools (Standards and agreements like OIE Aquatic ensure pathogen exclusion (sterilization of influent
Code, relevant technical guidelines, SOPs and BMPs) water, pathogen free feed, prevention of pathogen
and implementation tools (risk analysis, diagnostics, transfer through men, material and vectors)
quarantine & health certification, surveillance & • Disinfection, treatment and pathogen eradication
disease reporting, contingency plans and disease methods to contain and eradicate disease
control strategies. Strong national coordination, outbreaks due to pathogens of concern
good leadership, involvement of stakeholders and
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appropriate monitoring and review systems are Thus in mariculture, development of species specific
essential for its successful implementation (Subasinghe and location specific health management models
& Bondad-Reantaso 2006). with broader management approach for the control
of farm/cage level environmental deterioration,
Regional co-operation pathogens (Virus, bacteria, parasites and fungi)
introduction and disease outbreaks is imperative to
Many countries in a region can share common social, ensure the sustainability and economic viability.

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Livestock Disease Surveillance
M. R. Gajendragad
Emeritus Scientist
National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI)
Yelahanka, Bengaluru 560 064
e-mail: [email protected]

Every country in the world has some sort of animal potential for international trade places additional
disease surveillance system. Surveillance is needed constraints on both importing and exporting countries
to understand the health status of the animals in with regard to disease surveillance and the monitoring
the country, so that problems can be identified and of animal health.
actions can be taken. However, different countries
have very different surveillance needs and surveillance The four major purposes of surveillance are
capabilities: a wealthy country with few diseases
that depends on exports of animals and animal 1. Demostration of freedom from disease
products will have sophisticated surveillance systems 2. Early detection of disease
to protect trade. A poor country with uncontrolled 3. Measuring the quantum of disease
land borders with multiple other countries that 4. Finding cases of disease.
have regular outbreaks of epidemic diseases will be

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unable to maintain sophisticated surveillance systems The underlying principle is that surveillance is an
and will aim primarily at minimizing the impact of essential, potentially complex health management tool.
major animal diseases (Cameron, 2012). Thus, world
trade in animals and animal products has expanded Disease monitoring
dramatically over the last decade and will continue
to increase. Political changes in the world and world
and surveillance
trade agreements opened new markets for a wide Disease surveillance and monitoring are often used
variety of livestock products. However, the increased interchangeably; however, many definitions are

Table 1 Definitions of disease monitoring and surveillance (adapted from Christensen J. 2001)

Textbooks Monitoring Surveillance

Martin et al. 1987 (page 259) Animal disease monitoring describes The term "disease surveillance" is used to describe
the ongoing efforts directed at assessing a more active system and implies that some form of
the health and disease status of a given directed action will be taken if the data indicate a
population disease level above a certain threshold.
Thrusfield, 1995 (page 22) Monitoring is the making of routine Surveillance is a more intensive form of data recording
observations on health, productivity and than monitoring.
environmental factors and the recording and
transmission of these observations.
(page 358 and 360) The routine collection of information on An intensive form of monitoring (q.v.), designed so that
disease, productivity, and other characteristics action can be taken to improve the health status of a
possibly related to them in a population. population, and therefore frequently used in disease
control campaigns.
Noordhuizen et al., 1997 (page 379) Monitoring refers to a continuous, dynamic Surveillance refers to a specific extension of monitoring
process of collecting data about health and where obtained information is utilised and measures
disease and their determinants in a given are taken if certain threshold values related to disease
animal population over a defined time period status have been passed. It, therefore, is part of disease
(descriptive epidemiology). control programmes.

245
 provided by various authors as shown at Table 1. Objectives of Surveillance (Doherr and
Audige 2001, and Dufour and Hendrikx, 2009 )
The World Organization for Animal Health, OIE
(Office International des Epizooties) defines • To detect the appearance of an exotic disease in
surveillance as “Means the continuous investigation a given region, so as to enable its early control;
of a given population to detect the occurrence of • To determine the true importance of a disease
disease for control purposes, which may involve (incidence, prevalence, economic losses, etc.) and
testing of a part of the population, while Monitoring follow the course of the situation, so that a decision
constitutes on-going programmes directed at the as to appropriate control (or not) can be taken.
detection of changes in the prevalence of disease • To enable the establishment action priorities.
in a given population and in its environment”. In • To evaluate results of control programmes by
other word, It is an observational method based monitoring decline of the disease.
on continuous recording to follow health status or • Monitoring of risk factors and other information
risk factors in a defined population, and particularly related to monitoring and surveillance activities.
to detect the appearance of pathological processes • Meeting a strategic goal of most veterinary
and study their development over time and in regulatory agencies
space, with a view to adopting appropriate control
measures. To simplify further, Surveillance implies To attain the above mentioned objectives surveillance
anxiously waiting for a disease that we hope will has three requirements viz.,
never appear.
1. Early warning :To improve the awareness and
The Centers for Disease Control and Prevention (CDC) knowledge of the distribution of disease or
USA, has promoted the following definition. infection and which might permit forecasting the
further evolution of an outbreak.
The epidemiological surveillance is the ongoing 2. Early reaction: rapid and effective containment
systematic collection, analysis, and interpretation of of, and leading to, the elimination of a disease
outcome using the specific data essential to the planning, outbreak, thus preventing it from turning into a
implementation and evaluation of public health practice, serious epidemic.
closely integrated with the timely dissemination of these 3. Coordination: For global eradication
data to those who need to know. of an identified animal disease such as
transboundary disease.
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Fig. 1. Types of Surveillances

246
 Farmer

Management Animal Health

Decission Worker

Analysis and
Veterinerian

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Feedback

Epidemiology
Unit

Fig. 2. Generalised Data generation pattern in India

Surveillance is a manpower intensive activity and Surveillance programs may be developed at a number
substantial cost is involved. Large number of of different levels viz.,
personnel is required to gather data, compile and
collate, analyse the data, and inform the authority Farm or village level: which include monitoring of
for appropriate activities. Thus, early warning or economically significant variables to the individual.
information helps in early reaction. These together
improve the control/ eradication programmes Region or state: this may involve testing to establish
by better coordination. Hence, surveillance freedom from particular diseases which may give
activities pose a challenge of amalgamating and individuals collective financial advantages over
coordinating various agencies involved to carry competitors, such as brucellosis freedom, or FMD
out one activity. free areas,

Based on the type of surveillance activity carried out, National: such programs are usually very costly if
surveillance can be broadly classified as in Fig.1. active. This is specially required for prioritizing the

247
 livestock diseases. This knowledge will in turn help identification of most common diseases in the
in assessing the vaccine requirements, formulating country, locations from where they are reported
control / eradication strategies and the knowledge of frequently and species of livestock involved.
economic impact the disease causes. This is carried
out involving multiple agencies, both governmental Active surveillance
and non-governmental. A few of them may be taken
up to assess the Transboundary animal diseases (TAD) Active surveillance is defined as any activity that
together by neighbouring countries. is frequent, intensive and aims at establishing
the presence or absence of a specific disease.
The data for surveillance programs may be generated (FAO: Manual of livestock disease surveillance and
by a number of methods which include clinical information systems). In active surveillance, usually the
evaluations, laboratory reports, slaughter inspection veterinary authorities make active efforts to collect the
data, screening tests, or owner reports.(Fig. 2) information needed. Since the information is collected
by technical / trained personnel, the information will
Types of Surveillance be of appropriate quality.

The data can be used for calculations of incidence /

Passive surveillance prevalence rates and proportions.
This is the routine surveillance conducted to
understand the extent of livestock diseases and The advantages of active surveillance are that it
detect the changes in status over a specified area. The identifies high risk pockets and their epidemiological
area could be region or country. Passive surveillance assessment, close integration of field activity
is an important key element in early warning of a with laboratory investigations, trade benefits for
disease. The word “Passive” entail characterization commercial livestock industry, establishment of rapid
of technique and not a sign of lowered importance communication systems, associated risk factors and
of the work. Passive surveillance through a disease socio-economic data. Active surveillance strengthen
reporting system is the main method of collecting the passive surveillance programme like targeted
information on livestock diseases currently used serological surveys.
in most countries. Passive disease surveillance is
collection of data on disease incidents from sources The disadvantages are cost involved, time consuming
from farmers, field veterinary officers, based on and requires trained manpower.
the clinical samples submitted to laboratories and
their results. Thus, Passive surveillance is a system Serological surveillance
wherein the reports gathered from various sources Serosurveillance is an important component of
are collected and compiled. any comprehensive surveillance system for vaccine
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preventable diseases. It is the gold standard for

The disadvantages of passive surveillance are that measuring immunity in a population, thereby
they are not able to provide information on the total complementing traditional disease surveillance
amount of disease, cannot provide representative methods. Serological surveys should be carefully
information on the level of disease in the population, designed to yield statistically valid information
or the geographical pattern of disease. Passive disease on the disease status of animal populations.
reports are not reliable enough to be used to calculate Serosurveillance is essential to declare a country
rates or proportions. They will not necessarily inform free from a particular disease. The advantage of
about the absence of the disease in a country. The this method is that large number of samples can be
results of passive surveillance cannot be used to screened at a time. The major disadvantage is that it
demonstrate the disease status to trading partners. needs large resources from collection of sera to its
despatch to the diagnostic laboratory. The sterility
The advantages of passive surveillance include of the sample is to be maintained so also the cold

248
chain. It is not possible to differentiate the vaccinal Event-based (media-based) surveillance
antibodies to antibodies developed due to infection Surveillance that complements indicator based
unless sophisticated tests are employed. This may surveillance by continuously scanning the Internet
lead to misinterpretation. and other communication media to detect certain
information that may lead to the recognition of
Following types of surveillance are less used or used emerging threats. It uses unstructured data which
for a limited objective. then need to be studied and verified and which
cannot be summarized as an indicator.
Early warning surveillance (epidemiological
watch, epidemiovigilance) Sentinel Surveillance
A sentinel is one who stands guard to warn when
Surveillance of health indicators and diseases in something happens. Sentinel herds act as indicators
defined populations in order to increase the likelihood for the rest of the population to warn that disease
of timely detection of undefined (new) or unexpected is present. In this type of surveillance, certain herds
(exotic or reemerging) threats. These are surveillance are maintained as sentinel herds and are observed
systems which aim to achieve the early detection of for any sign, either clinical or serological, for the
these threats. presence of disease(s) that are known to be absent
in that particular country/ locality. If the herds show
Indicator-based surveillance any sign then it is considered as the disease is being
Traditional communicable disease surveillance which introduced in the country/ locality.
relies on the collection of data about the occurrence
of pre-defined diseases or conditions using agreed Aggregation Points (Abattoirs, Markets,
case definitions; these data are analysed to produce Watering Points, Dip Tanks)

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indicators that point towards the existence of a threat.
Abattoir surveillance and surveillance at other
Indicator-based surveillance may be hazard-specific aggregation points is commonly used as a form
or general and includes the use of clinical or other of either active or passive surveillance. This section
data for syndromic surveillance. deals primarily with abattoir surveillance, as animal
inspection is normally carried out for public health
Hazard-specific surveillance reasons, providing potentially valuable passive animal
Surveillance that is focused on one or more pre-defined disease surveillance data. Other animal aggregation
hazards (disease, condition, biological, chemical points such as markets or dip tanks act as convenient
or physical agent, or event) often using specific and practical sites for active surveillance. Instead of
diagnostic tests (e.g. molecular diagnostic methods). conducting a representative survey, examination and
specimen collection can be done at the one location.
Syndromic surveillance This makes the surveillance faster and less expensive,
Surveillance that uses health-related information but the population under surveillance is no longer
(clinical signs or other data) that may precede or completely representative of the overall population.
substitute for formal diagnosis; this information Any possible biases must be taken into consideration
may be used to indicate a sufficient probability of when interpreting the results.
a change in the health of the population to deserve
further investigation or to enable a timely assessment Abattoir surveillance
of the impact of health threats which may require The primary advantages of abattoir surveillance are
action. This type of surveillance is not usually focused that it
on a particular hazard and can be used to detect
a variety of diseases or pathogens including new • is inexpensive. Animals are being processed and
(emerging) diseases so is particularly applicable for inspected for other purposes, so the costs are
early warning surveillance. primarily only related to data capture and any

249
 laboratory tests performed of necessity in the case of emerging and exotic.
• is able to cover a very large number of animals 2. To prioritize the diseases for control/ eradication for
• allows easy collection of diagnostic specimens, such policy decisions and to take up research projects
as blood or tissue samples, for laboratory testing 3. To declare a region or nation free from disease
• provides a relatively constant supply of 4. To ascertain the baseline level of any disease in
surveillance data an country
• enables data to be collected from a relatively small 5. To describe any changes in the health of
number of abattoirs locations, which slaughter livestock population
animals from a large number of farms or villages 6. To understand any threat to the health of livestock
(thereby decreasing the data collection costs). population which may lead to change in the
population structure
Syndromic Surveillance 7. To estimate the economic losses due to diseases
and also the gain in applying the control measures.
Various forms of syndromic surveillance have been To study the cost effectiveness of the control /
used for many years. However recent interest from eradication programme
the field of human surveillance has lead to a great
deal of interest and research in the area. Surveillance will help the policy makers to formulate
strategies for the following:
A syndrome is defined as a collection of signs
that indicate the presence of a disease. Syndromic • Management of outbreaks
surveillance is therefore concerned not with the • Informing trade
detection and reporting of disease, but of the signs • Prioritisation
and groups of signs that are associated with disease. • Informing control
These signs may be clinical signs (such as fever,
lameness, diarrhoea), or less traditional signs. For Disease surveillance:
instance, a decrease in the feed consumption at the
Indian Scenario
pen level in a piggery may be considered as a sign of
disease; an increase in antibiotic feed additive sales Currently in India, the main method of collecting
from a supplier may be another. information on livestock diseases is through a passive
disease reporting system. When an animal is noticed
Syndromic surveillance involves the identification sick, the owner may contact the veterinary authorities,
of specific signs or groups of signs, and analysis who may then either submits a disease report, or
of the patterns of these signs, in space and time. sends a specimen to a diagnostic laboratory. As such,
The purpose is not to diagnose a specific disease, in this system, the information is not collected about
but to detect abnormal patterns of signs that all cases or all the diseases, and the populations at
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may be due to one of a large number of diseases. risk are not recorded. Hence, this system of passive
When an abnormal pattern is detected, a disease monitoring is being practiced because the veterinary
investigation follows, in order to diagnose the services in the country have the primary objective of
actual cause of the disease. providing free treatment to the ailing animals.

Why do we Reportedly only the occurrence of 10-20 percent

of actual outbreaks is only recorded. These reports
need Surveillance? collected and compiled by the animal husbandry
The main reasons for requirement of disease department of the state governments are further
surveillance in any country are as follows get compiled at the national level by Department of
Animal Husbandry Dairying and Fisheries Ministry of
1. Early detection or early warning of a disease, Agriculture, Government of India, which publishes
either endemic, or emerging or exotic. It is more these as Animal disease surveillance Bulletins.

250
Although not comprehensive, these reports/bulletins Suggested readings
never the less provide the following information based
on clinical diagnosis. Bryan, R.T., Pinner, R.W., and Berkelman, R.L. (1994). Emerging
infectious diseases in the United States. Annals of the New
York Academy of Science 740, 346–361
• Which of the diseases are present in the country, Cameron, A. (2012) Manual of Basic Animal Disease Surveillance Dr.
and where located Angus Cameron Manual of Basic Animal Disease Surveillance
AFRICAN UNION Interafrican Bureau for Animal Resources
• Provides information to respond to the (IBAR) Nairobi, Kenya
disease outbreaks Christensen J. (2001) Epidemiological concepts regarding disease
monitoring and surveillance. Acta vet. scand. 2001, Suppl.
• Meet the basic disease reporting requirements of OIE
94, 11-16.
Frisén, M. (1992). Evaluation of methods for statistical surveillance.
Finally, for any disease monitoring and surveillance Statistics in Medicine 11, 1489–1502
Hueston, W. D. (1993) Assessment of national systems for the
programme to be successful, availability of good surveillance and monitoring of animal health. Rev. sci. tech.
diagnostics is a prerequisite. Choice of diagnostic test Off. int. Epiz., 12 (4), 1187-1196. Linda H (2012) Animal
Health Surveillance Terminology. Final Report from Pre-
depends on the disease, its prevalence, no of samples
ICAHS Workshop http://www.animalhealthsurveillance.
to be screened purpose of survey etc. Currently, arrays com/ uploads/ Main/ICAHS%20workshop%20%20final%20
of diagnostic tests starting from conventional tests to report%20v1%201%20October%202012.pdf
Myers, M.F., D.J. Rogers, J. Cox, A. Flahault and S.I. Hay (2000)
molecular one are being used for disease surveillance Forecasting Disease Risk for Increased Epidemic Preparedness
and monitoring. in Public Health. Advances in Parasitology 47 309- 330
Paskin R. (1999) Manual on livestock disease surveillance and
information systems. FAO. 71 pp.
Wilson, M.E. (1994). Detection, surveillance and response to
emerging diseases. Annals of the New York Academy of
Science 740, 336–340.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

251
 Disease Diagnostic Techniques in
Aquaculture
K. V. Rajendran
Principal Scientist
ICAR-Central Institute of Fisheries Education, Off Yari Road, Versova, Andheri (W), Mumbai- 400061
e-mail: [email protected]

Introduction
emphasis on molecular diagnostics, especially PCR-
Aquaculture, the fastest growing food producing based diagnosis.
sector in the world and also a significant source
of livelihood to millions of people world over, is Disease Diagnosis
constrained by the limitations imposed by several
infectious diseases. Aquaculture in India, which is
in Aquaculture
almost synonymous with shrimp culture, has been Control and prevention of diseases in aquaculture
witnessing rampant disease outbreaks which led to is a function of management, and diagnosis of
catastrophic mortalities. Since the early Nineties, diseases forms the fundamental step in the health
Indian shrimp aquaculture has been plagued by management programme. To diagnose a disease is
various diseases, among which white spot disease to recognise the occurrence of an abnormality and
(WSD) caused by white spot syndrome virus (WSSV) to identify the disease causing agent. In aquaculture,
is the most dreaded one. The disease continues to failure in accurately diagnosing diseases would
cause heavy mortality resulting in huge economic lead not only to large-scale mortality but also to
loss to the farmers and valuable foreign exchange indiscriminate use of drugs and chemicals thereby
for the country. According to Stentiford (2011), causing environmental contamination, drug residual
WSSV has become an endemic barrier to production effect and often occurrence of drug-resistant
in all known production zones through its global pathogens. Therefore, accuracy/specificity forms
distribution with traded crustaceans. According to one of the fundamental factors in disease diagnosis.
Lightner et al., 2012, global production loss due It is quite obvious from the literature on aquatic
to WSD was predicted to be $ 8 bn. However, they animal diseases that diagnosis of aquatic animal
have stated that the actual losses could be as high as pathogens has evolved over a period of time and
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$ 15 bn. The example of WSSV shows how impactful has advanced from microscopic characterisation and
a disease could be in aquaculture. Recognising the morphological description of pathogens to molecular
negative impact of diseases, disease diagnosis in characterisation and probe-based diagnosis.
aquaculture and their prevention and control or in
other words aquatic animal health management Traditional diagnostic methods for pathogens such
have become the central theme of aquaculture as viruses, parasites, bacteria and fungi include
research for many years. Besides developing vaccines, microscopic characterisation and morphological
therapeutics and other strategies to manage description using light microscopy, histopathology
diseases, developing and applying various rapid and and electron microscopy. In the traditional diagnostic
sensitive diagnostic techniques to aquatic animal methods, history and gross signs of the disease,
pathogens have been the corner stone of health examination of wet mount preparations and
management. This article describes the evolution tissue impression smears also play important roles.
of the diagnostic procedures in aquaculture with Histopathology is an important diagnostic tool

252
for tentative as well as confirmatory diagnosis of Consequently, real-time PCR assays employing either
most of the infections in aquatic animals. Besides, SYBR-green or Taqman probe fluorescence detection
histological technique is an essential component of system have been developed to detect and quantify
diagnostic protocols for infectious diseases using several aquatic animal pathogens.
gene probes and immunohistochemistry. Although
histopathology supported by parasitological, Antibody-based
bacteriological and virological methods is a widely
used proven technology, the major disadvantages
Diagnostic Techniques
of the technique are that it requires high level of As mentioned earlier, conventional diagnostic
expertise and is often time-consuming. Besides, the methods rely solely on the microscopic examination
technique is not very specific and cannot be employed and its visual recognition of pathogens, which requires
if the pathogen is present in very low number. As a a great deal of experience with the organisms that
next level, however, antibody-based diagnosis, both often change so dramatically in their morphology
polyclonal and monoclonal, on different formats such during the course of their development. Microscopic
as ELISA, dot-blot and lateral flow chromatographic examination of gross pathology is also quite
assay, is being widely used. Several rapid methods challenging as many pathogens can produce similar
have been developed for the detection of pathogens pathology. Therefore, in the evolution of disease
in fish, shellfish, molluscs and their environment diagnostic procedures in aquaculture, antibody-
though immuno- and molecular diagnostics (Adams based (protein-based) immunodiagnosis plays a
et al., 2008). significant role. This method has the advantage over
other traditional methods in that it can detect sub-
As the diagnostic techniques evolved over the years, clinical/latent/carrier state of infection and can also
application of modern biotechnology found a discriminate the antigenic differences. This technique

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

prominent place in characterising many pathogens is relatively rapid and more specific and sensitive.
at molecular level and developing molecular probes Further refinement of conventional immunodiagnostic
for the detection of many pathogens. Thus, an array techniques has resulted in the development of
of rapid and sensitive genomic probes using non- monoclonal antibody-based techniques and this
radioactive labels which could be used either in a has increased the accuracy of detection and has
dot-blot or in situ hybridization format are available allowed studying the pathogenesis of diseases
for various aquatic animal pathogens. Simultaneously, (Bartholomew, 1989). Nevertheless, the specificity
the development of highly sensitive DNA amplification of antibodies also limits their usefulness because
method based on polymerase chain reaction (PCR) major antigens are not conserved among life-stages
has resulted in tremendous improvement in the of certain pathogens (Bartholomew et al., 1995).
diagnosis of diseases in aquaculture. These tests are Many routine immunodiagnostic tools have been
finding increasing application in routine screening developed for a variety of pathogens infecting fish and
of broodstock as well as larval stages, epidemiogical shellfish such as agglutination (slide/latex); fluorescent
studies, development of specific-pathogen-free stock antibody test (FAT/IFAT); immunohistochemistry (IHC);
and many other areas of aquaculture. However, enzyme linked immunosorbent assay (ELISA); and
although various PCR tests have been used and dot-blot (dot-blot/dip-stick/western blot) (Adams
can provide sensitive and accurate diagnosis, risk 1999; Adams 2004; Adams et al., 1995, Mialhe et
of misdiagnosis (false positive and false negative) is al., 1995). Further, immunochromatography/lateral
considerable in these tests. As an improved version flow method was reported to have great scope in
of the conventional PCR, over the years, real-time the detection of aquatic animal pathogens (Adams
PCR is increasingly being used in pathogen detection and Thompson, 2006), and accordingly many tests
and quantification. Real-time PCR has the advantage have been reported (Pantoja and Lightner, 2001;
of being simple, sensitive, highly reproducible and Flegel, 2006; Sithigorngul et al., 2006, 2007, 2011;
amenable to high throughput screening. The test Chaivisuthangkura et al., 2013). Further, monoclonal-
also facilitate in accurately quantifying infection level. antibody-based assays have been developed for

253
 various aquatic animal pathogens, especially shrimp in solutions or on solid support (dot-blot) or even on
viruses (Sithigorngul et al., 2000, 2002; Poulos et sections of tissues fixed on slide (in-situ hybridisation).
al., 2001; Liu et al., 2002; Chaivisuthangkura et al., In-situ hybridisation has the advantage in that non-
2010; Siriwattanarat et al., 2013; Chaivisuthangkura specific tissue effects which may result in false-positive
et al., 2014). However, certain inherent limitations of diagnosis in dot-blot assay can be distinguished
protein-based diagnostics prompted the researchers from specific histological lesions (Lightner, 1996).
to look for more exquisitely sensitive and specific Many reports are available on the in-situ hybridation
detection tools. probe-based detection of viral pathogens of shrimp
(Wongteerasupaya et al., 1996; Tang and Lightner,
Molecular Diagnosis 1999; Pantoja and Lightner, 2001; Soowannayan et
al., 2003; Flegel, 2006). Although in situ hybridization
Molecular diagnosis works on the fundamental assays have advantages in that it can detect the
understanding that every properly classified species pathogens as well as it can be used to demonstrate the
of organism has some unique molecular sequences/ tissue tropism and sequential progression of infection.
signatures that distinguish it from other species. And, However, the major disadvantage of the ISH is its low
each organism’s genetic composition is essentially sensitivity compared to PCR.
a finger-print that can be used for its identification
(Tenover, 1988). Molecular diagnostic procedures have Polymerase Chain
been available since the 1970’s, when researchers first
began using cloned DNA probes to detect viral nucleic
Reaction (PCR)
acids. However, Mosely et al. (1980) first used DNA In order to refine the sensitivity of molecular diagnostic
probes for enterotoxigenic E. coli. Since then, large tests to detect the pathogens in very low numbers it
number of DNA probes to detect various pathogens was imperative to find out an amplification method.
have been developed. Accordingly, Saiki et al., (1985, 1988) and Mullis
and Faloona (1987) developed a simple method for
Nucleic acid probes are fragments of DNA labeled in making multiple copies of a DNA sequence. PCR is
some fashion that can seek out and bind with high a technique of in vitro amplification of specific DNA
specificity to stretches of DNA or RNA that have sequences by the simultaneous primer extension of
complementary sequences (hybridization reaction). complementary strands of DNA. In other words, it is
These probes may be either RNA strands directed a simple rapid and sensitive method for selectively
toward DNA targets or labeled DNA sequences directed amplifying defined sequences/regions of DNA/RNA
towards RNA targets. Initially, molecular diagnostic from an initial complex source of nucleic acid. Copying
methods were not widely accepted because these tests of a specific genetic target is accomplished in a 3 step
used radioisotope detection methods. Radioisotope reaction: (i) Denaturing the target DNA at elevated
detection methods have been employed for detection temperature (90-95 oC) (ii) Cooling the reaction to
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of aquatic animal pathogens (Bruce et al., 1993), promote annealing of oligonucleotide primers that
however, they are more expensive and labour-intensive are complementary to either strand of target DNA (
when compared to traditional antibody detection ii) Extending the bound primers by DNA polymerase
methods. However, in recent years non-radioactive action resulting in the replication of target sequence.
probes have become increasingly popular and with The reaction is repeated in successive cycles of heating
high specificity probes labeled with markers such as and cooling, referred to as thermal cycles. Each cycle
biotin and digoxigenin are available. These probes have doubles the input target sequence. As the newly
advantages over the radioactive-labeled probes in that synthesized DNA copies can also serve as template
they are safer and have longer shelf-lives. Nucleic acid in subsequent replication, after a series of cycles an
hybridization reaction consists of four components; exponential amplification can be achieved. This final
the probe, the target DNA/RNA (in the sample), the PCR product can be characterised in many ways and
reporter molecule (the label on the probe) and the the most common is to determine the size of the
hybridisation method. Hybridisation can be performed amplified fragments using gel electrophoresis.

254
PCR diagnosis is the most widely used diagnostic tool and the intended amplified product may be of similar
in aquatic animal pathogen detection. It forms one length. If there is a significant difference between the
of the crucial components of screening of shrimp amplicon lengths, there is a possibility that shorter
broodstock and larvae for viral pathogens. Several product will have more amplified products at the
PCR-based assays have been reported for a variety of expense of the longer product. Multiplex PCR assays
shrimp viruses (Wongteerasupaya et al., 1997; Belcher have been developed for the simultaneous detection
and Young, 1998; Cowley et al., 2004; Lo et al., 1996b; of many shrimp viruses (Tsai et al., 2002; Xie et al.,
Pantoja and Lightner, 2000; Anonymous, 2014; Nunan 2007; Khawsak et al., 2008; Sibonga et al., 2013).
et al., 1998; Sritunyalucksana et al., 2006).
Reverse transcriptase PCR
PCR Methodologies or RT-PCR
There are a variety for procedural variations of a If the target sequence to be detected is RNA such
standard PCR available, and these are developed as RNA viruses of shrimp (YHV, GAV, MoV, TSV),
for specific purposes. However, the present article the conventional PCR step would precede a reverse
discusses only about a few such methodologies, transcription (RT) step by which RNA is enzymatically
which are commonly employed in the detection of converted to complementary DNA (cDNA). An oligo
shrimp viruses. These are nested and semi-nested PCR, deoxynucleotide primer hybridizes to mRNA and is
multiplex PCR and reverse transcriptase PCR or RT-PCR. extended by an RNA-dependent DNA polymerase.
The newly synthesized single-stranded cDNA can be
Nested and semi-nested PCR amplified using specific primers in a conventional PCR.
The RT-step can be carried out using either an oligo
This is based on employing an additional set of (dT) primer or random hexamer or a gene-specific

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

primers to increase the efficiency of the standard one- primer. The choice of primer depends on the specific
step PCR. After the initial amplification of the target application. Reports on the RT-PCR detection of RNA
sequence, an aliquot of the primary PCR product is viruses of shrimp include Wongteerasupaya et al.,
subjected to a second round of amplification using 1997; Nunan et al., 1998; Cowley et al., 2004, 2005).
another set of primers designed to amplify an internal
part of the amplified product. Primary product can Advantages and disadvantages
also be amplified using a semi-nested format in which
of PCR
one of the primers of primary PCR and a second
primer complementary to an internal region are In comparison, PCR has the advantage over other
used. Both these methods give additional specificity methods that use nucleic acid probes. PCR is more
and increased sensitivity to the PCR and accordingly sensitive in that on an average less than 10 target
many PCR tests have been reported for the sensitive molecules are sufficient to get a positive result,
detection of shrimp viruses (Lo et al., 1996; Kimura whereas in the case of most of the probes, sensitivity
et al., 1996; Kiatpathomchai et al., 2001). is approximately 104 to 105 molecules of the target.
Presently, PCR methods are available for a variety of
Multiplex PCR pathogens. Apart from the sensitivity and rapidity of
diagnosis, principal advantage of molecular diagnostic
Multiplex PCR is employed in simultaneously amplifying methods is in the detection of non-culturable agents.
more than one segment of target DNA in the same Further, DNA amplification can assist in detecting the
reaction. This is achieved by designing primers, which pathogens that are present in low numbers and also
can amplify different regions of the same template in handling tiny volume of specimen. It can also be
DNA (a particular virus) or primers that can amplify used to detect latent infection and thereby identifying
two entirely different DNA templates (two distinct the reservoir hosts of infection that is significant in
viruses). However, to amplify, the primers should be epizootiology, besides it can be used to differentiate
designed in such a way that their Tm should be similar antigenically similar pathogens.

255
 These methods are cost-intensive procedures. The are used in the later cycling reaction for strand
methods do not provide an accurate quantitative displacement DNA synthesis. The LAMP reaction is
assessment of infection level. Further, the test will initiated by an inner primer containing sequences
not give any indication of whether the pathogen of sense and anti-sense strands of the target DNA.
present in the hosts is replicating/live or dead. This is followed by the release of a single-stranded
Therefore, the carrier status of the host and the DNA through the priming by an outer primer. This
viability of the pathogen cannot be assessed by single-stranded DNA will serve as a template for
this method. Since the molecular tools are based DNA synthesis primed by the second inner and outer
on specific genetic sequence, any change in the primers that can hybridize at the other end of the
genome of the pathogen through mutation might target. This process will result in the formation of a
render the assays non-functional. As PCR is a very stem-loop DNA structure. In the subsequent step of
sensitive tool, it is prone to contamination. The LAMP cycling, one inner primer will hybridise to the
contamination which occurs during PCR processing loop on the product and initiate strand displacement
will lead to false-positive results. PCR method may DNA synthesis which will result in the original stem-
also lead to false negative results due to either a loop DNA and a new stem-loop. Cycling continues
wrong selection of tissue for nucleic acid extraction for a period of approximately 1 h and results in the
or faulty processing leading to low/no quantity accumulation of 1010 copies of the target. The final
or low quality of nucleic acid or due to low level products of the reaction are stem-loop DNA with
of pathogens present in the host. Like any other several inverted repeats of the target and cauliflower-
molecular methods, PCR also will have difficulty in like structures with multiple loops.
detecting new pathogens as the exclusive use of the
technique would overlook such infections. End products of LAMP reaction can be visualised
through several methods. The most common
Loop Mediated Isothermal method of visualization is by agarose gel
electrophoresis. Similar to PCR detection, agarose
Amplification (LAMP) gel is stained with intercalating dyes such as
Loop-mediated isothermal amplification (LAMP) ethidium bromide or SYBR Green I. On the agarose
is a novel nucleic acid amplification method that gel, LAMP product will appear as smear at the
amplifies DNA with high specificity, efficiency and top and bands at the base of the gel, as the end
rapidity under isothermal conditions (Natomi et al., products of LAMP consist of stem-loop DNA and
2000). When combined with reverse transcription, cauliflower-like structures with multiple loops of
this method can also amplify RNA sequences with various lengths. Since, one of the characteristics
high efficiency. The method relies on auto-cycling of the LAMP reaction is its ability to synthesise
strand displacement DNA synthesis using a DNA extremely large amount of DNA, addition of
polymerase with a high strand displacement activity intercalating dye, SYBR Green I, into the reaction
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and a set of four specially designed primers. These tube itself would help in visualising the product
four primers, termed as inner and outer primers, under a UV- transilluminator (Natomi et al.,
recognise six distinct sequences of the target DNA, 2000). This method is useful in the field-level
which improves the specificity of the reaction. The application. Another method is also based on the
reaction is carried out at isothermal condition, as accumulation of large amount of pyrophosphate
the denaturation of strands takes place by strand ion byproduct of the reaction, which will yield
displacement. LAMP technique has been employed white precipitate of magnesium pyrophosphate in
in the detection of many shrimp viruses including the reaction mixture. Hence, detection of presence
RT-LAMP for RNA viruses (Kono et al., 2004; Mekata or absence of white precipitate will provide an easy
et al., 2006; He et al., 2010; Nicolasora et al., 2014). distinction of whether the target DNA is amplified
during the reaction. Further, since the increase in
In the initial stages of LAMP reaction, all the four turbidity of the reaction mixture according to the
primers are involved, however, only the inner primers production of the precipitate, correlates to the

256
amount of target DNA synthesized, a colorimetric is based on the theory that there is a quantitative
estimation of the turbidity in real-time is also being relationship between the input target DNA and the
used as an efficient method of visualizing the amount of PCR product generated at any given cycle
amplified product. number. Real-time PCR detects the accumulation
of amplicon at every cycle and the data are then
Advantages and disadvantages measured at the exponential phase of the PCR.

of LAMP
Advantages and disadvantages of
LAMP amplifies the target DNA under isothermal
real-time PCR
amplification with high efficiency (end product
generated is 1010 compared to 107 produced in a PCR Real-time PCR technology has enormous advantages in
reaction); The detection limit of LAMP is comparable the detection and quantification of pathogens owing to
to PCR; No significant influence of the co-presence its increased sensitivity, reproducibility, and quantitative
of non-target DNA; LAMP allows simple, easy and accuracy, apart from the decreased hands-on-time and
selective detection; Lamp is highly specific for the less chances of contamination. The major advantage
target sequence, as it employs four primers targeting of this procedure is that it gives quantitative data on
multiple sequences; LAMP is simple and easy to starting copy number and has the sensitivity limit of
perform, as it requires (after appropriate primers are single copy of the genome. Further, it is amenable to
prepared) only a regular laboratory water bath or high throughput and turnaround. In the diagnostic
heat block for the reaction; By incorporating reverse format, the greatest advantage of real-time PCR is that
transcription, LAMP can be used for amplifying RNA as there is no post-PCR manipulation involved in the
as well. LAMP reaction is vulnerable to contamination visualisation of the result, cross-contamination and
because amplification of the target DNA is very high false-positive result can be minimised. The assay is the

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

at the final stage. Further, multiplexing of the reaction key tool used in gene expression studies. In short, real-
is not possible with LAMP. time PCR, due to its increased sensitivity, reproducibility,
and quantitative accuracy, apart from the decreased
Real-time PCR hands-on-time and less chances of contamination,
is a very important tool in the health management
Molecular diagnosis, especially PCR-based diagnosis in aquaculture. Accordingly, real-time PCR assays
has made tremendous impact in the field of aquatic have been developed and applied for detection and
animal health management. However, PCR has quantification of large number of shrimp viruses (Dhar
advanced from the endpoint detection of conventional et al., 2001, 2002; Tang and Lightner, 2002, 2004;
PCR to the detection of PCR amplification during Jang et al., 2009; Yan et al., 2010; Yadav et al., 2015).
the early phases (exponential phase) of the reaction
and following the entire amplification in real- Conclusion
time. Development of robust and high throughput
quantification technique, based on real-time PCR, Diagnostic techniques used in aquatic animal
which detects even a single copy of the genome, pathogen detection have advanced tremendously
has been reported for many aquatic viruses. And, over the last three decades. Molecular methods such
measuring the kinetics of the reaction in the early as conventional PCR, dot-blot hybridisation, in situ
phases of PCR provides a clear advantage over the hybridisation, LAMP and real-time PCR have now
conventional PCR. There are mainly two different been developed for a wide range of pathogens.
chemistries involved in the real-time assay; SYBR green Further, these techniques have been adopted and
(A highly specific double-stranded DNA minor grove- widely used routinely in hatchery, farms and large
binding dye which can detect all double-stranded number diagnostic laboratories because of their
DNA, not single-stranded and primers) and TaqMan exquisite sensitivity and specificity. New advanced
probe-based assays (A fluorogenic 5’ exonuclease variants of the technologies are also being reported
assay). Quantification of pathogen by real-time PCR regularly. However, PCR has been considered as the

257
 Kimura, T. et al., 1996. Fish Pathol. 31, 93–98.
gold standard and it is the key diagnostic tool widely Kono, T. et al., 2004. J Virol Methods. 115(1):59-65.
employed today. La Fauce K.A. et al., 2007. J. Virol. Methods, 140, (1-2), 10-16.
Lightner, D.V. et al., 2012. J. Invertebr. Pathol. 110, 174–183.
Liu, W. et al., 2002. Dis Aquat Organ 49: 11-18
Suggested readings Lo, C.F. et al., 1996. Dis. Aquat. Org. 25, 133–141.
Mekata, T. et al., 2006. J Virol Methods. 135(2):151-156.
Mialhe, E. et al., 1995. Mol. Mar. Bio. and Biotech. 4: 275-283.
Adams, A. 1999. pp. 1-12. In Karunasagar, I., Karunasagar, I. and Mosely, S.L. et al., 1980. J. Infect. Dis., 142, 892-898.
Reilly, A. (eds.). Aquaculture and Biotechnology, Oxford and Mouillesseaux K.P. et al., 2003. J. Virol. Methods. 111 (2), 121-127.
IHB Publishing Co. PVT. LTD. Nicolasora, A.D.D. et al., 2014. Philippine Science Letters. 7(2); 309-
Adams. A. 2004. Bull. Euro. Assoc. Fish Pathol. 24, 33-37. 316.
Adams, A. et al. 2008. pp. 71-88. In Bondad-Reantaso, M.G., Notomi, T. et al., 2000. Nucleic Acids Res. 28(12):E63.
Mohan, C.V., Crumlish, Margaret and Subasinghe, R.P. (eds.). Nunan, L.M. et al., 1998. Dis. Aquat. Org. 34, 87–91.
Diseases in Asian Aquaculture VI. Fish Health Section, Asian Pantoja, C.R., Lightner, D.V., 2000. Dis. Aquat. Org. 39,177–182.
Fisheries Society, Manila, Philippines. 505 pp. Pantoja, C.R., Lightner, D.V., 2001. Dis. Aquat. Org. 44, 87–96.
Adams, A and Thompson, K.D. 2006. Trends in Biotechnology Poulos, B.T. et al., 2001. Dis Aquat Organ. 47: 13-23.
24: 201-205. Qing-yu Cheng. et al., 2007. Virologica Sinica. 22 (1); 61-67.
Adams, A. et al., 1995. J. Fish Shellfish Immunol. 5:537-547. Rajendran K.V. et al., 2006. J. Virol. Methods. 137, 265-271.
Anonymous, 2014. Manual of Diagnostic Tests for Aquatic Animals. Sibonga, M.F.J. et al., 2013. International Journal of the Bioflux
Office International des Epizooties, Paris. Society. 5(3); 142-145.
Belcher, C.R., Young, P.R., 1998. J. Virol. Methods 74, 21–29. Sithigorngul, P. et al., 2000. Dis. Aquat. Org. 42, 27–34.
Bruce, L.D. et al., 1993. Dis Aquat Org. 17, 215-221. Sithigorngul, P. et al., 2002. Dis. Aquat. Org. 49, 71–76.
Chaivisuthangkura P, et al., 2010. Aquaculture 299: 15-20. Sithigorngul W. et al., 2006. Dis Aquat Org. 72: 101–106.
Chaivisuthangkura, P. et al., 2013. Arch Virol DOI 10.1007/s00705- Sithigorngul, W. et al., 2007. J Virol Methods. 140:193–199.
013-1680-0 Sithigorngul, P. et al., 2011. J Virol Methods. 173:85–91.
Chaivisuthangkura, P. et al., 2014. World J Virol 12; 3(1): 1-10. Siriwattanarat, R. et al., 2013. Arch Virol. 158: 967-979.
Cowley, J.A. et al., 2004. J Virol Methods. 117(1):49-59. Soowannayan, C. et al., 2002. Fish. Sci. 68 (Supplement 1), 805–
Cowley, J. A. Et al., 2005. Dis Aquat Organ. 66 (2): 91-104. 809.
Dhar, A.K. et al., 2001. Journal of Clinical Microbiology, Sritunyalucksana, K. et al., 2006. Virus Res. 118, 31–38.
DOI: 10.1128/JCM.39.8.2835–2845. Stentiford, G. D. 2011. J. Invertebr. Pathol. 106: 3–5.
Dhar, A.K. et al., 2002. J Virol Methods. 104(1):69-82. Tang, K.F, Lightner, DV. 2001. Dis Aquat Organ. 44(2):79-85.
Durand S.V., Lightner D.V. 2002. J. Fish Dis. 25, 381-389. Tang K.F. et al., 2004. J. Viro. Methods 11, 109-114.
Edawards K., Logan J., Saunders N. (Eds.). 2004. Real-time PCR: Tsai, J. M. et al., 2002. Dis. Aquat. Org. 50:9–12.
An Essential Guide. Horizon Bioscience, Norfolk, UK, p. 346. Wongteerasupaya, C. et al., 1996. Aquaculture. 143, 23–32.
Flegel, T.W. 2006. Aquaculture 258, 1–33. Wongteerasupaya, C. et al., 1997. Dis. Aquat. Org. 31, 181–186.
He, L. et al., 2010. Bing Du Xue Bao. 26(6):490-495. Xie, Z. et al., 2007. Dis Aquat Org. 76:77-80.
Jang, I.K. et al., 2009. Aquaculture 287; 40–45. Yan, D.C. et al., 2010. J Fish Dis. 33(6):507-511.
Khawsak, P. et al., 2008. Mol Cell Probes 22:177-183. Yadav et al., 2015. Mole. Cell. Probes. 29: 442-448.
Kiatpathomchai, W. et al., 2001. Dis Aquat Org. 47: 235–239.
Central Marine Fisheries Research Institute

258
Diseases in Fish Hatcheries
P. Rameshkumar
Scientist
Mandapam Research Center of CMFRI, Mandapam,Tamil Nadu
e-mail: [email protected]

Introduction fish, infectious and non-infectious diseases. Infectious

Sustainable aquaculture production can only occur diseases are caused by pathogenic organisms
when fish are healthy and free from disease. Fish present in the environment or carried by other fish.
disease management is a combination of preventing In contrast, non-infectious diseases are caused by
the onset of disease and measures to reduce environmental problems, nutritional deficiencies,
losses from disease when it occurs. Fish cultured or genetic anomalies; they are not contagious and
in hatcheries or floating cages become particularly usually cannot be cured by medications.
susceptible to disease when various environmental
parameters such as temperature, salinity, dissolved Non-infectious diseases:
oxygen and suspended particles fluctuate suddenly
or widely, or following rough, although often Non-infectious diseases can be broadly categorized
unavoidable, handling operation. Once conditions as environmental, nutritional, or genetic.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

suitable for pathological changes develop, progress
to disease in the warm water environment is rapid. • A hygienic fish culture environment is essential to
Early detection of behavioral changes and clinical the health and productivity of farming operations.
signs in the cultured fish are critical for proper The reasons for this include:
diagnosis of the disease. • Disease risks are increased in poor and
polluted environments.
Disease rarely results from simple contact between • Quality of the product depends on clean and
the fish and a potential pathogen. Environmental healthy environments.
problems, such as poor water quality, or other
stressors often contribute to the outbreak of Infectious diseases
disease. The cobia, Rachycentron canadum, is
distributed worldwide in tropical and subtropical • Infectious diseases are broadly categorized as
water. Cobia, Pompano and Grouper culture offers parasitic, bacterial, viral, or fungal diseases.
great possibilities in aquaculture because of its fast
growth rate and commercial interest. In India, the Predisposing Factors
first sea cage farming trial with hatchery produced
fingerlings of cobia was carried out during 2010, and • Fish stocks living under stressful conditions become
the first success in breeding and seed production of less able to defend against a pathogen and hence
silver pompano was achieved during in 2011 at the will become sick more readily. Fish that are well
Regional Centre of Central Marine Fisheries Research cared for generally do not become sick even in
Institute (CMFRI), Mandapam, Tamil Nadu. the presence of a pathogen. The most common
error in fish husbandry is overstocking. This leads
Types of Fish Diseases to problems such as:
• Fish to fish aggression
There are two broad categories of disease that affect • Increased fish and feed wastes

259
 • Ease of disease spread, stressors. Among the important stress-inducing factors
• Increased concentration of pathogens are those with strong psychological components that
• Resultant poor water quality cause fright, excitement and discomfort. Stress can
• High fish density, stress, and ease of transmission be induced by such activities as handling, transport
increase susceptibility of the fish population to and weighing. Moreover, crowding at high densities
diseases and parasites. also produces a variety of stimuli that cause stress
• In marine hatcheries diseases present in wild fish .Outbreaks of diseases are associated with depressed
can infect cultured fish and spread rapidly through oxygen levels. Predisposing risk factors include also
the population. overcrowding, organic pollution and hypoxia.

Stress Common Disease

Fish in husbandry are exposed to a multitude of
in Hatchery
Fish bacterial infections can occur as a bacteremia,
Common Disease and Disorders which implies the presence of bacterial organisms in
Encountered in the Hatchery the bloodstream without clinical signs. Others occur
as a septicaemia, which indicates that bacteria and
i) Diseases of Cobia (Rachycentron canadum)
toxins are actually present in the circulatory system
S.No Bacterial disease Causative organism
and usually precipitate disease and clinical signs.
1 Pasteurellosis Photobacterium damsella sub Inflammation, hemorrhage and necrosis are clinical
sp pisicida
signs associated with septicemia
2 Streptococcosis S. iniae
3 Vibriosis V. anguillarum, V. alginolyticus
i) Vibriosis
4 Mycobacterium infection MY. Sp, Aeromonas hydrophila

5 Viral disease Lymphocystis Irido virus Vibriosis, a disease caused by numerous species of
vibrio, is a primary disease of marine fish in salt &
6 Amyloodiniosis Amyloodinium ocellatum
brackish waters and creates huge economic loss in
ii) Common Diseases of Pompano (Trachinotus blochii) the mariculture industry, affects large number of
S.No Disease Causative agent fish and shellfish species, both cultured and feral.
Vibrio alginolyticus is a Gram-negative facultative
1 White spot disease Ciliate protozoan,
Cryptocaryon irritans anaerobic bacterium, which was formerly regarded
2 Cardiac myxosporidiosis Myxosporidian protozoan, as an opportunistic pathogen causing vibriosis in
Henneguya sp marine fish and shellfish. Vibriosis characterized
3 Monogenetic trematode Bicotylophora trachinoti- gills mainly by the haemorrhage erosion of gill lamellae,
infestation Benedenia sp- body
fluid accumulation in the perinoteal cavity and
Central Marine Fisheries Research Institute

4 Parasitic dermatitis Sea lice ( Calligus elongatus)

(infestation) hemorrhagic septicaemia. The clinical signs were
skin discolouration, red necrotic lesions in the
5 Amyloodiniosis Amyloodinium ocellatum
abdominal muscles erythema at the base of the
fins, vent and in the mouth, abdominal distention,
iii) Common Diseases of Ornamental fishes
and exopthalmia.
S.No Disease Causative agent
1 Red pest, Fin Rot Gram negative bacteria Treatment
2 Fish tuberculosis Mycobacterium sps
3 External Gas Bubble Commonly caused by excess gas • Oxytetracycline @ 100mg/kg biomass/day USFDA
disease in the system, brought about by approved drug in aquaculture.
super-saturation of gas in high • Agrimin(Virbac, Mumbai) forte as mineral mixture
pressure water mains
and immune modulator filled in the empty capsules
4 Amyloodiniosis Amyloodinium ocellatum
and given along with the feed.

260
Common Parasitic Diseases • Formalin dip 200ppm for 10-15 min
• Removing the fish to a separate tank and allowing
in Hatchery the tank to run fish-free for a month is probably
necessary. This will allow the organism to run
i) Amyloodiniosis through its life cycle and die out due to the lack
Amyloodiniosis is otherwise called as the Marine of a host.
Velvet disease. The dinoflagellate Amyloodinium
ocellatum is one of the most important pathogenic ii) Caligus infestation
ectoprarasite affecting the cultured marine and
brackish water fish, causing Amyloodiniosis. There Caligus (Müller,1785) is the largest genus of parasitic
are different stages in the life cycle of Amyloodinium. copepods, containing more than 250 species.Sea lice
Trophont, Tomont, and Dinospores are the 3 stages are marine ectoparasites that feed on the mucus,
in the life of Amyloodinium. In the Trophont stage epidermal tissue and blood of host marine fish.
is feeds as a parasite. It attaches itself to the fish The affected fishes showed frequent surfacing and
with the help of rhizoids and feeds on the host. As it anorexia. Sea lice caused physical and enzymatic
grows it comes to the next stage of life called Tomont damage at the sites of attachment and resulting in
at which its size will be around 350 micrometers. abrasion like lesions that varying in their sizes about
At this stage it disengages from its host and starts 1mm to 3mm in dia. The skin surface also showed
reproduction. Reproduction is by repeatedly dividing moderate to severe ulcers measuring that measuring
itself until there are 256 offsprings. To complete this 2mm to 4mm in dia.
stage it needs around 3 days after which it hatches
tiny dinospores and these dinospores start infecting Treatment
the host for about 15 days after which they go to

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

the next stage in their lifecycle. • Fresh water dipping, RO water dipping, formalin
dip treatment and decreasing salinity leads to
Amyloodiniosis was recorded in Pompano brood stock, detachment of the parasite.
Cobia fingerlings and some ornamental fishes. The • Ulcerated skin treated with Povidone Iodine
pathogen was identified as Amyloodinium ocellatum. ointment or solution as antiseptic to enhance the
The infective stage of dinospores was located in gill healing process.
surfaces. The infection has finally resulted in acute
mortality of the fishes. Fish Health Management
Treatment Fish health management is a term used in
aquaculture to describe management practices
• Only the free-swimming dinoflagellate form of which are designed to prevent fish disease.
the organism (the dinospore) is susceptible to Successful fish health management begins with
treatment. The encysted form is not susceptible prevention of disease rather than treatment.
to any treatment. Prevention of fish disease is accomplished through
• Fresh water dip for 2-3m. This fresh water dip will good water quality management, nutrition, and
remove the Amyloodinium from the fishes which sanitation. The hatchery management practices
are in the stage of dinospores. You need several that help prevent the introduction and spread of
fresh water dip treatment to remove other stages disease. Fish diseases caused by parasites, bacteria
of Amyloodinium. or viruses can be spread from tank to tank or from
• The most common treatment is the use of copper hatchery to hatchery by the transfer of infected
in water. Water that has free copper at the level of fish and by animals, people, equipment and water
0.2 mg/l is used in treating fish that are affected by contaminated by contact with infected fish or fish
Amyloodinium. Ionic copper level of between 0.15 pathogens. To prevent the introduction of new
and 0.2 parts per million for a minimum of 14 days diseases onto a fish hatchery, there should be no

261
 contact between the fish on the farm/cage and any from quarantine facilities. The equipment used in
potential disease carriers. quarantine facilities should not be moved to non-
quarantined areas until it has been disinfected. A
Hatchery equipment should be cleaned and quarantine period must last until the fish are exposed
disinfected before each use. Workers should disinfect to the full range of seasonal water temperatures at
clothing, boots and other gear before having contact which any disease of concern can be detected. If
with healthy fish. Sanitation is particularly important possible, new fish should be quarantined in the
in preventing the spread of disease between tanks or isolated place as far as practical from the rest of
hatchery when sick fish are present. the hatchery.

Potential sources of disease agents: Equipment

• Fish – carrier or diseased, wild or farmed; For the best results in killing pathogens, you must
• Equipment – nets, boats, diving gear, and clean, disinfect and dry equipment before it is used
feeding materials elsewhere on or off the hatchery. This is especially
critical for equipment that has been used to handle,
Disinfection of equipment harvest or transport sick fish. Equipment must be
thoroughly scrubbed clean with a brush and detergent
Well operated fish hatchery requires a tank or facility and then rinsed to remove any dirt and detergent
to permit regular disinfection of equipment such residue. Then an appropriate disinfectant should be
as dipnets, siphons, etc .Tanks also require regular applied and left on the equipment long enough to kill
disinfection to avoid disease out breaks. Chorine can disease organisms. Rinsing after disinfection ensures
be used to disinfect tanks and equipment, formalin that no residues are left behind. Drying equipment
can also be used and mentioned, ultra violent light in the sun will destroy bacteria or viruses that may
sterilizers arè required tò kill bacteria and micro have survived.
organisms that could affect the fish.
Table 2. Methods of cleaning and disinfecting
Aims of disinfection: S.No Item Method of cleaning
1 Plastic buckets, Scrub clean with detergent,Povidone
Rubber boots, iodine 100ppm solution
• Prevent disease incursions in biosecurity programs wet suits
• Routine hygiene measures to reduce build-up of 2 Netting (Dip nets) clean with detergent, 100 to 150 ppm
disease agents on hatchery Povidone iodine, Rinse and dry 50ppm
formalin- Rinse and dry
• Eradicate disease agents from outbreaks 3 Transport tanks Scrub and clean with iodine 200 to
215ppm.Rinse and dry
Quarantine 4 Tank bottom, Drain out all water.Remove all fish and
Central Marine Fisheries Research Institute

water vegetations. Add hydrated lime(……. )

5 Infected fish Remove the dead fish. Use USFDA
Quarantine is must for the newly arrived or wild brood approved drug Oxytetracyclin @
stock fishes as well as the fishes shifting from cage 7-10mg/kg .Body biomass. Used in the
feed with binder.
to hatchery. Wild fishes may act as the carrier for the
many of the viral, bacterial and parasitic diseases.
Salinity reduction and adaptation of the fishes to
Feed storage
the moderate salinity or even fresh water dipping A system of feed management needs to be put in
may dislodge the protozoa or ectoparasites from the place to ensure feeding of older feed stocks first,
skin or mucous. Along with the freshwater dip some to minimise wastage or feeding of expired feeds.
USFDA approved disinfectant like povidone iodine, Fish feeds generally contain a high percentage of
formaldehyde also can be used as disinfectant. unsaturated fatty acids (up to 40%) which may
become rancid (oxidised) with storage or exposure to
No fish or water should be allowed to escape high temperature. Feed may also become mouldy from

262
storage in high humidity. Rancid fats or mouldy feed approved algicide, and can kill fish if used improperly.
can cause disease in fish, and even if not contributing The relationship between toxicity of copper sulfate
to overt clinical disease, it can affect growth. The high and alkalinity is very important (alkalinity is the total
protein content feed able to ferment or develop toxin concentration of alkaline substances in the water
if not storing in the ideal temperature. The store place expressed as equivalent calcium carbonate). In water
should keep it dry without moisture content. Feeing with an alkalinity less than 50 milligrams per liter
should done in the early morning at the rate of 5to (mg/L), copper sulphate can be very toxic to fish and
8% of the body weight, twice in a day. should not be used unless a bioassay has been run
in the water first with a limited number of the fish
The feed return should take immediately once after to be treated. Since copper sulfate is an algicide,
the fish feed as maximum as. The high content of consideration must be given to dissolved oxygen
the protein content of the feed leads to formation in a tank to be treated. If a tank already has low
of invisible. dissolved oxygen, an alternate treatment should
be used. Copper sulfate will only aggravate low
Treatment dissolved oxygen problems by killing the primary
source of oxygen (the algae) and by adding a large
A good practice is to maintain only those chemicals biological oxygen demand in the form of dead and
that do have specific approval for aquaculture uses decomposing algae.
at the production facility. The presence of non-
approved chemicals at an aquaculture facility may • Formalin is approved for use in the treatment of
imply their use to an inspector even if they are never several external parasites. It is commonly used as
used. Regulations concerning approved chemicals for an indefinite pond treatment at 15 milligrams per
use in aquaculture are continuously being updated. liter (mg/L). Formalin will remove 1 mg/L dissolved

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

oxygen for every 5 mg/L of formalin used as a
All chemicals have precautions and considerations treatment. Therefore, if dissolved oxygen in a pond
associated with their use. If an aquaculturists has no is low, aeration must be provided or a different
experience with a particular chemical, a small group of treatment should be used. Formalin must be stored
fish should be treated first, as a test before the entire lot at temperatures above 40º F because it will form
is treated, to avoid potentially heavy losses due to toxicity very toxic paraformaldehyde at low temperatures.
associated with overtreatment. Extreme caution should
be practiced when applying any chemical treatment. • Potassium Permanganate (KMnO 4) is
Indiscriminate use of antibiotics leads to drugs resistance approved for use in Aquaculture as an oxidizer
and loss of immunity to the cultured fishes and detoxified. It has been used effectively against
a number of external disease organisms of fish. The
• Terramycin is an antibiotic used to treat systemic normal treatment is 2-8 milligrams per liter (mg/L).
(internal) bacterial infections. It is approved by Ideally, one would like to maintain a “wine red”
the U. S. Food and Drug Administration (FDA) color in the water for a 12 hour period to ensure
for the treatment of sensitive bacteria of the an effective treatment. A preliminary test can be
genera Aeromonas, Pseudomonas, vibriosis, and performed with a small volume of culture water
Hemophilus in cobia Pompano and Grouper. It is to determine the appropriate dose for the system.
used as a feed additive at a rate of 2.5 grams of
drug (active ingredient)/l00 pounds of fish weight/ Farm hygiene is vital to maintaining fish
day for 5 days. A 21-day withdrawal period is health.
required before the fish may be slaughtered and
used for human consumption. It involves routine activities carried out by the farmer
to ensure the following:
• Copper Sulfate (CuSO4) is used to treat a variety
of external parasites of fish. It is also an effective and • Cleaning of utensils and equipment used to handle

263
 or feed fish. Culture conditions can cause disease agents to build
• Water quality testing and correction of poor water up, e.g. high stocking density, low water exchange
quality includes the following: rates, increased total bacterial loads; and
• Measure dissolved oxygen and water
• Chemistry values e.g. salinity, temperature, pH, Feeding regime can cause organic wastes build-up
ammonia, nitrite and nitrates. in culture system, e.g. tank/pond bottoms, sea-
• Measure bacterial counts e.g. Vibrio spp. counts cage bottoms
of the water
• Aeration to maintain dissolved oxygen While some of the factors that predispose to disease
• Cleaning of the farm seabed and fallowing or cannot be controlled, the disease may be managed
rotation of sites Minimising organic pollution from by altering management.
fish wastes and feed wastes.
Disease may be precipitated by stressful events.
Preventive measures Understanding effects of disease on fish hosts helps
with treatment & control.
• Preventing the introduction of pathogens
• Maintenance of good water quality Vaccination may be necessary in preventing diseases
• Avoidance or reduction of environmental stressors predisposed by unavoidable management procedures
• Adequate nutrition such as transport, handling and grading. Studies have
• Isolation of cultured animals from feral stocks shown that high parasite loads can lead to vaccination
• Immunization failures. Vaccination programs must be carried out
with management of other potential pathogens.
Three steps to solve a disease problem
Suggested readings
• Determining that a problem exists.
• Identifying the cause of the disease or source of Bowser.P.R. and Buttner.J.K. (1993) General Fish Health
the distress Management College of Veterinary Medicine, Cornell University
NRAC Bulletin No. 111.
• Successfully curing the fish and eliminating the Inese Briede. (2010) The prevalent bacterial fish diseases in fish
disease or cause of distress. hatcheries of Latvia. Environmental and Experimental Biology
8: 103–106.
Kirjusina M., Briede I, Bondad-Reantaso M.G. 2007. Extension
Summary Manual on Some Important Viruses, Parasites and Bacteria of
Aquatic Animals in Latvia. NDC/LZRA/FAO. Riga. 69 p.
Peters G., Faisal M., Lang T., Ahmed I. 1998. Stress caused by
Arriving at an accurate and correct diagnosis has social interaction and its effect on susceptibility to Aeromonas
wide implications on disease management. Parasites hydrophila infection in rainbow trout Salmo gairdneri. Diseases
Aquat. Org. 4: 83–89.
have different life cycles, bacteria have different
Central Marine Fisheries Research Institute

Rameshkumar, P., C. Kalidas, G. Tamilmani, M. Sakthivel, A.K.

susceptibility to drugs and depending on the impact Abdulnazar, V. Ashokmaharshi, S.K. Srinivasa Rao and G.
of these disease agents on the fish tissues, different Gopakumar. 2014. Microbiological and histopathological
investigations of Vibrio alginolyticus infection in cobia
management strategies may be necessary. Rachycentron canadum linnaeus, 1766 cultured in sea cage.
Indian J. Fish., 61: 124-127, 2014.
Roberts, R.J. 1989. The bacteriology of teleosts. In: Fish. Pathology.
Immune incompetency can be age related or in R. J. Roberts, 2nd ed, Bailliere Tindall, London, pp. 289-319.
fish after periods of stressful events, e.g. transport, Sadler. J and Andrew Goodwin.2007. Disease Prevention on Fish
handling, temperature extremes; Farms. SRAC Publication No. 4703.
Sharma, S.R.K., G. Rathore, D.K. Verma, N. Sadhu and K.K.
Philipose. 2011. Vibrio alginolyticus infection in Asian seabass
Chronic exposure to seemingly benign environment (Lates calcarifer, Bloch) reared in open sea floating cages in
India. Aquac. Res., 1-7.
stress or infectious agents can deplete the fish
immune system;

264
Autopsy Procedure in Fish
S. R. Krupesha Sharma* and N. K. Sanil
Principal Scientist
Marine Biotechnology Division
Central Marine Fisheries Research Institute, Kochi - 682018, Kerala, India
e-mail: [email protected]

Introduction Autopsy procedure:

1. For fish necropsy, an ailing fish or fish which is
An autopsy can be defined as an examination of a body just dead is advisable because decomposition
of the fish after death in order to determine the cause of after death results in anatomical changes not
death or the changes produced by the disease. Examination due to disease. Before the autopsy, fish should
of an ailing fish before death due to disease is termed as be examined for the presence of behavioural
necropsy. The necropsy would help the fish pathologists abnormalities like flashing, spiral swimming,
to determine the causes of morbidity and mortality in fish. finrot, etc. and external abnormalities like presence
It may be noted that unlike terrestrial animals, majority of body ulcers, frayed fins, discoloration of the
of disease of fish are related closely to water quality body, abscesses, abrasions, fungus on the body
(including water source, ammonia levels, nitrite, nitrate, surface, excess mucus on gills and body surface,
pH, temperature, dissolved oxygen, hardness, alkalinity, protozoan or metazoan parasites, malformations,

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

salinity and presence of heavy metals and other toxins) exophthalmia, spinal deformities, etc.
and management issues ( history of water exchanges, 2. When external lesions like ulcers or abrasions
cleaning, filtration backwash, etc, disinfection, and fish are present, inoculations should be made into
quarantine before introduction of new stocks, feeding suitable media supplemented with sodium
with live feeds, trash fish, compound feed, feed storage). chloride, wherever necessary.
Hence, an accurate information pertaining to water quality 3. Peripheral blood smear should be made by
and husbandry practices are critical prior to necropsy. collecting a drop of blood on a microscopic
slide by cutting the caudal peduncle. The blood
Materials required for autopsy examination of fish: smear may be stained with suitable stains
like any of the Romanowsky stains which are
1. Compound microscope, dissecting microscope neutral stains consisting of a mixture of oxidized
and magnifying lamp methylene blue and Eosin Y.
2. Latex gloves 4. In order to conduct the post mortem examinations,
3. Scissors (blunt tips and with pointed tips) fish should be placed on their right side. By using
4. Scalpel with blade a scalpel blade, skin scrapings and gill impression
5. Rat toothed forceps wet mounts can be taken on a microscopic slide
6. Autopsy tray or Petri-dish (in case of juveniles) and examined under the microscope after placing
7. Microscopic slides with cover glasses few drops of PBS for the presence of parasites.
8. Syringe with hypodermic needle Gill impressions should be examined immediately
9. Culture swabs for microbiology since bronchial epithelium gets deteriorated due
10. Microbiological media (Tryptose soya broth with to post mortem changes which may lead to post
2% NaCl) mortem artefact.
11. Sterile loops 5. The body surface of the fish may be disinfected by
12. Ten percent neutral buffered formalin pouring 70% ethanol. The tools used for autopsy
for histopathology may also be disinfected in a beaker containing

265
 absolute ethanol. and its colour and consistency. Examine the swim
6. Lift the operculum and examine gills. Observe the bladder for thickening, haemorrhages, necrosis,
gills and record the colour, presence of parasites, and parasites.
whitish nodules, excess mucus, haemorrhages, 9. If bacterial isolations are to be made, the tissue
7. Approach the abdominal cavity by making an can be charred with a heated scalpel and a sterile
incision from the anal opening upto pectoral bacteriological loop may be pierced into the
fin. The skin flap may be pulled with the help charred area and inoculated on to suitable media.
of a pair of scissors thus exposing the internal 10. If viral isolations are to be made, the tissues
organs like liver, stomach and intestines, kidney can be placed in a suitable tissue culture media
and air bladder. While doing so, make sure that and refrigerated.
the air bladder remains inflated and alimentary 11. Squash of lower intestine should be made on
tract remains intact. microscopic slide and examined for type of food
8. The viscera and adjacent organs (heart, kidney, and pathogens like hexamita.
air bladder, liver, kidney, pyloric ceacae and GI 12. In case nervous signs are reported, small portion
tract) may be examined carefully for the presence of the brain and optic nerve should be placed in
of abnormalities like discoloration, enlargement, “RNA later” for molecular studies. Small pieces
haemorrhage, ascitis, cysts, parasites, neoplastic of tissues may also be preserved in 95% ethanol
growth, etc. Record the colour of the internal for molecular studies.
organs, haemorrhages, enlargement of spleen if 13. Representative samples of all organs
any, presence of white spots or whitish nodules should be placed in 10% buffered formalin
on liver, spleen and kidney, hardening of the gall for histopathology.
bladder, presence of fluid in the peritoneal cavity
Central Marine Fisheries Research Institute

266
Bacterial Diseases of Marine Fish and
Shellfish
S. R. Krupesha Sharma*, M. A. Pradeep and N. K. Sanil
Principal Scientist
Marine Biotechnology Division
Central Marine Fisheries Research Institute, Kochi- 682018, Kerala, India
e-mail: [email protected]

The demand for mariculture of high value fishes in sea from either cultured fish to wild fish or vice-versa.
cages has been rapidly increasing in the recent years.
Most prominent farmed fish species across the globe Diseases of farmed fish occurring due to bacterial
include Atlantic salmon, sea bass, sea bream, turbot, pathology have been one of the major important
mullet and tuna. In India, after the successful breeding limiting factors in mariculture practises. As regards to
and seed production, demand for cage farming of the diseases caused by bacteria in marine fish farming,
cobia is also increasing. Other than cobia, Asian although many pathogenic bacteria have been
seabass is also regarded as a potential marine fish described in the majority of the existing taxonomic
species for sea cage farming in India. groups, fairly small numbers are responsible for major
economic losses in the culture system globally. Majority
Increased intensification of mariculture production of the bacterial pathogens described in the culture

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

involving high density culture in low volume invite systems are normally present in wild fish populations.
the risk of diseases associated with rearing stress and However, in natural environments, in the absence of
fish welfare. According to the Code of Conduct for stressful conditions, they seldom cause mortality due
Responsible Fisheries of the Food and Agriculture to disease outbreak. High stocking density and poor
Organization (FAO), farming implies some sort of environmental parameters act as stressful situations for
intervention in the rearing process to enhance the occurrence of diseases in culture system.
production, such as regular stocking, feeding,
protection from predators, etc. Rearing of high value Vibriosis
fish in high density is also allied with manifestation
of altered host-parasite interactions resulting from Vibriosis is one of the most prevalent bacterial diseases
hosts being reared in new geographic locations. of cultured finfish caused by bacteria belonging to
The altered host-parasite association may also the genus Vibrio. Vibrios are gram negative bacteria,
result from aboriginal hosts being reared in diverse ubiquitous to marine and estuarine environment
environmental conditions (Kent, 2000). In case of as well as marine fish farms. There are around 34
marine cage culture, interactions between wild and recognized species within the genus Vibrio. Within
cultured fish populations are of greatest disquiet for the family Vibrionaceae, the species causing the most
both aquaculturists and environmentalists. economically serious diseases in marine fish and shrimp
culture include Vibrio anguillarum, V. alginolyticus, V.
Diseases occur due to intricate interactions between harveyi, V. parahemolyticus and V. spendidus. Other
host, pathogen and environment (Snieszko, 1974). species in like V. ponticus and V. ordalii are also
Olivier (2002) suggested presence of pathogen in seldom reported to cause mortalities in fish culture
both fish and environment (water), presence of system. Vibriosis caused by V. anguillarum is a major
susceptible host, viability of the pathogen (number problem in seabass farming. However, V. alginolyticus
and longevity) in the environment, and viable infection has been suggested to be a pathogen of humans
rout as essential pre-requisites for a disease to spread and several marine fish species like like seabream and

267
 Asian seabass. This species has been reported to be
the causal agent of outbreak of vibriosis in grouper.
An outbreak of Vibriosis caused by V. alginolyticus in
Asian seabass cultured in marine cages in Indian has
been reported (Sharma et al., 2013). The outbreak of
the disease in this case was associated with increased
water temperature. Isolation of V. ordalii, which has
been established to accommodate strains formerly
classified as V. anguillarum biotype 2 (Schieve and
Crosa, 1981), has been reported predominantly
from North America, Japan and Australia affecting
salmonid fishes. Mutharia et al. (2002) found that Microscopic lesions in case of vibriosis also reflect
cross-reactions subsist between V. ordalii and V. the haemorragic nature of the disease. Histologically,
anguillarum serotype O2 using polyclonal antisera, but bacteria invading the dermis, subcutaneous adipose
immunoblot analysis with absorbed antisera reveal tissue, and the underlying musculature are evident.
that the LPS of both species do not have identical Affected tissues are necrotic and heavily infiltrated
antigenic properties. Most of the pathogenic Vibrios by granulocytes. Gill filaments and lamellae are
are normal inhabitants of sea water and sediment. also infiltrated by neutrophills with haemorrhage.
Hence, these organisms are opportunistic in nature Liver shows hypertrophy of the bile ducts, necrosis,
causing disease when fish are subjected to stress. haemorrhage and congestion. In myocardium, loss of
cross striations and infiltration of polymorphoneuclear
Vibrio harveyi, another member of the family cells in to the endocardium is noticed. Kidneys reveal
Vibrionaceae, is a luminous marine bacterium which characteristic lesions of acute glomerulonephritis
is a normal microflora of warm marine environment, with increased expression of melano-macrophage
body of fish and shellfish, light-emitting organs of centres (Fig.1-B). Gastric mucosa contains engorged
marine fish and cephalopods, and intestinal microflora capillaries and loss of tubular glands. Extensive tissue
of marine vertebrates and invertebrates. The organism lesions in vibriosis are primarily due to the release
is predominantly responsible for the occurrence of of proteinases and other extra-cellular enzymes
luminous vibriosis, which affects a wide range of produced by the bacteria.
marine invertebrates, especially penaeid shrimp and
phyllosoma larvae of the rock lobster resulting in Diagnosis: Vibrios are gram negative rods
severe economic losses. Pathological manifestations characteristically curved or comma shaped. This
of vibriosis caused by V. harveyi in the cage farmed morphological appearance may not be always
mangrove red snappers associated with increased observed when organisms are selected for gram
water temperature and handling stress is also reported staining from solid media. Specific media like
Central Marine Fisheries Research Institute

(Sharma et al., 2014). Thiosulfate-citrate-bilesalts-sucrose agar (TCBS) agar

may be used for selective growth of Vibrios. Species
Pathology: In case of vibriosis, the pathogen
may enter the host orally, through skin lesion
and gill surface consequent to wound caused
by ectoparasites and protozoa. Fish affected by
classical vibriosis show typical signs of a generalized
haemorrhagic septicaemia with the presence of
haemorrhagic lesions at the base of fins, ulcerations
on the body surface, especially in chronic cases,
exophthalmia and corneal opacity (Fig. 1-A). Ailing
fish are often anorexic with pale gills due to anaemia
arising from haemorrhages.

268
level identification can be done by biochemical tests, production considerably. The disease is characterized
PCR using specific primers and 16S rDNA amplification by the presence of whitish nodules on liver, spleen
using universal primers and sequencing. and kidney. Severe mortalities occur in pasteurellosis
when water temperature is above 18– 20ºC. Below
Treatment and prevention: Even though Vibrios are this temperature, fish can harbour the pathogen for
susceptible to majority of broad-spectrum antibiotics, prolonged periods without causing clinical infection.
limitations exist based on the farming system. Since This disease was first discovered in natural populations
Vibrios are opportunistic pathogens, vibriosis can of white perch (Morone americanus) and striped
be best managed by proper husbandry practices. bass. The disease affects various species of fishes
Handling, transportation, overcrowding, low dissolved like yellow tail juveniles (Seriola quinqueradiata),
oxygen and increased water temperature make ayu (Plecoglossus altivelis), black seabream (Mylio
the farmed fish susceptible to vibriosis. Periodical macrocephalus), red seabream (Acanthopagrus
enumeration of the bacterial load of water and schlegeli), oval file fish (Navodan modestus) and red
sediment would help in preventing outbreaks. grouper (Epinephelus okaara).

Due to diversity of Vibrios and their serovars, the Pathology: Fish pasteurellosis is a septicaemic disease
advancement in vaccine development against vibriosis and manifests as an acute or chronic form. Pale
has been dawdling, and commercial vaccine is not gills, dark pigmentation and presence of petechial
currently available. However, attempts have been made haemorrhages on the body surface and fin base are
to vaccinate fish against different Vibrio spp. using oral, normally observed in acute form. Enlarged spleen and
killed and sub-unit vaccines. In case of oral vaccination, mottled liver are seen internally. In case of chronic
the vaccine is either mixed with the feed, top dressed form, nodules resembling tubercles are seen in spleen
on the feed, or bio-encapsulated. Bio-encapsulation and kidney.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

is used when fish fry are to be vaccinated. In case of
bio-encapsulation, live feed, such as artemia nauplii, Another disease caused by Photobactrium damselae
copepods or rotifers incubated in a suspension of subsp. damselae is also responsible for mortality
vaccine are fed to the fish fry. Oral vaccination causes in many cultured marine fish species. This disease
no stress to the fish. However, they have a very short has been reported from India in cage farmed cobia.
term stability once mixed with the feed. More recently, The lesions are haemorrhagic in nature resembling
the outer membrane proteins molecules are used for the lesions found in vibriosis. The pathological
development of subunit vaccines due to the exposed manifestations in both the infections are primarily
epitopes on the bacterial surface and conserved nature due to the extra cellular products (ECP) secreted by
in different serovars. It has also been demonstrated the bacteria. Both pathogens are normal inhabitants
that outer membrane proteins molecules like OMP -K of marine environment.
acts as protective antigen against fish vibriosis caused
by V. alginolyticus. Disease transmission in case of photobacteriosis
occurs through direct contact and ingestion. The
Photobacteriosis bacteria are unable to survive in fresh or brackish
water. Predisposing factors for the outbreak normally
Photobacteriosis, also known as fish pasteurellosis, include rise in water temperature.
is caused by the halophilic bacteria Photobacterium
damselae subsp. piscicida. Gauthier et al. (1995) Diagnosis: The pathogen can be isolated and
included the fish pathogen Pasteurella piscicida in cultured on marine agar and ordinary media
the species P. damselae according to phylogenetic supplemented with sodium chloride. The organism
analyses of 16S rDNA sequences and DNA⁄DNA can be confirmed by biochemical tests, 16S rDNA
relatedness, and named as Photobacterium sequencing and slide agglutination. Photobacterium
damselae subsp. piscicida. Pasteurellosis has been damselae subsp. piscicida has to be differentiated
a serious disease in Japan affecting the aquaculture from Photobactrium damselae subsp. damselae using

269
 a multiplex PCR that combines specific primers for 16S preparations obtained from gills or lesions. Phase
rRNA and urease genes (Osorio et al., 2000). contrast microscopy is advantageous in bacterial
identification. The bacteria cannot be easily isolated
Treatment and control: Several commercial vaccines on conventional media. This pathogen grows only in
against P. damselae subsp. piscicida are available, specific media since it needs an absolute requirement
wherever the disease is more prevalent. Efficacy of of seawater as well as low concentration of nutrients.
these vaccines depends on the species of fish, fish size, Some of the specific media devised for the isolation of
etc. Since outbreak of pasteurellosis normally occurs this pathogen include Flexibacter Maritimus Medium
during larval stages to fingerling stage, a vaccination (FMM). FMM has been regarded as most effective
programme involving dip immunisation during the media. The bacteria can be grown at 13 to 34ºC.
larval stage with a booster dose when fish reaches a Colonies are rhizoid with uneven edges. A nested PCR
size of 1–2 g is advocated. which is rapid and sensitive was also developed for
an accurate diagnosis (Cepeda et al., 2003).
Flexibacteriosis
Treatment and prevention: Hydrogen peroxide
Flexibacteriosis, also known as gliding bacterial disease used at a dose of 240 ppm would be beneficial
of sea fish, eroded mouth syndrome, and black in treating the infection. Many of the antiobiotics
patch necrosis, is a disease of marine fish caused are also effective in treating the fish infected with
by Tenacibaculum maritimum (formerly Flexibacter T. maritimum. However, they have to be used with
maritimus). T. maritimum exists exclusively in the marine caution depending on the type of antibiotic, farming
environment. The disease is normally distributed in system and environmental factors. A formalin killed
cultured and wild fish in North America, Australia, vaccine was also tried in Japanese flounder to reduce
Europe and Japan. Turbot, sole, seabream, seabass, the effects of infection.
red seabream and black seabream are the cultured
fishes affected by T. maritimum. The disease occurs Streptococcosis
in a more severe form in young fish when compared
to adults. The severity of the disease increases with Streprococcosis is a re-emerging disease of both fresh
increase in water temperature. The predisposing factors and marine fish caused by gram positive bacteria
in a farming system include injuries and skin abrasions. characterized by central nervous system damage
Stress induced by husbandry practices like inferior followed by exophthalmia and meningoencephalitis.
water quality, increased water temperature, handling Streptococcosis is also known as “pop-eye”, since one
etc., may also predispose the fish to flexibacteriosis. of the most characteristic clinical sign found in this
disease is the accumulation of muco-purulent exudates
Pathology: Affected fish are weak, develop pinkish around the eyes. Streptococcosis, a problem in both
ulcers on the skin with loss of scales. Affected fish farmed and wild marine fish, has been reported from
Central Marine Fisheries Research Institute

also have eroded and haemorrhagic mouth and frayed USA, Japan, and Spain. In Japan and Spain, this
fins with tail rot. White necrotic lesions develop on disease forms a major limiting factor for the marine
gills of the affected fish. Microscopically, long, thin fish production of yellowtail turbot. Fish of all the size
basophilic bacteria can be seen in sections taken from are susceptible. The sea water and sediment harbours
skin and muscle lesions. Colonization of the bacteria the pathogen which can be isolated from these sources
on the scale pockets, loss of scales and dermatitis round the year. It has been reported that this pathogen
are also noticed. Congestion and haemorrhage in can survive in frozen products for at least 6 months.
the superficial dermis is also evident histologically. Warm water streptococcosis which occurs when water
temperatures are above 15ºC is normally caused by L.
Diagnosis: The initial presumptive diagnosis of garvieae, S. iniae, S. agalactiae and S. parauberis. Cold
marine flexibacteriosis include microscopic observation water streptococcosis seen when water temperatures
of accumulations of long, thin, rod shaped flexing are below 15ºC is generally caused by L. piscium and V.
bacteria in wet mounts or Gram-negative stained salmoninarum. Pathogens responsible for warm water

270
streptococcosis are of zoonotic importance since they isolates are then characterized either biochemically or
can cause disease in humans. Horizontal transmission serologically. Significant characteristics are spherical
can occur through injuries and abrasions. or ovoid colony morphology and formation of
pairs or chains. All strains of streptococci are Gram
Streptococci capable of causing disease in marine fish positive, oxidase and catalase negative, non-motile
falls into three categories: alpha-haemolytic, beta- and non-sporulating. Slide agglutination and
haemolytic, and non-haemolytic. The majority of immunofluorescent techniques are widely used for
disease epizootics are generally caused by streptococci diagnosis of streptococcosis. PCR based diagnosis
belonging to alpha-haemolytic group. Among these including 16S rRNA amplification and sequencing
fish streptococci, L. garvieae, S. iniae and S. parauberis can also be employed.
can be regarded as the main aetiological agents
causing diseases in marine aquaculture. Treatment and prevention: Good husbandry
practises including avoiding over-crowding, excess
L. garvieae infects marine fish like yellowtail in Japan. feeding, handling and the timely removal of
S. Iniae is an important fish pathogen causing disease diseased or dead fish would help to minimise the
and mortality in many cultured fish species in both economic losses due to streptococcosis. Apart from
tropical and sub tropical environments. S. iniae is the this, vaccination, use of chemotherapeutics and
main aetiological agent of streptococcosis in tilapia in immunostimulants has also been tried. Many of
USA and rainbow trout in Israel. However, S. iniae was the isolated strains were sensitive to antibiotics like
also isolated from marine fish including yellowtail, erythromycin, tetracycline, ampicillin and doxycycline.
flounder, European seabass, and Asian seabass. There Attempts have been made to develop vaccines against
have been no reports from India. S. parauberis is streptococcosis in fish. Intra-peritoneal route was
reported to be endemic to cultured turbot. found to be most effective. -1-3-glucans used as

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

immunostimulants is also found to be effective.
Pathology: The lesions caused by different
streptococcal species in diverse host species are Mycobacteriosis
similar. The lesions are of general septecemic in nature
affecting liver, spleen, eye, brain and kidney. The
(Fish tuberculosis)
eyes show severe exophthalmia with granulametous Mycobacteriosis is a chronic wasting granulometous
inflammation. Granulomas with the presence of disease of fish caused primarily by Mycobacterium
bacteria may also be seen in pericardium, alimentary marinum. Some species of mycobacterium are
tract, peritoneum, brain, ovary, testes and spleen. found in soil and water. Mycobacteriosis was earlier
The most significant clinical signs are exophthalmia, known as fish tuberculosis or piscine tuberculosis
distended abdomen, haemorrhages in the eyes, because the causative pathogen is taxonomically
opercula, fin base, ulceration of the body surface and similar to Mycobacterium tuberculosis, which
darkening of the skin. Internally, the abdominal cavity caused tuberculosis in humans. But other than
is filled with variable amounts of purulent exudate. acid-fast staining characteristics and taxonomic
A yellowish exudate often covering the peritoneum position, the organisms causing tuberculosis in
and the pericardium may be seen. Yellowish exudates fish and human are dissimilar. In cultured fish,
may also be seen in cranial cavity. Haemorrhages are mycobacteriosis was reported in salmon, pejerrey,
found on all visceral organs. snakehead fish, turbot, tilapia, European seabass
and red drum. Mycobacteriosis caused by M.
Diagnosis: Fish streptococci can be generally marinum represents a significant threat especially
isolated from internal like spleen, liver and brain for seabass cultured on the Mediterranean and the
using brain heart infusion agar or tryptone soya Red Sea coast of Israel. Mycobacteriosis is regarded
agar supplemented with 1% yeast extract or 0.5% as a serious threat to the turbot culture in Europe.
glucose, and growth is enhanced on blood agar. In general mycobacteriosis is more dangerous to
The incubation period is 24 days at 25-30ºC. The marine fishes when compared to fresh water fishes.

271
 Incidence of mycobacteriosis in higher in aquarium Shrimp vibriosis
fish since these fish are kept for long time and also
mycobacteriosis being a chronic disease. Shrimp vibriosis also known as sea gull syndrome,
is caused by numerous etiological agents like V.
Ingestion, trans-ovarian transmission and direct harveyi, V. vulnificus, V. parahaemolyticus and V.
contact from water or infected fish have been alginolyticus causing severe economic losses in
suggested as the possible routes of transmission shrimp hatcheries and post larvae rearing ponds.
in case of mycobacteriosis. Also, high density of Infections of the exoskeleton extend into digestive
fish in an intensive culture system would be a tract, including the hepatopancreas, and finally
responsible for an outbreak of mycobacteriosis due septicaemia occurs leading to severe mortality.
to increased opportunity for transmission through These bacteria are Gram-negative, motile, rod-
the water column, faecal products or cannibalism shaped bacteria that require supplementation of
(Hedrick et al. 1987). sodium chloride in the media for their growth.
Among the Vibrio spp. which cause disease in
Pathology: Clinical signs include emaciation, stunted shrimp, V. harveyi, is one of the primary etiologic
growth, exophthalmia and slowed swimming. agents of that causes mass mortalities in Penaeus
Internal lesions in mycobacteriosis vary depending monodon larval rearing ponds. Epizootics of
on the fish species involved but typically consists vibriosis occur in all life stages of the shrimp, but
of greyish-white granulomas in the spleen, kidney are more common and lethal in shrimp hatcheries.
and liver. Microscopically, these typical granulomas Most of the Vibrio spp. form part of the natural
consist of central area of necrosis surrounded microflora of wild and cultured shrimps and cause
by macrophages, epithelioid cells and fibrous disease when natural defence mechanisms are
connective tissue. Granulomas are generally seen suppressed due to various stress factors like inferior
in spleen, liver and kidney during initial stages of the water quality, deteriorating environmental factors
disease, but later may spread to all internal organs and overcrowding.
in more advanced cases. Externally, loss of scales
and haemorrhages extending to the musculature is Pathology: Most of the Vibrio spp. form part
seen in advanced cases. of the natural microflora of wild and cultured
shrimps and cause disease when natural defence
Diagnosis: Presumptive diagnosis can be made mechanisms are suppressed due to various stress
by clinical signs, gross and microscopic pathology. factors like inferior water quality, deteriorating
However, these are many a times are inconsistent environmental factors and overcrowding. Clinical
and hence definitive diagnosis is not possible base signs of shrimp vibriosis include high mortality,
on pathology. Smears from spleen and kidney can be shrimp congregating in surface of pond edge and
stained with Ziehl-Neelsen stains so as to identify the presence of luminescence which can be appreciated
Central Marine Fisheries Research Institute

acid-fast short bacilli. Isolation on specific media can also in darkness. Gross lesions include melanosis of the
be helpful. Methods presently under research genetic shell which appear as black spots on the cuticle and
techniques, high-performance liquid chromatography delayed clotting. Post-larvae infected with Vibrio
(HPLC), and capillary gas chromatography for fatty-acid spp. have cloudy hepatopancreas. Gills are mostly
methyl-ester (FAME) analysis. brown in colour. Histopathologically, atrophy of the
hepatopancreas with multifocal necrosis associated
Treatment and prevention: Since no approved with haemocytic infiltration is evident. Presence
drugs or anti-mycobacterial agents are available, of localized haemocytic nodules in the lymphoid
depopulation and proper disinfection is the most organ, heart and connective tissues of the gills,
commonly adapted policy in case of culture conditions. hepatopancreas, antennal gland, telson and muscle
However, disinfection may not be always successful are also observed. Vibriosis in P. monodon is also
due to the resistance acquired by the pathogen to associated with the appearance of “spheroids” in the
many disinfectants (Jacobs et al., 2009). lymphoid organ. Large numbers of gram negative

272
bacteria are present in the hemolymph. Adult Suggested readings
shrimps infected with vibriosis are hypoxic, show
reddening of the body surface, reduced feed intake Cepeda, C., Garcia-Marquez, S. and Santos, Y. (2003). Detection
of Flexibacter maritimus in fish tissue using nested PCR
and move slowly mostly at the edges and surface of amplification. J. Fish Dis., 26: 65-70.
pond. Some of the Vibrio spp. also causes red-leg Gauthier, G., Lafay, B., Ruimy, R., Breittmeyer, V., Nicolas, J.L.,
disease which is characterised by red discolouration Gauthier, M. and Christen, R. (1995) Small-subunit rRNA
sequences and whole DNA relatedness concur for the
of the pleopods and gills associated with mortality reassignment of Pasteurella piscicida (Sniezko et al.) Janssen
up to 95% during summer months when the water and Surgalla to the genus Photobacterium as Photobacterium
damselae subsp. piscicida. Int J Syst Bacteriol., 45: 139–144.
temperature is high. Hedrick R.P., McDowell T. & Groff J. (1987) Mycobacteriosis in
cultured striped bass from California. J. Wildlife Dis. 23: 391–
Diagnosis: Vibrio infection can be readily diagnosed 395
Kent, M.L. (2000) Marine netpen farming leads to infections with
based on clinical signs and demonstration of rod- some unusual parasites. Int. J. Parasitol., 30:321–326.
shaped Vibrio bacteria in lesions, nodules or Mutharia, L.W., Raymond, B.T., Dekievit, T.R., Stevenson, R.M.W.
(1992) Antibody specificities of polyclonal rabbit and rainbow
haemolymph. Haemolymph may be inoculated trout antisera against Vibrio ordalii and serotype O2 strains of
on TCBS agar plate. Luminescent colonies may be Vibrio anguillarum. Can. J. Microbiol. 39: 492–499.
observed after 6 to 12 hr when inoculated onto Olivier, G. (2002) Disease interactions between wild and cultured
fish—perspectives from the American Northeast (Atlantic
tryptone soya agar. provinces). Bull. Eur. Assoc. Fish Pathol., 22: 103–109.
Osorio, C.R., Toranzo, A.E., Romalde, J.L., Barja, J.L. (2000)
Multiplex PCR assay for ureC and 16s rRNA genes clearly
Treatment and prevention: Luminiscent vibriosis is discriminates between both subspecies of Photobacterium
normally prevented in the hatcheries using appropriate damselae. Dis. Aquat. Organ., 40, 177-183.
chemicals so that bacterial load of the rearing units Schieve, M.H., Crosa, J.H. (1981) Molecular characterization
of Vibrio anguillarum biotype 2. Can. J. Microbiol., 27:
or the incoming water can be reduced. In shrimp 1011– 1018.
hatcheries, washing eggs with iodine and formaldehyde Sharma, S.R.K., Gaurav Rathore, Dev K Verma, Sadhu, N and

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Philipose, K.K. (2013) Vibrio alginolyticus infection in Asian
and preventing contamination of eggs by excreta is seabass (Lates calcarifer, Bloch) reared in open sea floating
advocated. In case of pond culture, increase in daily cages in India. Aquacult. Res., 44: 86–92.
water exchanges and reduction in pond biomass Sharma, S.R.K., Pradeep M.A., Loka, J., Sadhu, N., Dube, P.N. and
Philipose, K.K. (2014) Association of Vibrio harveyi in mortality
by partial harvesting would help in reducing the of mangrove red snapper (Lutjanus argentimaculatus, Forsskål,
mortality in case of an outbreak. Probiotics may also 1775) cultured in open sea cages. Indian J. Fish., 44: 86–92.
Snieszko, S.F. (1974) The effects of environmental stress on the
be administered through water or feeds. outbreaks of infectious diseases of fish. J. Fish Biol., 6: 197–208.

273
 Viral Diseases of Marine Fish and Shellfish
S. R. Krupesha Sharma*, M. A. Pradeep and N. K. Sanil
Principal Scientist
Marine Biotechnology Division, Central Marine Fisheries Research Institute, Kochi- 682018, Kerala, India
e-mail: [email protected]

Viruses are microorganisms that can replicate only virus and calci virus. However, the most lethal viral
inside the living cells of other organisms. Viruses can disease causing enormous loss to finfish farming is
infect all types of life forms including animals, plants the disease caused by betanodavirus. As for as shrimp
and microorganisms, including bacteria and archaea. farming is concerned, the major viral diseases of P.
Virus particles or virions normally consist of three monodon include white spot syndrome and yellow
components: i) genetic material made up of head disease that can also cause serious mortalities
either DNA or RNA which carries the genetic in P. vannamei farming.
information; ii) a  coat protein that protects these
genetic materials; iii) a lipid envelope that surrounds Viral nervous necrosis
the protein coat which may be absent in some viruses.
The shape of viruses may be helical or icosahedral or Betanodavirus is one of the genera making up the
more complex structures. family Nodaviridae which is the etiological agent
of viral nervous necrosis (VNN) also known as
Viruses do not multiply through cell division. They encephalomyelitis and vacuolating encephalopathy
normally make use of the host cell genetic material and retinopathy. This virus has remained as a
and metabolic pathways to produce multiple copies major threat for the establishment and expansion
of themselves. The life cycle of viruses differs greatly of Asian seabass (Lates calcarifer) and striped
between species but there are six basic stages in their jack (Pseudocaranx dentex). The disease was first
life cycle which include attachment, penetration, documented in 1990 in hatchery-reared Japanese
uncoating, replication, assembly and release. parrotfish (Oplegnathus fasciatus) in Japan and
Asian sea bass in Australia. Later, it was reported
The aquaculture as an industry has expanded globally in turbot (Scophthalmus maximus), European sea
with an increase in both productions in terms of bass (Dicentrarchus labrax), redspotted grouper
biomass and also in number of fish species being (Epinephelus akaara), striped jack (Pseudocaranx
cultured. Intensification of aquaculture operations dentex) and more recently in cultured warm-water
Central Marine Fisheries Research Institute

globally has provided new opportunities for the and cold-water marine fish species throughout the
transmission of fish viruses and hence the occurrence world (Munday et al., 2002). In India, mortality
of diseases caused by viruses forms a major limiting caused by betanodavirus infection in hatchery
factor for the sustainable aquaculture production. produced larvae of Asian seabass was first reported by
Azad et al. (2005). An Indian strain of betanodavirus
Fish viruses have been the subject of research interest belonging to RGNNV group was isolated from Asian
in the past two decades. Compared to diseases caused seabass juveniles reared in a brackish water farm in
by fresh water fish viruses, there have not been Bhimavaram in Andhra Pradesh in 2012. Outbreak of
extensive studies on marine fish viruses. Establishment mortality due to nodavirus infection in Asian seabass
of various fish cell lines lead to path breaking research juveniles cultured in fresh water cages in the south
in fish virology in the recent years. Major viral groups west coast of India has also been reported. Mortality
under which fish viruses can be classified include of Asian seabass juveniles cultured in indoor cement
herpes virus, iridovirus, rhabdo virus, reo virus, noda tanks as well as open sea cages and in cobia cultured

274
in cages in India associated with RGNNV was also been described in all Species. The lesions include
recorded (unpublished report). vacuolation of the cellular components of the retina
especially the bipolar and ganglionic nuclear layers.
Betanodaviruses can infect fish species belonging to
tropical, sub-tropical, or temperate waters. These Under transmission electron microscopy, fish
viruses can multiply at an optimum temperature betanodaviruses appeare icosahedral, non-enveloped
depending on the strain of the virus. For RGNNV, with a mean diameter of about 25 nm. The virions
the optimum temperature requirement is 25–30ºC may be membrane bound by endoplasmic reticulum
while for SJNNV, it is 20–25ºC. or are free in the cytoplasm and may present as
paracrystalline arrays. Cells containing virions normally
Mostly, betanodaviruses are a concern in marine include neurones, astrocytes, oligodendrocytes and
fish species. The species susceptible cobia, sea bass, microglia cells.
seabream, bluefin tuna, grouper, halibut, surgeonfish,
lined surgeonfish and tiger puffer. The freshwater fish Diagnosis: The diagnostic methods for fish
species susceptible to betanodaviruses include tilapia nodaviruses have been extensively studied. According
and the guppy. the Munday et al. (2002), VNN can be diagnosed by:

Pathology: In farming system stress factors like high 1. Demonstration of characteristic vacuolar lesions in
density, transportation, high temperature can act as the brain or retina by light microscopy.
predisposing factors making the fish susceptible to 2. Detection of virions and viral antigens by electron
VNN. Although young fishes are more susceptible, microscopy and serology
older fishes may also get infected especially when 3. Detection of viral nucleotides by molecular
water temperature is high. techniques including RT PCR, RT Nested PCR,

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

cloning and sequencing by designing the primers
During the acute stage of the disease, when the for the strain of interest.
mortality is very high, especially in juveniles, there 4. Tissue culture of virus in a suitable cell line.
would be no gross lesions on the body surface or
gills. However, affected juveniles and older fish show Treatment and prevention: Betanodaviruses are
a abnormal swimming behaviors such as spiral, highly resistant to various environmental conditions
whirling, floating with inflation of swim bladder, or and they can survive for a long time in sea water.
laying down at rest, circling on their own axis. This The disease can also be reproduced by simple
erratic swimming behaviour may not be noticed in co-habitation of the healthy fish with infected fish.
infected fish larvae. Grossly, the brain is oedematous Control measures are including imposing strict bio-
and in many cases severely congested (Fig. 2-A). security to exclude the virus from the farm premises.
The broodstocks should be tested for the presence
Microscopically, lesions are characterized by severe of viruses in the gonadal tissues and VNN specific
vacuolation and necrosis of the central nervous antibodies in serum and any positive fish should be
system (Fig.2-B). In general, the anterior brain is more culled at first.
severely affected when compared to the posterior
part of the brain and spinal cord. Larvae of the fish White spot syndrome
are more severely affected by betanodaviruses than
juveniles. The most characteristic lesion in the fish More than 1100 viruses of invertebrates have
larvae is the presence of vacuoles in the grey matter been reported so far. Most important groups of
of the brain which are intracytoplasmic. Basophilic, viruses reported in Crustacea include Reoviridae,
intra cytoplasmic inclusions have been reported in Picornaviridae, Parvoviridae, Togaviridae, Baculoviridae,
brain cells of Asian seabass. Paramyxoviridae, Rhabdoviridae and lridoviridae.

Lesions in the retina of the infected fish have also White spot syndrome  (WSS) is a  viral  infection

275
 of  penaeid shrimp caused by a double stranded mesodermal origin including gills, haemocytes and
DNA virus belonging genus Whispovirus within the haemotopoietic tissue, lymphoid organ, connective
Nimaviridae family which is a most devastating virus tissues, subcuticular epidermis, stomach, foregut and
of cultured shrimp characterized by severe mortality hindgut epithelium, heart, striated muscle, midgut
and appearance of white spots on the carapace of and ovary walls, antennal gland and the nervous
the infected fish. All decapod crustaceans including tissues. These inclusion bodies are strikingly distinct
prawns, lobsters and crabs from marine, brackish or and bigger than the Cowdry A-type inclusions which
freshwater environments are susceptible to infection. are seen in infectious hypodermal and haematopoietic
WSSV is the largest DNA virus of which whole genome necrosis virus infection.
sequencing has been done.
Diagnosis: The disease can be diagnosed and
WSSV virions are ovoid or ellipsoid to bacilliform in confirmed by using DNA probes, PCR using WSSV
shape and measure 80–120 nm in diameter and 250– primers, nested PCR, monoclonal antibody based kits
380 nm in length. Most noteworthy feature is the which can be used at field level.
presence of flagella like extension at one end of the
virion. Under laboratory conditions, WSS virus is viable Treatment and prevention: Even though
for at least 30 days at 30ºC in seawater and the virus research results are available on immunity against
is viable in ponds for at least 3–4 days. WSSV in shrimp injected with inactivated WSSV
virions or recombinant structural protein, or by
The virus is normally transmitted by horizontal using RNA interference (RNAi), or by administering
transmission through water and feed infected. Vertical orally bacterially expressed VP28 dsRNA, there are
transmission is also possible brooders to offspring. still no field data for either the vaccination or the
RNAi approach.
Pathology: Shrimp with acute WSS show a rapid
reduction in food consumption, lethargic movements Yellow Head Disease
and have a loose cuticle with the presence of white
spots which measure 0.5 to 2.0 mm in diameter. Yellow head diseases is a viral disease of shrimp
The targets organs for the virus are the cells of caused by most virulent shrimp virus, yellow head virus
ectodermal and mesodermal origin, including those genotype-1 characterized by yellowish discolouration
of the epidermis, gills, foregut, hindgut, antennal of the cephalothorax and mass mortality. Yellow head
gland, lymphoid organ, muscle, eye-stalk, heart, virus (YHV) can remain viable in aerated seawater for
gonads, haematopoietic cells and cells associated up to 72 hours. YHV is an enveloped, rod-shaped,
with the nervous system. Hence the death is normally ssRNA virus with a helical nucleocapsid and prominent
due to multi-organ dysfunction. These white spots glycoprotein projections on the virion surface.
are apparent on the inside surface of the carapace.
Central Marine Fisheries Research Institute

The white spots signify abnormal deposits of calcium Yellow head disease (YHD) outbreaks have been
salts by the cuticular epidermis. Shrimp showing these reported in the black tiger prawn (P. monodon) and
signs show high mortality, sometimes up to 100%, in the white Pacific shrimp (P. vannamei) and pacific blue
3 to 10 days of the onset of clinical signs. In a typical prawn (P. stylirostris) and few other shrimp species. P.
shrimp farm, WSSV infected shrimp gather near the monodon which are beyond PL15 are susceptible to
pond edge and display clinical signs 1 or 2 days before YHV. Like WSSV, YHV also targets tissues of ectodermal
the first mortalities occur. and mesodermal origin including lymphoid organ,
haemocytes, haematopoietic tissue, gill lamellae
White spot disease is histopathologically characterized and spongy connective tissue of the subcutis, gut,
by the presence of widespread and severe nuclear antennal gland, gonads, nerve tracts and ganglia. YHV
hypertrophy, chromatin margination and eosinophilic infection can be transmitted horizontally by injection,
to basophilic large intranuclear inclusions with ingestion of infected tissue, or by co-habitation of
focal necrosis in most tissues of ectodermal and healthy shrimp with infected shrimp. Homologous

276
genetic recombination is also an attribute of the a healthy shrimp. Unusually high feeding activity by
yellow head virus complex. The prevalence and the shrimp followed by a sudden cessation of feeding
geographic distribution of these recombinant viruses may occur within 2 to 4 days of the appearance of
indicate that the reason of swiftly increasing genetic gross clinical signs of disease and mortality.
diversity of the virus is primarily due to international
trade in live shrimp. Microscopically moderate to large numbers of deeply
basophilic, evenly stained, spherical, intra-cytoplasmic
Pathology: YHV genotype-1 can cause upto 100% inclusions of approximately 2 µm in diameter can
mortality in P. monodon within 3–5 days of the first be seen in tissues of the lymphoid organ, stomach,
appearance of clinical signs. Stress induced by sudden and gills.
changes in pH or dissolved oxygen levels can be the
predisposing factor for an outbreak of the disease Diagnosis: YHD can be diagnosed by gross
in shrimp farms. Moribund shrimp may congregate and microscopic lesions and RT-PCR using YHV
at pond edges near the surface In shrimp farms, specific primers.
infection can result in mass mortality especially in
early to late juvenile stages. Clinical symptoms of the Suggested readings
disease may include cessation of feeding, aggregation
of infected shrimp at pond edges and bleached Azad I.S., Shekhar M.S., Thirunavukkarasu A.R., Poornima M.,
appearance. Moribund shrimp may exhibit a bleached Kailasam M., Rajan J.J.S., Ali S.A., Abraham M. & Ravichandran
P. (2005) Nodavirus infection causes mortalities in hatchery
overall appearance and a yellowish discoloration of produced larvae of Lates calcarifer, first report from India. Dis.
the cephalothorax. This is caused by the underlying Aqua. Org. 63: 113–118.
Munday B.L., Kwang J. and Moody, N. (2002) Betanoda virus
yellow hepatopancreas, which may be remarkably soft infections of teleost fish: a review. J. Fish Dis., 25:127-142.
when compared with the brown hepatopancreas of

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277
 Microbiological Staining Techniques
S. R. Krupesha Sharma* and M.A. Pradeep
Principal Scientist
Marine Biotechnology Division
Central Marine Fisheries Research Institute, Kochi- 682018, Kerala, India
e-mail: [email protected]

Etiological agents of bacterial diseases of marine fish less peptidoglycan and they have lipopolysaccharide
and shellfish can be identified by various microbiological containing endotoxin.
methods. The samples should always be taken from
the fish under aseptic condition and they are first Preparation of reagents
inoculated onto non-specific media, then the various • Primary Stain : Crystal Violet
microbial diagnostic methods are used. The commonly
used microbiological diagnostic tools include: Solution A:
• Crystal violet : 2g
• Staining • Ethanol, 95% : 20 ml
• Motility test
• Culturing Solution B
• Biochemical test • Ammonium oxalate : 0.8 g
• Distilled water : 80 ml
Staining
Mix both A and B solutions to obtain
Different staining methods are in use to identify working crystal violet reagent.
various bacterial pathogens. Gram staining is the Mordant: Gram’s Iodine
most important and most commonly used method. • Iodine : 1.0 g
• Potassium iodide : 2.0 g
Gram stain  • Distilled water : 300 ml
The Gram stain classifies bacteria according to whether • Decolourizing agent : Ethanol, 95%
they retain crystal violet stain (gram-positive-blue) or • Counter stain : Safranin
not (gram-negative-red). If the bacteria can retain
crystal violet, then ther are classified as Gram +ve Stock solution
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and takes purple colour. If they do not crystal violet, • Safranin O : 2.5 g
then they are classified as Gram -ve and appear pink. • 95% Ethanol : 95 ml
The morphology of the bacteria, whether they are tod
shaped or cocci or cocco-bacillary and arrangements of Working Solution:
bacterial cells in the form of clumps, diploids or chains • Stock Solution : 10 ml
can also be ascertained by Grams staining. • Distilled water W : 90 ml

Principle Procedure
The basic principle of Gram staining is the properties 1. Air-dry and heat-fixe the smear.
of cell walls of some bacteria to retain the crystal 2. Pour crystal violet and wait for 1 min.
violet dye. The cell walls for Gram positive bacteria 3. Wash slide gently using tap water for 2 seconds
have a large amount of peptidoglycan and lower lipid 4. Flood slide with the mordant and wait for1 min
content. The cell wall of Gram negative bacteria has 5. Wash slide gently with tap water for 2 seconds.

278
6. Flood slide with decolorizing agent. Wait for about Neutrophils: dark purple nuclei, pale pink cytoplasm,
15 seconds until no more decolorizing agent run reddish-lilac small granules
off from slide Eosinophils: blue nuclei, pale pink cytoplasm, red to
7. Flood slide with safranin and wait for 1 min. orange-red large granules
Wash slide gently with tap water until no colour Basophils: purple to dark blue nucleus
appears in the effluent and dry the slide. Observe Lymphocytes: deep bluish purple nuclei
under oil immersion Platelets: violet to purple granules

Interpretation
Motility (Hanging Drop Method)
Gram negative: pink/red and gram positive: blue/
purple Procedure
1. Place a small drop of bacterial culture in a broth
Wright’s stain at the center of a coverslip
2. Place a small drop of water at each corner of
These stains are used for detection of parasites in blood, the coverslip
phagocytes and tissue cells, intracellular inclusions 3. Invert a cavity slide with a central depression over
formed by viruses and also some intracellular bacteria. the coverslip
4. The coverslip will stick to the slide and when the
Procedure
slide is inverted the drop of bacterial culture will
Make thin smears across a sterile slide by means of be suspended in the well
a second slide or cover glass. Air dry. 5. Examine microscopically for motile organisms

Staining:
Culturing bacteria pathogen in

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Place 1.0 ml of the Wright Stain Solution upon the
selective medium
smear (1 min). Add 2.0 ml distilled water (2 min).
Rinse stained smear with water or the Phosphate Some bacteria have the ability to grow in selective media
buffer pH 6.5 until the edges show faintly pinkish-red. which can be used to identify the pathogen. One of the
Blot dry very carefully. Stain may be adjusted by selective media is TCBS on which only vibrio can grow.
further dilution or in the timing of either before or General media is one on which any aerobic bacteria can
after dilution in the above procedure. grow. For e.g., TSA or nutrient agar. In a general purpose
media, bacterial cultures produce colonies. The form,
Interpretation:
elevation and margin of the colonies can also be used
Red blood cells: red to pink to identify the bacteria (Fig.1)

Fig.1. Bacterial colony characteristics

279
 Histopathology
N.K. Sanil
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Histology is the microscopic examination and Compared to that of homeotherms the rate of autolysis
study of biological cells/ tissues while the term of fish tissues is rapid and hence, must be fixed
Histopathology refers to the study of diseased cells/ immediately to prevent any degenerative changes.
tissues. It helps to understand the fine changes or For satisfactory histological preparations, only freshly
abnormalities in cells/tissues caused by pathogens/ killed or moribund fish should be considered.
diseases. Histopathology involves a series of
procedures starting from fixation. Types of fixation
The primary objective of fixation is to preserve the Various method are employed to achieve fixation,
morphology of the tissues in a condition as close as the most common processes are chemical or physical
possible to that existing during life and hence proper means. Physical methods include heating, micro-waving
fixation is fundamental to all satisfactory histological and cryo-preservation (freeze drying). However, heat
preparations. Unless fixation is carried out in a proper fixation is mainly used to fix smears of micro organisms
way or if it is delayed, a tissue can be irreversibly and microwave fixation, which is another form of heat
damaged and any effort to rectify in the later stages fixation, is widely practiced. Cryo-preservation is not
will not help. The moment any tissue is deprived of used routinely for diagnostic tissue preparations.
its blood supply, autolysis sets in, leading to tissue
digestion by intracellular enzymes released following Microwave fixation can be used for tissue fixation and
cell death, and subsequent bacterial decomposition or heat generated during the process is responsible for
putrefaction. Fixation aims to arrest these degenerative tissue fixation. Apart from increasing diffusion rates
processes with minimal damage to the tissue heat will increase molecular kinetics and speed up
architecture. Loss and diffusion of soluble substances chemical reactions. Fresh tissue, in saline or other
in the cells/tissues can be avoided by precipitation or isotonic medium, can be irradiated to produce
coagulation or by cross-linking them to other insoluble primary fixation or alternatively, specimens can be
structural components. Fixation also help in protecting initially placed in buffered formalin or other fixative
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the tissues from the harmful effects of tissue processing and later microwaved to assist the fixative action
including dehydration and infiltration, at the same time of the fixing agent (microwave-assisted fixation).
retaining the reactivity to stains and other reagents. Microwave-assisted fixation is more commonly used
Normally the process of fixation will produce a number than primary microwave fixation. Microwaves have
of changes to the tissues including shrinkage, swelling little effect on tissue beyond a depth of 4 mm and
and hardening of various components. Fixation in hence for primary microwave fixation tissue slices
10% buffered formalin causes slight swelling initially, should not exceed 3 mm in thickness and they
but subsequently during processing this end up to should immediately be sliced to 2 mm and placed in
about 20%–30% shrinkage in volume. The choice of 70% ethanol after microwaving. For best results in
fixative depends on the precise requirement, because microwave assisted fixation 2 mm thick slices should
the fixative used can influence the degree to which be prepared from tissues initially fixed in formalin
individual elements will stain with various histochemical prior to microwave treatment.
and immuno-histochemical reagents.

280
Chemical fixation is carried out by immersing of these agents are highly reactive and bind to a
the specimen in the fixative (immersion fixation) variety of chemical groups in tissues, often affecting/
or, by perfusing the vascular system with fixative modifying the charge at the site of attachment, thereby
(perfusion fixation). In some cases, fixatives such as affecting the subsequent staining characteristics of
paraformaldehyde and osmium tetroxide can also be the particular protein as well as altering its molecular
used to vapour-fix freeze-dried tissues. Usually fixative conformation and solubility. For example, tissue fixed
solutions may contain a single fixative agent dissolved with formaldehyde stains poorly with eosin because
in a solvent such as water or alcohol or a buffer solution formaldehyde reacts extensively with amino groups
to stabilize pH. There is no ideal fixative and hence, to form methylene bridges and thus these groups
a mix of fixative solutions containing different fixing are no longer available to bind negatively charged
agents in combination is used, so that the deficiency dye molecules such as those of eosin. The ability to
in one can be compensated by the addition of others. form cross-links can vary considerably. For example
The process of fixation is in fact “a complex series of glutaraldehyde is more efficient in forming cross-links
chemical events” the various tissue elements (peptides and hence preferred for electron microscopy. It also
and proteins, lipids and phospholipids (membranes), explains why glutaraldehyde-fixed tissues stain poorly
carbohydrates and carbohydrate complexes, various with conventional dye-staining methods.
types of RNA and DNA) will chemically react with
the fixative, get ‘stabilised’ by cross-linking, while Factors influencing
other unfixed components may get trapped within
the matrix formed by various fixed elements.
chemical fixation
Various factors are known to influence the rate and
Generally fixatives can be classified into “coagulant” effectiveness of tissue fixation.
or “non-coagulant” based on their effect on soluble

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

proteins in solution. Coagulant fixatives produce a Temperature: Increase in temperature will increase
permeable meshwork of protein strands whereas non- the rate of diffusion of the fixative into the tissue and
coagulant fixatives which are additive in nature, form enhance the rate of chemical reaction between the
extensive cross-links producing a less permeable gel. fixative and tissue elements. At the same time, this can
The two major mechanisms underlying the fixation also adversely affect fixation by increasing the rate of
of proteins and protein complexes are denaturation tissue degeneration in unfixed areas of the specimen.
and addition and cross-link formation. Fixation for light microscopy is usually carried out at
room temperature. Microwave fixation may involve
In denaturation, dehydrating agents like alcohols or the use of higher temperatures, up to 65ºC, but for
acetone remove and replace free water in cells/tissues relatively short periods.
thereby altering the tertiary structure of proteins by
destabilizing hydrophobic bonding. Hydrophobic Time:  During fixation the fixative has to penetrate
areas, on the inside of protein molecules, are relieved the specimen by diffusion and react with the tissues.
from the repulsion of water and occupy a greater Both diffusion time and reaction time depend on
area. In hydrophilic areas, hydrogen bonds which the particular reagent used and the optimum time
bind water molecules are also destabilized. These will vary depending on the fixative used. Incomplete
changes in the conformation of protein molecules fixation can lead to poor quality sections showing
leads to changes in their solubility, thereby rendering tissue distortion and poor quality staining because
water soluble proteins insoluble, a change that can poorly fixed tissue does not process well.
be reversed if returned to an aqueous environment.
Penetration rate: The penetration rate of a fixing
In the case of Addition and cross-link formation, the agent depends on its diffusion characteristics and
non-coagulant fixing agents chemically react with varies from agent to agent. It can be expressed as
proteins and other cell/tissue components and forms d = K√t, where d is the depth of penetration, K is
inter-molecular and intra-molecular cross-links. Most the coefficient of diffusion (specific for each fixative),

281
 and t is the time.  In simple terms, the coefficient of Formaldehyde: Formaldehyde (CH2O) is the gaseous
diffusion (K) is the distance traversed by the fixative in aldehyde, dissolved in water to saturation (at 37% –
millimeters in one hour. For 10% formalin K = 0.78. 40% w/v) and this generally referred to as “formalin”.
This means that formalin should not be expected to For fixation, one part formalin is usually diluted
penetrate more than 1 mm in an hour. with nine parts of water or buffer. This produces
a 10% formalin solution which contains about 4%
Specimen size:  Good fixation depends on the formaldehyde w/v, an optimal concentration for
dimensions of the specimen/tissue. A specimen fixation. To avoid the formation of paraformaldehyde
should not be more than 4 mm thick, the most ideal (highly polymerised form of formaldehyde which gets
thickness for fixation & processing is 3 mm. deposited as a white precipitate in concentrated
formaldehyde solutions), small amount of methanol
Volume ratio: It is important to have an excess (up to 15%) is added to proprietary solutions.
volume of fixative in relation to the total volume of Unbuffered formalin will slowly oxidize to formic
tissue because with additive fixatives the effective acid, lowering the pH and react with hemoglobin
concentration of reagent is depleted as fixation in tissues producing acid formaldehyde hematin
proceeds and in a small total volume this could have a brown-black granular artefact pigment which
an effect on fixation quality. The minimum advised is deposited in blood-rich tissues. This often gets
ratio of fixative to tissue is 20:1. confused with micro organisms or other pathological
pigments. For this reason 10% formalin solutions are
pH: Though the pH of a fixative may not affect the usually buffered to pH 6.8 – 7.2.
quality of preservation in light microscopy as most of
the formulations have low pH, especially with fixatives Formaldehyde reacts with the side-chains of
containing acetic or picric acids. However pH can proteins to form reactive hydroxy-methyl groups.
be important in the case of formaldehyde solutions, It can penetrate nuclear proteins and nucleic
where breakdown of formaldehyde to form formic acids stabilizing the nucleic acid-protein shell and
acid produces an acidic solution which in turn reacts modifying the nucleotides by reacting with free
with hemoglobin to produces an artefact pigment amino groups. Formaldehyde can also react with
(acid formaldehyde hematin). The most popular some groups in unsaturated lipids particularly if
formaldehyde solution in use today is therefore calcium ions are present, but does not react with
buffered to pH 6.8 – 7.2 for this reason. carbohydrates.   Though washing can reverse some
of these reactions, cross-linkages remain.  The ability
Osmolality: The osmotic effects exerted by the to preserve the peptides of cellular proteins has made
fixative are more visible at the ultrastructural level, formaldehyde the best general purpose fixative.
because the phospholipid membranes get easily
damaged by excessively hypotonic/hypertonic Glutaraldehyde: or glutaric dialdehyde (CHO(CH2)3CHO)
Central Marine Fisheries Research Institute

solutions. Generally the osmolality of the vehicle is regarded as a bifunctional aldehyde, possessing
(buffer) is more and it is always better to adjust it to aldehyde groups at either end of the molecule and
that of tissue fluid (eg. formalin in isotonic saline). To react with the same chemical groups as formaldehyde
avoid the damage to tissues caused by non-isotonic does. Glutaraldehyde fixed tissues will be more
fluids such as water before actual fixation starts, it extensively cross-linked (irreversibly), though this may
is better to keep them moist with gauze soaked in adversely affect certain immunohistochemical staining,
isotonic saline for a short time, but never immerse in it provides excellent ultrastructural preservation
saline for extended periods. and hence considered as the best primary fixative
for electron microscopy. The rate of penetration is
Fixing agents rather slow and tissue thickness should be less than
1mm for satisfactory fixation. For electron microscopy
There are a number of reagents that are regularly glutaraldehyde is available in sealed ampoules and can
used to fix tissues. be added to a suitable buffer at pH 7.2 – 7.4 (usually

282
cacodylate, phosphate or maleate) to produce a 3% Acetone: Acetone (CH3COCH3) though resemble
glutaraldehyde concentration for use. alcohol in its activity, is generally used as a fixative and
dehydrant for tissue processing, particularly of small
Other commonly used fixatives include specimens. Generally not used in tissue processors
because it may affect seals and other components
Mercuric chloride: Mercuric chloride (HgCl2) is a of the equipment.
powerful protein coagulant, react with amines, amides,
amino acids and sulphydryl groups, produceing cross- Acetic acid: Acetic acid (CH3COOH) is coagulant that
links. It reacts with phosphate residues of nucleic acids reacts with nucleic acids but generally does not fix
and effectively fixes nucleoproteins. Disadvantages proteins. Hence is incorporated in various compound
include corrosive nature, toxicity and formation of fixatives to preserve nucleic acids and to counter
greenish-brown artefact in tissues. Treatment with the shrinkage caused by other ingredients such as
Lugol’s iodine during processing or sections prior ethanol. Though penetration is rapid, it can lyse red
to staining, followed by treatment with sodium blood cells.
thiosulphate is widely used to clear this.
Histopathology procedures for finfishes: The most
Zinc salts: Zinc sulphate (ZnSO4) and zinc chloride widely used fixatives are formaldehyde and Bouin’s
(ZnCl2) are used to replace mercuric chloride in many fixative. Small fish/fry can be directly dropped into
formulated fixatives. Zinc enhances fixation and the fixative while in larger samples, incisions should
staining, particularly of nuclei, as in the case with made into the abdomen so that the fixative reaches
mercuric chloride, are less toxic than mercury salts. the internal organs/viscera. It is always advisable to
make incisions in the tissue (4 mm size) and transfer
Picric acid: Picric acid or trinitro phenol into a vial of fixative (20 times the volume of tissue)

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

(C6H2(NO2)3OH) Picric acid is a coagulant fixative
which changes the charges on the ionisable side Histopathology procedures for Bivalves: Bivalves
chains of proteins and disrupts electrostatic and less than 6 cm in length (shucked) can be fixed whole,
hydrogen bonds and is always used in combination by dropping into preservative. Animals must be
with other agents. It imparts a yellow colour to shucked cleanly from the shell by severing adductor
tissues during fixation and has to be washed from muscles prior to fixation. For perfect fixation, larger
tissues with 70% ethanol before processing and bivalves require 3 incisions (anterior, mid, posterior)
residues can alter the staining characteristics of made across the surface of the animal about mid-way
the tissues. through the tissues. Never cut completely through the
animal so that tissues do not get mixed. Common
Potassium dichromate: Potassium dichromate fixatives used for bivalves are Helly’s fixative, Bouin’s
(K2Cr2O7) acts as a coagulant at pH < 3.4 – 3.8, and fixative or Davidson’s fixative. Small bivalves can
is a component of several compound fixatives. either be embedded whole or cut longitudinally on
the median axis and both tissue halves placed face
Ethanol and methanol: Ethanol (CH3CH2OH) and down within a cassette.
methanol (CH3OH) are coagulants that denature
proteins, they replace water in the tissues by Bivalve Larvae: Are fixed in a test tube of Helly’s
disrupting hydrophobic and hydrogen bonding fixative and centrifuged @ 1,500 rpm for 10 minutes.
thereby altering their tertiary structure and their Discard supernatant and embed the larvae in an
solubility in water. Fixation starts at a concentration agar plug. Remove the plug from the test tube for
of 50 – 60% for ethanol and >80% for methanol. dehydration and embedding in wax in the usual
Methanol is commonly used to fix blood films and manner (trim if necessary).
95% ethanol is used as a fixative for cytology smears
but combinations of these with other agents are Histology of Shrimp: The chitinous exoskeleton of
widely used. shrimp prevents adequate penetration of fixatives.

283
 Hence, the fixative must be injected into the internal • Allow sufficient time for fixation.
areas of each animal before dropping the whole shrimp
into the fixative. The fixative is injected using a 10-ml Right choice of a correctly
syringe with an appropriately sized needle. Subsequently,
formulated fixative
the cuticle is slit from the last (6th) abdominal
segment to the base of the rostrum. The incision in the • Always use quality reagents.
cephalothoracic region should be lateral to the dorsal • Unstable fixatives should be made up from stock
midline and that in the abdominal region should be solutions immediately before use.
mid-lateral. This will help to break the cuticle and allow • Never reuse fixatives
sufficient fixative penetration. After injection and body • Avoid metal lids and containers.
incisions, the animal can be dropped whole into the
fixative and after 48 hrs the animal is transferred to The fixed tissues are washed, dehydrated in ethanol
70% ethyl alcohol. Commonly used fixatives for shellfish: series, cleared, embedded in paraffin and sectioned
Davidson’s fixative, Buffered formalin & Helly’s Fixative. at 5-7 μm thickness. The sections are then stained
with Haematoxylin & Eosin and observed under
Getting best results the microscope.

in fixation
Staining
The key to good fixation are:
Hematoxylin and Eosin (H&E) is the most widely
Fresh tissue used stain in routine histology and histopathology
studies.  Hematoxylin is a natural dye obtained from
• Fix as soon as possible, the moment cells are the heartwood of Haematoxylon campechianum, a
deprived of blood supply, degeneration starts. tree commonly found in Central America. In its pure
• If unable to fix immediately better refrigerate, but form haematoxylin is a colourless or slightly beige
never freeze the tissues.  powder which cannot stain and the active staining
• Do not allow specimens to dry, desiccation/shrinkage agent is an oxidation product, haematein (usually at
of surfaces may result in permanent damage. an acid pH). Haematein can be produced naturally
• Distortion/other mechanical damage will result in through exposure to air and sunlight or UV light
altered morphology, making interpretation difficult (a process referred to as ‘ripening’) but is a time
• Precise labelling is absolutely essential. consuming process. The use of a chemical oxidants
hastens the process and various agents like potassium
Proper penetration of fixative permanganate, iodine, sodium iodate, sodium
periodate, potassium periodate, hydrogen peroxide
• Always place specimens in containers with fixative or mercuric oxide etc are used. Mayer’s and Ehrlich’s
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so that good penetration from all sides is assured. haematoxylin use sodium iodate, whereas Harris’
Cavities/hollow organs should be slit open/exposed haematoxylin relies upon both vigorous boiling and
to allow access of fixative. addition of mercuric oxide to generate haematein.
• Fixation by perfusion of vascular system of whole The advantage of chemical oxidation is–a working
organs or small experimental animals may produce dye can be produced immediately, but care should
uniform fixation. be taken to avoid over-oxidizing the haematoxylin.
• Thickness of specimen/tissue should not
exceed 4mm Though the actual staining is done by haematein, a
• Gentle agitation/swirling of the specimen mordant for haematoxylin is added to stain tissues
during its first few minutes in fixative will help effectively. Various metal salts are used as mordants
better penetration. with haematoxylin, but those with aluminium, iron
• Volume of fixative should be in excess (20:1 or tungsten are better. A combination of mordant
at least). and dye is known as a ‘lake’ and the haematoxylin-

284
mordant lakes are often positively charged, behaving • 95% ethanol–2 minutes
as cationic dyes at low pH. Haematoxylin is the most • 50% ethanol–2 minutes
widely used natural dye in histology/histopathology • Water–2 minutes
and among the various hematoxylin preparations used
in histology, Gill’s hematoxylin, Harris’s hematoxylin Staining
and Mayer’s hematoxylin are the most popular. • Immerse slides in Hematoxylin solution for 10-
Hematoxylin acts like a basic dye with a purplish 20 minutes.
blue colour. It stains acidic, or basophilic, structure • Rinse 2 minutes in water or Scott’s
including the cell nucleus (which contains DNA and Tapwater substitute.
nucleoprotein), and organelles that contain RNA such • Differentiate with 2-5 dips in 70% ethanol
as ribosomes and the rough endoplasmic reticulum. containing 0.1% HCl.
In a clinical histology laboratory, all specimens are • Immerse for 2 minutes in Scott’s Tapwater substitute.
initially stained with H&E and special or advanced • Stain in 1% aqueous Eosin with 0.1 acetic acid
staining is carried out if required. for 10 minutes.

Haematoxylins can be used as either progressive or Dehydrate, clear and mount

regressive stains. Solutions such as Mayer’s, and Gill’s,
will not overstain and can be used progressively. a) Immerse slides in the following sequence of baths:
Progressive reactions provide a mechanism for
highly selective staining but often require extended • 50% ethanol–2 minutes
reaction times to maximize dye-tissue binding • 95% ethanol–2 minutes
and produce sections of high contrast. On the • 100% ethanol–2 minutes
other hand, regressive techniques are designed • Xylene I–10 minutes

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

to overstain and the solution will stain a range of • Xylene II–10 minutes
tissue components including nuclei. The overstained • b) Mount coverslip with DPX
sections are differentiated carefully using a weak
acid solution (normally prepared in 70% ethanol to Results
improve control) to achieve acceptable level of nuclear
staining. Staining with Ehrlich’s & Harris haematoxylin • Nuclei–blue. 
is done using regressive staining. • Other tissue components–shades of red and pink.

Eosin is an acidic dye that is typically reddish or pink H&E stains almost all the cell structures including
and which requires an acidic environment to work. In cytoplasm, nucleus and organelles as well as extra-
solution the dye molecule is negatively charged and cellular components and can reveal changes in
stains basic, or acidophilic, structures which include the general organization of the tissues/cells and
the cytoplasm, cell walls, and extracellular fibres. abnormalities or specific indicators/clues of diseases/
pathogens thereby aiding diagnosis. Thus H&E
Staining procedure: staining provides a clear basic picture of the tissue
and hence forms a critical part of histopathology.
Dewax and rehydrate slides Various other advanced staining procedures/
techniques are widely employed in histopathology
a) Immerse slides in the following sequence of baths: to stain particular tissues/structures depending on
the specific requirement. In clinical histology, all
• Xylene I–10 minutes specimens are initially stained with H&E and special
• Xylene II–10 minutes or advanced staining is carried out only if additional
• Xylene III–10 minutes information/details are required to get more details
• 100% ethanol–2 minutes to clear any ambiguities/doubts.

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 Electron Microscopy
N.K. Sanil
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Microscopy is the science of visualizing objects that Histology uses light microscopy and is still an
cannot be seen with the naked eye. Optical or light invaluable tool in disease diagnosis, the advantage
microscopy involves passing visible light through being – its less sophisticated nature and simplicity.
a series of lenses to allow a magnified view of However, in many cases it cannot provide enough
the sample. Light microscopy has advanced much evidence to make a confirmatory diagnosis. In the
since its discovery and has attained the limit of case of viral infections, one can observe CPEs, lesions
resolution imposed by the nature of light in the or inclusions, which are only suggestive of a specific
past century itself. The major limitation of light viral infection through histopathology. Moreover, due
microscopy is that, diffraction limits its resolution to the limited magnification and resolution, ultra
to approximately 0.2 micrometres, providing a structural/sub cellular changes and minute pathogens/
useful magnification of ~1000x which is good for stages cannot be observed. Whereas TEM can clearly
obtaining information up to cellular level. When it provide information on the morphology of pathogens,
comes to the study of diseases, understanding the sub cellular changes / particles / structures etc.
pathogen and the pathogenesis at cellular levels are
very important and for this far higher resolutions Sero-diagnostic methods play an important role in
are necessary. Electron Microscopes are instruments disease diagnosis, especially in field conditions and
that use a beam of highly energetic electrons to serology still remains the mainstay of viral diagnosis.
examine objects on a very fine scale and function The tests are generally based on specific antibodies
exactly like their optical counterparts. With its very (immunoprobes) and can detect sub clinical / latent
high resolving and magnifying powers, electron / carrier states of infection. However, the draw backs
microscopy allows visualizing structures within of serological tests are (a) highly variable sensitivity
individual cells and can identify cell types/organelles/ & specificity (b) many viruses often produce clinical
pathogens which are far beyond the reach of light disease before the appearance of antibodies (c) less
microscopy. Modern electron microscopes are useful in the case of latent viruses (d) antigenic cross-
capable of providing magnifications up to 1200000 reactivity between related viruses may lead to false
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X with a resolving power of less than 0.1 nm. positive results and (e) less effective in invertebrates
Transmission electron microscopy (TEM) can reveal which does not produce antibodies.
ultra structural details at cellular levels, whereas
Scanning electron microscopy (SEM) can show the Microbiological methods are widely used for the
morphology of minute structures/organisms in its diagnosis of bacterial infections and involve culture,
three-dimensional state. Combining the TEM and isolation and identification of the pathogens. But
SEM, it has become possible to study and classify the procedure is tedious and time consuming and
the viruses and virus like organisms. may even take weeks in some cases. In the case of
non-culturable organisms/pathogens, microbiological
Commonly employed methods for disease diagnosis methods may fail to provide conclusive results.
include histology, serology, microbiology, molecular
diagnostics and electron microscopy and each method Molecular biology tools involve the detection of
has its own advantages and disadvantages. genetic material of pathogens using molecular probes

286
and is presently considered as the most important tool microscopy provides primary information on the
in disease diagnosis. Advantages of molecular tools target tissues, electron microscopy can be employed
include (a) extremely high sensitivity (b) easy to set to visualize the pathogens and study its morphology.
up and (c) fast turnaround time. Disadvantages are Electron microscopy can also provide information
(a) expensive (b) extremely liable to contamination (c) on the ultrastructural modifications/changes at sub-
high degree of operator skill required (d) quantitative cellular levels caused by the pathogen. So compared
assay difficult and (e) difficulty in interpreting positive to other diagnostic methods, E M benefits from an
results, especially with latent viruses and (f) though “open view”, it also reveals double infections and the
they are more sensitive, are only capable of identifying presence of agents that might not otherwise have
the presence of previously identified agents and thus been considered. Finally, since the test entails the
fail to identify new/emerging pathogens. visualization of the virus itself, rather than a color
change or agglutination reaction, false positive tests
Electron microscopy can be an important adjunct resulting from cross-reactions of reagents with similar
to conventional culture and serologic techniques in materials are not likely. Hence electron microscope
diagnosing viral diseases. Though detection of viruses can be considered as the ultimate tool in identifying
by E M requires relatively large numbers of virions, and the etiology of emerging diseases.
cannot provide information on the specific serotypes
within a virus family, it has the distinct advantage of Two types of preparations are primarily used for
being simple and rapid. Some viruses do not grow in routine EM virus identification, negative staining
tissue culture or grow only after special manipulation, and thin sectioning, although specialized research
and may not survive if transportation conditions to techniques such as specific antibody aggregation or
the lab are not optimal. Naturally, culturing would labeling with electron-dense tags, in situ labeling,
miss these agents. Additionally, a wide variety of cryomicroscopy, and high-voltage microscopy have

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

agents can be visualized by E M; because specific also been used to classify viruses and describe virus-
reagents such as antibodies, antigens, or nucleic host relationships. With the simple negative staining
acid and protein probes are not required, one is not preparation available, E M allows the rapid and direct
limited to the availability of these reagents, and prior detection of an etiological agent on a sample from a
knowledge of the virus identity for reagent selection is patient, or from diagnostic cell cultures.
not required. Diagnostic electron microscopy has two
advantages over enzyme-linked immunosorbent assay Negative staining of liquid samples is fairly rapid, and
and nucleic acid amplification tests. After a simple can provide an answer within a few minutes to a
and fast negative stain preparation, the undirected, couple of hours. It enables the examiner to view cell
“open view” of electron microscopy allows rapid particles and organelles in isolation. The isolated cell/
morphologic identification and differential diagnosis particle is placed in a “puddle” of staining material,
of different agents contained in the specimen. usually uranyl acetate or phosphotungstic acid, and
is then supported on a thin, plastic/formvar film. The
The biggest advantage of electron microscopy lies stain molecules deposit into surface crevices in the
in the fact that it provides direct visual evidence specimen during the drying process and typically
of various pathogens/biological processes, while produce a “ghost” image in which the specimen
most of the other techniques are indirect and in appears light against a dark background.
some instances non-specific. Electron microscopic
diagnosis is uniquely suited for rapid identification Sensitivity and specificity of E M may be further
of infectious agents. A specimen can be ready enhanced by immuno electron microscopy, which
for examination and an experienced virologist or includes classical immune electron microscopy and
technologist can identify, by electron microscopy, a solid phase immuno electron microscopy. In classical
viral pathogen morphologically within 10 minutes immuno electron microscopy, the sample is treated
of arrival in the electron microscopy laboratory. with specific anti-sera before being subjected to
Once the histopathological observations using light electron microscopy. The viral particles present will

287
 be agglutinated and thus congregate together by the tissues in the case of small animals. The desired size of
antibody, making them easily visible. In solid phase the tissue to achieve proper fixation is about 1 mm.
immuno electron microscopy the grid is coated with Very small animals and larvae less than 2 mm size are
specific anti-sera. The virus particles present in the fixed whole in ice-cold fixative in live condition. The
sample will be absorbed onto the grid by the antibody sample vial should be labelled properly.
thus enhancing the visibility under the microscope.
Fixatives: Fixatives help to preserve the structures
Advantages: The most important among the in the living cell and prevent changes induced by
benefits offered by the electron microscope is autolysis. There is no single ideal fixative and so a
undoubtedly the very high resolution. Since timely combination of fixatives is preferred depending
and accurate diagnosis forms the first step in the on the type and nature of the tissues. In electron
health management of farmed fishes and shellfishes, microscopy, 2 to 4 % Glutaraldehyde is used as the
the right diagnosis defines the very success of disease primary fixative which is excellent in fixing nucleic
control. Though E M has an important role in the acids, nuclear proteins and carbohydrates but not
diagnosis of viral infections, it is equally useful in lipids. Poor contrast and slow penetration are the
the diagnosis and understanding the pathogens as limiting factors of glutaraldehyde fixative. Osmium
well as the pathological changes caused by various tetroxide is used for secondary fixation. It acts as
other pathogenic organisms. As a confirmatory both fixative as well as stain, fixes nucleic acids,
diagnostic method for many of the existing and carbohydrates and lipids and provides contrast and
emerging diseases, especially of viral origin, electron fast penetration. The combination of glutaraldehyde
microscopy still remains an indispensable tool in the and Osmium tetroxide as primary and secondary
field of disease investigation and control. To exploit fixatives, gives the desired results in contrast and
the full potential of diagnostic electron microscopy, resolution. Fixatives are prepared in a suitable buffer
it should be quality controlled, applied as a frontline for two reasons, to maintain the pH (7.2 to 7.4)
method, and be coordinated and run in parallel with and to maintain the osmolality, in order to minimize
other diagnostic techniques. the swelling or shrinkage of the tissues which may
otherwise lead to artifacts. The most commonly used
Disadvantages: However, the disadvantages of E buffer is Sodium Cacodylate buffer, while phosphate
M in the diagnosis of infections are (a) detection buffer is also used.
of viruses by electron microscopy requires relatively
large numbers of virus particles (b) possibility of false Primary fixation: Tissues are fixed in 2 to 4%
negatives, if concentration is very low (c) provides no Glutaraldehyde in 0.1 M Cacodylate buffer (in the
information regarding specific serotypes within a virus case of marine species, 3 to 5% NaCl or sucrose can
family and (d) factors like high cost of operation and be added to the fixative). For proper penetration
infrastructure, need for skilled technical personnel, of the fixative, the tissues should not exceed 1 mm
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laborious and time-consuming procedures, expertise in size. Fixation is carried out for 4 to 6 hrs (varies
needed for interpretation etc. restricts the use of depending on the nature of the tissues), at 4ºC. After
electron microscopy as a routine diagnostic tool. fixation, the fixative is drained and tissues washed
thrice (15 mts each) with buffer. In case of larger
Specimen processing tissues, further trimming is done if required and
washed with fresh buffer.
Sampling: Soon after the death of the organism,
post mortem changes sets in, making the tissue Secondary Fixation or Post fixation: For secondary
unsuitable for ultra-structure studies. Hence for or post fixation, the washed tissues are transferred
electron microscopy, always live animals are preferred. to 1% Osmium tetroxide (OsO4 ) in 0.1 M cacodylate
The animals are sacrificed, the desired tissues/samples buffer, kept for 1- 2 hrs at 4ºC (above 4ºC, OsO4
dissected out and immediately placed in cold fixative disintegrates). OsO4 treatment turns the tissues black.
or perfusion carried out to ensure uniform fixation of OsO4 is drained and tissues washed two to three times

288
with buffer, for 15 min each, or until free of a black objective requires an evaluation of the methods,
precipitate formed from excess OsO4. Samples can subjected to a careful examination of pertinent
be stored in buffer under refrigeration until further literature. There is no schedule that will work for all
processing is desired. (Since OsO4 is highly toxic, care tissues and conditions.
must be taken while handling. Always use gloves and
carry out all operations under a hood). Trimming: The resin blocks are trimmed to remove
the unwanted areas using a glass knife fitted to
Dehydration: Dehydration is done through graded an Ultramicrotome.
alcohol or acetone series to remove the water from
the tissues. Sectioning and staining: To achieve high resolution
for electron microscopy, the sections should be
Dehydration can be done as follows very thin (60 nm) and are prepared using an
ultramicrotome. The resin blocks are trimmed using
30% Acetone two changes, 15 mts each at 4ºC a glass knife. Standard procedures are followed
50% Acetone two changes, 15 mts each at 4ºC
for obtaining semi-thin and ultra-thin sections for
70% Acetone two changes, 15min each at 4ºC, (can be
stored in 70% acetone indefinitely) light and electron microscopy respectively. Semi-
80% Acetone two changes, 15 mts each at 4ºC thin sections are first taken, stained with methylene
90% Acetone two changes, 15 mts each at 4ºC blue and observed under a light microscope for
95% Acetone two changes, 15min each, 4ºC determining the area for ultra-thin sectioning. The
100% Acetone two changes, 15 min each, 4ºC
blocks are again trimmed and ultra-thin sections
100% Acetone two changes, 30 mts each at room
temperature taken. These sections are floated on distilled water,
Propylene oxide two changes of 15 min each at room stretched to remove the wrinkles and collected over
temperature the matt/dull surface of the copper/nickel grid.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

* Analar grade acetone is used to ensure proper dehydration

Staining: Double staining with Uranyl acetate

Infiltration and Embedding: Fixed and dehydrated and Lead citrate is employed for routine electron
tissues are infiltrated with liquid plastic resins and microscopy studies. The sections are first stained with
then cast into blocks. The purpose of embedding is Uranyl acetate. A drop of Uranyl acetate (saturated
to allow future ultra thin sectioning of the material. solution in 50% ethanol) is taken on a clean glass
Commercially available Plastic resins like Epon or Spurr slide and the grid with the section side down is kept
are used for embedding. The media is mixed as per the on to the stain drop and is covered with any opaque
instructions, under a fume hood. Prepare fresh media object to ensure darkness to carry out the staining
2 to 3 hrs. prior to use, as it will absorb water vapor effectively. After 10–15 mts, the grid is taken out and
from surroundings and the components will begin to washed 3–4 times in double distilled water (ensuring
polymerize. Mixture of embedding medium (Spurr’s that the sections are not washed away) and dried
medium) and acetone is prepared in various grades with a filter paper. The grids are then stained with
(mix. A – medium and acetone in the ratio 1: 3, mix. Lead citrate for 1–4 mts., washed well and dried.
B – medium and acetone in the ratio 2 : 2 and mix. In the case of particulate specimen, the specimen is
C – medium and acetone in the ratio 3 : 1) and the taken on formvar-coated grids, subjected to negative
tissue kept in each for 1 to 2 hrs each or as specified staining using 1-3% Phosphotungstic acid and dried.
(period varies with the medium used) for infiltration.
For embedding, medium is prepared as instructed, Observation and photography: The grid carrying
poured into readymade moulds made of plastic or the stained section is loaded into the electron
silicon rubber and infiltrated tissues transferred to it, microscope, the image observed and recorded on
taking care not to trap any air bubbles. The moulds photographic plates/film or digitally. In order to study
are then kept in an incubator at 70ºC for 12 to 24 hrs. and interpret EM results one has to have a thorough
knowledge about the ultrastructure of the normal
Each tissue with reference to the experimental cells and the pathogen.

289
 Scanning Electron Microscopy: The scanning SEM samples. The dried specimen is then coated with
electron microscope, like the TEM consists of an a thin conductive metal film (Gold, Palladium etc.)
electron optical column, a vacuum system and using an ion coater to prevent charging artifacts
electronics and works under the same principle and to stabilize the specimen mechanically. Variable
as that of the TEM. The electron gun produces pressure SEM’s can operate without high vacuum thus
an extremely fine beam of electrons, which are avoiding the time consuming specimen preparation
focused into a fine spot less than 4 nm on the techniques as well as reduce specimen damage
specimen and scanned in a rectangular raster over caused during coating.
the specimen. The secondary electrons produced
by the interaction of the electron beam with the Benefits: The most important among the benefits
specimen surface as well as the backscattered offered by the electron microscope is undoubtedly
primary electrons (depending upon the topography) the very high resolution (as low as 0.1 nm) and
are detected using a suitable sensor/detector. The magnification (up to 12, 00,000 X) in TEM and 0.4
signals from the detector are electronically amplified nm resolution with a magnification of up to 800,000X
to modulate the brightness of a Cathode Ray Tube in SEM. Since timely and accurate diagnosis forms the
(CRT) so as to produce an image, which can be first step in the health management of farmed fishes
recorded photographically. and shellfishes, the right diagnosis defines the very
success of disease control. Factors like high cost of
Specimen preparation for S E M: The specimen operation and infrastructure, need for skilled technical
is first fixed with glutaraldehyde as in the case of personnel, laborious and time-consuming procedures,
TEM and washed well in buffer. Post fixation with thorough knowledge needed for interpretation etc.
OsO4 is optional. The specimen is subjected to restricts the use of electron microscopy as a routine
dehydration using ascending grades of acetone as diagnostic tool, but as a confirmatory diagnostic
in TEM processing. The dehydrated specimen for SEM method for many of the existing and emerging
has to be dried without causing any shrinkage. Except diseases, especially of viral origin, electron microscopy
in the case of fine particulate specimen, critical point still remains an indispensable tool in the field of
drying or freeze drying is usually preferred for drying disease investigation and control.
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290
Fish Immunological Techniques
K. J. Reshma
Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction • Does not require extensive sample preparation

The vertebrate immune system is considered to be • Do not require expensive instrumentation
the strategically advanced protective system which • Mostly based on simple photo-fluro-
is capable of responding to many of the infective luminometric detection
challenges that arise in the body and its environment. • Measurements may be either qualitative/
Such immune response from the immune system quantitative
follows two phases. The first and foremost phase is the
recognition of the pathogen or other foreign material Methods of Analysis:
and second phase involves the effort of the body
to eliminate the disturbance. An immune response All immunochemical methods are based on a highly
from the body falls into two categories; the innate specific and sensitive reaction between an antigen
immunity and the adaptive or acquired immunity. The and an antibody. Structurally, antibodies are often

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

innate immunity forms the first line of defence against visualized as Y-shaped molecules, each containing
any infectious agent. The adaptive immunity is the 4 polypeptides – 2 identical polypeptide units called
specific counterpart which becomes more and more heavy chains and another 2 called light chains. It
effective over several days after initial activation. The has a domain called Fab, the site where it binds to
most important cell types in the acquired immunity
are B and T lymphocytes. The B lymphocytes produce
antibodies on antigenic stimulation. An antigen is any
foreign substance capable of producing an immune
response leading to the production of antibodies
which is specific to particular antigen. Immunologists
developed a number of techniques based on the
selective reversible and covalent binding of antibody
to specific antigen. Such immunological techniques
forms integral to many clinical, pharmaceutical
and basic scientific investigations. The widely used
immunological techniques, their principles and
application are described in this chapter.

Characteristics of Uroteuthis (Photololigo) duvauceli

an antigen.
immunological techniques:
• Simple, rapid and robust The region of an antigen binding to an antibody
• Highly sensitive is called an epitope. The measure of the strength
• Easily automated – Applicable to regular of the binding is called affinity, and it is usually
clinical laboratories expressed in terms of the concentration of an

291
 antibody-antigen complex measured at equilibrium. 1. Monoclonal antibody – products of single clone of
It is measured by quantitative precipitin curve (basis plasma cells by B lymphocytes; mostly prepared in
for many immunochemical techniques) proposed by laboratory. They are directed against single epitope
Heidelberger and Kendall in 1935. – identical copies with same structure and antigen
specificity. They have excellent specificity but poor
Quantitative precipitin curve: It describes the ability to precipitate antigen.
relationship between the antigen concentration and 2. Polyclonal antibody – they are conventional, i.e.,
the amount of precipitate for a constant quantity of produced by immunization of animals with antigen.
Thus antibody consists of mixture of monoclonal
antibodies having specificity for complex antigens.
Sometimes monoclonal is called as “monovalent”
and polyclonal as “polyvalent” which indicates the
antigen specificity.

Applications of
immunological tests
• Antigen – Antibody interactions: Where the high
specificity of the antibody is used to identify,
isolate or quantify the antigen
• To identify the cell populations: Cell populations
are characterised by their surface markers,
using techniques of immunofluorescence
and immunohistochemistry
• To isolate cell populations: Also done by the
surface markers of the cell, using fluorescence
activated cell sorting, panning and density
dependant centrifugations
• Principal assay for lymphocyte functions: Assays
an antibody. Three zones can be distinguished from for antibody/cytokine production, proliferation in
the precipitin curve: response to antigen or by cytotoxicity

I. Antibody excess zone – first phase where less Techniques based on antigen-
antigen is present in sample
antibody interactions:
II. Equivalence zone – both antigen and antibody are
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cross-linked forming precipitate; no free antigen These methods exploit the property of antibody
or antibody is present – antigen complex and use antibodies as reagent
III. Antigen excess zone – amount of precipitate to detect and quantify antigens. This method is
reduces due to high antigen concentration subdivided to two steps

The precipitin curve forms the basis of most of the Particle method: Can be divided
immunological techniques that can be performed
into two categories
in laboratories
Precipitation of the large
Types of Antibody Used immune complexes:
The antibodies used for the methods are produced The reaction which occurs when specific antibody
by various ways: combines with soluble antigen is known as the

292
precipitation reaction. This reaction requires that Immunoelectrophoresis
the antigen contain multiple binding sites (epitopes) Immunoelectrophoresis combines two techniques;
for the antibody or anti-sera used. This allows for electrophoresis and immunodiffusion. It is a simple,
cross-linking to occur with the formation of large quick and reproducible method for determining the
macromolecular species. These large complexes concentration of antigen (Ag) in an unknown sample.
become insoluble and subsequently precipitate. The Various concentrations of antigen are located side
precipitate may be read visually in a gel such as in by side in small circular wells along the edge of an
immunofixation electrophoresis or may be measured agarose gel that contains the specific antibody (Ab). On
by an increase in light scatter in a nephelometer. Some electrophoresis, the antigen begins to migrate towards
common techniques where precipitation reaction is the anode and interacts with antibody molecules to
being used are described below: form a soluble antigen-antibody complex. However,
as the samples electrophorese farther through the
Immunodiffusion gel, more antibody molecules are encountered that
interact with the antigen and when the “equivalence
Immunodiffusion in gel encompasses a number of point” is reached. The Ag-Ab complex precipitates
techniques that are used for the analysis of antigen in the form of a rocket shape. Higher the amount of
antibody interactions. Immunodiffusion in gel is antigen loaded in the well, farther the antigen will
classified as single diffusion and double diffusion. travel through the gel. Hence, with increasing antigen
In ouchterlony double diffusion, both antigen and concentration, a series of rockets of increasing heights
antibody are allowed to diffuse into the gel. This are seen that is proportional to amount of antigen in
assay is frequently used for comparing different the well. Therefore, a direct measurement of the height
antigen preparation. In this test, different antigen of rocket will reflect upon the antigen concentration.
preparations, each containing single antigenic

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

species are allowed to diffuse from separate wells
against the antiserum. Depending on the similarity
between the antigens, different geometrical
patterns are produced between the antigen and
antiserum wells. The patterns of lines form can be
interpreted to determine whether the antigens are
same or different.
Dçifferent patterns of lines obtained on Ouchterlony double diffusion
Pattern of identity: A
The antibodies in the antiserum react
with both the antigen resulting in
a smooth line of precipitate. The
antibodies cannot distinguish between
the two antigens. i.e.) the two antigens
are immunologically identical.
A standard graph of antigen concentration versus peak
Pattern of identity: B
In the ‘pattern of partial identity’, the height is then constructed and from the peak height
antibodies in the antiserum react more of the unknown sample, concentration of antigen
with one of the antigens than the other.
is determined.
The ‘spur’ is thought to result from the
determinants present in one antigen
but lacking in the other antigen. Agglutinations
Pattern of identity: C
In the ‘pattern of non-identity’, none of
the antibodies in the antiserum react The reaction which occurs when antibodies react
with antigenic determinants that may with particulate antigen is known as an agglutination
be present in both the antigen i.e.
reaction. Agglutination reactions may be designed
the two antigens are immunologically
unrelated as far as that antiserum is with antigen or antibody bound to any large particle.
concerned. Materials may include starch particles, synthetic

293
 latex beads or red cells. The common thread in all (dose-response) curve from which the unknowns
types of agglutination systems is that the reaction is are calculated by interpolation.
read visibly by the clumping of the particles when
antibody crosslink with antigen. ELISA

Label methods Is a biochemical technique used mainly in immunology

to detect the presence of an antibody or an antigen
Involves the use of a label on either the antibody or in a sample. The technique is divided into;
antigen to identify antigen antibody complex formation
Competitive ELISA: The labelled antigen competes
Radioimmuno assay (RIA) for primary antibody binding sites with the sample
antigen (unlabeled). The more antigen in the sample,
Radioimmunoassays (RIAs) use antibodies to detect the less labelled antigen is retained in the well and
and quantitative the amount of antigen (analyte) in the weaker the signal)
a sample. These assays are typically very sensitive
and specific. It is possible to detect as low as a Sandwich ELISA (also called direct ELISA):
few picograms of analyte in the experimental tube The ELISA plate is coated with Antibody to detect
when using antibodies of high affinity (Kd = 10-8 - specific antigen. Prepare a surface to which a known
10-11 M). The basic principle of radioimmunoassay quantity of capture antibody is bound and block
is competitive binding, where a radioactive antigen any non specific binding sites on the surface. On
(«tracer») competes with a non-radioactive antigen applying the antigen-containing sample to the plate
for a fixed number of antibody or receptor binding followed by a chemical which is converted by the
sites. When unlabeled antigen from standards enzyme into a coloured product, it will develop a
or samples and a fixed amount of tracer (labeled colour. The absorbency of the plate wells is measured
antigen) are allowed to react with a constant and to determine the presence and quantity of antigen.
limiting amount of antibody, decreasing amounts
of tracer are bound to the antibody as the amount Indirect ELISA: In this technique, an antigen is
of unlabeled antigen is increased. Results obtained passively adsorbed to solid phase by incubation and
for the standards are used to construct a standard antibodies are added and incubated which are specific
Central Marine Fisheries Research Institute

294
and will bind to antigen on the solid phase. Excess (FITC) or tetramethyl rhodamine isothiocyanate
antibodies or nonbinding components are washed (TRITC). These labeled antibodies bind (directly or
away after incubation phase. A secondary antibody indirectly) to the antigen of interest which allows for
labelled with enzyme (conjugate) directed against antigen detection through fluorescence techniques.
the particular species in which the original antibodies The fluorescence can then be quantified using a
were produced (anti-species) were allowed to bind flow cytometer, array scanner or automated imaging
to any antibodies which are attached to antigen. instrument, or visualized using fluorescence or
Excess conjugate is washed away after a period of confocal microscopy.
incubation. Substrate/ chromophore are added to this
and a colour develops as a result of enzyme present.
After a period of incubation the colour development
is stopped and read by spectrophotometer.

After reading the results the standard curve is drawn

where the concentration is blotted on the X-axis and
the absorbance on the Y-axis. This standard curve is
used to determine the unknown concentration of
each sample by finding the opposite concentration
to the absorbance.

Western blot The two main methods of immunofluorescent labeling

are direct and indirect. Less frequently used is direct
Western blotting, also known as immunoblotting immunofluorescence whereby the antibody against

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

or protein blotting is a core technique in cell and the molecule of interest is chemically conjugated to a
molecular biology. In most basic terms, it is used fluorescent dye. In indirect immunofluorescence, the
to detect the presence of a specific protein in a antibody specific for the molecule of interest (called
complex mixture extracted from cells. The western the primary antibody) is unlabeled, and a second
blotting procedure relies upon three key elements anti-immunoglobulin antibody directed toward the
to accomplish this task: the separation of protein constant portion of the first antibody (called the
mixtures by size using gel electrophoresis; the efficient secondary antibody) is tagged with the fluorescent dye.
transfer of separated proteins to a solid support;
and the specific detection of a target protein by FACS
appropriately matched antibodies. Once detected,
the target protein will be visualized as a band on a The traditional method for sorting cells is a
blotting membrane, X-ray film, or an imaging system. fluorescence-activated cell sorting (FACS) machine. A
suspension of cells containing varying amount of the
protein of interest are placed into a flask. Antibodies
that bond with the specific protein are coated with
a fluorescent dye. Therefore, cells that contain the
protein will also hold the fluorescent dye. A nozzle
at the end of the flask is set to vibrate, which forms
drops that contain only one cell at a time. The cells
pass through the focus of a laser, operating at the
wavelength of absorption for the fluorescent dye that
Immunoflourescence is being used. Therefore, cells that hold the protein of
interest will fluoresce. Traditionally, the fluorescence
In IF techniques, antibodies are chemically conjugated will be measured by a photomultiplier tube whose
to fluorescent dyes such as fluorescein isothiocyanate signal is being monitored by a computer. The

295
 side of the plates will collect the cells. Therefore, the
population has been sorted based upon the presence
of the specific protein.

Immunohistochemistry

Immunohistochemistry (IHC) combines anatomical,

immunological and biochemical techniques to
identify discrete tissue components by the interaction
of target antigens with specific antibodies tagged
with a visible label. IHC makes it possible to visualize
the distribution and localization of specific cellular
components within cells and in the proper tissue
context. Immunohistochemistry is a technique that
uses antibodies (matching molecules) that can seek
out, identify and attach themselves to these markers
on cells. The antibodies themselves can be seen under
the microscope, which helps the technician make
precise identification.
Central Marine Fisheries Research Institute

computer controls an electric wire which generates an

Compliment fixation test
electric charge in the cell. If the cell fluoresces (it holds
the protein), it will be positively charged. Otherwise, A known volume of antigen is mixed with the
it will be negatively charged. All of the cells then pass test serum to be assayed for antibody and Ag/Ab
through two charged plates. The positively charged complexes are allowed to form. At the same time a
cells will be forced towards the negative plate and the control tube in which no Ag is added is also prepared.
negatively charged cells will be forced towards the If no Ag/Ab complexes are present in the tube none
positive plate. Two collection tubes placed at either of the complement will be fixed. However, if Ag/

296
Ab complexes are present, they will fix complement all the complement was still present (i.e. no Ag/

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

and thereby reduce the amount of complement in Ab complexes formed between the Ag and Ab in
the tube. After allowing for complement fixation by question), all the RBC will be lysed by the compliment.
any Ag/Ab complexes, a standard amount of red
blood cells, which have been pre-coated with anti- Suggested readings
erythrocyte antibodies, is added to the first tube. The
amount of antibody-coated RBC is predetermined Roitt, I., Brostoff, J., Male, D., 2001. Immunology. Mosby
to be just enough to completely use up all the publications, pp. 417-433
Actor, J. K., 2007.Immunology and microbiology. Elsvier, pp. 73-80
complement initially added if it were still there. If Hay, F. C., Westwood, O.M. R.,2002. Practical immunology.
Blackwellscience publications, pp. 163-203

297
 Hybridoma Technology and its Use in
Disease Diagnosis and Therapy
K. Pani Prasad
Principal Scientist
ICAR-Central Institute of Fisheries Education
Off Yari Road, Versova, Andheri (W), Mumbai- 400061
e-mail: [email protected]

Introduction Applications
In a landmark discovery, Kohler and Milstein (1975) The usefulness of monoclonal antibodies stem from
developed a technique that allows the growth of three characteristics their specificity of binding, their
clonal population of cells secreting antibodies with homogeneity and their ability to be produced in
a defined specificity. In this technique an antibody- unlimited quantities. The production of monoclonal
secreting cell, isolated from an immunized animal antibodies allows the isolation of reagents with a
is fused with a myeloma cell, a type of B-cell unique, chosen specificity. Because all of the antibodies
tumor. These hybrid cells or hybridomas can be produced by descendants of one hybridoma cell
maintained in vitro and will continue to secrete are identical, monoclonal antibodies are powerful
antibodies with defined specificity. Antibodies reagents for testing for the presence of a desired
that are produced by hybridomas are known as epitope. These characteristics make them attractive
monoclonal antibodies. for using them in diagnosis as well as therapeutic
agents in various diseases.
Stages of Hybridomas Technology
Hybridoma production
Antigen
For the production of monoclonal antibodies,
animals are injected with an antigen preparation,
and once a good humoral response has appeared Mouse
in the immunized animal, an appropriate screening
procedure is developed. The sera from the test bleeds
Central Marine Fisheries Research Institute

are used to develop and validate the screening B Cells

procedure. For fusion, antibody-secreting cells are
prepared from the immunized animal, mixed with the
Myeloma
myeloma cells and fused in the presence of a suitable Cells
fusogen. Mostly PEG (polyethylene glycol) is used
for this purpose. After the fusion, cells are diluted in
selective medium (HAT: hypoxanthene, aminoptrein Hybridomas
and thymidine medium) which allows only the growth
of fused myeloma-antibody secreting cells. Now
hybridomas are tested and cells from positive wells
are grown and then single cell cloned. These single-
cell cloned hybridoma produces antibodies that are Monoclonal
termed as monoclonal antibodies. antibody

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Diagnostic uses therefore can provide information on protein
relatedness, structure, function, synthesis, processing,
Antibodies produced in the mouse or rat are most cellular or tissue distribution and on association
commonly used for diagnostic purposes as they between molecules.
are more readily produced. The major advantage
of monoclonal antibodies over conventional sera The procedure for production of monoclonal antibody
in diagnostic uses is their high specificity, which is now well established. The advantages of mabs are
enhances the accuracy and speed of diagnosis and that they are homogenous and they
their ready availability for an infinite period at standard
titer thus antibodies to common serum analytes such recognise one antigenic determinant which is not
as protein hormones or alphafetoprotein are already possible with conventional polyclonal sera e.g.
commercially marketed and are slowly replacing detection of different strains of viruses. Mabs can also
conventional sera. A very large number of monoclonal reveal relationships between viruses at structural and
antibodies have been produced to a wide range biological levels. They can be used after preliminary
of viruses such as influenza (Gerhard et al. 1981), tests for reactivity with individual viral peptides such
hepatitis (Shih, Wands et al. 1981), polio (Fergeuson as in Western blotting and virus neutralisation etc.
et al. 1982), Epstein-Barr virus (Hoffman et al. 1980)
and rabies (Wiktor and Koprowski, 1978). Their high In order to obtain mab the virus antigen employed
specificity has led to accurate identification between for the induction should be free from extraneous
similar strins of virus such as Simplex types I and II. proteins. Characterization of viruses with mabs is
They are also used for early diagnosis of the IgM done through analysis of individual proteins. Active
production in affected patients. areas of the molecule, its synthesis and processing
can be monitored by mabs. Mabs distinguish between

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

The considerable potential of monoclonal antibodies related proteins and can be used to provide a genetic
in the study of parasitic diseases such as malaria, marker in recombinant experiments.
leishmaniasis and schistosomiasis has already
been widely exploited. Monoclonal antibodies for Therapeutic uses
immunological tissue typing are also produced by
several laboratories. Due to various problems that have been encountered
in the production of human monoclonal antibodies,
Tumor diagnosis is undoubtedly the field in which majority of the monoclonal antibodies used for
there has been most interest in monoclonal antibodies therapeutic applications, to date, have been raised
at the present time. Efforts are on the way to produce in mouse. Murine antibodies have the obvious
monoclonal antibodies, which react solely with tumor disadvantage of being foreign to the human
associated antigens and can consequently be used as system and likely therefore to loose their efficacy on
wide ranging diagnostic tools. However antibodies continual application as a host response is mounted.
to tissue or cell type specific antigens are already However, the treatment of bone marrow allogenic
generated and these have great potential in the or autologous transplants is one such area in which
detection of tumors and their metastases. monoclonal antibodies have been shown to be of
value in either reducing graft versus host disease or in
Characterization of removing leukemia cells from autografts. Orthoclone
(marketed by Janssen Pharmaceutica) is such a murine
viruses using Monoclonal monoclonal antibody which reverses graft rejection by
antibodies (Mabs) blocking the function of CD3 molecule (mw 20,000
dalton), found on the membrane of human T-cells
Using Mab technology, it is possible to produce that has been associated in vitro with the antigen
a large amount of homogenous antibody against recognition structure of T-cells and is essential for
many antigenic epitopes on a virus. A single Mab signal transduction.

299
 Human monoclonal antibodies are likely to prove murine antibody (4D5) that binds to HER2. In case of
of considerable value in immunosupression in heart human breast carcinoma, it inhibits the proliferation
and kidney transplant recipients as well as in the of human tumor cells that over express HER2. In the
prevention of both graft versus host and host versus long term there may be possibility of specific immune
graft disease in bone marrow transplantation. Despite therapy for autoimmune disease such as the use of
facing various problems, some laboratories have anti-idiotype antibodies.
succeeded in producing human monoclonal antibody.
For example HERCEPTIN (marketed by Genetec Inc) is Suggested readings
a recombinant DNA-derived humanized monoclonal
antibody that selectively binds with high affinity to the Campbell Ailsa M. 1986. Monoclonal antibody technology,
extracellular domain of the human epidermal growth Elsevier, Oxford
Harlow E., Lane D. 1988. Antibodies: A laboratory manual, Cold
factor receptor 2 protein, HER2. The antibody is an Spring Harbor laboratory.
IgG1 kappa that contains human framework regions Kohler G., and C. Milstein. 1975. Continuous cultures of fused
cells secreting antibody of pre-defined specificity, Nature
with the complementary-determining regions of a 256: 495-497.
Central Marine Fisheries Research Institute

300
Antibiotic Susceptibility Test -Applications
in Fisheries Science
T. G. Sumithra
Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction fish and during severe outbreak in commercial fish

farms but only after the isolation into contained
Antibiotic susceptibility test (ABST) or antibiogram environment preferably, in a quarantine/hospital
is an in-vitro test which determines the susceptibility tank. Legalities must also be considered when
of a microbe against different antibiotic agents. The selecting antibiotics for food fish, as they have fewer
tests are performed under standardized conditions options than ornamental fish. The antibiotic treated
to get reproducible results. Clinical signs produced fish should be released into routine farms or will
by the organism in a disease sometimes helps in be used for consumption only after the prescribed
identifying the microorganism, but it is not always withdrawal period. Thus, ABST results of pathogen
possible to determine reliably which antibiotic will help in selecting the most suitable antibiotic
is to be used for treatment, as different strains for treating an infection. However, only approved

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

of the same bacterial species differ substantially antibiotics for fish have to be used for treatment in
in antibiogram. In medical and veterinary field, aquacultures systems. Affected fish should not be
antibiotic susceptibility pattern of the causative treated with antibiotics before taking the samples
agent helps the clinician in selecting the most for analysis as it may result in poor culture yields.
effective antibiotic which has least toxicity to While waiting for the culture results, the fish health
host. Susceptibility testing of individual isolates specialist may suggest a broad-spectrum antibiotic
is very important in some species that possess that can be used until culture and sensitivity tests
acquired resistance mechanisms (eg: members have been completed. However, ability of antibiotics
of the Enterobacteriaceae, Pseudomonas sp., to eliminate a fish disease, depends on whether the
Staphylococcus sp., Enterococcus sp. and disease actually has a bacterial component, whether
Streptococcus pneumoniae). However, results of the proper dosage and treatment intervals being
antimicrobial susceptibility test should be combined used, and whether the other contributing stress
with clinical information and experience while factors (poor water quality, drastic temperature
selecting the most appropriate antibiotic for change, nutrition, and handling or transport) are
the treatment. removed or reduced etc.

Applications in As indiscriminate use of antibiotics without knowing

the susceptible pattern will lead to the emergence
fisheries science of antibiotic resistant strains, doing treatment after
ABST helps in selecting an antibiotic that will knowing the ABST pattern, can help to control the use
successfully and economically treat an infection of antibiotics in fisheries field. In an attempt to avoid
during a disease outbreak in aquaculture systems: the emergence of antibiotic resistant pathogens, some
Antibiotics can be used for the treatment of infection farmers are rotating the antibiotics they use. However,
in case of ornamental and pet fish. Similarly, the best solution is to positively identify the bacteria
antibiotics can be used for treatment of high value and their antibiogram pattern, by running culture

301
 and sensitivity tests, and thereby avoid unnecessary, single tray containing 96 wells 12 antibiotics can
costly, and potentially harmful treatments. be tested in a range of 8 two-fold dilutions. Frozen
or dried microdilution panels can be purchased
The occurrence of antibiotic resistant bacteria is from various commercial suppliers (outside
increasing in aquatic and marine environments. Thus, India). The cost of such preprepared ABST panels
results of ABST pattern of bacteria in water basins, vary from approximately $10 to $22 each. The
sediments and bivalves can be used to investigate advantages of this procedure include generation
the occurrence and distribution of antibiotic resistant of MICs, convenience of having preprepared panels
bacteria in various aquatic environments. and, economy of reagents and space due to the
miniaturization of the test. However, there is some
The anthropogenic impacts on coastal areas through inflexibility of antibiotic selections available in
inflow of domestic effluents can also be evaluated standard commercial panels.
by measuring the occurrence of bacterial resistance
to antimicrobial agents. Disc diffusion method: Due to the convenience,
efficiency and cost, the agar disc diffusion test is
METHODS OF ANTIBIOGRAM: There are different probably the most widely used method for ABST.  This
methods for performing the antibiotic susceptibility method is also known as Kirby–Bauer method of
test for a microbe. disc diffusion.

Tube dilution method (Macrobroth dilution Materials required

method): This is one of the earliest antimicrobial • Sterile Mueller-Hinton agar (MHA) plate
susceptibility testing methods. This procedure • Sterile cotton swabs
involves preparing two-fold dilutions of antibiotics • Antibiotic discs
in a broth medium dispensed in test tubes. The • 4-6 h old culture of test organism
antibiotic containing tubes are then inoculated
with a standardized bacterial suspension. Procedure
Following the overnight incubation, the tubes are
examined for visible bacterial growth as indicated Adjust the turbidity of the culture to 0.5 McFarland
by turbidity. The lowest concentration of antibiotic opacity tube (1-2 x 108 CFU/ml) using sterile saline.
that prevents bacterial growth represents the
minimal inhibitory concentration (MIC). The Then, a sterile swab is dipped into the turbidity
precision of this method is considered to be only adjusted culture, squeezed the excess fluid against
plus or minus 1 two-fold concentration, due to the side of the tube and inoculated into MHA plate so
errors during the manually preparation of serial that a complete lawn is formed on the agar surface.
dilutions of the antibiotics. The advantage of Dip the sterile swab in the turbidity adjusted culture,
Central Marine Fisheries Research Institute

this technique is the generation of a quantitative squeeze the excess fluid against the side of the tube
result. The principal disadvantages of this method and inoculate into MHA plate so that a complete lawn
are, it is tedious, manual task of preparing the is formed on the agar surface. Let the plate stand for
antibiotic solutions for each test, the possibility 10 min to imbibe the broth inoculum.
of errors in preparation of the antibiotic dilutions
during each test and the requirement of larger Select the required antibiotic discs and place it at
amount of reagents and space for each test. equidistance using a sterile forceps in such a way
that the zone of inhibition of two antibiotics shall not
Microdilution method: The miniaturization overlap each other. Incubate at suitable temperature
and mechanization by use of disposable, plastic (optimum temperature for that pathogen)
microdilution trays has made the broth dilution for overnight.
test as practical and popular. Microdilution test uses
about 0.05 to 0.1 ml total broth volume. Using a Measure the zone of inhibition of bacterial growth

302
around each disc and consult the interpretive chart • Allows flexibility in selection of discs for testing
to determine whether the organism is resistant (R), • It is the least costly of all susceptibility methods
fully susceptible (S) or intermediately susceptible (I). (approximately $2.50–$5 per test for materials)

Interpretation of Disadvantages
Susceptibility Test Results • Lack of mechanization or automation of the test
If the bacterial isolate is susceptible to a particular • Some fastidious bacteria cannot be accurately
antibiotic, a clear area of “no growth” can be tested by this method
observed around that particular disc.  The zone
around an antibiotic disc that has no growth E-TEST: E-test (AB Biodisk, Solna, Sweden) is a
is referred to as the zone of inhibition and this commercially available method that utilizes plastic
approximates the minimum antibiotic concentration strips that are impregnated on the underside with
sufficient to prevent the growth of that isolate.  a dried antibiotic concentration gradient. The
This zone is then measured in mm and compared concentration gradients are marked on the upper
to a standard interpretation chart and the isolate surface with a concentration scale. As many as
is then categorized as susceptible, intermediately 5 or 6 strips can be placed in a radial fashion on
susceptible or resistant.  The zone diameters of each the surface of a 150-mm agar plate that has been
drug are interpreted using the criteria published inoculated with a standardized organism suspension
by the Clinical and Laboratory Standards Institute like that used for a disc diffusion test. After overnight
(CLSI, formerly the National Committee for Clinical incubation, the tests are read by viewing the strips
Laboratory Standards or NCCLS) or those included from the top of the plate. The MIC is determined
in the US Food and Drug Administration (FDA). The by the intersection of the lower part of the ellipse

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

results of the disc diffusion test are “qualitative,” shaped growth inhibition area with the test strip.
in that a category of susceptibility (ie, susceptible, This method provides a convenient quantitative test
intermediate, or resistant) is derived from the test of antibiotic resistance.  However, a separate strip is
rather than an MIC. However, some commercially needed for each antibiotic, and therefore the cost
available zone reader systems claim to calculate of this method can be high. The gradient diffusion
an approximate MIC with some organisms and method has intrinsic flexibility for selecting the drugs.
antibiotics, by comparing zone sizes with standard E test strips cost approximately $2–$3 each. Hence,
curves of that species and drug stored in an this method is best suited to situations in which MIC
algorithm. Generally, reporting the ABST result as for only 1 or 2 drugs is needed.
susceptible, intermediate, or resistant provides the
clinician with the information necessary to select Mechanism-specific tests: Resistance may
appropriate therapy. It is important that the tables also be established through tests that directly
used for susceptibility test interpretations represent detect the presence of a particular resistance
the most current criteria. Indeed, the CLSI documents mechanism.  For example, beta lactamase detection
are reviewed and updated frequently, usually once can be accomplished using an assay such as the
per year. Use of old or outdated information from chromogenic cephalosporinase test (Cefinase disk by
the original editions of FDA-approved drug labels BD Microbiology Systems, Cockeysville, MD and BBL
or older CLSI tables could represent a serious DrySlide Nitrocefin, Becton Dickinson, Sparks, MD).
shortcoming in the reporting of patients’ results. Another example is detection for chloramphenicol
modifying enzyme chloramphenicol acetyltransferase
Advantages of the disc method (CAT) utilizing commercial colorimetric assays such as
a CAT reagent kit (Remel, Lenexa, Kansas).
• The test does not require any special equipment
• The results that can be easily interpreted by Genotypic methods: Since resistance traits are
all clinicians genetically encoded, we can sometimes test for

303
 the specific genes that confer antibiotic resistance.  according to the procedures defined by CLSI or by the
However, although nucleic acid-based detections manufacturers of the commercial products. However,
systems are generally rapid and sensitive, it is there is a need for development of new automated
important to remember that the presence of a instruments that could provide faster results and
resistance gene does not necessarily equate to also save money by virtue of lower reagent costs
treatment failure, because resistance is also dependent and reduced labor requirements. To accomplish
on the mode and level of expression of these genes. this, it will likely be necessary to explore different
Some of the most common molecular techniques methodologic approaches for detection of bacterial
utilized for antimicrobial resistance detection are PCR, growth. The direct detection of resistance genes by
Modifications of PCR and DNA hybridization.  polymerase chain reaction or similar techniques has
limited utility, because only a few resistance genes
Current Test Methods and are firmly associated with phenotypic resistance.
Thus, it seems likely that phenotypic measures of
Future Directions the level of susceptibility of bacterial isolates to
The antimicrobial susceptibility testing methods antimicrobial agents will continue to be relevant
described here provide reliable results when used for years to come.
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304
Immunization of Fish: A Tool for
Aquaculture Health Management
T. G. Sumithra
Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction treating diseases in fish. Feeding infected fish with

antibiotic-medicated food is a universal practice
Aquaculture is the fastest emerging global food followed by many aquaculture farmers for health
industry among all other food animal producing management. However, this is usually costly, and may
sectors. Nevertheless, the economical loses imposed be ineffective as sick fish may be anorectic. More
by high mortality rates due to incidences and outbreaks significantly, frequent use of antimicrobial compounds
of infectious diseases form a major challenge to may lead to the development of antimicrobial
develop productive, feasible and sustainable resistance, posing serious challenges to health and
aquaculture systems. Numerous reasons including national security and thus, cannot be encouraged.
intensive aquaculture practices, introduction of new Therefore, immunization of fish is becoming an
fish species into farming practices, increased trade in increasingly important part of aquaculture as it is

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

ornamental fish and increased interaction between a cost effective method for controlling different
wild fish and farmed fish have altogether caused threatening diseases.
increased economic losses, due to infectious diseases
in the aquaculture systems. Literature proposed Overview of fish immune system: Fish immunology
that among the aetiological agents responsible for has a more recent history than human and veterinary
periodical disease outbreaks in fish, 54.9% are caused immunology. It is still a young and maturing science
by bacteria, 22.6% by viruses, 3.1% by mycotic agents but amazing advances can be made in future. Fish are
and 19.4% are by parasitic agents (Dhar et al., 2014). reported to have a functional immune system similar
The Office International des Epizooties (OIE)/World to mammals comprising of two arms of immunity
Organization for Animal Health has listed that viral such as innate and adaptive immunity. The major
diseases including epizootic hematopoietic necrosis, koi difference from other vertebrates is that their immune
herpesvirus disease, red sea bream iridovirus disease, response is temperature dependent. The innate arms
infectious hematopoietic necrosis virus, infectious of fish immunity which does not require any previous
salmon anemia virus, spring viremia of carp, and viral exposure to the particular agent includes physical
hemorrhagic septicemia as the agents causing major barriers (skin and mucus layers), specialized cells
catastrophe for large scale aquaculture industry. (macrophages and natural killer cells and particular
soluble molecules such as complement and interferon.
Therefore, prevention and control of fish diseases has The mucosa-associated lymphoid tissues are sub-
got high priority in aquaculture industry. The farming divided into gut, skin and gill. Recent studies have also
practices of fish (in both freshwater and seawater confirmed the presence of functional homologues of
aquaculture) in developed countries have already mammalian cytokines in fish.
demonstrated that effective disease management
is the key to profitability in commercial aquaculture In adaptive immunity, fish above the level of
systems. On contrast, unlike treating human or other the Agnatha display typical vertebrate adaptive
animal diseases, only few drugs are available for immune responses characterized by presence of

305
 immunoglobulins, T-cell receptors, cytokines, and Vaccination: In this method vaccine is mixed with
major histocompatibility complex molecules. However, feed. It is the easiest method for mass vaccination of
the immune system of fish is quite different in its all sizes of fish and it saves labour and avoids stress.
efficiency and complexity from that of higher However, large quantities of antigen are required.
vertebrates. Acquired immunity in fish includes both Moreover, immune protection is generally weak and
humoral and cell mediated response. The primary and of short duration
secondary lymphoid organs in mammals are present
in fish, except lymphatic nodules and bone marrow. Currently, it is widely accepted that only injection
and immersion routes give enough protection to be
The anterior portion of kidney is most likely the used as the primary route of fish immunization in
source of histocompatibility complex. In teleost fish, commercial production. The less immune response
progenitor T-cells migrates from the kidney to the in oral vaccination can be improved by protecting
thymus for T-cell maturation and for self, non-self- the antigens from digestion and decomposition,
recognition. B-lymphocytes originate and mature during the passage through gut. Some promising
within the kidney. B-cells of fish produce antibody results have been obtained using microencapsulation
when stimulated. of antigens. From the economic point of view, oral
vaccination will be the ideal route to be employed in
Routes of immunization for fish: For vaccination fish vaccination program which requires one or more
in developed countries, fish are usually transported booster immunizations.
in pipes from the rearing tanks to an anaesthetic
bath and the anaesthetized fish are then immunized. Current status of fish vaccines: As per the
Vaccines are usually administered to fish by any latest review, now, vaccines are accessible in many
of the three methods: injection (intraperitoneal/ countries for more than 17 fish species which can
intramuscular), immersion (short bath/long bath/spray induce protection for more than 22 different kinds of
vaccination), or oral administration. All these methods bacterial diseases as well as 8 viral diseases (Dadar et
have their own advantages and disadvantages with al., 2017). The range of bacterial infections for which
respect to the level of protection, side effects, vaccines are commercially available now comprises
practicality and cost-efficiency. classical vibriosis (V. anguillarum and V. ordalii),
cold-water vibriosis (V. salmonicida), furunculosis
Advantages and disadvantages of immersion (Aeromonas salmonicida subsp. salmonicida),
vaccination: Advantages include: suitable for mass yersiniosis (Y. ruckeri), pasteurellosis (Photobacterium
vaccination and for all sizes of fish, reduced stress damselae supsp. piscicida), edwardsiellosis (E.
for fish during immunization procedures, lower ictaluri), winter ulcer (Moritella viscosa), and
labour costs and less risk to vaccination team. Major streptococcosis/lactococcosis (S. iniae, L. garviae).
disadvantages are large amount of vaccine are The viral diseases for which commercial vaccines are
Central Marine Fisheries Research Institute

required and there will be only a low level protection. available include Koi herpesvirus, Iridovirus, red sea
Duration of immune-protection is also short. bream iridiovirus, salmon alphaviruses, infectious
hematopoietic necrosis virus, spring viremia of carp
Advantages and disadvantages of injection: This virus, infectious salmon anemia virus and infectious
is the most common vaccine delivery method in fish pancreatic necrosis virus.
as it is highly efficient in generating both humoral and
cellular immune response. However, it is unsuitable for The major producers of fish vaccines are Intervet
small fish and needs sophisticated machinery or large International, Novartis Animal Health, Schering-Plough
skilled workforce. It also causes significant handling Animal Health, Pharmaq and Bayer Animal Health. The
stress so that risk of post vaccination mortality and key commercial markets are currently the salmon and
local reactions are more. trout industries in Northern Europe, Canada, USA,
and Chile. Commercial vaccines are also available for
Advantages and disadvantages of oral catfish industry in USA and for European seabream,

306
seabass and tilapia. Some locally developed vaccines that the immune system of fish has a defined and
are also available in countries such as China, Japan, limited capacity to respond to individual antigens,
Russia, Spain and Germany (Sommerset et al., 2005). it becomes increasingly difficult to formulate these
complex mixtures into effective commercial products.
Important considerations during The other limitation is many fish species are too
vulnerable to handle the stress induced during
fish immunization
the vaccination so that oral vaccination should be
• Species of fish to be immunized considered in future as the most desirable method
• Size of fish at vaccination- Smaller the fish, more for immunizing fish. However, the limited protection
stress and higher risk of local reactions by this route challenges the fish immunization.
• Temperature and salinity at the time of vaccination- Another problem in fish immunization is that the
Higher the temperature higher risk of local reactions major disease problems appear during the larval or
• Onset of immunity: It is temperature dependent fry stages during which the animal is small enough
• Diseases to be controlled and when these to be vaccinated or will not have functional immune
diseases occur system. The apparent lack of maternal immunity in
• Adjuvants-Various adjuvants are tried in fish in fish limits the possibilities to protect offspring by
which oil based adjuvants give best protection parental vaccination.
• Types of vaccines: Killed/Attenuated live/Gene
deleted live/Toxoid/Subunit vaccines as DNA vaccine/ Future prospects
recombinant vector vaccine/recombinant protein/
conjugate/
and conclusion
• Stress: Stress caused by environments, crowding, Vaccines have become a reputable, verified, and
handling and transport, can induce immune cost-effective method for reducing the occurrence

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

suppression and can be a limiting factors for of diseases and usage of antibiotics in fish
vaccine efficacy production. However, to achieve further progress in
• Nutrition fish vaccinology, an increased collaborative research
• Cost benefit between the microbiologist and vaccinologist is
• Farming technology needed. The fish vaccination may be benefitted in
future by increased knowledge of their immune
Limitations in fish system and pathogens, through the application of
comparative genomic and transcriptome analysis.
vaccine development This would facilitate to open new generation
As with all veterinary vaccines, cost effectiveness in vaccines in aquaculture systems. Improvement in
the field is an essential limitation to commercial fish oral immunization by better delivery systems may
vaccine development. Ideal vaccines for aquaculture also benefit the future fish vaccinology. Development
must be effective in preventing death, be inexpensive of novel non-mineral oil adjuvants lacking side
to produce and license, provide sustained immunity effects, development of polyvalent vaccines and
of long duration, stable, will not interfere the standardization of immunization calendar appropriate
diagnosis and be easily administered. In case of fish for each economically important fish species etc.
they generally need a larger antigen dose compared can be possible novel approaches for fish vaccine
to terrestrial animals so that cost effective vaccines development. Also, vaccines designed to augment
are difficult to develop. In the past ten years, cell mediated immunity must be targeted against viral
commercial vaccine products for fish have more fish diseases. Novel expression systems, improvements
often consisted of mixtures of multiple vaccines. in adjuvants, better immunostimulants, progress
Considering the fact that not all antigens stimulate in DNA vaccinology and development of passive
protective immune response, that antigens vary in immunization etc. may also benefit fish immunization
their immune-dominance relative to each other and strategies in future.

307
 Polyphasic Taxonomy as a Consensus
Methodology for Bacterial Identification
Anusree V. Nair
Technical Assistant
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction popular choice for classifying bacteria and several

microbes, which were previously placed under invalid
Microbial taxonomy or microbial systematics deals with taxa have now been resolved into new genera and
the classification, identification and nomenclature of species. It helps to yield good quality of phenotypic
microorganisms. Microbial systematic is a vast intuitive and genotypic data which is required to obtain a
science but has become increasingly objective due to better understanding of microbial diversity. This field
the introduction of the new concepts and methods. is rapidly expanding and therefore it is imperative to
Classification of microorganisms on the basis of update readers with the recent technical advances.
traditional microbiological methods (morphological,
physiological and biochemical) creates a blurred image Identification of Bacteria
about their taxonomic status and thus needs further
clarification. It should be based on a more pragmatic
approach of deploying a number of methods for
Morphological identification
the complete characterization of microbes. The new Gram Staining: This technique is used to differentiate
advancements, mainly in molecular systematics, gram positive and gram negative bacteria. Gram
stimulated the need to compare established and positive bacteria stains as purple colour. Gram
more recent approaches to microbial classification negative bacteria stains as pink color. This method
which helped the integrated use of genotypic helps to differentiate their shapes (rod, cocci, comma,
and phenotypic information. Hence, the methods etc.) and their arrangements (cluster, tetrad, etc.)
now employed for bacterial systematics include,
the complete 16S rRNA gene sequencing and its Cultural identification
comparative analysis by phylogenetic trees, DNA-DNA
hybridization studies with related organisms, analyses Cultural characteristics of the isolates can be used to
Central Marine Fisheries Research Institute

of molecular markers and signature pattern(s), help identify the bacterial species. Colony characters
biochemical assays, physiological and morphological such as colony colour, form, elevation and margin
tests. Collectively these genotypic, chemotaxonomic has to be noted.
and phenotypic methods for determining taxonomic
position of microbes constitute what is known as Biochemical identification
the ‘polyphasic approach’ for bacterial systematics.
Polyphasic approach is a recent trend in microbial The major tests followed in a routine microbiology
taxonomy, which provides natural and authentic lab include
system of classification of microbes. The term coined
by Colwell in 1970, refers to the integration of a) KOH Test (String formation)
genotypic, chemotypic and phenotypic information
of a microbe in order to perform reliable grouping Based on the differences in the chemistry of the
of the organism. This approach is currently the most bacterial cell wall, the cell wall of gram negative

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bacteria gets easily disrupted when exposed to dilute c) Triple Sugar Iron Agar Test
alkali solution resulting in the formation of string. TSI Agar is used for the determination of carbohydrate
Absence of string formation is observed for gram fermentation and hydrogen sulfide production in the
positive bacteria. identification of gram-negative bacilli. Carbohydrate
fermentation is detected by the presence of gas and
Procedure a visible colour change (from red to yellow) of the pH
A loopful of 24-hour-old growth from a colony of indicator, phenol red. The production of hydrogen
the organism was emulsify on the surface of a clean sulfide is indicated by the presence of a precipitate
glass slide in a suspension of 3%potassium hydroxide. that blackens the medium in the butt of the tube.
The suspension was mix continuously for 10 seconds To facilitate the detection of organisms that only
after which the toothpick pulled from the suspension. ferment dextrose, the dextrose concentration is one-
tenth the concentration of lactose or sucrose. The
Interpretation small amount of acid produced in the slant of the
String formation:–gram negative bacteria (formation tube during dextrose fermentation oxidizes rapidly,
of a string within 10 sec of mixing the bacteria). causing to remain red or revert to an alkaline pH.
Absence of string formation:–gram positive bacteria. In contrast, the acid reaction (yellow) is maintained
in the butt of the tube because it is under lower
b) Oxidation Fermentation (OF) reaction oxygen tension.

Oxidative organisms can only metabolize glucose Procedure

or other carbohydrates under aerobic conditions • Inoculate a slant of triple sugar iron agar slant by
ie. oxygen is the ultimate hydrogen acceptor. Other using a straight needle.
organisms ferment glucose and the hydrogen • First stab the butt down to the bottom, withdraw

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

acceptor is then another substrate. e.g., Sulphur. This the needle & streak the surface of the slant.
fermentative process is independent of oxygen and • Read the results after incubation at 37ºC for18-
cultures of organisms may be aerobic or anaerobic. 24 h.
The end product of metabolizing a carbohydrate is
acid. The method described, is sometimes referred to Interpretation
as the Hugh and Leifson test, is a semi-solid medium Yellow coloration of the slope:–Oxidative reaction;
in tubes, containing the carbohydrate under test Red coloration of the slope:–Alkaline reaction;
(usually glucose) and a pH indicator Yellow coloration throughout the tube or in the
butt:–Fermentative reaction; Blackening of the butt:–
Procedure Hydrogen sulphide production; Split or gas bubble
• Heat two tubes of Hugh Leifson’s medium in in the butt:–Gas production
boiling water for 10 minutes to drive off the
oxygen, cool and inoculate by stabbing with a d) Oxidase Test
straight wire.
• Incubate one tube aerobically and either incubate The oxidase test is based on the bacterial production of
the second tube anaerobically or seal the surface an intracellular oxidase enzyme. The oxidase reaction
with a layer of sterile liquid paraffin oil to create is due to the presence of a cytochrome oxidase system
an anaerobic condition. which activates the oxidation of reduced cytochrome
• Incubate at 37ºC for 24-48 hours. by molecular oxygen. The cytochrome oxidase
enzyme is able to oxidize the substrate tetramethyl-p-
Interpretation phenylenediamine dihydrochloride, forming a colored
Oxidation: Acid in aerobic tube only (yellow colour in end product, indophenol. The dark- purple end
aerobic tube, green in anaerobic), Fermentation: Acid product will be visible if a small amount of growth
in both tubes (yellow colour), Neither fermentation from a strain that produces the enzyme is rubbed on
nor Oxidation: No colour change. substrate-impregnated filter paper.

309
 Procedure Procedure
• A filter paper moistened with 2-3 drops of • Inoculate the test organisms in a required amino
Wurster’s reagent (1%tetramethyl-p-phenylene acid decarboxylation broth (Moller Decarboxylase
diamine dihydrochloride (TPDD). Broth with corresponding amino acids)
• Pick a colony grown freshly on nutrient agar and • Incubate for 24 hours at 37ºC and observe the
make a compact smear on a filter paper color change of the medium.
• Observe for a colour change to blue or purple
within 10 secs (timing is critical). Interpretation
Colour change of the medium to violet or colour is
Interpretation taken as positive result.
Blue to purple colour formation within 10 to 30 sec
is consider as positive reaction and no colour change g) Indole production Test
is negative reaction
This test is done to determine if bacteria can
e) Catalase Test breakdown the amino acid tryptophan into indole,
which is present in tryptone broth. This liberated
The catalase test is used to detect the presence of indole reacts with Kovacs’ reagent to produce red
catalase enzyme, by the decomposition of hydrogen colour at the top of the medium.
peroxide to release oxygen and water. Hydrogen
peroxide is produced by some bacteria as an oxidative Procedure
end product of the aerobic break down of sugars. If • Inoculate the culture in to tryptone broth and
allowed to accumulate it is highly toxic to bacteria and incubate for24 to 48 hrs.
can result in cell death. Aerobes, facultative aerobes • After incubation add about 0.5 ml of Kovacs’
and microaerophilic organisms degraded this product reagent to each tube.
using catalase enzyme. • Observe the colour of ring formed at the top of
the medium.
Procedure
• Prepare a thick smear of the organism from a 24 Interpretation
hr culture on a clean slide A red coloured ring at the top of the medium is taken
• A drop of hydrogen peroxide is placed on the slide as positive result.
• Observe the immediate formation of gas bubbles.
h) Methyl Red test
Interpretation
Immediate formation of gas bubbles indicated the This is used to determine whether the bacteria can
liberation of oxygen is the positive reaction. convert glucose to acidic products like lactate, acetate,
Central Marine Fisheries Research Institute

and formate. The production of acid from glucose

f) Aminoacid decarboxylases test has lowered and held pH at about 4.2 or below and
this is detected using methyl red indicator.
The aminoacid decarboxylase test demonstrates
the bacterial decarboxylation of amino acid Procedure
lysine, arginine, and ornithine. In this test, the • Inoculate pure culture to the MR VP broth and
decarboxylation or the elimination of a molecule of incubate for 24- 48 hours at 37ºC.
carbon dioxide from the amino acid results in the • Add a few drops of methyl red indicator and
formation of an amine with one carbon atom less. observe the colour change.
Alkaline degradation products are produced in the
course of decarboxylation and subsequently color of Interpretation
the media changes to violet or purple. Bromocresol A definite red colour of the medium can considered
purple is the dye using in the test as positive.

310
i) Voges-Proskauer Test broth medium. The medium was basically a nutrient
broth supplemented with 0.1% potassium nitrate as
Some organisms, after producing acids from glucose, the nitrate substrate. Following incubation, the ability
are capable of converting acids to acetylmethyl of organisms to reduce nitrate to nitrite is determined
carbinol or 2,3butanediol, which are neutral by the addition of two reagents. Solution A, which is
substances. Aeration in the presence of alkali then sulfanilic acid, followed by solution B, which is alpha-
converts the products to diacetyl, which in turn naphthylamine. Following reduction, the addition of
reacts with the peptone constitutents producing a solution A & B will produce a cherry red colour.
red colouration.
Procedure
Procedure • Culture is inoculated into the autoclaved nitrate
• Inoculate pure culture to the MR VP broth and broth and incubate for 24 hours at 37ºC.
incubate for 24- 48 hours at 37ºC. • Add 2 drops of each nitrate solution A and nitrate
• Add 4 drops of Barrits reagent A and 2 drops of solution B.
Barrits reagent B to the broth & shake well. • Observe the colour change of the media.
• Observe the broth for colour change after 3 hours.
Interpretation
Interpretation Appearance of a blood red colour indicates the
Red colour formation is positive result. positive reaction. No characteristic colour change
for negative reaction.
j) Citrate utilization
Molecular identification–16S
This test demonstrates the ability of the microbes to

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

utilize citrate as a sole source of carbon and oxygen.
rDNA Gene Amplification
Utilization of citrate and growth in citrate agar results Total genomic DNA was extracted from bacterial
in an alkaline reaction, which changes the colour of cultures grown in nutrient broth using phenol-
the medium provided. In this Simmon’s citrate agar chloroform extraction method. Purified colony was
medium, bromothymol blue indicator is used which inoculated to 5 ml of LB broth, incubated overnight in
changes from green to bright blue on utilization a shaking incubator at 30ºC. After the incubation, the
of citrate. cells were harvested by centrifugation at 10,000 rpm
for 15 min. The pellet was re-suspended in 450 μl TEG
Procedure buffer with lysozyme (5 mg/ml of TEG buffer) mixture
• Prepare Simmon’s citrate agar medium in the form and vortexed well. A volume of 50 μl of 10% SDS was
of agar slopes in the tubes. added and mixed well. Sample was kept on ice for 10
• Inoculate the slope by streaking over the surface min followed by incubation at room temperature for
with a loopful of culture. 15 min and then in water bath at 60ºC for 10 min.
• Incubate for 24-48 hours and observe the result. To this, 10 μl of Proteinase-K was added, vortexed
and incubated in water bath at 60ºC for 1 h (until
Interpretation the proteins got digested). Equal amount of phenol-
Colour change from green to bright blue indicates chloroform-isoamyl alcohol (25: 24: 1) was added,
a positive result. mixed gently by inverting and centrifuged at 10,000
rpm for 15 min at 4ºC. The aqueous phase was
k) Nitrate reduction collected in another micro-centrifuge tube without
disturbing the interphase and lower phase. To the
The reduction of nitrates by some aerobic and aqueous phase, 1/10th volume of 3 M sodium acetate
anaerobic microorganisms occurs in the absence was added followed by 2.5 times ice cold absolute
of molecular oxygen. Nitrate reduction can be ethanol and incubated at -20ºC overnight so that
determined by cultivation of organisms in nitrate the DNA got precipitated and was again centrifuged

311
 for 15 min at 10,000 rpm. The supernatant was Conclusion
discarded and the pellet was rinsed with 500 μl
70% ethanol, centrifuged again at 10,000rpm for In this regard, a polyphasic taxonomic approach is
15 min at 4ºC and the tubes were air dried after advantageous because it exploits simultaneously
discarding the supernatant. The DNA samples were both conventional as well as molecular identification
then re-suspended in 30 μl of DNA dissolving buffer techniques. The major advantages of the polyphasic
(TE/elution buffer) and stored at -20ºC. Further, purity approach is that certain groups of bacteria as
of DNA was checked by agarose gel electrophoresis case studies to arrive at a consensus approach to
(AGE) and quantified using Biophotometer. microbial identification. Now-a-days there is an
increasing access to microbial genomes due to the
Amplification of 16S rRNA gene accumulation of numerous DNA sequences which
ultimately leads to a higher level of accuracy and
16S rRNA gene amplification was carried out reliability of results. Though polyphasic taxonomy is a
using universal prokaryotic primers; NP1F useful technique to meet the challenge of identifying
5 ’ G A GT T T G ATC C T G G C TC A - 3 ’ a n d N P 1 R any unknown strain, new mathematical and
5’-ACGGCTACCTTGTTACGACTT-3’. Each polymerase informative strategies should be developed for the
chain reaction (PCR) mixture consisted of 1 μl of possible development of a synthetic taxonomy. Thus,
template DNA, 2.5 μl of 1X Taq buffer, 0.5 μl of the conventional biochemical basis of identification
dNTP mix, 0.5 μl of Forward and Reverse Primer and practices cannot be replaced completely and the
1.25 U of DNA polymerase. The PCR programme vast majority of the data should lead to a perfectly
of each for sample included initial denaturation reliable and stable identification and classification
at (95ºC for 5 min) followed by 35 cycles of system. Though many advanced techniques are
denaturation (95ºC for 30 sec), annealing (58ºC available now-a-days for the identification of any
for 1 min) and extension (72ºC for 1.30 min). Final unknown bacterium, a far less number of the total
extension was carried out at (68ºC for 5 min). The number of microbial species have been discovered
PCR products were then characterized by submarine and identified till now; many of them are yet to
gel electrophoresis (1% agarose gel). The amplified be cultured under laboratory conditions and some
products were purified and sequenced. The of them may possess certain unique characteristic
obtained sequences were then subjected to BLAST features. Microbial taxonomy and biosystematics
search (NCBI) and the bacteria were identified. The is a major modern discipline which needs further
16S rRNA gene sequences were analysed and the financial and intellectual support to determine
relative phylogenetic positions were determined its role in biotechnology, biodiversity, agriculture,
by searching GenBank database using BLASTn medical science and environmental science.
algorithm (Altschul et al. 1997).
Central Marine Fisheries Research Institute

312
Marine Chemistry
Laboratory- Safety and Hazards
Kajal Chakraborty
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction Material Safety Data

A key element of planning an experiment is Sheets (MSDSs)
assessing the hazards and potential risks associated
with the chemicals and laboratory operations to be Federal regulations (OSHA Hazard Communication
used. The primary responsibility for proper hazard Standard 1910.1200) require that manufacturers and
evaluations and risk assessments lies with the person distributors of hazardous chemicals provide users
performing the experiment. The actual evaluations with material safety data sheets (MSDSs), which
and assessments may be performed by trained are designed to provide the information needed to
laboratory personnel, but these should be checked protect users from any hazards that may be associated
and authorized by the supervisor. The supervisor is with the product (Globally Harmonized System for
also responsible for ensuring that everyone involved Hazard Communication). MSDSs have become the
in an experiment and those nearby understand the primary vehicle through which the potential hazards

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

evaluations and assessments. Some organizations of materials obtained from commercial sources are
have environmental health and safety offices, with communicated to trained laboratory personnel.
industrial hygiene specialists to advise trained Institutions are required by law (OSHA Hazard
laboratory personnel and their supervisors in risk Communication Standard) to retain and make readily
assessment. As part of a culture of safety, the available the MSDSs provided by chemical suppliers.
supervisor and scholars in the laboratory must work
cooperatively to create a safe environment and to OSHA recommends the general 16-part format
ensure that hazards are appropriately identified and created by the American National Standards Institute
assessed prior to beginning work. All laboratory (ANSI Z400.1). The information typically found in an
personnel should be familiar with and have ready MSDS follows:
access to their institution’s chemical hygiene plan.
In some laboratories, chemical hygiene plans • Supplier (with address and phone number) and
include standard operating procedures for work date MSDS was prepared or revised. 
with specific chemical substances, and the chemical 1. Chemical. 
hygiene plan may be sufficient as the primary 2. Physical and chemical properties. 
source of information used for risk assessment 3. Physical hazards related to flammability, reactivity,
and experiment planning. However, most chemical and explosion hazards.
hygiene plans provide only general procedures 4. Toxicity data. 
for handling chemicals, and prudent experiment 5. Health hazards-Acute and chronic health
planning requires that laboratory personnel consult hazards together with the signs and symptoms
additional sources for information on the properties of exposure.
of the substances that will be encountered in the 6. Storage and handling procedures. 
proposed experiment. Many laboratories require 7. Emergency and first-aid procedures. 
documentation of specific hazards and controls 8. Disposal considerations. 
for a proposed experiment. 9. Transportation information. 

315
 MSDSs remain the best single source of information • flammable aerosols
for the purpose of evaluating the hazards and • oxidizing gases
assessing the risks of chemical substances. However, • gases under pressure
laboratory personnel should recognize the limitations • flammable liquids
of MSDSs as applied to laboratory-scale operations. • flammable solids
If MSDSs are not adequate, specific laboratory • self-reactive substances
operating procedures should be available for the • pyrophoric liquids
specific laboratory manipulations to be employed: • pyrophoric solids
• self-heating substances
1. The quality of MSDSs produced by different • substances which, in contact with water, emit
chemical suppliers varies widely. flammable gases
2. Unique morphology of solid hazardous chemicals • oxidizing liquids
may not be addressed in MSDSs; for example, • oxidizing solids
an MSDS for nano-size titanium dioxide may not • organic peroxides
present the unique toxicity considerations for • corrosive to metals
these ultra-fine particulates.
3. MSDSs must describe control measures and Health hazards include
precautions for work on a variety of scales. • acute toxicity
4. Many MSDSs comprehensively list all conceivable • skin corrosion or irritation
health hazards associated with a substance • serious eye damage or eye irritation
without differentiating which are most • respiratory or skin sensitization
significant and which are most likely to actually • germ cell mutagenicity
be encountered. As a result, trained laboratory • carcinogenicity
personnel may not distinguish highly hazardous • reproductive toxicology
materials from moderately hazardous and • target organ systemic toxicity-single exposure
relatively harmless ones. • target organ systemic toxicity-repeated exposure
• aspiration hazard
Globally Harmonized System (GHS)
Environmental hazard includes
for Hazard Communication
• Hazardous to the aquatic environment: acute
The GHS of Classification and Labeling of Chemicals aquatic toxicity or chronic aquatic toxicity with
is an internationally recognized system for hazard bioaccumulation potential rapid degradability.
classification and communication (available at http://
www.unece.org.) It was developed with support Laboratory Chemical Safety
from the International Labour Organization (ILO), Summaries (LCSSs)
Central Marine Fisheries Research Institute

the Organisation for Economic Co-operation and

Development, and the United Nations Sub-Committee Although MSDSs are invaluable resources, they
of Experts on the Transport of Dangerous Goods with suffer some limitations as applied to risk assessment
the goal of standardizing hazard communication in the specific context of the laboratory. LCSSs
to improve the safety of international trade provide information on chemicals in the context of
and commerce. laboratory use. These documents are summaries and
are not intended to be comprehensive or to fulfill
GHS recognizes 16 types of physical hazards, 10 types the needs of all conceivable users of a chemical.
of health hazard, and an environmental hazard. LCSS gives essential information required to assess
the risks associated with the use of a particular
Physical hazards include chemical in the laboratory. LCSSs also contain a
• explosives concise critical discussion, presented in a style readily
• flammable gases understandable to trained laboratory personnel, of

316
the toxicity, flammability, reactivity, and explosivity CDC/NIOSH, 2007). This volume is updated
of the chemical; recommendations for the handling, regularly and is found on the NIOSH Web site
storage, and disposal of the title substance; and first- (http://www.cdc.gov/niosh). These charts are
aid and emergency response procedures. quick guides to chemical properties, reactivities,
exposure routes and limits, and first-aid measures.
Labels 3. A Comprehensive Guide to the Hazardous
Properties of Chemical Substances, 3rd edition
Commercial suppliers are required by law (OSHA (Patnaik, 2007). This particularly valuable guide
Hazard Communication Standard) to provide their is written at a level appropriate for typical
chemicals in containers with precautionary labels. laboratory personnel. It covers more than 1,500
Labels usually present concise and non-technical substances; sections in each entry include uses
summaries of the principal hazards associated with and exposure risk, physical properties, health
their contents. It is of note that precautionary labels do hazards, exposure limits, fire and explosion
not replace MSDSs and LCSSs as the primary sources hazards, and disposal or destruction. Entries are
of information for risk assessment in the laboratory. organized into chapters according to functional
However, labels serve as valuable reminders of the key group classes, and each chapter begins with a
hazards associated with the substance. general discussion of the properties and hazards
of the class.
Additional Sources of Information 4. 2009 TLVs and BEIs: Based on the
Documentation of the Threshold Limit Values
The resources described above provide the foundation for Chemical Substances and Physical Agents
for risk assessment of chemicals in the laboratory. and Biological Exposure Indices. A booklet
Although MSDSs and LCSSs include information on listing ACGIH threshold limit values (TLVs)

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

toxic effects, in some situations laboratory personnel and short-term exposure limits (STELs). These
should seek additional more detailed information. values are under continuous review, and this
This step is particularly important when laboratory booklet is updated annually. The multivolume
personnel are planning to use chemicals that have publication Documentation of the Threshold Limit
a high degree of acute or chronic toxicity or when Values and Biological Exposure Indices reviews
it is anticipated that work will be conducted with the data that were used to establish the TLVs.
a particular toxic substance frequently or over an 5. Fire Protection for Laboratories Using
extended period of time. Chemicals (NFPA, 2004). This is the national
fire safety code pertaining to laboratory use of
The following annotated list provides references on chemicals. It describes the basic requirements
the hazardous properties of chemicals and which are for fire protection of life and property in
useful for assessing risks in the laboratory. the laboratory
6. Fire Protection Guide to Hazardous Materials,
1. International Chemical Safety Cards from the 13th edition (NFPA, 2001). This resource
International Programme on Chemical Safety contains hazard data on hundreds of chemicals
(IPCS, 2009). The IPCS is a joint activity of the and guidance on handling and storage of, and
ILO, the United Nations Environment Programme, emergency procedures for those chemicals.
and the World Health Organization. The cards 7. Hazardous Chemicals Handbook, 2nd edition
contain hazard and exposure information from (Carson and Mumford, 2002). This book is
recognized sources and undergo international geared toward an industrial audience. It provides
peer review. They are available in 18 languages basic information about chemical hazards and
and can be found online through the NIOSH Web synthesizes technical guidance from a number of
site, www.cdc.gov/niosh, or through the ILO Web authorities in chemical safety. The chapters are
site, www.ilo.org. organized by hazard (e.g., “Toxic Chemicals,”
2. NIOSH Pocket Guide to Chemical Hazards (HHS/ “Reactive Chemicals,” and “Cryogens”).

317
 A number of Web-based resources also exist. Some and behaviors in a safe way. Such training is especially
of these are NIOSH Databases and Information useful for learning emergency response procedures.
Resources (www.cdc.gov/niosh) and TOXNET through Another effective tool, particularly when trying to build
the National Library of Medicine (NLM; www.nlm. awareness of a given safety concern, is case studies.
nih.gov). Prior to beginning any laboratory activity, it is important
to ensure that personnel have enough training to safely
The National Library of perform required tasks. If new equipment, materials, or
techniques are to be used, a risk assessment should be
Medicine Databases
performed, and any knowledge gaps should be filled
The databases supplied by NLM are easy to use and before beginning work.
free to access via the Web. TOXNET is an online
collection of toxicological and environmental health Toxic effects of
databases. TOXLINE, for example, is an online database
laboratory chemicals
that accesses journals and other resources for current
toxicological information on drugs and chemicals. The chemicals encountered in the laboratory have a
It covers data published from 1900 to the present. broad spectrum of physical, chemical, and toxicological
Databases accessible through TOXNET include the properties and physiological effects. The risks
Hazardous Substance Data Base (HSDB) Carcinogenic associated with chemicals must be well understood
Potency Database (CPDB), the Developmental and prior to their use in an experiment. The risk of toxic
Reproductive Toxicology Database (DART), the Genetic effects is related to both the extent of exposure and
Toxicology Data Bank (GENE-TOX), the Integrated the inherent toxicity of a chemical. As discussed in
Risk Information System (IRIS), the Chemical detail below, extent of exposure is determined by
Carcinogenesis Research Information System (CCRIS), the dose, the duration and frequency of exposure,
and the International Toxicity Estimates for Risk (ITER). and the route of exposure. Exposure to even large
Other databases supplied by NLM that provide doses of chemicals with little inherent toxicity, such
access to toxicological information are PubMed, as phosphate buffer, presents low risk. In contrast,
which includes access to MEDLINE, PubChem, and even small quantities of chemicals with high inherent
ChemIDPlus. Free text searching is available on most toxicity or corrosivity may cause significant adverse
of the databases. Another source of toxicity data is effects. The duration and frequency of exposure are
Chemical Abstracts Service (CAS). In addition to the also critical factors in determining whether a chemical
NLM, several services provide CAS, including DIALOG, will produce harmful effects. A single exposure to
ORBIT, STN, and SciFinder. Searching procedures some chemicals is sufficient to produce an adverse
for CAS depend on the various services supplying health effect; for other chemicals repeated exposure is
the database. Additional information can be found required to produce toxic effects. For most substances,
on the CAS Web site, www.cas.org. Searching any the route of exposure (through the skin, the eyes, the
Central Marine Fisheries Research Institute

database listed above is best done using the CAS gastrointestinal tract, or the respiratory tract) is also
registry number for the particular chemical. an important consideration in risk assessment. For
chemicals that are systemic toxicants, the internal dose
Training to the target organ is a critical factor. Exposure to
acute toxicants can be guided by well-defined toxicity
One important source of information for laboratory parameters based on animal studies and often human
personnel is training sessions, and the critical place exposure from accidental poisoning. The analogous
it holds in creating a safe environment should not quantitative data needed to make decisions about the
be underestimated. Facts are only as useful as one’s neurotoxicity and immunogenicity of various chemicals
ability to interpret and apply them to a given problem, is often unavailable.
and training provides context for their use. Hands-on,
scenario-based training is ideal because it provides When considering possible toxicity hazards while
the participants with the chance to practice activities planning an experiment, recognizing that the

318
combination of the toxic effects of two substances chemical. The dose is the amount of the chemical
may be significantly greater than the toxic effect of and the response is the effect of the chemical. This
either substance alone is important. Because most relationship is unique for each chemical, although
chemical reactions produce mixtures of substances for similar types of chemicals, the dose-response
with combined toxicities that have never been relationships are often similar. Among the thousands
evaluated, it is prudent to assume that mixtures of of laboratory chemicals, a wide spectrum of doses
different substances (i.e., chemical reaction mixtures) exists that are required to produce toxic effects and
will be more toxic than their most toxic ingredient. even death. For most chemicals, a threshold dose
Furthermore, chemical reactions involving two or has been established (by rule or by consensus) below
more substances may form reaction products that which a chemical is not considered to be harmful to
are significantly more toxic than the starting reactants. most individuals.
This possibility of generating toxic reaction products
may not be anticipated by trained laboratory personnel Some chemicals (e.g., dioxin) produce death in
in cases where the reactants are mixed unintentionally. laboratory animals exposed to microgram doses
For example, inadvertent mixing of formaldehyde (a and therefore are extremely toxic. Other substances,
common tissue fixative) and hydrogen chloride results however, have no harmful effects following doses
in the generation of bis(chloromethyl)ether, a potent in excess of several grams. One way to evaluate
human carcinogen. the acute toxicity (i.e., the toxicity occurring after
a single exposure) of laboratory chemicals involves
All laboratory personnel must understand certain their lethal dose 50 (LD50) or lethal concentration 50
basic principles of toxicology and recognize the major (LC50) value. The LD50 is defined as the amount of a
classes of toxic and corrosive chemicals. The next chemical that when ingested, injected, or applied to
sections of this chapter summarize the key concepts the skin of a test animal under controlled laboratory

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

involved in assessing the risks associated with the use conditions kills one-half (50%) of the animals. The
of toxic chemicals in the laboratory. LD50 is usually expressed in milligrams or grams per
kilogram of body weight. For volatile chemicals
Dose-Response Relationships (i.e., chemicals with sufficient vapor pressure that
inhalation is an important route of chemical entry
Toxicology is the study of the adverse effects of into the body), the LC50 is often reported instead of
chemicals on living systems. The basic tenets of the LD50. The LC50 is the concentration of the chemical
toxicology are that no substance is entirely safe and in air that will kill 50% of the test animals exposed
that all chemicals result in some toxic effects if a high to it. The LC50 is given in parts per million, milligrams
enough amount (dose) of the substance comes in per liter, or milligrams per cubic meter. Also reported
contact with a living system. For example, water, a are LCLO and LDLO values, which are defined as the
vital substance for life, results in death if a sufficiently lowest concentration or dose that causes the death
large amount (i.e., gallons) is ingested at one time. of test animals. In general, the larger the LD50 or LC50,
On the other hand, sodium cyanide, a highly lethal the more chemical it takes to kill the test animals
chemical, produces no permanent (acute) effects if and, therefore, the lower the toxicity of the chemical.
a living system is exposed to a sufficiently low dose. Although lethal dose values may vary among animal
The single most important factor that determines species and between animals and humans, chemicals
whether a substance is harmful (or, conversely, safe) that are highly toxic to animals are generally highly
to an individual is the relationship between the toxic to humans.
amount (and concentration) of the chemical reaching
the target organ, and the toxic effect it produces. Assessing Risks of Exposure to
For all chemicals, there is a range of concentrations
Toxic Laboratory Chemicals
that result in a graded effect between the extremes
of no effect and death. In toxicology, this range is Exposure to a harmful chemical results in local toxic
referred to as the dose-response relationship for the effects, systemic toxic effects, or both. Local effects

319
 involve injury at the site of first contact; the eyes, implicated as a potential cause of cancer in humans),
the skin, the nose and lungs, and the digestive tract reproductive toxins, and compounds with a high
are typical sites of local reactions. Examples of local degree of acute toxicity. When working with these
effects include (1) inhalation of hazardous materials substances for the first time, it is prudent to consult
causing toxic effects in the nose and lungs; (2) contact with a safety professional prior to beginning work.
with harmful materials on the skin or eyes leading to This will provide a second set of trained eyes to review
effects ranging from mild irritation to severe tissue the safety protocols in place and will help ensure that
damage; and (3) ingestion of caustic substances any special emergency response requirements can
causing burns and ulcers in the mouth, esophagus, be met in the event of exposure of personnel to the
stomach, and intestines. Systemic effects, by contrast, material or accidental release.
occur after the toxicant has been absorbed from the
site of contact into the bloodstream and distributed The following are the most common classes of toxic
throughout the body. Some chemicals produce substances encountered in laboratories.
adverse effects on all tissues of the body, but others
tend to selectively injure a particular tissue or organ Acute Toxicants
without affecting others. The affected organ (e.g.,
liver, lungs, kidney, and central nervous system) is Acute toxicity is the ability of a chemical to cause a
referred to as the target organ of toxicity, although harmful effect after a single exposure. Acutely toxic
it is not necessarily the organ where the highest agents cause local toxic effects, systemic toxic effects,
concentration of the chemical is found. Hundreds or both, and this class of toxicants includes corrosive
of systemic toxic effects of chemicals are known; they chemicals, irritants, and allergens (sensitizers). In
result from single (acute) exposures or from repeated assessing the risks associated with acute toxicants, it
or long-duration (chronic) exposures that become is useful to classify a substance according to the acute
evident only after a long latency period. toxicity hazard level as shown in the following table.

Hazard Level Toxicity Rating Oral LD50 (rats, per kg) Skin Contact Inhalation LC50 (rats, Inhalation LC50 (rats,
LD50 (rabbits, per kg) ppm for 1 h) mg/m3 for 1 h)
High Highly toxic <50 mg <200 mg <200 <2,000

Medium Moderately toxic 50 to 500 mg 200 mg to 1 g 200 to 2,000 2,000 to 20,000

Low Slightly toxic 500 mg to 5 g 1 to 5 g 2,000 to 20,000 20,000 to 200,000

Laboratory chemicals are grouped into several classes Acute Toxicity Hazard Level
of toxic substances, and many chemicals display more
than one type of toxicity. The first step in assessing the Special attention is given to any substance classified
risks associated with a planned laboratory experiment according to the above criteria as having a high level
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involves identifying which chemicals in the proposed of acute toxicity hazard. Chemicals with a high level
experiment are potentially hazardous substances. The of acute toxicity make up one of the categories of
term “health hazard” includes chemicals that are PHSs defined by the OSHA Laboratory Standard.
carcinogens, toxic or highly toxic agents, reproductive The following table lists some of the most common
toxins, irritants, corrosives, sensitizers, hepatotoxins, chemicals with a high level of acute toxicity that are
nephrotoxins, neurotoxins, agents that act on the encountered in the laboratory.
hematopoietic systems, and agents that damage the
lungs, skin, eyes, or mucous membranes. The OSHA Examples of Compounds with a High Level of
Laboratory Standard further requires that certain Acute Toxicity
chemicals be identified as particularly hazardous Acrolein Methyl fluorosulfonate
substances (commonly known as PHSs) and handled Arsine Nickel carbonyl
using special additional procedures. PHSs include
Chlorine Nitrogen dioxide
chemicals that are select carcinogens (those strongly

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Diazomethane Osmium tetroxide
inorganic cyanides are examples of such substances.
Diborane (gas) Ozone

Dimethyl mercury Phosgene Neurotoxins

Hydrogen cyanide Sodium azide
Neurotoxic chemicals induce an adverse effect on the
Hydrogen fluoride Sodium cyanide (and other cyanide salts)
structure or function of the central or peripheral nervous
system, which can be permanent or reversible. The
Types of Toxins detection of neurotoxic effects may require specialized
laboratory techniques, but often they are inferred from
Irritants, Corrosive Substances, behavior such as slurred speech and staggered gait.
Many neurotoxins are chronically toxic substances with
Allergens, and Sensitizers
adverse effects that are not immediately apparent.
Lethal dose and other quantitative toxicological Some chemical neurotoxins that may be found in
parameters generally provide little guidance in the laboratory are mercury (inorganic and organic),
assessing the risks associated with corrosives, organophosphate pesticides, carbon disulfide, xylene,
irritants, allergens, and sensitizers because these tricholoroethylene, and n-hexane.
toxic substances exert their harmful effects locally.
It would be very useful for the chemical research Reproductive and
community if a quantitative measure for such effects
Developmental Toxins
were developed. When planning an experiment that
involves corrosive substances, basic prudent handling Reproductive toxins are defined by the OSHA Laboratory
practices should be reviewed to ensure that the skin, Standard as substances that cause chromosomal
face, and eyes are protected adequately by the proper damage (mutagens) and substances with lethal or

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

choice of corrosion-resistant gloves and protective teratogenic (malformation) effects on fetuses. These
clothing and eyewear, including, in some cases, face substances have adverse effects on various aspects of
shields. Similarly, LD50 and LC50 data are not indicators reproduction, including fertility, gestation, lactation,
of the irritant effects of chemicals, and therefore and general reproductive performance, and can affect
special attention should be paid to the identification both men and women. Various reproductive hazards
of irritant chemicals by consulting LCSSs, MSDSs, and have been noted following exposure to halogenated
other sources of information. Allergens and sensitizers hydrocarbons, nitro aromatics, arylamines, ethylene
are another class of acute toxicants with effects that glycol derivatives, mercury, bromine, carbon disulfide,
are not included in LD50 or LC50 data. and other chemical reagents.

Asphyxiants Carcinogens
Asphyxiants are substances that interfere with the A carcinogen is a substance capable of causing cancer.
transport of an adequate supply of oxygen to vital Cancer, in the simplest sense, is the uncontrolled
organs of the body. The brain is the organ most growth of cells and can occur in any organ. The
easily affected by oxygen starvation, and exposure to mechanism by which cancer develops is not well
asphyxiants leads to rapid collapse and death. Simple understood, but the current thinking is that some
asphyxiants are substances that displace oxygen from chemicals interact directly with DNA, the genetic
the air being breathed to such an extent that adverse material in all cells, to result in permanent alterations.
effects result. Acetylene, carbon dioxide, argon, Other chemical carcinogens modify DNA indirectly
helium, ethane, nitrogen, and methane are common by changing the way cells grow. Carcinogens are
asphyxiants. Certain other chemicals have the ability chronically toxic substances; that is, they cause
to combine with hemoglobin, thus reducing the damage after repeated or long-duration exposure,
capacity of the blood to transport oxygen. Carbon and their effects may become evident only after a long
monoxide, hydrogen cyanide, and certain organic and latency period. Carcinogens are particularly insidious

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 toxins because they may have no immediate apparent
Flammability Characteristics
harmful effects.

Flash Point
Flammable, reactive, and
The flash point is the lowest temperature at which
explosive hazards
a liquid has a sufficient vapor pressure to form an
In addition to the hazards due to the toxic effects of ignitable mixture with air near the surface of the liquid.
chemicals, hazards due to flammability, explosivity, Note that many common organic liquids have a flash
and reactivity need to be considered in risk assessment. point below room temperature: for example, acetone
Reactive hazards arise when the release of energy (-18ºC), benzene (-11.1ºC), diethyl ether (-45ºC),
from a chemical reaction occurs in quantities or at and methyl alcohol (11.1ºC). The degree of hazard
rates too great for the energy to be absorbed by the Table 1. NFPA Fire Hazard Ratings, Flash Points (FP),
immediate environment of the reacting system, and Boiling Points (bp), Ignition Temperatures, and
material damage results. Flammable Limits of Some Common Laboratory
Chemicals
NFPA Flammable
The following outline provides a summary of the steps Flash Boiling Ignition Limits (% by
Flamm
Point Point Tempera- volume)
that laboratory personnel should use to assess the ability
(ºC) (ºC) ture (ºC)
Ratinga Lower Upper
risks of managing physical hazards in the laboratory.
Acetaldehyde 4 -39 21 175 4 60
Acetic acid 2 39 118 463 4 19.9
• Identify chemicals to be used and circumstances (glacial)
of use.  Acetone 3 -20 56 465 2.5 12.8
• Consult sources of information. Consult an up-to- Acetonitrile 3 6 82 524 3 16
date laboratory chemical safety summary, material Carbon 4 -30 46 90 1.3 50
disulfide
safety data sheet, or NIOSH Pocket Guide to
Cyclohexane 3 -20 82 245 1.3 8
Chemical Hazards (HHS/CDC/NIOSH, 2007). Diethylamine 3 -23 57 312 1.8 10.1
• Evaluate type of physical, flammable, explosive, or Diethyl ether 4 -45 35 180 1.9 36
reactive hazard(s) posed by the chemicals.  Dimethyl 2 95 189 215 2.6 42
• Evaluate the hazards posed by chemical changes sulfoxide
Ethyl alcohol 3 13 78 363 3.3 19
over the course of the experiment. 
Heptane 3 -4 98 204 1.05 6.7
• Evaluate type of physical hazard(s) posed by the Hexane 3 -22 69 225 1.1 7.5
equipment required.  Hydrogen 4 -252 500 4 75
• Select appropriate procedures to minimize risk.  Isopropyl 3 12 83 399 2 12.7
• Prepare for contingencies. Be aware of institutional alcohol @ 200
(93)
procedures in the event of emergencies Methyl alcohol 3 11 64 464 6 36
and accidents.
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Methyl ethyl 3 -9 80 404 1.4 @ 11.4

ketone 200 @ 200
(93) (93)
Flammable Hazards Pentane 4 <-40 36 260 1.5 7.8
Styrene 3 31 146 490 0.9 6.8
Flammable substances, those that readily catch fire Tetrahydrofuran 3 -14 66 321 2 11.8
and burn in air may be solid, liquid, or gaseous. Toluene 3 4 111 480 1.1 7.1
The most common fire hazard in the laboratory is a p-Xylene 3 25 138 528 1.1 7
flammable liquid or the vapor produced from such a a
0, will not burn under typical fire conditions; 1, must be preheated to
liquid. An additional hazard is that compounds can burn, liquids with FP ≥ 93.4ºC (200ºF); 2, ignitable when moderately
heated, liquids with FP between 37.8ºC (100ºF) and 93.4ºC (200ºF); 3,
enflame so rapidly that it produces an explosion. ignitable at ambient temperature, liquids with FP < 22.8ºC (73ºF), bp ≥
Proper use of substances that cause fire requires 37.8ºC (100ºF) or FP between 22.8ºC and 37.8ºC (100ºF); 4, extremely
flammable, readily dispersed in air, and burns readily, liquids with FP <
knowledge of their tendencies to vaporize, 22.8ºC (73ºF), bp < 37.8ºC (100ºF).
SOURCE: Adapted with permission from Fire Guide to Hazardous
ignite, or burn under the variety of conditions in
Materials (13th Edition), Copyright © 2001, National Fire Protection
the laboratory. Association.

322
associated with a flammable liquid also depends moisture and the thermodynamics of metal oxide or
on other properties, such as its ignition point and metal nitride formation. Other reducing agents, such
boiling point. At ambient pressure and temperature, as metal hydrides, alloys of reactive metals, low-valent
an acetone spill produces a concentration as high as metal salts, and iron sulfides, are also pyrophoric.
23.7% acetone in air. Although it is not particularly
toxic, with a flash point of -18ºC and upper and Explosive Hazards
lower flammable limits of 2.6% and 12.8% acetone
in air, respectively clearly an acetone spill produces An explosive is any chemical compound or
an extreme fire hazard. mechanical mixture that, when subjected to heat,
impact, friction, detonation, or other suitable
Ignition Temperature initiation, undergoes rapid chemical change,
evolving large volumes of gases that exert pressure
The ignition temperature (autoignition temperature) on the surrounding medium. Hydrogen and chlorine
of a substance, whether solid, liquid, or gaseous, react explosively in the presence of light. Acids,
is the minimum temperature required to initiate or bases, and other substances catalyze the explosive
cause self-sustained combustion independent of the polymerization of acrolein, and many metal ions
heat source. The lower the ignition temperature, can catalyze the violent decomposition of hydrogen
the greater the potential for a fire started by typical peroxide. Shock-sensitive materials include acetylides,
laboratory equipment. For instance, carbon disulfide azides, nitrogen triiodide, organic nitrates, nitro
has an ignition temperature of 90ºC, and it can be compounds, perchlorate salts (especially those of
set off by a steam line or a glowing light bulb. Diethyl heavy metals such as ruthenium and osmium), many
ether has an ignition temperature of 160ºC and can organic peroxides, and compounds containing diazo,
be ignited by a hot plate. halamine, nitroso, and ozonide functional groups.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Reactive Hazards The following table lists a number of explosive
compounds. Some are set off by the action of a metal
spatula on the solid; some are so sensitive that they
Water Reactives are set off by the action of their own crystal formation.
Water-reactive materials are those that react violently Diazomethane (CH2N2) and organic azides, for
with water. Alkali metals (e.g., lithium, sodium, and example, may decompose explosively when exposed
potassium), many organometallic compounds, and to a ground glass joint or other sharp surfaces.
some hydrides react with water to produce heat and
flammable hydrogen gas, which ignites or combines Functional Groups in Some
explosively with atmospheric oxygen. Some anhydrous
metal halides (e.g., aluminum bromide), oxides (e.g.,
Explosive Compounds
calcium oxide), and nonmetal oxides (e.g., sulfur
trioxide), and halides (e.g., phosphorus pentachloride) Azos, Peroxides,
react exothermically with water, resulting in a violent and Peroxidizables
reaction if there is insufficient coolant water to
dissipate the heat produced. Organic azo compounds and peroxides are among the
most hazardous substances handled in the chemical
Pyrophorics laboratory but are also common reagents that often
are used as free radical sources and oxidants. They are
For pyrophoric materials, oxidation of the compound generally low-power explosives that are sensitive to
by oxygen or moisture in air proceeds so rapidly shock, sparks, or other accidental ignition. They are
that ignition occurs. Many finely divided metals are far more shock sensitive than most primary explosives
pyrophoric, and their degree of reactivity depends on such as TNT. Inventories of these chemicals should be
particle size, as well as factors such as the presence of limited and subject to routine inspection. Liquids or

323
 Structural Feature Compound Structural Feature Compound

Acetylenic compounds Arenediazoates

Metal acetylides Arenediazo aryl sulfides

Haloacetylene derivatives Bis-arenediazo oxides

Bis-arenediazo sulfides
Diazirines

Trizazenes (R = H, —CN, -OH, —NO)

Diazo compounds

High-nitrogen compounds, tetrazoles

Nitroso compounds

Alkylhydroperoxides
Nitroalkanes, C-nitro and polynitroaryl
compounds

Peroxyacids
Polynitroalkyl compounds

Peroxides (cyclic, diacyl, dialkyl)

Acyl or alkyl nitrites

Acyl or alkyl nitrates Peroxyesters

Aminechromium peroxocomplexes
1,2-Epoxides

Azides (acyl, halogen, nonmetal,

Metal fulminates or aci-nitro salts organic)

Diazoniumsulfides and derivatives,

Fluorodinitromethyl compounds “xanthates”

Hydrazinium salts, oxosalts of

N-Metal derivatives
Central Marine Fisheries Research Institute

nitrogenous bases

N-Nitroso compounds Hydroxylammonium salts

N-Nitro compounds Diazonium carboxylates or salts

Alkyl perchlorates, chlorite salts, halogen

Azo compounds oxides, hypohalites, perchloric acid,
perchloryl compounds

SOURCE: Carson and Mumford (2002). Reprinted from Hazardous Chemicals Handbook (Second Edition), Carson, P. and Mumford, C. “Reactive
Chemicals”, p. 228, Copyright 2002, with permission from Elsevier.

324
solutions of these compounds should not be cooled to Essentially all compounds containing C—H bonds pose
the point at which the material freezes or crystallizes the risk of peroxide formation if contaminated with
from solution, however, because this significantly various radical initiators, photosensitizers, or catalysts.
increases the risk of explosion. Refrigerators and For instance, secondary alcohols such as isopropanol
freezers storing such compounds should have a form peroxides when exposed to normal fluorescent
backup power supply in the event of electricity loss. lighting and contaminated with photosensitizers,
Users should be familiar with the hazards of these such as benzophenone. Acetaldehyde, under normal
materials and trained in their proper handling. conditions, autoxidizes to form acetic acid.

Certain common laboratory chemicals form peroxides Physical hazards

on exposure to oxygen in air (see the following table).
Over time, some chemicals continue to build peroxides Compressed Gases
to potentially dangerous levels, whereas others
accumulate a relatively low equilibrium concentration Compressed gases can expose the trained laboratory
of peroxide, which becomes dangerous only after personnel to both mechanical and chemical hazards,
being concentrated by evaporation or distillation. depending on the gas. Hazards can result from the
flammability, reactivity, or toxicity of the gas; from
Classes of Chemicals That Can the possibility of asphyxiation; and from the gas
Form Peroxides compression itself, which could lead to a rupture of
Class A: Chemicals that form explosive levels of peroxides without the tank or valve.
concentration
Isopropyl ether Sodium amide (sodamide)
Nonflammable Cryogens
Butadiene Tetrafluoroethylene

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Chlorobutadiene (chloroprene) Divinyl acetylene
Nonflammable cryogens (chiefly liquid nitrogen) can
Potassium amide Vinylidene chloride
cause tissue damage from extreme cold because of
Potassium metal
contact with either liquid or boil-off gases. In poorly
Class B: These chemicals are a peroxide hazard on concentration ventilated areas, inhalation of gas due to boil off
(distillation/evaporation). A test for peroxide should be performed if
concentration is intended or suspected.
or spills can result in asphyxiation. Another hazard
is explosion from liquid oxygen condensation in
Acetal Dioxane (p-dioxane)
vacuum traps or from ice plug formation or lack of
Cumene Ethylene glycol dimethyl
functioning vent valves in storage Dewars. Because
Cyclohexene ether (glyme)
1 volume of liquid nitrogen at atmospheric pressure
Cyclooctene Furan
vaporizes to 694 volumes of nitrogen gas at 20ºC,
Cyclopentene Methyl acetylene
the warming of such a cryogenic liquid in a sealed
Diaacetylene Methyl cyclopentane
container produces enormous pressure, which can
Dicyclopentadiene Methyl-isobutyl ketone
rupture the vessel.
Diethylene glycol dimethyl Tetrahydrofuran
ether (diglyme) Tetrahydronaphthalene
High-Pressure Reactions
Diethyl ether Vinyl ethers

Class C: Unsaturated monomers that may autopolymerize as a result Experiments that generate high pressures or are
of peroxide accumulation if inhibitors have been removed or are
depleted carried out at pressures above 1 atm can lead to
Acrylic acid Styrene explosion from equipment failure. For example,
Butadiene Vinyl acetate hydrogenation reactions are frequently carried
Chlorotrifluoroethylene Vinyl chloride out at elevated pressures, and a potential hazard
Ethyl acrylate Vinyl pyridine is the formation of explosive O2/H2 mixtures and
Methyl methacrylate the reactivity/pyrophoricity of the catalyst. High
* These lists are illustrative, not comprehensive. SOURCES: Jackson et al. pressures can also be associated with the use of
(1970) and Kelly (1996). supercritical fluids.

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 Vacuum Work laboratory settings. The insulation on wires can
Precautions to be taken when working with vacuum be eroded by corrosive chemicals, organic solvent
lines and other glassware used at sub ambient vapors, or ozone (from ultraviolet lights, copying
pressure are mainly concerned with the substantial machines, and so forth). Eroded insulation on
danger of injury in the event of glass breakage. The electrical equipment in wet locations such as
degree of hazard does not depend significantly on cold rooms or cooling baths must be repaired
the magnitude of the vacuum because the external immediately. In addition, sparks from electrical
pressure leading to implosion is always 1 atmosphere. equipment can serve as an ignition source in the
Thus, evacuated systems using aspirators merit as presence of flammable vapor. Operation of certain
much respect as high-vacuum systems. Injury due to equipment (e.g., electrophoresis equipment) may
flying glass is not the only hazard in vacuum work. involve high voltages and stored electrical energy.
Additional dangers can result from the possible toxicity
of the chemicals contained in the vacuum system, as Magnetic Fields
well as from fire following breakage of a flask (e.g.,
of a solvent stored over sodium or potassium). Increasingly, instruments that generate large static
magnetic fields (e.g., NMR spectrometers) are present
Ultraviolet, Visible, and Near- in research laboratories. Such magnets typically have
Infrared Radiation fields of 14,000 to 235,000 G (1.4 to 23.5 T), far above
that of Earth’s magnetic field, which is approximately
Ultraviolet, visible, and infrared radiation from lamps 0.5 G. The magnitude of these large static magnetic
and lasers in the laboratory can produce a number fields falls off rapidly with distance. Many instruments
of hazards. Medium-pressure Hanovia 450 Hg lamps now have internal shielding, which reduces the strength
are commonly used for ultraviolet irradiation in of the magnetic field outside of the instrument. Strong
photochemical experiments. Ultraviolet lights used attraction occurs when the magnetic field is greater
in biosafety cabinets, as decontamination devices, than 50 to 100 G and increases by the seventh power
or in light boxes to visualize DNA can cause serious as the separation is reduced. However, this highly
skin and corneal burns. Powerful arc lamps can nonlinear falloff of magnetic field with distance results
cause eye damage and blindness within seconds. in an insidious hazard.
When incorrectly used, the light from lasers poses a
hazard to the eyes of the operators and other people Ergonomic Hazards in the Laboratory
present in the room and is also a potential fire hazard.
Depending on the type of laser, the associated hazards General workplace hazards also apply in the
can include mutagenic, carcinogenic, or otherwise laboratory. For example, laboratory personnel are
toxic laser dyes and solvents; flammable solvents; often involved in actions such as pipetting and
ultraviolet or visible radiation from the pump lamps; computer work that can result in repetitive-motion
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and electric shock from lamp power supplies. injuries. Working at a bench or at a microscope
without considering posture can result in back
Electrical Hazards strain, and some instruments require additional
in-room ventilation that may raise the background
The electrocution hazards of electrically powered noise level to uncomfortable or hazardous levels.
instruments, tools, and other equipment are almost With these and other issues such as high or low
eliminated by taking reasonable precautions, and room temperatures and exposure to vibrations,
the presence of electrically powered equipment it is important to be aware of and to control
in the laboratory need not pose a significant risk. such issues to reduce occupational injuries. For
But, in the laboratory these safety features should example, microscope users may find that using a
not be defeated by thoughtless or ill-informed camera to view images on a screen, rather than
modification. Equipment malfunctions can lead direct viewing through the eyepiece, reduces back
to electrical fires. Some special concerns arise in and eye strain. The Centers for Disease Control

326
and Prevention (CDC) and the National Institutes deficient viral vectors, for protein expression or other in
of Health have information on their Web sites vitro applications. Risk assessment for biological toxins is
(www.cdc.gov and www.nih.gov, respectively) similar to that for chemical agents and is based primarily
describing specific ergonomic concerns for on the potency of the toxin, the amount used, and the
laboratories and proposed solutions. The CDC procedures in which the toxin is used.
provides a downloadable self-assessment form
to aid in evaluating these hazards. NIOSH (www. Suggested readings
cdc.gov/niosh) and OSHA (www.osha.gov) provide
information about vibration, noise levels, and other OSHA Hazard Communication Standard 1910.1200
workplace hazards. Dedicated website: http://www.unece.org
International Chemical Safety Cards from the International
Programme on Chemical Safety IPCS, 2009
Bio hazards Dedicated website: www.cdc.gov/niosh
Dedicated website: www.ilo.org
NIOSH Pocket Guide to Chemical Hazards. HHS/ CDC/
Biohazards are a concern in laboratories in which NIOSH, 2007
microorganisms, or material contaminated with them, Dedicated website: http://www.cdc.gov/niosh
A Comprehensive Guide to the Hazardous Properties of Chemical
are handled. Anyone who is likely to come in contact Substances, 3rd edition (Patnaik, 2007).
with blood or potentially infectious materials at work is Hazardous Chemicals Handbook, 2nd edition, Carson and
Mumford, 2002
covered under OSHA’s Bloodborne Pathogen Standard. NIOSH Databases and Information Resources, www.cdc.gov/niosh
These hazards are usually present in clinical and infectious TOXNET through the National Library of Medicine (NLM; dedicated
disease research laboratories but may also be present in website: www.nlm.nih.gov
Dedicated website: www.cas.org
any laboratory in which bodily fluids, tissues, or primary NIOSH Pocket Guide to Chemical Hazards (HHS/CDC/NIOSH, 2007
or immortalized cell lines of human or animal origin are Dedicated website: www.cdc.gov
Dedicated website: www.nih.gov
handled. Biohazards are also present in any laboratory

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Dedicated website: www.cdc.gov/niosh
that uses microorganisms, including replication- Dedicated website: www.osha.gov

327
 General Biochemical Methodologies
Kajal Chakraborty
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction up-to-date by entering the name of each

All biochemistry experimental activities in a research experiment and page number.
laboratory are replete with techniques that must 3. Use the right-hand pages only for writing your
be carried out on an almost daily basis. This lecture experimental notes. The left-hand pages may
outlines the theoretical and practical aspects of some be used as scratch paper for your own personal
of these general and routine procedures. notes, reminders, or calculations not appropriate
for the main entry. Each entry for an experiment
Keeping records or project must begin with a title and date.

and communicating
Using biochemical reagents
experimental results
and solutions
The biochemistry laboratory experience is not finished
when you complete the experimental procedure and Water Purity
leave the laboratory. Reports are most easily prepared
outside of the lab using notes taken in a laboratory Water is the most common and widely-used substance
notebook while the experiment is in progress. These in the biochemistry laboratory. Applications of water
notes usually include procedural details, preparation usage include: (1) solvent for preparing most buffer
of all reagents and solutions, setup of equipment, and reagent solutions; (2) column chromatography;
collection of data, and your thought processes and (3) high-performance liquid chromatography; (4)
observations during the experiment. Experiments are tissue culture; and (5) washing glassware. Both
often complex and move rather quickly, and it would the quality and quantity of water required must be
be impossible to write down your data and not a considered for each lab application. Ordinary tap
good practice to record results on scraps of paper water is relatively abundant, but its quality is very
Central Marine Fisheries Research Institute

or on paper towels that may easily become lost or low. It contains a variety of impurities including
destroyed. The lab notebook will also come in handy particulate matter (sand, silt, etc.); dissolved organics,
if you need to troubleshoot or repeat an experiment inorganics, and gases; and microorganisms (bacteria,
because of inconsistent results. Your instructor may viruses, protozoa, and algae). In addition, the natural
have his or her own rules for preparation of the lab degradation of microorganisms leads to the presence
notebook, but here are some useful guidelines: of byproducts called pyrogens. Tap water should never
1. The notebook should be hardbound with ruled be used for the preparation of reagent solutions or
pages; writing should be done with pen. This for any sensitive procedures. For most laboratory
provides a permanent, durable record and the procedures, it is recommended that some form of
potential for construction of tables, graphs, purified water be used. There are five basic water
charts, etc. Number each page of the book. purification technologies—distillation, ion-exchange,
2. Save the first few pages of the book for activated carbon adsorption, reverse osmosis, and
construction of a table of contents. Keep this membrane filtration. Most academic and industrial

328
research laboratories are equipped with “in-house” experiments, it is best to refrain from acetone washing.
purified water, which typically is produced by a Glassware and plastic ware should be rinsed well with
combination of the above purifying purified water and dried in an oven designated for
glassware, not one used for drying chemicals. Never
processes and piped throughout all the labs in a clean cuvettes or any optically polished glassware with
building. The water quality necessary will depend on ethanolic KOH or other strong base, as this will cause
the solutions to be prepared and on the biochemical etching. All glass cuvettes should be cleaned carefully
procedures to be investigated. For most procedures with hot tap water or 0.5% detergent solution, in a
carried out in the biochemistry lab, water purified sonicator bath or in a cuvette washer, followed by
by ion-exchange, reverse osmosis, or distillation is thorough rinsing with purified water.
usually acceptable.
Concentrations
Cleaning and Calculations
Laboratory Glassware The concentrations for solutions used in the
The results of your experimental work will depend, to biochemistry laboratory may be expressed in several
a great extent, on the cleanliness of your equipment, different units. The most common units are:
especially glassware used for preparing and
transferring solutions. There are at least two important Molarity (M): concentration based on the number of
reasons for this: (1) many of the chemicals and moles of solute per liter of solution. A 1 M solution of
biochemicals will be used in milligram, microgram, the amino acid alanine contains 1 mole, or 89.1 g, of
or even nanogram amounts. Any contamination, alanine in a solution volume of 1 liter. In biochemistry,
whether on the inner walls of a beaker, in a pipet, or it is more common to use concentration ranges that

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

in a glass cuvette, could be a significant percentage of are millimolar micromolar or nanomolar
the total experimental sample; (2) many biochemicals
and biochemical processes are sensitive to one or more Percent by weight (% wt/wt): concentration
of the following common contaminants: metal ions, based on the number of grams of solute per 100 g
detergents, and organic residues. In fact, the objective of solution.
of many experiments is to investigate the effect of a
metal ion, organic molecule, or other chemical agent Preparing and
on a biochemical process. Contaminated glassware
will virtually ensure failure in these activities.
Storing Solutions
In general, solid solutes should be weighed on
The preferred method for cleaning glassware is weighing paper or plastic weighing boats, with
to begin with hot tap water. Rinse the glassware the use of an electronic analytical or top-loading
at least 10 times with this; then rinse 4–6 times balance. Liquids are more conveniently dispensed
with distilled or de-ionized water. Occasionally it is by volumetric techniques; however, this assumes
necessary to use a detergent for cleaning. Use a dilute that the density is known. If a small amount of a
detergent solution (0.5% in water) followed by 5–10 liquid is to be weighed, it should be added to a
water rinses with distilled or de-ionized water. Dry tared flask by means of a disposable Pasteur pipet
equipment is required for most processes carried out with a latex bulb. The hazardous properties of all
in the biochemistry laboratory. When you needed dry materials should be known before use and the proper
glassware in the organic laboratory, you probably safety precautions obeyed. The storage conditions
rinsed the glassware with acetone, which rapidly of reagents and solutions in the biochemistry lab
evaporated, leaving a dry surface. Unfortunately, this are especially critical. Although some will remain
technique coats the surface with an organic residue stable indefinitely at room temperature, it is good
consisting of nonvolatile contaminants found in the practice to store all solutions in a closed container.
acetone. Because this residue could interfere with your Often it is necessary to store some solutions in a

329
 refrigerator at this inhibits bacterial growth and slows the top graduated mark. Then, remove the bulb and
decomposition of the reagents. Some solutions may quickly grasp the pipet with your index finger over
require storage below If these are aqueous solutions the top end of the pipet. The level of solution in the
or others that will freeze, be sure there is room for pipet will fall slightly, but should not fall below the
expansion inside the container. Stored solutions top graduated mark. If it does fall too low, use the
must always have a label containing the name and bulb to refill.
concentration of the solution, the date prepared,
and the name of the preparer. All stored containers, Special procedures are required for cleaning glass
whether at room temperature, or below freezing, pipets. Immediately after use, every pipet should be
must be properly sealed. This reduces contamination placed, tip up, in a vertical cylinder containing warm
by bacteria and vapors in the laboratory air (carbon tap water or a dilute detergent solution (less than
dioxide, ammonia, HCl, etc.). Volumetric flasks, of 0.5%). The pipet must be completely covered with
course, have glass stoppers, but test tubes, Erlenmeyer solution. This ensures that any reagent remaining in
flasks, bottles, and other containers should be sealed the pipet is forced out through the tip. If reagent
with screw caps, corks, or hydrocarbon foil (Parafilm). solutions are allowed to dry inside a pipet, the tips
Remember that hydrocarbon foil, a wax, is dissolved can easily become clogged and are very difficult to
by solutions containing nonpolar organic solvents open. After several pipets have accumulated in the
like chloroform, diethyl ether, and acetone. Bottles of water or detergent solution, the pipets should be
pure chemicals and reagents should also be properly transferred to a pipet rinser. Pipet rinsers continually
stored. Many manufacturers now include the best cycle fresh water through the pipets. Immediately
storage conditions for a reagent on the label. The after detergent wash, tap water may be used to rinse
common conditions are: store at room temperature; the pipets, but distilled water should be used for the
store at store below or store in a desiccator at room final rinse. Pipets may then be dried in an oven.
temperature, or below. Many biochemical reagents
form hydrates by taking up moisture from the air. pH, buffers, electrodes,
If the water content of a reagent increases, the
molecular weight and purity of the reagent change.
and biosensors
Most biological processes in the cell take place in a
Quantitative transfer water-based environment. Water is an amphoteric
substance; that is, it may serve as a proton donor
of liquids (acid) or a proton acceptor (base). The following
Practical biochemistry is highly reliant on analytical equation shows the ionic equilibrium of water.
methods. Many analytical techniques must be
mastered, but few are as important as the quantitative H2O = H+ + OH-
transfer of solutions. Some type of pipet will almost
Central Marine Fisheries Research Institute

always be used in liquid transfer. The use of any pipet In pure water, in other words, the pH or is 7.
requires some means of drawing reagent into the
pipet. Liquids should never be drawn into a pipet by [The pH scale runs from 0 (very acidic) to 14 (very
mouth suction on the end of the pipet! Small latex basic)]. Acidic and basic molecules, when dissolved in
bulbs are available for use with disposable pipets. water in a biological cell or test tube, react with either
For volumetric and graduated pipets, two types of hydrogen ions or hydroxide ions to shift the equilibrium
bulbs are available. One type features a special conical of the equation and result in a pH change of the
fitting that accommodates common sizes of pipets. To solution. Biochemical processes occurring in cells and
use these, first place the pipet tip below the surface of tissues depend on strict regulation of the hydrogen
the liquid. Squeeze the bulb with the left hand (if you ion concentration. Natural acids and bases are often
are a right-handed pipettor) and then hold it tightly generated in cells by normal biological processes and
to the end of the pipet. Slowly release the pressure they must be neutralized by buffers. Biological pH is
on the bulb to allow liquid to rise to 2 or 3 cm above maintained at a constant value by naturally-occurring

330
buffer systems such as phosphate. When biological HA represents a weak acid and represents its
processes are studied in vitro, artificial media must be conjugate base; represents the rate constant for
prepared that mimic the cell’s natural environment. dissociation of the acid and k2 the rate constant for
Because of the dependence of biochemical reactions association of the conjugate base and hydrogen ion.
on pH, the accurate determination of hydrogen ion
concentration has always been of major interest.

Measurement of pH The equilibrium constant, for the weak acid HA is

defined by Equation 3.4.
A pH measurement is usually taken by immersing a glass
or plastic combination electrode into a solution and which can be rearranged to define the
reading the pH directly from a meter. At one time, pH following equation,
measurements required two electrodes, a pH-dependent
glass electrode sensitive to ions and a pH-independent
calomel reference electrode. The potential difference
that develops between the two electrodes is measured
as a voltage, as defined by the following equation. The is often reported as pH, which is in a similar
fashion, is represented by the following equation,
V = E const + (2.303 RT/F) * pH

where
• V = voltage of the completed circuit
• Econstant = potential of reference electrode This equation is termed as the Henderson-Hasselbalch

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

• R = the gas constant equation, which defines the relationship between
• T = the absolute temperature pH and the ratio of acid and conjugate base
• F = the Faraday constant concentrations. The salt of the acid is also referred
to as the proton acceptor (A) and the acid (HA) as
A pH meter is standardized with buffer solutions of the proton donor (D). The Henderson-Hasselbalch
known pH before a measurement of an unknown equation is of great value in buffer chemistry because
solution is taken. It should be noted from the above it can be used to calculate the pH of a solution if the
equation that the voltage depends on temperature. molar ratio of buffer ions and the of HA are known.
Hence, pH meters must have some means for Also, the molar ratio of HA to that is necessary to
temperature correction. Older instruments usually prepare a buffer solution at a specific pH can be
have a knob labeled “temperature control,” which calculated if the is known.
is adjusted by the user to the temperature of the
measured solution. Newer pH meters automatically A solution containing both HA and has the capacity
display a temperature-corrected pH value. to resist changes in pH; i.e., it acts as a buffer. If
acid were added to the buffer solution, it would be
Biochemical Buffers neutralized by in solution:

Buffer ions are used to maintain solutions at constant

pH values. Weak acids and bases do not completely
dissociate in solution, but exist as equilibrium mixtures Base added to the buffer solution would be neutralized
as described in the following equation. by reaction with HA:

331
 Biosensors product is the result of coordination of peptide
nitrogen atoms with the reagents, and the amount
Electrodes or electrode-like devices are currently of product formed depends on the concentration
being developed for the specific measurement of of protein. In practice, a calibration curve must be
physiologically important molecules such as urea, prepared by using a standard protein solution. An
carbohydrates, enzymes, antibodies, and metabolic aqueous solution of bovine serum albumin (BSA) is
products. This type of device, now referred to as a a commonly used standard. Various known amounts
biosensor, is an analytical tool or system consisting of this solution are treated with the biuret reagent,
of an immobilized biological material (such as and the color is allowed to develop. Measurements
an enzyme, antibody, whole cell, organelle, or of absorbance at 540 nm are made against a blank
combinations thereof) in intimate contact with containing biuret reagent and buffer or water. The
a suitable transducer device that will convert the A540 data are plotted versus protein concentration
biochemical signal into a quantifiable electric signal. (mg/mL) or amount of protein (mg). Unknown protein
The important components of a biosensor are: samples are treated with biuret reagent and measured
after color development. The protein concentration
1. A reaction center consisting of a membrane or gel is determined from the standard curve. The biuret
containing the biochemical system to be studied, assay has several advantages, including speed, similar
2. A transducer, color development with different proteins, and few
3. An amplifier, and interfering substances. Its primary disadvantage is its
4. A computer system for data acquisition lack of sensitivity.
and processing.
The Lowry protein assay is one of the more sensitive
When biomolecules in the reaction center interact, assays and has been widely used. The principle behind
a physicochemical change occurs. This change in color development is identical to that of the biuret
the molecular system, which may be a modification assay except that a second reagent (Folin-Ciocalteu) is
of concentration, absorbance, mass, conductance, added to increase the amount of color development.
or redox state, is converted into an electrical signal Two reactions account for the intense blue color that
by the transducer. The signal is then amplified and develops: (1) the coordination of peptide bonds with
displayed on a computer screen. Each biosensor is alkaline copper (biuret reaction), and (2) the reduction
specifically designed for a particular type of molecule of the Folin-Ciocalteu reagent (phosphomolybdate-
or biological reaction. phosphotungstate) of the biuret assay. A standard
curve is prepared with bovine serum albumin or other
Measurement of protein solutions pure protein, and the concentration of unknown
protein solutions is determined from the graph. The
Biochemical research often requires the quantitative obvious advantage of the Lowry assay is its sensitivity,
Central Marine Fisheries Research Institute

measurement of protein concentrations in solutions. which is up to 100 times greater than that of the
Several techniques for such measurement have been biuret assay; however, more time is required for the
developed; however, most have limitations because Lowry assay.
either they are not sensitive enough or they are based
on reactions with specific amino acids in the protein. Techniques for
Since the amino acid content varies from protein to
protein, no single assay will be suitable for all proteins.
sample preparation
In this section, we discuss different assays that are
widely used. Dialysis

When substances containing two or more peptide One of the oldest procedures applied to the
bonds react with the biuret reagent, alkaline copper purification and characterization of biomolecules
sulfate, a purple complex is formed. The colored is dialysis, an operation used to separate dissolved

332
molecules on the basis of their molecular size. The suitable separation. The other methods require 1 to 2
technique involves sealing an aqueous solution hours or less. Ultrafiltration involves the separation of
containing both macromolecules and small molecules molecular species on the basis of size, shape, and/or
in a porous membrane. The sealed membrane is charge. The solution to be separated is forced through
placed in a large container of low-ionic-strength a membrane by an external force. Membranes may be
buffer. The membrane pores are too small to allow chosen for optimum flow rate, molecular specificity,
diffusion of macromolecules of molecular weight and molecular weight cutoff. Two applications of
greater than about 10,000. Smaller molecules membrane filtration are obvious: (1) desalting buffers
diffuse freely through the openings. The passage of or other solutions, and (2) clarification of turbid
smaller molecules continues until their concentrations solutions by removal of micron- or submicron-sized
inside the dialysis tubing and outside in the large particles. Ultrafiltration membranes have molecular
volume of buffer are equal. Thus, the concentration weight cutoffs in the range of 100 to 1,000,000. They
of small molecules inside the membrane is reduced. are usually composed of two layers: (1) a thin (0.1–
Equilibrium is volume dependent and is reached 0.5), surface, semipermeable membrane made from a
after 4 to 6 hours. If the outside solution (dialysate) variety of materials including cellulose acetate, nylon,
is replaced with fresh buffer after equilibrium is and polyvinylidene, and (2) a thicker, inert, support
reached, the concentration of small molecules base. These filters function by retaining particles on
inside the membrane will be further reduced by the surfaces, not within the base matrix. Membrane
continued dialysis. Dialysis membranes are available filters of these materials can be manufactured
in a variety of materials and sizes. The most common with a predetermined and accurately controlled
materials are collodion, cellophane, and cellulose. pore size. These filters require suction, pressure, or
Recent modifications in membrane construction centrifugal force for liquid flow. A typical flow rate
make a range of pore sizes available. Spectrum for the commonly used membrane is 57 mLmin-1

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Laboratories offers Spectra/Por membrane tubing cm-2 at 10 psi. Ultrafiltration devices are available for
with complete molecular weight cutoffs ranging macroseparations (up to 50 L) or for microseparations
from 100 to 300,000. Dialysis is most commonly (milli- to microliters).
used to remove salts and other small molecules from
solutions of macromolecules. During the separation Suggested readings
and purification of biomolecules, small molecules
are added to selectively precipitate or dissolve the S. Sawhney and R. Singh (Editors), Introductory Practical
desired molecule. For example, proteins are often Biochemistry, (2005), Alpha Science International (Oxford,
U. K.).
precipitated by addition of organic solvents or D. Sheehan, Physical Biochemistry: Principles and Applications,
salts such as ammonium or sodium sulfate. Since 2nd ed. (2009),
Wiley-Blackwell (Hoboken, NJ).
the presence of organics or salts usually interferes J. Walker, Editor, The Protein Protocols Handbook, 2nd ed. (2002),
with further purification and characterization of the Humana Press (Totowa, NJ).
molecule, they must be removed. Dialysis is a simple, K. Wilson and J. Walker, Editors, Principles and Techniques of
Practical Biochemistry, 5th ed. (2000), Cambridge University
inexpensive, and effective method for removing all Press (Cambridge, U.K.).
small molecules, ionic or nonionic. Dialysis is also P. Smith et al., Anal. Biochem. 150, 76–85 (1985). “Measurement
of Protein Using Bicinchoninic Acid.”
useful for removing small ions and molecules that J. Walker, Editor, The Protein Protocols Handbook, 2nd ed. (2002),
are weakly bound to biomolecules. Humana Press, Inc.(Totowa, NJ), pp. 3–21.
Dedicated website: http://www.ruf.rice.edu/~bioslabs/methods/
protein/abs280.html
Ultrafiltration Dedicated website: http://www.ruf.rice.edu/~bioslabs/methods/
protein/protcurve.html
K. Barker, At the Bench: A Laboratory Navigator, 2nd ed. (2005),
Although dialysis is still used occasionally as a Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY).
purification tool, it has been largely replaced by S. Borman, Chem. Eng. News, (2006), “A Boost for Biosensors,”
ultrafiltration and gel filtration. The major disadvantage August 7, pp. 48–49.
R. Boyer, Concepts in Biochemistry, 3rd ed. (2006), John Wiley &
of dialysis that is overcome by the newer methods Sons (Hoboken, NJ), pp. 36–62.
is that it may take several days of dialysis to attain a R. Curtright et al., Biochem. Mol. Biol. Educ. 32, 71–77 (2004).

333
 “Facilitating Student Understanding of Buffering by an Practical Biochemistry,
Integration of Mathematics and Chemical Concepts.” 5th ed. (2000), Cambridge University Press (Cambridge, U.K.),
R. Garrett and C. Grisham, Biochemistry, 4th ed. (2010), Brooks/ pp. 37–42.
Cole (Belmont, CA). Dedicated website: http://www.hannainst.com
N. Good and S. Izawa, in Methods in Enzymology, Vol. XXIV, A. Complete information on the selection, use, and care of pH meters
San Pietro, Editor (1972), Academic Press (New York), pp. and electrodes.
53–68. “Hydrogen Ion Buffers for Photosynthesis Research.” http://www.ysi.com
D. E. Gueffroy, Editor, Buffers—A Guide for the Preparation and Dedicated website: http://www.ornl.gov/sci/biosensors
Use of Buffers in Biological Systems (1993), Calbiochem- Virtual poster session on biosensors in biomedicine, sponsored by
Novabiochem Corp., P.O. Box 12087, La Jolla, CA92039–2087. the Oak Ridge National Lab.
D. Nelson and M. Cox, Lehninger Principles of Biochemistry, 5th Dedicated website: http://en.wikipedia.org/wiki/Biosensor
ed. (2009), Freeman (New York). Dedicated website: http://en.wikipedia.org/wiki/Buffer_solution
J. Risley, J. Chem. Educ. 68, 1054 (1991). “Preparing Solutions in Dedicated website: http://www.ysilifesciences.com/index.
the Biochemistry Lab.” php?page=ysi-5300a-biological-oxygen-monitor
D. Voet and J. Voet, Biochemistry, 3rd ed. (2005) John Wiley & Dedicated website: http://www.thermo.com/com/cda/product/
Sons (Hoboken, NJ). detail/0,1055,23574,00.html
D. Voet, J. Voet, and C. Pratt, Fundamentals of Biochemistry, 3rd Dedicated website: http://www.thermo.com/eThermo/CMA/PDFs/
ed. (2008), John Wiley & Sons (Hoboken, NJ). Various/File_10350.pdf
K. Wilson and J. Walker, Editors, Principles and Techniques of Dedicated website: http://www.labconco.com
Central Marine Fisheries Research Institute

334
Introduction to Marine Bio-prospecting
Kajal Chakraborty
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction to the same extent despite the fact that the sea
Marine bioprospecting can be described as targeted covers more than 70% of the Earth’s surface. The
and systematic search for bioactive compounds within evolution of the marine environment started several
marine organisms. This may include bacteria, fungi million years earlier than that of the land, and the
and viruses and larger organisms such as sea plants, marine biodiversity is thought to be greater than
shellfish and fish. The result of the bioprospecting that found on land. Up to this point in time, little is
could be a purified molecule that is produced known about the molecules and genetic properties
biologically or synthetically, or the entire organism. The of the marine species. This applies particularly to
purpose of marine bioprospecting, from a business organisms from cold waters. Up to now, research
perspective, is to find the components and purified has mainly concentrated on life forms from tropical
compounds that may be included as components and temperate regions. In the future, we will probably
in products or processes. Marine bioprospecting, see a shift in focus to mapping biological material

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

therefore, is not an industry in the traditional from northern waters. In addition, there is also
sense, but it may procure different compounds that increasing interest in life at sea, as products isolated
may be used in many different industries. Marine from marine organisms tend to be more bioactive
bioprospecting makes exploitation of bioactive than equivalent bioactive compounds isolated
compounds from marine organisms possible. from land. In relation to future economy related
Collection and extensive analysis/preparation of the to biotechnology, also called “the bioeconomy”,
collected material is necessary before the substance the OECD estimates that the industrial application
is suitable for further development to an end product of biotechnology will be of great significance until
or product component. The various phases within 2030. It is estimated that biotechnology-based output
marine bioprospecting can be divided as development could account for approximately 35% of the output
from collection of marine organisms, preparation, value of chemicals and other industrial products.
categorisation, storage and analysis to creation of a Correspondingly this will be 80% for pharmaceutical
substance, i.e. marine genetic and biological material and diagnostic products and 50% for agriculture/
with the potential for further use. food. Within the biotechnology area generally,
industrial processes could be the largest sector with
Applications 39%, while agriculture/food and health is estimated
at 36% and 25% respectively.
and importance
Humans have traditionally searched for natural Marine flora and fauna and
bioactive substances that can be used in medicines
and for other purposes. It has been asserted that
their potential use
over 65% of medicines used today are based on The great ocean forms more than 70% of the earth,
natural substances, most of which are derived from though we know to some extent of the coastal and
the biodiversity found on land. The biodiversity the upper layers of the ocean, the 80 percent of the
that can be found in the sea has not been explored ocean, which constitute 62 percent of the entire

335
 earth’s surface, are unknown. The marine environment (than terrestrial bioprospecting). The natural
provides a wide range of goods and services essential products isolated from marine sources tend to be
for human life. Other than food, marine ecosphere more highly bioactive than terrestrial counterparts
constitutes a vast reservoir of valuable compounds because they have to retain their potency despite
with wide range of bioactivities against several life- dilution in surrounding seawater to be effective in
threatening diseases. the “chemical warfare” that allows marine flora and
fauna to ward off would-be predators and animals
The “Marine Pharmacology Review 2003-2004” that might attempt to grow over and smother them.
shows initial pharmacological results of 166 marine Despite lesser attention paid to marine natural
chemicals with products historically, there are notable marine-derived
bio products that are commercially available and
• About 67 marine organisms showing antibacterial, IP protected.
antifungal, antimalarial, antituberculosis or
antiviral activities Bioactive Compounds from
• About 45 marine derived compounds reported
to have significant effects on the cardiovascular,
Marine Organisms
immune and nervous system as well as possessing Ocean is a potential source of bioactive compounds,
anti-inflammatory effects. which does not have a significant history of use in
• About 54 marine derived compounds, which act traditional medicine as in the case of terrestrial plants
on a variety of molecular targets with a potential (Kamboj, 1999). Previously, the research was focused
contribution to several pharmacological classes mainly on terrestrial plants because Number of marine
(Source: WWF). natural products: 1971- 2005 (Blunt, 2007) of their
easier availability. The isolation of biologically unique
There is a high degree of representation of terrestrial- molecules from marine organisms that are not found
derived bioproducts, and, therefore, the number of in terrestrial sources leads to a remarkable progress
marine natural products that have found their way in marine bioprospecting. The boom of marine
into pharmacies is thus far small. This has more to bioprospecting began in recent years and 18000 plus
do with the relative infancy of marine bioprospecting natural compounds from marine organisms have been
Central Marine Fisheries Research Institute

336
isolated as compared to 155000 terrestrial products
(Blunt, 2004; Mayer et al., 2007). Between 1969
and 1995, 63 marine substances were patented as
antitumour agents, accounting for half the marine
molecules patented for pharmaceutical purposes
(Mart´ınezPrat, 2002). There are a significant (and
growing) number of marine-derived compounds
with pharmaceutical potential in the pipeline. The
accompanying table in the next page (modified
from one included in Kijjoa and Sawangong, 2004)
presents the marinederived potential therapeutic
compounds used for drug discovery efforts. Many
of these are still undergoing preclinical evaluation,
but several others are currently being administered
to patients as part of clinical trials

Marine-Derived Drugs
The first modern marine-derived drugs dated back
more than 50 years. Werner Bergman extracted
the novel compounds spongothymidine and
spongouridine from the Caribbean sponge Tethya
crypta in the early 1950s. These compounds were

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

nucleosides similar to those forming the building
blocks of nucleic acids (DNA and RNA). These for HIV, sold under the names Retrovir. AZT use was
natural nucleoside analogs were discovered to have a major breakthrough in AIDS therapy in the 1990s
unexpected antiviral properties. that significantly altered the course of the illness.
This success story frommarine ecosystem represents
The arabinoside Vidarabine® (ARA-A) and an annual market of about $50 million. AZT works
Cytarabine® (ARA-C) (two of the first ever discovered by inhibiting the action of reverse transcriptase
marine drugs) are the compounds extracted from (Mitsuya et al., 1985; Yarchoan et al., 1986; Mitsuya
the marine sponge Tethya crypta. Vidarabine is et al., 1990).
patented, and is commonly prescribed for viral
infection as ophthalmic ointment, whereas patented
Cytarabine® (ARA-C) is a chemotherapy drug. This
medicine reduces the growth of cancer cells, and
can suppress the immune system. Cytarabine® is
sold under the trade name Cytosar-U® by Pharmacia
& Upjohn. It was FDA-approved for the treatment
of certain leukemias in 1969, making it the first
such approved marine-derived drug for use in
cancer chemotherapy. AZT (Zidovudine) was originally isolated from a marine
sponge and manufactured under the trademark Retrovir®
Azidothymidine (or Zidovudine, AZT) is an and was the first drug licensed for the treatment of
antiretroviral drug used for the treatment of HIV/ HIV infection.
AIDS based on a group of compounds (arabinosides)
extracted from the sponge Tethya crypta more than Anti-inflammatory and analgesic pseudo pterosins
40 years ago. AZT was the first approved treatment isolated from a Caribbean marine gorgonian

337
 (Pseudoterigorgia elisabethae), which led to the venom of predatory tropical cone snails (Conus spp).
development of bioproducts now used in Estee
Lauder skin care and cosmetics lines and currently Marine Natural Products
worth $3-4 million a year. Pseudopterosins belong
to a class of patented compounds known as tricyclic Natural products have long been used as foods,
diterpene glycosides (Kijjoa and Sawangwong, 2004; fragrances, pigments, insecticides, medicines, etc. Due
Kohl and Kerr, 2003) to their easy accessibility, terrestrial plants have served
as the major source of medicinally useful products,
Ziconotide (trade name Prialt®) is a synthetic form of especially for traditional or folk medicine. About 25%
a compound extracted from the venom of predatory of all pharmaceutical sales are drugs derived from plant
tropical cone snails (Conus spp). The conotoxins from natural products and an additional 12% are based on
the various species of cone snails alone represent microbially produced natural products. The marine
more than 100 patents and patent applications. environment covers a wide thermal range (from the
In December 2004, Prialt® was approved by the below freezing temperatures in Antarctic waters to
FDA (approval was granted to Irish pharmaceutical about 350ºC in deep hydrothermal vents), pressure
company Elan Corporation to market its product for range (1-1000 atm), nutrient range (oligotrophic
pain management) as a treatment for severe cases to eutrophic) and it has extensive photic and non-
of chronic pain in patients who require intrathecal photic zones. This extensive variability has facilitated
analgesia and conditions such as cancer and AIDS. extensive speciation at all phylogenetic levels, from
microorganisms to mammals. Despite the fact that
the biodiversity in the marine environment far exceeds
that of the terrestrial environment, research into the
use of marine natural products as pharmaceutical
agents is still in its infancy. This may be due to the
lack of ethno-medical history and the difficulties
involved in the collection of marine organisms. But
with the development of new diving techniques,
remote operated machines, etc., it is possible to
collect marine samples and during the past decade,
Pseudopterosins have been originally isolated over 5000 novel compounds have been isolated from
from marine soft coral species called a sea whip shallow waters to 900-m depths of the sea.
(Pseudopterogorgia elisabethae)
Conclusions
Ocean is a potential source of bioactive compounds,
Central Marine Fisheries Research Institute

which does not have a significant history of use in

traditional medicine as in the case of terrestrial plants
(Kamboj, 1999). Previously, the research was focused
mainly on terrestrial plants because Number of marine
natural products: 1971- 2005 (Blunt, 2007) of their
Cone snails are found in tropical seas, carnivorous easier availability. The isolation of biologically unique
molluscs known as cone snails sport venomous molecules from marine organisms that are not found
harpoons that can instantly paralyze small fish and in terrestrial sources leads to a remarkable progress
other prey. The snails’ venom contains hundreds of in marine bioprospecting. The boom of marine
compounds, some of which chemists have used to bioprospecting began in recent years and 18000 plus
create highly powerful, nonaddictive painkillers such natural compounds from marine organisms have been
as Ziconotide. Ziconotide (trade name Prialt®) is a isolated as compared to 155000 terrestrial products
synthetic form of a compound extracted from the (Blunt, 2004; Mayer et al., 2007). Between 1969

338
 Publishers. 1979. P. 128.
and 1995, 63 marine substances were patented as Dedicated website: http://www.artsdatabanken.no
antitumour agents, accounting for half the marine El-Shafei, H.A. (1997). Influence of l-sorbose and the cell-wall-
lytic Micrococcus sp on the major polymers of Aspergillus
molecules patented for pharmaceutical purposes fumigatus. Polymer Degradation and Stability, 57(2), 151-156.
(Mart´ınezPrat, 2002). There are a significant (and Erba, E., Bergamaschi, D., and Bassano, L. (2001). Ecteinascidin-743
growing) number of marine-derived compounds with (ET-743), a natural marine compound, with a unique
mechanism of action. Eur J Cancer. 37, 97-105.
pharmaceutical potential in the pipeline. Farooqi, A. H. A., Shukla, Y. N., Shulda, A., and Bhakuni, D. S.
(1990). Cytokinins from marine organism. Phytochmistry.
29(7), 2061-2063.
Suggested readings Faulkner, D.J. (2002). Marine natural products. Natural Products
Report. 19(1), 1-48.
Flodin, C., and Whitfield, F.B. (1999). Biosynthesis of bromophenols
Amador, M.L., Jimeno, J., Paz-Ares, L., Cortes-Funes, H., and
in marine algae. Water Sci Technol. 40, 53–58.
Hidalgo, M. (2003). Progress in the development and
Fuestani, N., Sugawara,T., and Matsunago, S. (1992). Potent
acquisition of anticancer agents from marine sources. Annals
antitumor metabolites from a marine sponge. J. Org. Chem.
of Oncology. 14, 1607-1615.
57, 3828-3832.
Anjaneyulu, A.S.R., Prakash, C.V.S., and Mallavadhani, U.V. (1991).
Garg, H.S., Sharma, M., Bhakuni, D.S., Pramanik, B.N., Plough, S.,
Two caulerpin analogues and a sesquiterpene from Caulerpa
and Bose, A.K. (1992). An antiviral sphingosine derivative from
racemosa. Phytochemistry. 30, 3041–3042.
the green alga, Ulva fasciata. Tetrahedron Lett. 33, 1641-44.
Balaban, N. and Dell’Acqua, G. (2005). Barriers on the road to
Gibson, R.D., and Gibson, S.L.M. (1981). Seatone in arthritis. British
new antibiotics. Scientist. 19, 42–43.
Medical Journal. 283, 1472.
Bansemir, A., Blume, M., Schröder, S., and Lindequist, U. (2006).
Ginarte, C.J. and Walter, P.G. (1997). Determinants of patent rights:
Screening of cultivated seaweeds. Antimicrobial Agents and
A cross national study. Research Policy. 26(3), 1997, 283-301.
Chemotherapy. 48, 3645–3654.
Grain. G. (1998). TRIPS versus CBD: Conflicts between the
Bergmann, W., and Burke, D.C. (1955). Contributions to the study
WTO regime of intellectual property rights and sustainable
of marine products. XXXIX. The nucleosides of sponges. III.
biodiversity. Global Trade and Biodiversity in Conflict, No.1,
Spongothymadine and spongouridine. J Org Chem. 20, 1501-
April 1998.
1507.
Gram, L., Melchiorsen, J., Spanggaard, B., Huber, I., and Nielsen,
Bergmann, W., and Feeney, R.J. (1951). Contributions to the study
T.F. (1999). Inhibition of Vibrio anguillarum by Pseudomonas
of marine products. XXXII. The nucleosides of sponges. J. Org.
fluorescens AH2, a possible probiotic treatment of fish. Appl.
Chem. 16, 981-987.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Environ. Microbiol. 65, 969–973.
Blunt, J.W., Copp, B.R., Munro, M.H.G., Northcote, P.T., and
Guerriero, A., Meinesz, A., D’Ambrosio, M., and Pietra F.
Prinsep, M.R. (2004). Marine natural products. Nat. Prod.
(1992). Isolation of toxic and potentially toxic sesqui- and
Rep. 21, 1-49.
monoterpenes from the tropical green seaweed Caulerpa
Blunt, J.W., Copp, B.R., Munro, M.H.G., Northcote, P.T., and
taxifolia which has invaded the region of Cap Martin and
Prinsep, M.R. (2004). Marine natural products. Natural Product
Monaco. Helv. Chim. Acta. 75, 689-695.
Reports, 21, 1-49.
Hamann, M.T., and Scheuer, P.J. (1993). Kahalalide F. a bioactive
Burres, N.S., and Clement, J.J. (1989). Antitumor activity and
depsipeptide from the sacoglossan mollusk Elysia rufescens and
mechanism of action of the novel marine natural products
the green alga Bryopsis sp. Journal of the American Chemical
mycalamide-A and –B and onnamide. Cancer Res. 49, 2935-
Society. 115, 5825-5826.
2940.
Handley, J.T., and Blackman, A.J. (2005). Secondary metabolites
Burres, N.S., Sazech, S., Gunavardana, G.P., and Clement, J.J.
from the marine alga Caulerpa brownii (Chlorophyta).
(1989). Antitumor activity and nucleic acid binding properties
Australian Journal of Chemistry. 58(1), 39-46.
of dercitin. Cancer Res. 49, 5267-5274.
Judulco, R., Brauers, G., Edrata, R.A., Ebel, R., Wray, S.V., and
Chakraborty, K., and Paulraj, R. (2010). Sesquiterpenoids with free
Proksh, P. (2002). New metabolites from sponge-derived fungi
radical scavenging properties from marine macroalga Ulva
Curvularia lunata and Cladosporium herbarum. Journal of
fasciata Delile. Food Chemistry. 122, 31-41.
Natural Products. 65, 730-733.
Chakraborty, K., Lipton, A.P., Paulraj, R., and Chakraborty, R.D.
Kamat, S.Y., Wahidulla, S., D’Souza, L., Naik, C.G., Ambiye, V.,
(2010). Guaiane sesquiterpenes from seaweed Ulva fasciata
Bhakuni, D.S. Jain, S., Geol, A.K., and Srimal, R.C. (1994).
Delile and their antibacterial properties. European Journal of
Bioactivity of marine organisms : Part VII – Effects of seaweed
Medicinal Chemistry. 45, 2237-44.
extracts on central nervous system. Indian J. Expt. Biol. 32, 418-
Chandran, B., Rameshkumar, G., and Ravichandran, S. (2009).
22.
Antimicrobial activity from the gill extraction of Perna viridis.
Kamboj, V.P. (1999). Bioactive agent from the ocean biota: In:
Global Journal of Biotechnology and Biochemistry. 4, 88-92.
Ocean science trends future directions. Somayajulu BLK (Ed).
Correa, C.M. (1998). Intellectual Property Rights and Aquatic
Indian National Science Academy. New Delhi, India, pp. 197-
Genetic Resources. In Pullin, R.S.V., Bartley D.M. and Kooiman,
227.
J. (Eds.) Towards Policies for Conservation and Sustainable
Katz, M. L., Mueller, L. V., Polyakov, M., and Weinstock, S. F. (2006).
Use of Aquatic Genetic Resources. ICLARM Conference
Where have all the antibiotic patents gone? Nat. Biotechnol.
Proceedings 59.
24, 1529–1531.
Couch, R.A., Ormrod, D.J., Miller T.E., and Watkins W.R. (1982).
Kijjoa, A., and Sawangwong, P. (2004). Drugs and cosmetics from
Anti-inflammatory activity in fractionated extracts of the green-
the sea. Mar. Drugs. 73-82.
lipped mussel. New Zealand Medical Journal. 95, 803-806.
Koehn, F.E., Sarath, G.P., Neil, D.N., and Cross, S.S. (1991).
Cragg, G.M., and Newman, D.J. (2001). Medicinals for the
Halitunal, an unusual diterpene aldehyde from the marine
millennia. NY Acad. Sci. 953a, 3–25.
alga Halimeda tuna. Tetrahedron Letters. 32(2), 169-172.
Croft, J.E. (1979). Relief from arthritis: a safe and effective
Kohl, A.C., and Kerr, R.G. (2003). Pseudopterosin biosynthesis:
treatment from the ocean. Wellingborough (UK): Thorsons
aromatization of the diterpene Cyclase Product,

339
 Elisabethatriene. Mar. Drugs 2003. P. 54-65. Pridmore, D., Rekhif, N., Pittet, A.C., Suri, B., and Mollet. B. (1996).
Mayer, A.M.S., Rodríguez, A.D., Berlinck, R.G.S., and Hamann, Variacin, a new lanthionine-containing bacteriocin produced by
M.T. (2007). Marine pharmacology in 2003–4: Marine Micrococcus varians: comparison to lacticin 481 of Lactococcus
compounds with anthelmintic antibacterial, anticoagulant, lactis. Appl. Environ. Microbiol. 62 (5), 1799-1802.
antifungal, anti-inflammatory, antimalarial, antiplatelet, Puglisi, M. P., Tan, L. T., Jensen, P. R., and Fenical W. (2004).
antiprotozoal, antituberculosis, and antiviral activities; Capisterones A and B from the tropical green alga Penicillus
affecting the cardiovascular, immune and nervous systems, and capitatus: Unexpected anti-fungal defenses targeting the
other miscellaneous mechanisms of action. In: Comparative marine pathogen Lindra thallasiae. Tetrahedron. 60, 7035-
Biochemistry and Physiology, Part C, Vol. 145 (2007), pp. 553– 7039.
581. Sakemi, S., Ichiba, T., Kohmoto, S., and Saucy, G. (1988). Isolation
Mayer, M.S. (1999). Marine pharmacology in 1998: Antitumor and structure elucidation of onnamide A, a new bioactive
and cytotoxic compounds. The Pharmacologist. 41, 159-164. metabolite of a marine sponge Theonella sp. Journal of
Miller, T.E., Dodd, J., Ormrod, D.J., and Geddes, R. (1993). Anti- American Chemical Society. 110, 4851-4853.
inflammatory activity of glycogen extracted from Perna Smith, P., and Davey, S. (1993). Evidence for the competitive
canaliculus. Agents Actions. 38, 139-42. exclusion of Aeromonas salmonicida from fish with
Mitsuya, H., Yarchoan, R., and Broder, S. (1990). Molecular targets stressinducible furunculosis by a fluorescent pseudomonad.
for AIDS therapy. Science. 249 (4976), 1533–44. Journal of Fish Disease. 16, 521–524.
Moellering, R. C. Jr. (1998). Problems with antimicrobial resistance Taylor, M.W., Radax, R., Steger, D., and Wagner, M. (2007).
in gram-positive cocci. Clin. Infect Dis. 26, 1177–1178. Sponge-associated microorganisms: evolution, ecology, and
Newman, D.J., and Cragg, G.M. (2004). Advanced preclinical and biotechnological potential. Microbiology and Molecular
clinical trials of natural products and related compounds from Biology Reviews. 71, 295-347.
marine sources. Current Medicinal Chemistry. 11, 1693-1713. Tincu, J.A., and Taylor, S.W. (2004). Antimicrobial peptides from
Perry, N.G., Blunt, J.W., and Munro, H.H.G. (1988). Mycalamide marine invertebrates. Antimicrobial Agents and Chemotherapy.
A, and antiviral compound from a New Zealand sponge of 48, 3645-3654.
the genus Mycale. Journal of American Chemical Society. The Bioeconomy to 2030: Designing a Policy Agenda”, OECD (2009)
110, 4850-4851. Torrento, M., and Torres, J. (1996). In vitro inhibition of Vibrio
Petitt, G.R., Cichacz, Z.A., Gao, F., Herald, C.L., and Boyd, M.R. harveyi by Pseudomonas sp isolated from aquatic environment.
(1993). Isolation and structure of the remarkable human cancer UPV J. Nat. Sci. 1, 130–138.
cell growth inhibitors spongistatins 2 and 3 from an Eastern Yarchoan, R., Klecker, R., Weinhold, K., Markham, P., Lyerly, H.,
India Ocean Spongia sp. J Chem Soc. (Lond Chem Commun) Durack, D., Gelmann, E., Lehrman, S., Blum, R., and Barry,
1, 1166-1168. D. (1986). Administration of 3‘-azido-3‘-deoxythymidine, an
Pettit, G. R., Collins, J.C., Herald, D.L., Doubek, D.L., Boyd, M. R., inhibitor of HTLV-III/LAV replication, to patients with AIDS or
Schmidt, J.M., Hooper, D.L., and Tackett, L.P. AIDS-related complex. Lancet. 1 (8481), 575–80.
(1992). Isolation and structure of cribostatins1 and 2 from blue
marine sponge, Cribrochalina sp. Can J Chem. 70, 1170-1175.
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Importance of Marine Organisms for
Prospecting Bio-molecules
Kajal Chakraborty
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
The marine environment covers a wide thermal range
(from the below freezing temperatures in Antarctic
waters to about 350ºC in deep hydrothermal
vents), pressure range (1-1000 atm), nutrient range
(oligotrophic to eutrophic) and it has extensive photic
and non-photic zones. This extensive variability has
facilitated extensive speciation at all phylogenetic levels,
from microorganisms to mammals. New metabolites
from marine organisms have resulted in the isolation

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

of more or less 10,000 metabolites, many of which
are endowed with pharmacodynamic properties. Rich
diversity of marine organisms represents an enormous
resource for the discovery of potential compounds
with valuable pharmaceutical and biomedical potential
(Kamboj, 1999). These bioactive compounds belong to
different chemical classes’ viz., terpenoids, steroids/sterol
glycosides, phenolics, amino acids, fatty alcohol esters,
glycolipids etc. Despite the fact that the biodiversity
in the marine environment far exceeds that of the
terrestrial environment, research into the use of marine
natural products as pharmaceutical agents is still in its
infancy. This may be due to the lack of ethno-medical
history and the difficulties involved in the collection of
marine organisms. But with the development of new
diving techniques, remote operated machines, etc., it is
possible to collect marine samples and during the past
decade, over 5000 novel compounds have been isolated
from shallow waters to 900-m depths of the sea.

Natural biologically active sources of known and novel bioactive compounds

including toxins with wide pharmaceutical
compounds from Marine Algae applications is unquestionable. Among the five
The fact that microalgae/cyanobacteria in general divisions of microalgae, studies of biomedical
and marine forms in particular are one of the richest natural products have been concentrated on only

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 two divisions, i.e., Cyanophyta (blue-green algae) Monocyclic diterpenes have been purified from the
and Pyrrophyta (dinoflagellates). Although several Tasmanian green alga Caulerpa trifaria (Handley &
metabolites have been isolated from cyanophytes, Blackman, 2000). The green alga, Caulerpa racemosa,
most of them are isolated from fresh water species, was reported to yield a bioactive sesquiterpene acid
which are cultured easily in comparison to marine (Anjaneyulu et al., 1991). Halitunal, a novel antiviral
organisms. Lyngbyatoxin-A and debromoaplysiatoxin diterpene aldehyde has been isolated from the
are two highly inflammatory but structurally marine alga, Halimeda tuna (Koehn et al., 1991).
different metabolites isolated from toxic strains of 2-Hydroxy-10-methylzeatin has been purified from
alga Lyngbya mausculata collected in Hawaii, and seaweeds, NIO-143, and the absolute configuration
anatoxin-a from Anabaena ciecinalis. Some of the of the said cytokinin has been determined by
marine cyanobacteria appear to be potential sources spectroscopic procedures (Farooqi et al., 1990).
for large-scale production of vitamins of commercial Kahalalide F, a cytotoxic, antiviral and antifungal cyclic
interest such as vitamins of the B complex group depsipeptide, was isolated from a Hawaiian species
and vitamin-E. The carotenoids and phycobiliprotein of Bryopsis sp. (Hamann & Scheuer, 1993). A method
pigments of cyanobacteria have commercial value to purify labdane diterpenois as major constituents
as natural food colouring agents, as feed additives, of dichloromethane-soluble fraction green alga Ulva
as enhancers of the color of egg yolks, to improve fasciata has been illustrated. Antimicrobial assay
the health and fertility of cattle, as drugs and in showed that the compounds labda-14-ene-3a,8a-
the cosmetic industries. Some anti-HIV activity has diol (ULV2) and labda-14-ene-8a-hydroxy-3-one
been observed with the compounds extracted from (ULV4) were inhibitory to the growth of Vibrio
Lyngbya lagerhaimanii and Phormidium tenue. More parahaemolyticus and Vibrio alginolyticus with
than 50% of the 100 isolates from marine sources minimum inhibitory concentrations of 30 μg/ml by
are potentially exploitable bioactive substances. The ULV2, and 40 μg/ml by ULV4, respectively against
substances tested for were either the ones that killed the former and 30 μg/ml by ULV2, and 80 μg/ml by
cancer cells by inducing apoptotic death. ULV4, respectively, against the latter (Chakraborty
et al., 2010). Two new guaiane sesquiterpene
Natural biologically active derivatives, guai-2-en-10a-ol (G1) and guai-2-en-10a-
methanol (G2), were chromatographically purified
compounds from seaweeds as major constituents of the CHCl3/CH3OH (1:1, v/v)
Seaweeds are abundant in the intertidal zones soluble fraction of Ulva fasciata. Acetylation of G2
and in clear tropical waters. However, they have furnished guai-2-en-10a-methyl methanoate (G3)
received comparatively less bioassay attention. with acetyl group at C11 position. Compounds G2
Seaweeds, popularly known as green algae, are and G3 exhibited significant inhibition to the growth
widely distributed in both inter-tidal and deep- of Vibrio parahaemolyticus with minimum inhibitory
water regions of the seas. These seaweeds are of concentrations of 25 and 35 mg/mL, respectively
Central Marine Fisheries Research Institute

immense pharmaceutical and agricultural value. A (Chakraborty et al., 2010). The antiinflammatory
wide range of compounds, particularly terpenes, agent produced by Ulva lactuca was identified as
polyphenolic compounds and steroids, have been 3-O-b-glucopyranosylstigmasta-5,25-diene (Awad,
reported from various seaweeds (Blunt et al., 2006), 2000). A survey of the metabolites of U. lactuca led
amongst which terpenoid compounds represent a to the proposal that 4-hydroxybenzoic acid is the most
major share. For example, Caulerpa brownii from likely biosynthetic precursor of 2,4,6-tribromophenol,
Australia was reported to yield a number of bioactive an antibacterial compound (Flodin & Whitfield, 1999).
novel diterpenoids and terpenoid esters (Handley & Two new antimicrobial terpenes, taxifolione and
Blackman, 2005). Capisterones A and B are triterpene 7,7-didehydro-6-hydroxy-6,7-dihydrocaulerpenyne,
sulphate esters that were isolated from the tropical were purified from Caulerpa taxifolia, a tropical green
green alga, Panicillus capitatus, and were found to alga from Cap Martin, France (Guerriero et al., 1993).
exhibit potent antifungal activity against the marine Neomeris annulata, from Kwajalein Atoll, was reported
algal pathogen Lindra thallasiae (Puglisi et al., 2004). to possess three brominated sesquiterpenes, shown

342
to deter fish feeding (Paul, Cronan Jr., & Cardellina II, ulcer if taken frequently. Therefore exploring the bivalve
1993). A product containing anti-inflammatory and molluscs for their anti-inflammatory and antioxidant
antioxidant principles from seaweeds (CadalminTM activities and development of product therefrom may
GAe) for use against inflammatory disorders with an significantly reduce adverse side effects resulting from
ecofriendly “green” technology has been developed taking NSAIDs. There are reports of dried flesh of
by CMFRI. The product know-how has been patented the New Zealand mussel Perna canaliculus possessig
(Chakraborty K et al. IP 2064/CHE/2010), and is polyunsaturated fatty acids (PUFAs) with possible anti-
under commercialisation. The active ingredients in inflammatory effects (Croft, 1979; Zwar, 1994; Gibson
CadalminTM GAe also suppress the build-up of uric & Gibson, 1981). The anti-inflammatory, antioxidant,
acid in hyperuricemic patients. and anti-prostaglandin activities were reported in
green lipped mussels of New Zealand (Couch et al.,
Bioactive metabolites 1982; Miller et al., 1993). Neosurugatoxin isolated
from Babylonia japonica is useful in characterizing
from molluscs two classes of acetylcholine receptors. Dolastatin,
More than 2600 scientific studies over the last 20 a cytotoxic peptide from Dolabella auricularia is an
years testify to the important contribution of toxins antineoplastic substance. Ulapualide-A, a sponge-
extracted from marine molluscs to medicine and derived macrolide isolated from the nudibranch
cellular biology. To date, only 100 out of a potential Hexabranchus sanguineus exhibits cytotoxic activity
50,000 toxins have been extracted and analyzed. The against L 1210 murine leukemia cells and antifungal
Conus species produce deadly nerve toxins. Some of activity, which exceeds that of clinically useful
the conotoxins block channels regulating the flow of amphotericin-B. Chromodorolide-A isolated from
potassium or sodium across the membranes of nerve Chromocloris cavae exhibits in vitro antimicrobial
or muscle cells; others bind to N-methyl-D-aspartate and cytotoxic activities. Onchidal from Onchidella

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

receptors to allow calcium ions into nerve cells; bieyi is a useful probe for identifying the active site
and some are specific antagonists of acetylcholine residues that contribute to binding and hydrolysis
receptors responsible for muscle contraction. Thus, of acetyl cholinesterase. A team of researchers from
conotoxin are valuable probes in physiological the University of Melbourne extracted the conotoxin
and pharmacological studies. Bivalve molluscs and from a cone-shell snail. It not only inhibits pain as
cephalopods are widely used in different parts of being 10,000 times more powerful than morphine,
the world for bioprospecting, but only recently but also accelerates the recovery of injured nerves.
they have been recognized as potential sources for The absolute stereochemistries of membrenones
bioactive compounds. In the marine environment, A–C, -dihydropyrone-containing polypropionates
where the animals are constantly exposed to the isolated from the skin of the Mediterranean
threat of biofouling, molluscs remain relatively free mollusc Pleurobranchus membranaceus, have been
of biofouling, due to the ability of these sedentary determined by stereocontrolled syntheses of the
organisms control fouling epibionts by effective enantiomers. The first synthesis of siphonarin-B
antimicrobial mechanisms (Tincu & Taylor, 2004; has confirmed the absolute stereochemistry of the
Bansemir et al., 2006; Mayer et al., 2007). Preliminary metabolite isolated from the molluscs Siphonaria
studies indicated marine bivalves and cephalopods zelandica and S. atra. Bursatellanin-P, a 60-kDa
as rich sources of structurally diverse compounds protein was purified from the purple ink of the sea
with antibacterial potential (Chandran et al., 2009). hare Bursatella leachii. The protein exhibited anti-HIV
There is evidence that bivalve molluscs are useful in activity. The first total syntheses of aplyolides B–E,
the treatment of inflammatory joint diseases (Couch ichthyotoxic macrolides isolated from the skin of sea
et al., 1982; Miller et al., 1993). Nonsteriodal anti- hare Aplysia depilans, have been reported confirming
inflammatory drugs (NSAIDs), viz., aspirin and the absolute stereochemistry reported for the
ibuprofen, are often used for inflammatory conditions. metabolites. Cephalopods, gastropods, and bivalve
However, most of these medications can produce the molluscs constitute a major share of marine fauna,
unfortunate side effects, which may lead to stomach and were reported to possess structurally diverse

343
 anti-stress metabolites with respect to antibacterial, in the world and most of them live in marine
antioxidant, and anti-inflammatory properties waters. A range of bioactive metabolites has been
(Chandran et al. 2009). A product (CadalminTM found in about 11 sponge genera. Three of these
GMe) developed by CMFRI containing 100% natural genera (Haliclona, Petrosia and Discodemia) produce
anti-inflammatory ingredients was prepared from powerful anti-cancer, anti-inflammatory agents.
green mussel Perna viridis to combat joint pain and The discovery of spongouridine, a potent tumor-
inflammatory diseases (Chakraborty et al., 2010a; inhibiting arabinosyl nucleoside in Caribbean sponge
Chakraborty et al. 2010b). Cryptotethia crypta, focused attention on sponges
as a source of biomedically important metabolites.
The compound manoalide from a Pacific sponge
has spawned more than 300 chemical analogs, with
a significant number of these going on to clinical
trials as anti-inflammatory agents. An aminoacridine
alkaloid, dercitin, has been isolated from the deep-
water sponge, Dercitus spp. that possesses cytotoxic
activities in the low nanomolar concentration range
and in animal studies, prolongs the life of mice-bearing
ascitic P388 tumours, and is also active against B16
melanoma cells and small cell Lewis lung carcinoma.
Halichondrin-B, a polyether macrolide from Japanese
sponge Theonella spp., has generated much interest
as a potential anticancer agent. The theopederins are
structurally related to mycalamide-A from marine
sponge, Mycale spp. collected in New Zealand and
onnamide-A from marine sponge, Theonella spp.
collected in Okinawa, which show in vitro cytotoxity
and in vivo antitumour activity in many leukemia and
solid tumour model systems. Isoquinolinequinone
metabolite cribostatin from the Indian Ocean
sponge Cribrochalina spp. shows selective activity
against all nine human melanoma cells in National
Critical Technologies (NCT) panel. Spongstatin, a
macrocytic lactone from the Indian Ocean collection
of Spongia spp., is the most potent substance known
against a subset of highly chemoresistant tumour
Central Marine Fisheries Research Institute

types in the NCT tumour panel. Two new -pyrones

(herbarin) along with a new phthalide, herbaric
acid, were isolated from two cultured strains of
the fungus Cladosporium herbarum isolated from
the sponges Aplysina aerophoba and Callyspongia
aerizusa collected in the French Mediterranean and
in Indonesian waters, respectively.

Polyunsaturated fatty acids

from marine fish
Metabolites from Sponges
Long-chain polyunsaturated fatty acids (LC-PUFAs),
Approximately 10,000 sponges have been described viz., eicosapentaenoic acid (EPA, 20:5 n3),

344
docosahexaenoic acid (DHA, 22:6 n3) and linolenic was winterized to crystallize the remaining saturated
acid (LA, 18:3 n3) are widely available in a large variety fatty acids, resulting in a further increase in the
of marine organisms, like microalgae, polychaetes, concentration of DHA and EPA. The polyunsaturated
fin fish and shellfish. These LC-PUFAs are recognised fatty acids EPA and AA have been purified from the
to have special pharmacological and physiological red microalga Porphyridium cruentum by the urea
effects on human/animal health (Harris, 1989). The inclusion method followed by silica gel column
n3 and n6 long-chain polyunsaturated fatty acids chromatography of the urea concentrate. The
(LCPUFAs), viz., eicosapentaenoic acid (EPA, 20:5n3), unique substrate specificity of microbial lipases has
docosahexaenoic acid (DHA, 22:6n3), and arachidonic been utilised for the enhancement of PUFA content
acid (AA, 20: 4n6), are essential fatty acids in the diet in fish oils by several groups (Harris, 1989; Matori
of animals and human beings, because they cannot et al., 1991). Isolates of Pseudomonas fluorescens
synthesize it de novo from precursor molecules have been found to produce enzymes active on
(Chakraborty et al., 2010). Therefore they require lipolytic substrates under alkaline conditions (Kojima
greater concentrations of PUFAs for their growth, & Shimizu, 2003). Lipase genes from Pseudomonas
reproduction and survival (Cahu et al., 1994). Diets have been cloned and expressed in Escherichia coli,
deficient in these PUFAs particularly EPA have been due to their potential industrial applications (Kojima
found to have a negative effect on various vital et al., 2003). P. fluorescens SIK W1 was found to
biochemical and physiological pathways (Chakraborty produce extremely heat-stable lipase that has
et al., 2009). The important natural sources of high lipolytic activity for short- to medium-chain
n3 LC-PUFAs are marine fish oils such as sardine, triacylglycerols (Chung et al., 1991). An extracellular
mackerel, cod, shark, and menhaden, which contain alkaline metallolipase with molecular weight of 74.8
PUFA levels of about 30%. For this reason, marine kDa derived from cultures of Bacillus licheniformis
fish oils are preferentially used as raw material to MTCC 6824 was found to enrich D5 olefinic double

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

prepare n3 PUFA concentrates. Additionally, it was bond fatty acids, viz., EPA and AA (Chakraborty et
reported that n3 PUFAs were moderately absorbed al., 2008b).
by the intestine as triglycerides and most promptly
absorbed when free fatty acids (FFA) were given Polyunsaturated Fatty Acid (PUFA)
orally. Therefore, it is convenient to prepare n3
Enriched Formulation
concentrates as FFA after chemical hydrolysis of
marine oils. Argentation silica gel chromatography A thermophilic and alkalophilic lipase from Bacillus
of urea inclusion adducts from cod liver oil yielded coagulans BTS-3 was purified and biochemically
highly pure DHA in the process, while for EPA, the characterised (Kumar et al., 2005). A lipase produced
recovery in the combined process was 29.6%. EPA by recombinant B. licheniformis was found to be
and DHA have been concentrated from shark liver stable at alkaline pH of 12.0 (Nthangeni et al.,
(Isurus oxyrinchus) in one single step, in which fish 2001). These results are in contrast to thermotolerant
liver oil was simultaneously extracted, saponified, lipases from Bacillus thermoacetenulatus and Bacillus
and concentrated. Additionally, the PUFA concentrate thermoleovorans, which display maximum activity

O Eicosapentaenoic acid

HO
Sardine oil used raw material to prepare PUFA concentrate

345
 at pH 8.0 (Lee et al., 1999; Rua et al., 1997). B. were significantly reduced, resulting in increases in
coagulans NCIMB 9365 has been reported to possess life expectancy. The majority of compounds that
an intracellular carboxylesterase (Molinari et al., were discovered during this period were isolated
1996). The substrate specificity of lipase has been from soil bacteria, most notably the filamentous
utilised for the recovery of EPA (D5) and DHA (D4) Actinobacteria. Microorganisms are a prolific source
from marine oils and c-linolenic acid (D6) from borage of structurally diverse bioactive metabolites and
seed oil (Morioka et al., 1987). DHA-rich triglycerides have yielded some of the most important products
were prepared from fish oil with lipases obtained of the pharmaceutical industry. Microbial secondary
from Candida cylindracea and Chromobacterium metabolites are now being used for applications
viscosum (Tanaka et al., 1992, 1994). Substrates other than antibacterial, antifungal and antiviral
containing D2–D7 isomers of 18:1 were resistant infections. It was during 1928s when Alexander
to pancreatic lipase-catalysed hydrolysis, resulting Fleming (Fleming, 1929) began the microbial drug
in higher concentrations of oleic acid, and the era when he discovered in a Petri dish seeded with
discrimination was the greatest for the D5 isomer Staphylococcus aureus that a compound (penicillin)
(Heimermann et al., 1973). An extracellular lipase produced by a fungus/mold killed the bacteria. Later,
purified from Pseudomonas fluorescens MTCC 2421 penicillin was isolated as a yellow powder and used as
was used to enrich sardine oil triglycerides with a potent antibacterial compound during the Second
eicosapentaenoic acid and linolenic acid to 35.28% World War. Following this extraordinary discovery
and 8.25%, respectively (Chakraborty et al., 2010). by Flemming, the antibiotics chloramphenicol and
An extracellular lipase derived from Bacillus circulans, streptomycin, were isolated. Naturally occurring
isolated from marine macroalga, Turbinaraia conoides, antibiotics are produced by fermentation, an old
was used to prepare n-3 polyunsaturated fatty acid technique that can be traced back almost 8000
(PUFA) concentrates from sardine oil triglycerides. years. Owing to technical improvements in screening
The enzyme was purified 132-fold with specific programs, and separation and isolation techniques,
activity of 386 LU/mg. The purified lipase was able the number of natural compounds discovered
to enrich sardine oil with 37.7 ± 1.98% 20:5n-3 and exceeds 1 million (Ecker et al. 2005). Among them,
5.11 ± 0.14% 18:3n-3 in the triglyceride fraction 50–60% are produced by plants (alkaloids, flavonoids,
(Chakraborty et al., 2010). terpenoids, steroids, carbohydrates, etc.) and 5%
have a microbial origin. Of all the reported natural
Marine Bacteria as a Source products, approximately 20–25% show biological
activity, and of these approximately 10% have been
of marine natural products obtained from microbes. Furthermore, from the 22
It has been argued that because of the high 500 biologically active compounds that have been
dilution effect of seawater, marine-derived bioactive obtained so far from microbes, 45% are produced by
compounds may have evolved great potency. This bacteria or bacteria-like microbes, 38% by fungi and
Central Marine Fisheries Research Institute

theory was supported in 2004 with the report of 17% by others (Berdy, 2005). The increasing role of
a first-in-class antimicrobial compound from a microorganisms in the production of antibiotics and
marine isolate Verrucosispora. Renewed interest in other drugs for treatment of serious diseases has been
marine microorganisms and their ability to produce dramatic. However, the development of resistance
antimicrobials has resulted in numerous reports in microbes to various life-thretening diseases and
of novel antimicrobial compounds. The period of in aquaculture has become a major problem and
antimicrobial drug discovery from the early 1940s requires renewed research effort to combat it.
to the 1960s is referred to as the Golden Age. Antimicrobial development after the Golden Age
During this time, the industrialization of penicillin was characterized by semi-synthetic modifications
production created the expertise and facilities to make of compounds that were already clinically proven.
significant quantities of antimicrobial compounds by The poor antimicrobial discovery rate from microbes,
fermentation. The clinical use of antibiotics heralded a coupled with the availability of chemically synthesized
health care miracle; deaths due to bacterial infections small molecule libraries, led to the abandonment of

346
microbial screening programmes in the majority of metabolites is important as chemical diversity in the
pharmaceutical companies. To date, small chemical construction process of an marine natural products
libraries have failed to deliver a new antimicrobial library. An optimization of ‘one strain, many active
compound to the clinic, prompting many to speculate compounds’ can be used together with ‘fingerprint’
that the withdrawal of microbial screening was methods (HPLC and nuclear magnetic resonance)
premature, exacerbating the threat of antibiotic including tandem analytical techniques such as
resistant bacteria. MS/MS, GC-EI/MS, HPLC-SPE-NMR, LC-MS-MS and
LC-NMR for the optimization/selection of culture
Microbial natural products media for high-throughput fermentation of novel
strains. Tormo et al. (2003) developed a method
Microbial natural products that have reached the for the selection of production media for bacterial
market without any chemical modifications are a strains based on their metabolite HPLC profiles, that
testimony to the remarkable ability of microorganisms yielded the highest metabolite diversity and least
to produce drug-like small molecules. Although overlapping HPLC profiles were selected for large-scale
still in clinical trails, a feature example of this is fermentation. Targeted high-throughput screening
salinosporamide A (NPI-0052), a novel anticancer methods are important for the speed and accuracy
agent found in the exploration of new marine of identification of novel antimicrobials. From these
environments (Fenical et al. 2009). In 2008, over evaluation models, many crude extracts or purified
1000 marine natural products were reported (Blunt compounds were obtained as positive hits. In addition
et al. 2010). However, out of the 19 microbial-derived for evaluation purposes, it is worthy to note that
drugs reported in 2008, no natural products from these screening assays also provide mode of action
marine microbes were present, signifying the novelty hypothesis from the crude extracts.
of their systematic exploration (Ganesan, 2008).

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Currently, >30 compounds of marine microbial Antibiotics from
origin are in clinical or preclinical studies for the
treatment of different types of cancer (Simmons et
marine microbes
al. 2005), clearly demonstrating that potential of During recent decades, we have seen an increasing
marine microorganisms as an essential resource in number of reports on the progressive development
the discovery of new antibiotic leads. The evolution of bacterial resistance to almost all available
of marine microbial natural product collections and antimicrobial agents. In the 1970s, the major
development of high-throughput screening methods problem was the multidrug resistance of Gram-
have attracted researchers to the use of natural negative bacteria, but later in the 1980s the
product libraries in drug discovery. These libraries Gram-positive bacteria became important, including
include subsections of crude extracts, pre-fractionated methicillin-resistant staphylococci, penicillin-
extracts (automated HPLC-MS fractionation) and resistant pneumococci and vancomycin-resistant
purified natural products. A research group in Ireland enterococci (Moellering, 1998). In the past, the
has developed a two-dimensional chromatographic solution to the problem has depended primarily
strategy that includes a protocol to generate on the development of novel antimicrobial agents.
purified marine natural product libraries that are However, the number of new classes of antimicrobial
accurately characterized by mass spectroscopy agents being developed has decreased dramatically
during production to expedite dereplication of known in recent years. The conventionally used antibiotics/
compounds and identification of novel chemotypes. drugs become resistant to most of the natural
Although the biosynthetic and regulative crosstalk antimicrobial agents that have been developed over
of secondary metabolite biosynthesis is complex the past 50 years (Hancock, 2007) thereby limiting
within and between microorganisms, all levels can the effectiveness of current antimicrobial drugs.
be influenced by imitating natural environmental In 2004, more than 70% of pathogenic bacteria
changes. Development and testing of new culture were estimated to be resistant to at least one of the
media for the maximum expression of secondary currently available antibiotics (Katz et al. 2006). The

347
 so-called ‘superbugs’ (organisms that are resistant to agents as a solution towards multiresistant antibiotic
most of the clinically used antibiotics) are emerging and drug molecules.
at a rapid rate. S. aureus, which is resistant to
methicillin, is responsible for many cases of infections Aquaculture grade
each year (Balaban et al. 2005). The incidence of
multidrug-resistant pathogenic bacteria is increasing.
antimicrobial chemicals
The Infectious Disease Society of America (IDSA) from marine microbes
reported in 2004 that in US hospitals alone, around
2 million people acquire bacterial infections per Disease caused by bacterial pathogens has been
year (dedicated website: http://www.idsociety.org/ widely recognized as a major cause of economic
Content.aspx). There are also other examples of loss in many commercially cultured fish and shellfish
Gram-positive (Enterococcus and Streptococcus) and species in India, with mortality of larval stages in
Gram-negative pathogens (Klebsiella, Escherichia, hatcheries and the growing stages in different
Enterobacter, Serratia, Citrobacter, Salmonella and mariculture systems. Pathogenic vibrios are involved in
Pseudomonas) (Cragg & Newman, 2001). Among significant mortalities in the larviculture and growout
them, Pseudomonas aeruginosa accounts for almost phases of famed finfish and shellfishes. In an attempt
80% of these opportunistic infections. They represent to control the proliferation of pathogenic vibrios, the
a serious problem in patients hospitalized with cancer, prophylactic and therapeutic use of antibiotics has
cystic fibrosis and burns, causing death in 50% of been practiced in commercial hatcheries, creating
cases. Other infections caused by Pseudomonas spp more serious problem of antibiotic resistance
include endocarditis, pneumonia and infections of among the microflora in the environment. With
the urinary tract, central nervous system, wounds, safety concerns about synthetic antibiotics, and
eyes, ears, skin and musculoskeletal system (Levin, the antibiotic resistance problems, considerable
& Bonten, 2004). New families of anti-infective interest has arisen in finding alternative natural
compounds are needed to enter the marketplace at sources (Gomez-Gil et al., 2000). Screening and
regular intervals to tackle the new diseases caused development of aquaculture-grade chemicals from
by evolving pathogens. At least 30 new diseases bacterial flora could be a highly promising approach
emerged in the 1980-2000s and they are growing. to produce these bioactive moleules. Members of the
Emerging infectious organisms often encounter hosts genus Pseudomonas and Bacillus either free living or
with no prior exposure to them and thus represent a associated with marine flora are common beneficial
novel challenge to the host’s immune system. Several bacterial candidates, and are known to produce a
viruses responsible for human epidemics have made a wide range of secondary metabolites (Raaijmakers
transition from animal host to humans and are now et al., 1997; Alavandi et al., 2004; Vijayan et al.,
transmitted from human to human. HIV, responsible 2006) inhibiting a wide range of pathogenic bacteria
for the acquired immunodeficiency syndrome (AIDS) (Rengpipat et al., 1998). The metabolites 6-oxo-de-
Central Marine Fisheries Research Institute

epidemic, is one example. Although it has not been O-methyllasiodiplodin, (E)-9-etheno-lasiodiplodin,

proven, it is suspected that severe acute respiratory lasiodiplodin, de-O-methyllasiodiplodin, and
syndrome (SARS), caused by the SARS coronavirus, 5-hydroxy-de-O-methyllasiodiplodin, were isolated
also evolved from a different species (Kremer et al. from the mycelium extracts of a microbe obtained
2000). One additional reason for developing new from South China Sea (Yang et al., 2006). Marine
antibiotics is related to their own toxicity. As with bacterial strain, Pseudomonas I-2, producing
other therapeutic agents, the use of antibiotics may inhibitory compounds against shrimp pathogenic
also cause side effects in patients. Some side effects vibrios including Vibrio harveyi, V. fluvialis, V.
are more severe and, depending on the antibiotic, parahaemolyticus, V. damsela and V. vulnificus was
may disrupt the hearing function (aminoglycosides), reported by Chaitanya et al. (2002) and Vijayan et
kidneys (aminoglycosides and polypeptides) or liver al., (2006). Bioactive compounds were isolated from
(rifampin). In recent times, several research groups are a marine bacterium Bacillus circulans (Chakraborty et
making concerted efforts to find novel antimicrobial al., 2010). Labda-14-ene-3a,8a-diol and labda-14-

348
ene-8a-hydroxy-3-one were found to be inhibitory have been elucidated by 1H NMR and 13C NMR
to the growth of Vibrio parahaemolyticus with spectra, including 2D NMR. Several bacterial flora
minimum inhibitory concentrations of 30-40 µg/ were isolated from marine ecosystem (Bacillus
mL (Chakraborty et al., 2010), and their structures subtilis, Bacillus amyloliquifaciens, Pseudomonas

Compound Name Structure Chemical Class Organism Company Status

HO
OMe
NH HO Me
O
O O
O O S
Me H Alkaloid Tunicate PharmaMar Approved
Trabectedin (ET-743) N
N
O
O OH

H2N
OH O

H
O O O
O
O . H H
Eribulin Mesylate H3C S OH
O OO
Macrolide Sponge Eisai Inc. Phase III
(E7389) O O

O O
H
NH2 S
OH Amino-steroid Shark Genaera Phase II
Squalamine lactate N
H H O

H N OH
H2 H

NH N Nereus Phar-
HN
NH Diketopipe-razine Fungus Phase II
Plinabulin (NPI-2358) maceuticals
O

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

O
HO
O
H
N
Nudi-
N Alkaloid PharmaMar Phase II
Zalypsis O
O OH
branch
NH
CF3
O

CH3 OH OMe O
H
N NH
H3C
CH3
OH OH O
O Amino acid Sponge Novartis Phase I
LAF389 O

OH OH
O O (CH2)24
HO
NH OH α -galactosyl
HO Sponge Kirin Phase I
KRN7000 O C13H27 ceramide

OH

H
OH
H O
Marizomib, N
O
Beta-lactone-gamma Nereus
Salinosporamide A; O
Bacterium Phase I
lactam Pharmaceuticals
NPI-0052) Me

Cl

O
O
O O HN Cl Cyano
O Cryptophycin —- Preclinical
LY355703, CRYPTO 52 bacterium
O N O O
H

Clinical status of marine derived antitumor agents, their chemical class and mode of action

349
 and Adami, H.O. (1995). Cancer Epidemiology, Biomarkers &
putida, and Pseudomonas aeroginosa) with potential Prevention. 4, 187–192
activities (> 20 mm inhibition zone) against Anfinsen. C. B, Edsall. J. T. and Richards, F.M. (1972). Advances in
Protein Chemistry. New York: Academic Press. pp. 99, 103.
pathogenic Vibrios (Chakraborty et al., 2010). Annamalai, N., Anburaj, R., Jayalakshmi, S & Thavasi, R. Research
The antibacterial component in the CHCl3 fraction Journal of Microbiology. (2007) 2(12): 978-982
of P. aerogenosa was found to be N-substituted Ayyappan. S, Moza. U, Gopalakrishnsn. A, Meenakumari. B,
Jena. J. K, and Pandey. A. K. (2011). Hanbook of fisheries
methyl octahydro-1-phenazinecarboxylate. The and aquaculture. Indian Council of Agricultural Research, New
other important antibacterial molecules were Delhi. Published by Directorate of knowledge management in
agriculture, ISBN 978-81-7164-106-2, 1116 pp.
found to be propyl 2-oxoacetate and phenethyl Bacque, E., Barriere, J. C. & Berthand, N. Curr. Med. Chem. Anti-
2-oxoacetate. About 4530 bacterial isolates were infect. Agents 4, 185–217 (2005).
purified from seaweeds and sediments, and 23 Balaban, N. & Dell’Acqua, G. Scientist 19, 42–43 (2005).
Bansemir, A., Blume, M., Schröder, S & Lindequist, U. Antimicrobial
isolates (B. subtilis MTCC 10402, 10403 & 10407, Agents and Chemotherapy (2006) 48: 3645–3654.
B. amyloliquifaciens 10456, P. putida MTCC 10458, Barber, M. S. Chim. Oggi. 19, 9–13 (2001)
Bauer, A.W., Kirby, W.M.M., Scherris, J.C & Turck, M. American
P. aeroginosa MTCC 10610) were found to be Journal of Clinical Pathology (1966) 45:493-496.
potential against pathogenic Vibrios. Pyocyanins, Bell. M. V, Henderson. R. J. and Sargent, J. R. Comparative
N-substituted phenazinecarboxylate, propyl/ Biochemistry and Physiology. 83B, 711-719 (1986).
Belluzzi. A. C, Brignola. M, Campieri. A, Pera. S, Boschi. and
phenethyl 2-oxoacetates were the major antibacterial Miglioli, M.(1996). The New England Journal of Medicine.
molecules in bacteria (Preetha et al. 2010). 334, 1557.
Bligh, E.G. & Dyer, W.J. Canadian Journal of Biochemistry &
Physiology (1959) 37: 911-917
Conclusions Blunt, J. W., B. R. Copp, M. H. G. Munro, P. T. Northcote, and M.
R. Prinsep. (2004). Nat. Prod. Res. 21 (1): 1–49.
Borel, J. F. Wien. Klin. (2002). Wochenschr. 114, 433–437
“Poison kills the poison,” the famous proverb is Brown, M. F. (1994). Chemistry and Physics of Lipids. 73, 159-180.
the basis for researchers in finding the biomedical Cahu. C. L, Guillaume. J. C, Stephan. G. and Chim. L. (1994).
Aquaculture, 126, 159–170.
metabolites from living organisms. Sea has got plenty Calder. P. C. (2006). American Journal of Clinical Nutrition.
of metabolites and other resources in living or dead 83, 1505S-1519S.
form. The main emphasis is given in the search of Chakraborty K et al. (2010a) IP 2065/CHE/2010)
Chakraborty K et al. (2010b) IP 2066/CHE/2010)
drugs for deadly human diseases as cancer and Chakraborty K, Lipton AP, Paulraj R, Vijayan KK (2010) Food
AIDS. Efforts of the researchers around the world Chemistry 119: 1399–1408
Chakraborty K, Vijayagopal P, Chakraborty RD, Vijayan KK (2010)
are continuing their efforts in finding new molecules Food Chemistry 120: 433-42
and drug candidates in the context of deadly disases Chakraborty. K, & Paulraj. R. (2007). Journal of Agricultural and
such as AIDS, cancer, cardiovascular disease, arthritis, Food Chemistry. 55, 7586–7595.
Chakraborty. K, & Paulraj. R. (2008). Food Chemistry. 109, 727–736.
diabetes, etc. Chakraborty. K, & Paulraj. R. (2008). Journal of Agricultural and
Food Chemistry. 56, 1428–1433.
Chakraborty. K. and Paulraj. R. (2007). Journal of Agricultural and
Suggested readings Food Chemistry. 55 7586-7595.
Chakraborty. R. D, Chakraborty. K. and Radhakrishnan. E. V. R.
(2007). Journal of Agricultural and Food Chemistry. 55, 4043-
Acebal, C., L. M. Cañedo, J. L. F. Puentes, J. P. Baz, F. Romero, F.
Central Marine Fisheries Research Institute

4051.
De La Calle, M. D. G. Grávalos, and P. Rodrigues. (1999). J.
Chandran, B., Rameshkumar, G & Ravichandran, S. (2009) Global
Antibiot. 52: 983-987.
Journal of Biotechnology and Biochemistry. 4: 88-92.
Aggarwala, D., Fernandez, M. L. & Solimanb, G. A. (2006).
Chandrashekar. K, and Deosthale. Y. G. (1993). Journal of Food
Metabolism 55, 794–802
Composition and Analysis, 6, 195-200.
Akoh. C. C, Jennings. B. H, and Lillard, D. A. (1996). Journal of the
Chapkin. R. S, Arrington. J. L, Apanasovich. T. V, Carroll. R. J.
American Oil Chemists Society. 73, 1059–1062
and McMurray. D. N. (2002). Clinical & Experimental
Alanis, A. J. (2005). Arch Med Res. 36(6):697-705.
Immunology. 130,12.
Alarcon, J., Aguila, S., Arancibia-Avila, P., Fuentes, O., Zamorano-
Chung, S.K., Osawa, T & Kawakishi, S. (1997) Bioscience
Ponce, E. & Hernandez, M. (2003). Z. Naturforsch. 58, 62–64
Biotechnology and Biochemistry. 61: 118-123.
Alavandi SV, Vijayan KK, Santiago TC, Poornima M, Jithendran
Chythanya, R, Karunasagar, I. and Karunasagar, I. (2008).
KP, Ali SA, Rajan JJS. (2004). Fish & Shellfish Immunology.
Aquaculture 208, 1–10
17, 115-120
Ciminiello, P., Fattorusso, E., Forino, M., Poletti, R & Viviani, R.
Alberts, A. W. (1980). Proc. Natl Acad. Sci. USA 77, 3957–3961
(2000) European Journal of Organic Chemistry 291: 204-213.
Alfred, J.R.B., Das, A.K & Sanyal, A.K. (1998). Zoological Survey of
Cobb, C.S & Ernst, E. (2006) Clinical Rheumatol. 25(3): pp.275-284.
India Calcutta. 104-11
Connolly. J. M, Coleman. M, and Rose. D. P. (1997). Nutrition and
Andersson. B. A. (1978). Progress in the Chemistry of Fats and
Cancer. 29, 114–119.
Other Lipids. 16, 279–308 (1997).
Connor. W.E. (2000). American Journal of Clinical Nutrition.
Andersson. S.O., Baron. J, Wolk. A, Lindgren. C, Bergstro¨M. R.
71, 171S-175S

350
Couch, R.A., Ormrod, D.J., Miller T.E &. Watkins W.R. (2001).New of the American Oil Chemists Society. 71, 1277–1280.
Zealand Medical Journal (1982) 95:803-806. Imamura, N., M. Nishijima, T. Takadera, K. Adachi, M. Sakai, and
Cragg, G. M., Newman, D. J. & Snader, K. M. (1997). J. Nat. Prod. H. Sano. J. Antibiot. 50: 8-12. (1997).
60, 52–60 Imura. K. and Okada. A. (1998). Nutrition 14 (1), 143–8.
Dancey, J. E. (2006). Cancer Biol. Ther. 5, 1065–1073 Ishida, H., Nozawa, A., Totoribe, K., Muramatsu, N., Nukaya, H.,
Dedicated website: http://www.idsociety.org/Content.aspx Tsuji, K., Yamaguchi, K., Ysumoto, T., Kaspa, H., Berkett, N &
Defer, D., Bourgougnon, N & Fleury, Y. (2009) Aquaculture 293: 1–7 Kosuge T. Tetrahedron letters (1995) 36: 725–728.
El-Shafei H.A., (1997). Polymer Degradation and Stability, 57( Iso. H, Kobayashi. M. and Ishihara J. (2006). Circulation 113 195–
2), 151-156 202.
Erickson. K. L. (1986). Progress in Clinical and Biological Research Jolly. C. A, Jiang. Y. H, Chapkin. R. S. and McMurray. D. N. (1997).
(New York, NY). 222, 555–586. Journal of Nutrition. 127,37.
Fan. Y, Ly. L. H, Barhoumi. R, McMurray. D. N. and Chapkin. R. S. Jones, R.N & Barry, A. L. Journal of Antimicrobial Chemotherapy
(2004). The Journal of Immunology. 173, 6151–6160. (1987) 19: 841-842.
FAO, Corporate document repository, http://www.fao.org Kamboj, V.P. Indian National Science Academy. New Delhi, India,
Farnsworth, N. R., Akerele, O., Bingel, A. S., Soejarto, D. D. & Guo, (1999) pp. 197-227.
(1985). Z. Bull. WHO 63, 965–981 Kamiya, H., Muromoto, K., Sakai, G.K., Endo, Y.M & Yamamzaki,
Faulkner, D.J. (2002). Nat Prod Rep. 19, 1-48. M. Toxicon (1989) 27: 1269-1277.
Faust. T, Lee. E, Redfern. J, and Feldman. M. (1989). Prostaglandins. Key. T. J. A, Silcocks. P. B, Davey. G. K, Appleby. P. N, and Bishop.
37, 493–504. D. T. (1997). British Journal of Cancer. 76, 678–687.
Fenical, W. (1993).Chem Rev. 1673-1683. Kosugi. Y, and Azuma. N. (1994). Journal of the American Oil
Fennema. O. R. (1996). Food Chemistry (3rd ed.). CRC Press. Chemists Society. 71, 1397–1403.
pp. 327–8. Kremer. J. M. (2000). American Journal of Clinical Nutrition. 71,349S.
Fleming, A. Br. (1929). J. Exp. Pathol. 10, 226–236 Kris-Etherton. P. M, Harris. W. S. and Appel. L. J. (2002). Circulation
Fowler. K. H, Chapkin. R. S. and McMurray. D. N. (1993). Journal 106, 2747–2757.
of Immunology. 151, 5186. Lanier. A. P, Bulkow. L. R, and Ireland. B. (1989). Public Health
Fowler. K. H, McMurray. D. N, Fan. Y. Y, Aukema. H. M, and Chapkin. Rep, 104, 658–664.
R. S. (1993). Biochimica et Biophysica Acta. 1210, 89. Leclercq, R. J. Antimicrob. Chemother. 48, 9–23 (2001).
Fürst. P. and Stehle. P. (2004). Journal of Nutrition. 134 (6 Lemos, M. L., A. E. Toranzo and J. L. Barja. Microbiol. Ecol. 11:
Suppl), 1558S–1565S 149-163. (1985).
Gatesoupe, F.J., 1999). Aquaculture, 180(1), 147-165. Levin, B. R. & Bonten, M. J. M. Proc. Natl Acad. Sci. USA 101,
Gerard, J., M. P., P. Haden, M. T. Kelly, and R. J. Andersen. J. Nat. 13101–13102 (2004).
Prod. 62: 80-85. (1999). Liang. T, and Liao. S. (1992). Biochemical Journal. 285, 285–562.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Ghosh. J. and Myers. C. E. (1997). Biochemical and Biophysical Lourenço. R. and Camilo. M. E. (2002). Nutrición Hospitalaria 17
Research Communications. 235, 418–423. (6), 262–70.
Gibson, R.D & Gibson, S.L.M. British Medical Journal (1981) Lowry, O.H., Rosebrough, N.J., Farr, A. L & Randall, R.J. Journal of
283: 1472. Biological Chemistry (1951) 193 (1): 265–75.
Gilroy. D. W, Lawrence. T, Perretti M, and Rossi. A. G. (2004). Mansour. M. P, Frampton. D. M. F, Nichols. P. D, Volkman. J. K,
Nature Reviews Drug Discovery. 3, 401-416. Blackburn. S. I. (2005). Journal of Applied Phycology. 17, 287-
Gomez-Gil, B., Roque, A., Turnbull, J.F. Aquaculture 191, 259– 300.
270 (2000). Maruya. D. Chemosphere (2003) 52 (2): 409-413.
Gopakumar. K. (1974). Journal of the Marine Biological Association. Mayer, A.M.S., Rodriguez, A.D., Berlinck, R.G.S & Hamann, M.T.
India, 16, 3. Comparative Biochemistry and Physiology, Part C: Toxicology
Gopakumar. K. (1975). Fishery Technology. 12(1), 21. and Pharmacology (2007) 145: 553–581.
Gopakumar. K. and Rajendranathan Nair. M. (1972). Journal of Mearns-Spragg A., M. Bregu, K. G. Boyd & J. G. Burgess, 1998.
the Science of Food and Agriculture. 23, 493. Lett. Apl. Microbiol. 27: 142–146.
Gram, L., Melchiorsen, J., Spanggaard, B., Huber, I., Nielsen, T.F., Metcalf, L.D., Schimtz, A. A & Pelka, J.R. Analytical Chemistry (1966)
1999. Appl. Environ. Microbiol. 65, 969–973. 38: 514-515
Grimble. R. F, Howell. W. M, O’Reilly. G, Turner. S. J, Markovic. O, Meydani. S. N, Endres. S, Woods. M. M, Goldin. B. R, Soo. C,
Hirrell. S, East. J. M. and Calder. P. C. (2002). American Journal Morrill-Labrode. A, Dinarello. C. A. and Gorbach. S. L. (1991).
of Clinical Nutrition.76, 454-459. Journal of Nutrition. 121,547.
Hans-Peter, G., S. Andrea, B. Michael, S. Meinhard, B. Thorsten. Miliou, H., Fintikaki, M., Tzitzinakis, M., Kountouris, T &
FEMS Microbiol Ecology 1619: 1-11. (2004). Verriopoulos, G. Aquaculture (2006) 256: 311–322.
Harris. W. S. (1989). Journal of Lipid Research. 30, 785–809. Miller, T.E., Dodd, J., Ormrod, D.J & Geddes, R. Agents Actions
Harrison. K. (1990). Journal of Shellfish Research. 9, 1–28. (1993) 38: 139-42.
He. K, Song. Y. and Daviglus. M. L. (2004). Circulation 109, 2705– Mishina. T, Watanabe. H, Araki. H, and Nakao. M. (1985). Prostate,
2711. 6, 423–436.
Hebert. J. R, Hurley. T. G, Olendzki. B. C, Teas. J, Ma. Y. and Moriarty, D.J.W., 1997. Aquaculture 151, 333–49.
Hampl, J. S. (1998). Journal of the National Cancer Institute. Nair. R. K. G. and Gopakumar. K. (1977). Journal of Food Science
90, 1637–1647. and Technology. 14, 268.
Hebert. J. R. and Rosen. A. (1996). Cancer Detection and Nair. V. P. G. and Gopakumar. K. (1978). Journal of Food Science.
Prevention. 20, 234–244. 43, 1162.
Heinrikson. L. and Meredith. S. C. (1984). Analytical Biochemistry. Nobmann. E. D, Byers. T, Lanier. A. P, Hankin. J. H, and Jackson. M.
136, 65-74. Y. (1992). American Journal of Clinical Nutrition. 55, 1024–
Hu, T., Curtis, J. M., Oshima, Y., Quilliam, M. A., Walter, J. 1032.
A., Watson-Wright, W. M & Wright, J. L. C. Chemical Nutting. P. A, Freeman. W. L, Risser. D. R, Helgerson. S. D, Paisano.
Communication (1995) 21: 59. R, Hisnanick. J, Beaver. S. K, Peters. I, Carney. J. P. and Speers.
Huang. K. H. and Akoh. C. C. (1994). Lipase-catalyzed incorporation M. A. (1993). American Journal of Public Health. 83, 1589–
of n3 polyunsaturated fatty acids into vegetable oils. Journal 1598.

351
 Pettit, G.R., Kamano, Y., Hurald, C.L., Tuinman, A.A & Boethner, Ternstrom. A, Lindberg. A. M, and Molin. G. (1993). Journal of
F.E.X. Journal of American Chemical Society (1987) 109: 6883- Applied Bacteriology. 75, 25–34.
6885. Terry. P, Lichtenstein. P, Feychting. M, Ahlbom. A, and Wolk. A.
Preetha, R, Jose, Seena, Sreedharan, P, Vijayan. K.K., Jayaprakash, (2001). Lancet. 357, 1764–1766.
N S, Philip, Rosamma and Bright Singh, I S (2010) Aquaculture Tincu, J.A & Taylor, S.W. Antimicrobial Agents and Chemotherapy
Research, 41 . pp. 1452-1461. (2004) 48(10):3645-3654.
Pridmore, D., Rekhif, N., Pittet, A.C., Suri, B., Mollet. B., 1996. Tocher, D.R. Glycerophospholipid metabolism. In: Hochachka, P.W.,
Appl. Environ. Microbiol., 62 (5), 1799-1802. Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology
Raaijmakers, J.M., Weller, D.M., Thomashow, L.S., 1997. Appl. of Fishes, Metabolic Biochemistry, vol. 4. Elsevier Press,
Environ. Microbiol. 63, 881–887. Amsterdam, (1995) pp. 119–157.
Rajaganapathi, J., Thyagrajan, S.P & Edward, J.K.P. Journal of Torrento, M., Torres, J., 1996. UPV J. Nat. Sci. 1, 130–138.
Experrimental Biology (2000) 38: 519-520. Uemura, D., Chou, T., Haino, T., Nagatsu, A., Fukuzawa, S., Zheng,
Re, R., Pellegrini, N., Proteggente, A.A., Pannala, A., Yang, M.M S & Chen, H. Journal of American Chemical Society (1995)
& Rice-Evans, C. Free Radical Biology and Medicine (1999) 117: 1155.
26: 1231-1237. Vazhappilly. R. and Chen. F. (1998). American Oil Chemists’ Society.
Reeds. P.J. (2000). The Journal of Nutrition 130 (7), 1835S–1840S. 75, 393-397.
Refsgaard. H. H. F, Brockhoff. P. B. and Jensen. B. (1998). Journal Vijayan, K K and Bright Singh, I S and Jayaprakash, N S and
of Agricultural and Food Chemistry. 46, 808-812. Alavandi, S V and Pai, Somnath and Preetha, R and Rajan, J J S
Rose. D. P, and Connolly. J. M. (1991). Prostate. 18, 243–254. and Santiago, T C (2006) Aquaculture, 251 (2-4). pp. 192-200.
Rose. D. P. (1997). American Journal of Clinical Nutrition. Vijayan, K K and Stalin Raj, V and Alavandi, S V and Selchar, V
66, 1513S-1522S. Thillai and Santiago, T C (2005) Aquaculture Research, 36.
Russo, G & Tringali, G. Review of International Oceanography and pp. 311-316.
Medicine (1983) 70/71:49-54. Waksman, S. A. & Woodruff, H. B. J. Bacteriol. 42, 231–49 (1941).
Saha, S., Roy, R.N., Sen, S.K., Ray, A.K., 2006. Aquaculture Research Wang, Y. B., Li, J. R., Lin, J., 2008. Aquaculture (in press).
37, 380-388. Whitehouse, M.W., Macrides, T.A., Kalafatis, N., Betts, W.H.,
Sakami. W. and Harrington. H. (1963). Annual Review of Haynes, D.R &. Broadbent, J. Inflammopharmacology (1997)
Biochemistry. 32, 355–98. 5: pp. 237-246.
Schmidt. E. B, Arnesen. H, De Caterina. R, Rasmussen. H. and Yen, G.C. & Chen, H.Y. Journal of Agricultural and Food Chemistry
Kristensen. S. D. (2005). Thrombosis Research. 115, 163–17. (1995) 43: pp. 27–37.
Schmidt. E. B. (1997). Danish Medical Bulletin. 44,1–22 Young. V.R. (1994). The Journal of Nutrition 124 (8
Serhan. C. N, Hong. S, Gronert. K, Colgan. S. P, Devchand. P. Suppl): 1517S–1523S.
R, Mirick. G. and Moussignac. R. L. (2002). Journal of Yuan, Y.V., Bone, D.E & Carrington, M.F. Food Chemistry (2005)
Experimental Medicine. 196(8), 1025–1037. 91:pp. 485–494.
Smith, P., Davey, S., 1993. J. Fish Dis. 16, 521–524. Zaccheo. T, Giudici. D, and di Salle. E. (1998). Prostate. 35, 237–
Smith. M. L, and Murphy. R. C. (2002). In Biochemistry of Lipids, 242.
Lipoproteins and Membranes, Vance, D. E, Vance, J, Eds, Zhang, L., An, R., Wang, J., Sun, N., Zhang, S., Hu, J., Kuai J., 2005.
Elsevier: Amsterdam. 341-371. Current Opinion in Microbiology, 8, 276–281.
Central Marine Fisheries Research Institute

352
Marine Organisms –Treasure House of
Valuable Products and their Chemical
Perspectives
I. Rajendran* and P. Vijayagopal
Principal Scientist
Mandapam Research Center of CMFRI
e-mail: [email protected]

Introduction like ET 473, ziconotide, bryostatin A, etc. With the

advancement in the instrumentation like 2D NMR,
The oceans and sea constitute more than 70% of HRMS, etc. it is also now possible to elucidate the
the earth’s surface. The biologically diverse and chemical structure of the compounds which are
ecologically complex marine kingdom comprising normally available in traces.
both flora and fauna may, therefore, be considered
as the largest reservoir of natural products on Secondary metabolites
earth. Marine ecosystem accommodates more
than 80% of earth’s phyla comprising 34 of the The biosynthesis and breakdown of proteins, fats,

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

total 36 phyla, out of which 13 are exclusive of nucleic acids and carbohydrates, which are essential to
marine origin. Marine biota excluding some culinary all living organisms, is known as primary metabolism
organisms are the store house for compounds of with the compounds involved in the pathways known
pharmaceutical and pharmacological importance, as “primary metabolites”. The mechanism by which
biochemical molecular probes for pharmacological an organism biosynthesizes compounds called
studies, models for drug development, anticancer ‛secondary metabolites’ (natural products) is often
agents, nutraceuticals, and other therapeutic found to be unique to an organism or is an expression
importance. They are the facilitators for the stability of the individuality of a species and is referred to as
of marine ecosystem. They include corals, sponges, “secondary metabolism”. Secondary metabolites are
micro and macro algae, coelenterates (soft and generally not essential for the growth, development
hard corals, gorgonians), bryozoans, molluscs, or reproduction of an organism and are produced
tunicates (ascidians), echinoderms, micro organisms either as a result of the organism adapting to its
and miscellaneous including soap fish, hydroid, surrounding environment or are produced to act as
annelid, sea grass, mangroves, etc. The compounds a possible defense mechanism against predators to
associated with them as secondary metabolites are assist in the survival of the organism.
now mostly attributed to the symbiotic bacteria,
fungi, cyanobacteria housing in sponges, algae, Marine natural
corals etc. with compelling evidence. eg. bacteria,
Candidatus Endobugula sertula of bryozoan, Bugula
products (MNPs)
neritina for the production of bryostatin 1. Chemical Marine natural products are compounds with novel
symbiosis is for host organism’s defense and food, structural features found typical of marine origin.
shelter for the parasites. This chemical warfare Many are having definite biological activity with
has been occuring for thousands of years for their unique structural features mainly of chemical interest.
existence/survival with their hostile environment. Any report of the new product from marine organisms
Few of them find place for successful clinical trials is followed by its synthesis reported by another group

353
 elsewhere. The compounds are the result of either Sesquiterpenes
metabolism by organism itself or of the symbiotic
microbiota present in inter or intra specific mode Agelasidine A
in the cells of the organisms. Some compounds
are indeed specific to particular genus useful for It is a sesquiterpene based taurocyamine derivative
chemotaxonomical classification. from a Pacific Sponge Agelas Sp.

NH NH2
The abundance of the source of MNPs from the
Cl
organisms is approximately in the order: O
NH2
O
S
Sponge > Coelenterates > Microorganisms &
Phytoplankton > Echinoderms = Tunicates > Red
algae > Molluscs ≥ Brown algae ≥ Green algae
> Bryozoans Agelasidine A

Since sponges cover most of the structurally featured The sesquiterpenes having unusual carbocyclic
compounds, the compounds retrieved from them skeleton are present in Cymbastela hooperi. The
have been taken for discussion. On the basis of compounds are 1R*,2S*,5R*,6R*,7S*,8R*)-1,5-
novelty in the structures, they are classified under dimethyl-7-(1’-methylethenyl)-tricyclo [6.2.0.3]
the major groups of organic compounds: decane (kelsoene).

1. Terpenoids H
14

2. Alkaloids 10 7
4 14
2 15 9
3. Heterocycles 1 5
5 9 13
4. Steroids 10
15 H 7 2
5. Polyacetylenes, peptides, polyethers, polyketides, H 11 O
H 1 O
16 3
macrolides and 13 11 12 12 OH

6. Glycosides & nucleosides

Kelsoene Furanosesquiterpene

Terpenoids Sesquiterpene with incorporated furanone moiety

has been reported from sponge, Dysidea herbacea.
Terpenes are the major class of compounds Germacrane sesquiterpenes (1Z,4Z)-7RH-11-
found among the sponge secondary metabolites. aminogermacra-1(10),4-diene.
Original compounds, artifacts, analogues and
functional derivatives are among the range of the The formation of the artifacts from the parent
Central Marine Fisheries Research Institute

terpenoids identified from sponge extracts. The compound puupehenone by methanol adduct is
terpenes isolated and characterized from sponges dicussed. Hyrtios sp. (Puupehenone Congeners)
generally include sesquiterpenes (C15), diterpenes
(C20), sesterterpenes (C25) and triterpenes (C30), OH

20 15
with functional groups comprising formamide, O

hydroquinone, epoxy, halogen substituted 1

10
16
carbonimides, peroxides, isocyano, furan, sulphate, 15
14
keto, aldehyde, hydroxyl, acetoxy, aromatic, 9 O 7
1
isonitrile, pyrrole, amino, guanidine, adenine, 12
H 13 4 5 11
pyran, etc. Devoid of one or two carbons result 4 NH2
in the norterpenes. Stereo isomers include either 14 13
12
enantiomers or diastereoisomers. The compounds 11
(1Z,4Z)-7alphaH-11-amino-
are cyclic and/or acyclic and rearranged form. (+)-Puupehenone germacra-1(10),4-diene

354
Diterpenes Alkaloids
They include agelasine and kalihinane.
Alkaloids are having unique structures different from
N
N
that of terrestrial origins. They include derivatives
H2N having heterocyclic structural units of bromopyrrole,
+
NMe pyrroloquinoline, pyrroloiminoquinone, bromoindole,
N
Cl- cyclic amine linked to a β-carboline, imidazole,
oxazoles, tryptophan, tyrosine, guanidine,
isoquinoline, pyridine, purine, etc
Ageline A

NHCHO Pyrroloquinoline alkaloids, Zyzzya fuliginosa;

H
Sventrin, Agelas sventres

O
R
O
N
H O

Cl

NH
Cl
Batzelline D, R=H
Kalihinene X - 1 beta H, 14 alpha Cl Batzelline C, R=CH3
Kalihinene Y - 1 alpha H, 14 alpha Cl Br
Kalihinene Y - 1 alpha H, 14 alpha Cl
4
3 15 N
Br 5
Sesterterpenes

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

H 11 NH2
N 2 6 N 8 13
Cavernosolide, Fasciospongia cavernosa, 35 10
N
H3C H
O
OAc
Sventrin
H
24
22 O Bromoindoles
23
1 14 16
H 1'
10 8 R1 N
2'
H O
25 19 H 1"
4 1 N R2
O 4' N 2"
H
8"
N3
H 4"

Cavernosolide O
Topsentin A, R1=H, R2=H
Topsentin B1, R1=H, R2=OH
Topsentin B2, R1=Br, R2=H
Triterpenes
Sodwanone K Cyclic amine linked to a β-carbolines
31
13 14
O 30
11 16

H
24 HO N 20 19
22 10 N 18
3 O H H
2 1
25 R
26 18 H 7 9
1O 5
H
OH
3
N 6 Cl
29 25 23
H 7 6 14
30

15 24
28 27 N H
10 H OH 36 31

28 35
27
Manzamine A HCl, R=H
Sodwanone K ent-18-Hydroxymanzamine A HCl, R=OH

355
 Imidazoles alkaloids Guanidine alkaloids

23
H
OMe OH H
17 H Cl H O
N N
18 22 H2N
29 14
OMe H
H2N N (CH2)14 O N
3 4
27 N 28 O 20
H
N H
5 O O
O 24 13,14,15-Isocrambescidin800
26 2
N 1 Isonaamidine E
N 25 N
H 6
Sulfamate Indoles
7 8
1
11 2
13 21 R2 N
12 OMe 13
12a 2a 3
14 R1 N 12b 2b
OH OH 12
8 6 11a
O 6
7a N 6a
O 7 1
7b 7
12 9 N OH 10 NH
N 8
10 O
11 Bengazole A
13 O Plakinidine A, R1=H, R2=CH3
O 14 27 Plakinidine B, R1, R2=CH3
(CH2)12CH3
Indolizidine alkaloid
Tryptophan and tyrosine alkaloids
14' 13' 12' 3
OMe H
H 1 CH3
1' N N
3 5 Cl
Br Br 11' 9 8a
H
5
O
1 8
HO Stellettamide B
Br
O
H 11
N 9 N
14

20
Steroidal alkaloids
18
O
Br 16 O NH2Me
Purealidin S

Isoquinoline alkaloids
Central Marine Fisheries Research Institute

OMe
N
HO

O H E O
D OH
NMe N
A B
N O
MeO HO H Plakinamine E
H H
O O O
O
12

O 14 10 1
N 7 H 3
Cribrostatin 4 MeO N NH2
13 N
O
O
20

O 18 Motuporamine A
Renierone

356
Heterocycles Polyacetylenes, polyethers,
Bengamides polyketides, peptides and macrolides
1
OH OMe O
R2
4 H H Acetylenic acids
2 6 9 N 16 N
15 10 13 14
1 7 8
OH OH O 13 HO
Callyspongenol A
22
Bengamide Y, R1=R2=H OR1
Bengamide Z, R1=H, R2=Me

Purine and nucleoside metabolites 13

9 7
O O
5 3
12 10 1
HO NH OH
6 H C14 Acetylenic acid
5 N
N 7 17'
8 O Br
2 9 15' 9'
O
N 4 N
H 7'
1'
Erinacean 28
OR
21
22
20
18
26
Heterocyclic macrocyclic lactones (Polyketide) 17
25

19 11 29
Fijianolides 27
1 14
10 30

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

R = 5
28
ax H
C Heq
Xestosterol ester of 18-bromooctadeca-(9E,17E)-diene-
5 9 7,15-diynoic acid
O

O Calyculinamide related compounds: Geometricin

13
O H 30 29 O
30 28
OH N
20 B 17
26

23 O
A OH OH OH OMe
3
27 O H ax H 7 11 O O 23
16
31
Fijianolide A 1 CONH2 35
18 20
37 36

HO O OH
Bengazoles are homologous fatty acid esters of a Geometricin A P 33 32

heterocyclic nucleus comprised of a bis(oxazolyl)- HO O

methanol further substituted with a hexanetetrol Polybrominated diphenyl ethers

side chain.
OR2 OH OR1 OR2 OR1
8 1
R3 1' O Br
7 6 1 6
O
12 6' 3
9 N OH
N Br Br

13 11 R4 Br
O
Polybrominated diphenyl ethers
Bengazole 1, R1=CO(CH2)14CH3, R2=H 3 = R1=R2=H, R3=R4=Br
Bengazole 2, R1=H, R2=CO(CH2)14CH3 7 = R1=R2=H, R3=Br, R4=H
Bengazole 3, R1=CO(CH2)12CH(CH3)2, R2=H
Bengazole 4, R1=H, R2=CO(CH2)12CH(CH3)2
Bengazole 5, R1=CO(CH2)13CH3, R2=H
Calyculins: Calyculin J- It is a spiro ketal of an
Bengazole 6, R1=H, R2=CO(CH2)13CH3 unprecedented skeleton bearing phosphate, oxazole,

357
 nitrile, and amide functionalities, Discodermia calyx; Clavosines A and B are closely related to calyculins
Hamigera tarangaensis[163] and calyculinamides
OH O 51 41
OH O
O 37
38 29 O
MeO N 33
MeO N 28
H H
NMe2 OH N OH N
39 40 26

O
HO
P
HO O O 43
HO OH O 42

O O P 44
NC 11 48 47 46
48 8 O R2
O H O O 20
O
10 3 7 11 16
OH OMe R1 1'
Br 47
50 49 OH OH OMe O OMe 8'
Calyculin J
45 MeO OMe
7' 6' 9'

Clavosine A, R1=H, R2=CONH2

Clavosine B, R1=CONH2 R2=H
Dysiherbaine: It is a cis fused hexahydrofuro[3,2-b]
pyran ring substituted with a 3-[2-aminopropanoic
acid] side chain, Dysidea herbacea Macrocyclic lactone/lactams
Bicyclic peptides

NH2Me
H1 NH3 H
OH
OOC 3 O 7
4
6 10
OH
OOC O 14
H

Dysiherbaine
17
N
Discodermolide O 8
7 1
1 S O O
O O
30 31 32
3
NH
O O 7 28 19
1
9 15
7 N
5 HN
O N 4
3 OH 12 OH O
25 26 29 2
27 6 O O
O
OH OH 24
NH2
NHSO3Na
Discodermolide Scleritodermin A

Polyether macrolide
Central Marine Fisheries Research Institute

Homohalichondrin B
Trp-6 MeGin-10
H O NH2
N Cys(O3H)-8
Ala-1 Val-4
SO3Na
O Phe-11
O H O O H O H O HMeN
H H H H H N N N N HN
O O O H N N N N N O
H O H O H O H O
O H H NH
O
Pro-3 Val-5 Thr-9
O O N
O O O O NH O Gly-12
Br X
H H H H H O O
O
H2N NH2 Asn-13
O BrPhe-2
O Arg-7 NH2
O
O Halicylindramide D
O
HO
R= O
HO H

Homohalichondrin B

358
Macrolactone Steroidal oligoglycosides

O
HO
HO OAc OMe R=
R

OH
OH
O O H O
HO O
O O
O O H H
OH
OH X= OH OH
OH
X O O O
H O
R1 HO HO OH OH
OH H
OH HO

O O Mycaloside A

HO
OH O

O OH
H OH
R2 OH

5-Desacetylaltohyrtin A, R1=OH, R2=Cl

Glycosides and nucleosides derivatives

Sterols, glycosides and 26

nucleosides derivatives Cl
23
22 O
O Cl OH O
21
7 1 O OH
18 N
3 O
HO OH
O 5 OH
H2N O

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

AcO O O
Rubroside A O

MeO
HO OH

OSO3Na

Acanthosterol sulfate J
Marine algae
They include both micro and macro algae and are
N easily renewable product sources of the oceans.
Unlike other organisms they are somewhat easily
H accessible for the harvest and product isolations.
OAc
N Major products are the sulphated polysaccharides
in general and in particular are the agar, algin and
Plakinamine I carrageenan depending on their sources of red,
26'
brown and red respectively. In the oriental countries
25' of Japan, China, Korea, etc. marine macro algae
27
24'
21 26
is the traditional staple food. Apart from the algal
24

O 22'
polysaccharides, minor products like, phenolics,
19
phlorotannins, lipids, terpenoids, sterols, vitamins and
HO
O minerals are also available. They have good medicinal
HO
3
properties as micro and macro nutrients essential for
OSO3Na

19'
the balanced diet. They act as the natural antioxidants
O and antimicrobial compounds.
Crellastatin A HO OH
With the explorations of their utility, algal products

359
 are increasingly felt applicable in various fields as, to express these genes in a convenient and suitable
biofuels, cosmetics, nutraceuticals, biofertilizers, host such as bacterium like E. coli. Sequencing these
functional foods, antifouling compounds, etc. The responsible genes of bacteria is indeed a challenging
polysaccharides are composed mainly of galactose, task. Inserting the gene cluster (~55,000 base pairs
fucose, guluronic acid, mannuronic acid as major in the case of bryostatin) into a suitable bacterium,
sugar units. With their extent and conformations will trigger the synthesis of the proteins that in turn
they form different polysaccharides among the three produce the desired compound. This approach will
algal groups. help save marine environment’s depletion of marine
biota by harvest for the want of required quantity of
Industrial important marine the product. Using DNA recombinant technology,
marine drugs are being attempted in a convenient
natural products (MNPs) culture systems. This is because marine microbes have
Cold active proteinase (psychrophilic enzyme) from very huge genome with one billion base pairs per
Atlantic cod is temperature and acid sensitive making cell with two picogram (10−12), which is obstacle for
them use in food processing industries. They have search of genes responsible for biosynthesis of these
higher catalytic activity at very low temperature. compounds. However researchers are unraveling the
Products of aquaculture importance – useful in feed possibilities to simulate the functions by recombinant
formulations as binder (agar), feed additives (micro technology for a target molecules.
nutrients) from algae, probiotic marine microbes for
aquaculture, culture and isolation technologies of the Conclusion
nutraceuticals from micro algae, immunostimulants
from marine sources for commercial important Marine organisms are the store house of marine
fish cultures. micro biota to have unique molecules. The associated
microbes are having prime role in the formation of
Biotechnological tool these secondary metabolites. Modern techniques
of biotechnology are increasingly felt essential for
for MNPs genetically modified organisms to get the desired
There is a serious problem regarding yield of product in bioprospecting the marine ecosystem.
such promising compounds to complete the
pharmacological trials, as most of the compounds Suggested readings
are present in very low quantity. This supply problem
can be ameliorated with the advent of modern Colegate, S.M.; Molyneux, R.J. 2008 Bioactive Natural Products:
biotechnological tools. Enzymes (proteins) are Detection, Isolation and Structure Determination; CRC Press:
Boca Raton, FL, USA, pp. 421–437
responsible for the production of these secondary Antonov AS, Afiyatullov SS, Kalinovsky AI et al. 2003, Mycalosides
metabolites and these are like cells’ machines to B-I, Eight New Spermostatic Steroid Oligoglycosides from the
Central Marine Fisheries Research Institute

Sponge Mycale laxissima. J Nat Prod 66:1082-1088

use raw materials like amino acids and sugars to Kim, S K and Chojnacka (Eds), K 2015 Marine Algae Extracts–
biosynthesize them. The responsible genes that encode Processes, Products, and Applications Vol I & II, Wiley-VCH
the production of these compounds will be identified Verlag GmbH & Co, KGaA

360
Classification of Organic Compounds with
Reference to Natural Products
I. Rajendran
Principal Scientist
Mandapam Research Center of CMFRI
e-mail: [email protected]

Introduction
isomer from a structural formula. A uniform variation
The aim of the classification of natural products was of the molecular entity in a series of compounds is
to record them depending on their medicinal value called homologous. Eg. Alkanes, the members have
and other biological activities. They were classified the uniform difference of –CH2 and common formula
into their structural groups with functional groups of CnH2n+2. Likewise it is treated for alkenes, alkynes,
responsible for the bio activity. Though they fall into alcohols, cyclic alkanes, with common formulae.
the organized literary structural groups of organic
compounds, their medicinal and physical properties Classification of
are taken into account for their classification. It is for
the convenience to identify their chemical structural
organic compounds

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

features and in turn correlate them to their bio The compounds isolated from marine natural
activity. With the classification of organic compounds resources by bioprospecting generally fall under the
having identical structural features, documentation of categories of carbohydrates, terpenoids, carotenoids,
a particular compound with respect to its chemistry polycyclic aromatic hydrocarbons (PAH), steroids,
and bio activity has become easy for further reference. heterocyclic compounds, peptides and proteins,
Proper documentation of the identified compounds alkaloids, polysaccharides, anthocyanins, nucleic
helps to dereplicate the known compounds in an acids, vitamins, pigments, toxins and prostaglandins.
extract during the exploration of an organism for Some groups like toxins and carbohydrates are special
a product of medicinal value. This dereplication is to marine resources with respect to their unique
necessary to save time and money in rediscovering structural features specific to marine origin. Though
the known compounds from an extract. each group has the spectrum of bioactivity, these
bioactivities overlap among one another leaving
IUPAC system of the option to have the compound quantitatively for
further utilization under renewable condition. So the
nomenclature and quantitative availability of the product depends on the
homologous series extent of the source to be renewed and/or cultured.
In this way the product leads have the difficulty in
In order to document the compounds with universal reproducibility from the same species of the source.
scientific names, chemists agree to common rule to Some of the lead compounds under each group will
adapt to avoid the controversy in naming an organic be seen in the following sections.
compound. In this way, the IUPAC nomenclature
system is a set of logical rules devised and used by Carbohydrates
organic chemists to circumvent problems caused by
arbitrary nomenclature. With these rules one can Carbohydrates are general term used to represent
write a unique name for every possible compound or sugars of monomer, oligo and polysaccharides with

361
 CHO
general formula Cx(H2O)y with the equivalence of
H OH OH
hydrogen and oxygen as that of water present in it
along with carbon. But number of other structural H OH HOOC H O
H
units are found out from the natural sources, HO H H H
which may not conform to the general formula. eg. H OH
H OH
formaldehyde, acetic acid. Their names end with ose; OH OH

carbohydrates with an aldehyde group are called COOH

aldoses and those with ketonic, ketoses. Molecules Fischer projection alpha-pyranose form

containing four carbons are tetrose, a pentose five,

Guluronic acid
a hexose six, etc. The basic units by which the macro
molecule are built, are the monomers. The macro acid and α-L-guluronic acid.
molecules depending on the extent of multiplicity
of the basic units are called oligosaccharides and Terpenoids
polysaccharides. The deoxy representation of the
sugar name means the replacement of a hydroxyl These are compounds formed generally by the basic
group by hydrogen. unit of isoprene joined together in “head to tail”
mode with the multiplicity of 5. Monoterpenoids
The agar and carrageenan are the polysaccharides are compounds having the formula of C10H16,
present in red seaweeds with galactan unit. The agarose diterpernoids of C15H24, diterpenoids of C20H32,
is composed of agarobiose repeating disaccharide sesterterpenoids of C25H40, triterpenoids of C30H48,
units alternating with 1, 3-linked-β-D-galactopyranose tetraterpenoids of C 40H 64 (separate class of
CHO caroternoids) and polyterpenoids of general formula,
H OH OH OH
(C5H8)n. compounds of marine origin in this class
of compounds limits to triterpenoids. They found
H O
HO H
H
mostly as oxygenated derivatives with alcohol,
HO H HO OH aldehyde, ketone functional groups. However in MNPs
H OH
H OH
heterocycles like furan, quinines are also linked to the
H H
terpenoid skeleton to form compounds. Some of the
CH2OH
CHO
H
Fischer projection Beta-Pyranose form OH
H
Galactose

and 1, 4-linked-3, 6-anhydro-α-L-galactopyranose. H

O

Algin is an anionic polysaccharide found in brown H

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seaweeds. It has mannuronic acid and guluronic acid O

as building blocks with 1 to 4 linked β-D-mannuronic Hyrtiosal OH O

CHO
H

HO H HOOC OH O
HO
HO H
HO OH
H H OH
H OH
H H
H H
H OH

COOH H

Fischer projection alpha-pyranose form

Scalarane sesterterpene d
Mannuronic acid
examples with good biological activities are:

362
 2' 5' isomers like naphthaquinones, anthraquinones are
NaO3SO OSO3Na
reported in MNPs.
1'

1 25
8
26
3
Steroids
6
13 They are crystalline compounds occurring in nature
27
in free or ester of higher fatty acids.
30
OH 24 O
18

HO
AcO
29

Adociasulfate 3
HO
OSO3Na
Carotenoids Acanthosterol sulfate J
They are polyenes the molecular formula of C40H56.
They are yellow or red pigments associated with
chlorophyll. Hydrocarbon based compounds are

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

beta-Carotene

known as the carotenes and that of oxygenated O

derivatives as xanthophylls.
OH

Polycyclic aromatic Oxygenated steroid

hydrocarbons (PAH)
They occur in plants, animals, of both land and marine
They may be of two types with the isolated rings origins. Some are of the typical sterols of marine
and of fused rings. Eg. Biphenyl, anthracene. They origin. Eg. A series of oxygenated steroids have been
isolated from green algae Codium arabicum.

Heterocyclic compounds
Biphenyl
They contain five or six membered heterocyclic rings
primarily occur in coal tar. Some of their structural 1
OH OMe O
R2
H
3 10 N 12 18 N
2
16
OH OH O
Pyrene
Anthracene OR1
Bengamide L, R1=CO(CH2)11CH(CH3)2, R2=H

363
 28 are extensively characterized from marine organisms
ax H
C Heq
and also land plants and microbes. Aciculitins A-C are
5 9
O bicyclic peptide that contain an unusual histidinotyrosine
O bridge with attachment to the bicyclic peptide.
13
O H
30
OH Polyhydroxylated lactone
20 B 17
23 O
A OH Alkaloids
27 O H ax 30 31 32

Fijianolide A
O O 7 28 19
H2N 1 5
9 15

N 3 OH 12 OH O
25 26 29
27
HN
O OH OH NH2
24

Discodermolide

They are the bases of plant, animal and marine origin

HN having a nitrogen atom predominantly. They are used as
NH medicine in small quantities. Some of the compounds
O of substituted alkaloids of marine origin are:
debromohymenialdisine

with aromaticity. The heteroatoms are oxygen, nitrogen Guanidine alkaloids

and sulphur with prefixes oxa, thia and aza respectively
while forming the names. Some heterocycles of marine 14' 13' 12' 3
H
origin are: Bengamides with anticancer activity. H 1 CH3
1' N N 5 Cl
11' 9 8a
H
O
Peptides and proteins 8

Stellettamide B

Proteins furnish a mixture of amino acids on hydrolysis

with acids, alkalis or enzymes with the intermediate
components of peptides of different and number
Pigments
of amino acids having different molecular weights. H
OH H
Amino acids are charges molecules of both positive and H Cl H O
N N
negative depending on the pH of the medium. Twenty H2N
amino acids are common and found in all proteins. H2N N (CH2)14 O
H
N
However amino acids of different structural features H
Central Marine Fisheries Research Institute

H
O O
H2NOC 13,14,15-Isocrambescidin800
E
F O D
CONH2
N
H
N They include number of compounds with chromophores
O H

HN N
O
OH
OH responsible for light energy absorption of visible
G HN NH
O
wavelength region. They are found in marine algae
O C and based on their presence, algae are classified into
NH HN
H red, green and brown algae. Eg. fucoxanthin – useful
O O OH O
O H antioxidant and in treatment of obesity.
OH N N N NH B
H H
HO O O
Toxins
OH
A
O I
HO
K

J Aciculitin A, R=C5H11 They are defensive chemicals of both marine and

Aciculitin B, R=C6H13
R Aciculitin C, R=C7H15 terrestrial origin; that of marine origin are called

364
ichthyotoxins. Phenero toxin and crypto toxin are two origin are of important in pharmological studies
types; the former having the venomous apparatus as molecular probes in retrieving biochemical
and the latter having the toxin incorporated in body reactions. Further unraveling the marine resources
tissues gained by food chain, symbiotic parasites and may throw light to the treasure of many more
biosynthetic transformations of the acquired chemicals products for the welfare of the mankind.

Ciguatoxin (CTX)

OH H H
O O H
H OH
H H
O H H O
O H H
O H
O H
H O O
O H O
H O H
H O
R1 H H H H
R2

R1 = -CH(OH)-CH2OH; R2 = OH
Tetrodotoxin (TTX)

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

O

HO OH

H2N O O
O
OH
HN
N
H HO
HO O
N HO 6 R2 OH
H H2N
N
H
R1 HO

R1 R2
TTX OH CH2OH

Suggested readings
Conclusion
Finar I L, 2005 Oranic chemistry, vol 1 The fundamental principles
6th edn, Pearson education (Singapore) Pte. Ltd, New Delhi.
Classification of organic compounds in the context Newman D J, Cragg G M 2004 Marine natural products and related
of bioactivity helps to document the compound in compounds in clinical and advanced preclinical trials, J. Nat.
a convenient way to refer to them for any assay Prod, 67:1216-1238
Jimeno J, Faircloth G, Fernandez Sousa-Faro J M, Scheuer P, Rinehart
or isomer synthesis or a lead compound for the K, 2004 New Marine Derived Anticancer Therapeutics ─ A
clinical trials. In this way compounds of marine Journey from the Sea to Clinical Trials, Mar. Drugs 2:14-29

365
 Physical and Chemical Methods for
Structural Elucidation and Identification
of Organic Compounds
I. Rajendran
Principal Scientist
Mandapam Research Center of CMFRI
e-mail: [email protected]

Introduction chromatographic techniques are involved depending

on the nature of the mixture that is handled. State-
Organic compounds are the derived from carbon of-art instrumentation is now available to use this
and are of plant or animal origin. Any interpretation technique for a successful separation of a compound.
and study are done with respect to the pure organic The quantity of the final pure compound may not be
compound only to know the molecular characteristics sufficient in many cases and marine bioprospecting
and its property. The pure compound is therefore in particular. Various separation methods have been
isolated from its original mixture or extract by dealt with in other chapter extensively. The suitable
various methods. Unlike inorganic salts which are method can be followed depending on the type and
ionic and simple molecules, organic molecules are quantity of compound isolated.
from simple to complex. Since it is available from
living systems, it is formed by regular biosynthetic Identification of
pathway either by the host or the microbes present
in the host organism. Macro molecules albeit simple
organic compounds –
with respect to some structural features, are complex Chemical methods
with secondary and tertiary structures which are
responsible for their biological functions. With the Qualitative analysis
latest developments in the structural interpretation
with the help of MALDI-TOF-MS/MS tandem The elements of an organic compound are: carbon,
mass spectrometry, it is now possible to deal with hydrogen, oxygen, nitrogen, sulphur, phosphorous,
molecules like peptides and acetogenins. and metals. Preliminary qualitative chemical analysis
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of the elements is done using cupric oxide for C and

Separation of H. Elements of N, halogens, and S are analyzed by
Lassaigne test. Common functional groups present
organic compounds in organic compound are analyzed/estimated by
Separation of organic compound is a foremost standard methods. In this way, estimation of phenols,
step to proceed further for the determination ketones, sugars, ascorbic acid, amino groups (aromatic
of the structure. Previously solvent extraction, amines), nitro groups (aromatic nitro compounds),
distillation, steam distillation were used. Later amino acids (glycine), etc. are done. The unknown
counter current separation, electrophoresis, dialysis, compound is subjected to various chemical reactions
molecular distillation, ultracentrifugation, etc. to study the plausible structure. The compound
Chromatography is most extensive method used for is also degraded with reagents to break them into
the isolation and purification of compounds from small molecules of known structures. Integration of
the reaction mixture or extract. Various forms of small molecules in a possible manner was done to

366
arrive at a probable structure. However execution of Some compounds with same physical and chemical
the structural determination methods depends on properties are not of mirror image arrangement of
the quantity of the compound isolated from natural their groups and they are diastereoisomers. Some
sources. If the quantity is low, analysis by chemical compounds do not possess rotation and are called
methods, degradative studies, etc. is limited and the meso- compound. The optical isomers can be
problem is explored with instrumental analysis. represented by

Stereochemistry Newman projection or saw-horse form. Racemic

mixture is a mechanical mixture of enantiomers.
Stereochemistry is the chemistry of groups and
atoms of a molecule with respect to their spatial Geometrical isomerism
arrangements. Stereoisomerism is exhibited by
molecules having the same molecular formula but It is also called cis-trans isomerism. Eg. Maleic acid
different spatial arrangement. It helps to understand and fumaric acids. The cis-compound has identical or
the reaction course because the orientation of groups similar atoms or groups on the same side.
determine the conditions of the reaction. There are
two types of stereoisomerism, optical isomerism and Identification of
geometrical isomerism (cis-trans).
organic compounds –
H H H R2
Physical methods
The pure organic compound obtained by
R1 R2 R1 H appropriate physical method of separation is

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

subjected to elemental as well as the functional
cis trans
group analysis to know the preliminary
composition and nature of the composition.
Optical isomerism: It is formed by the asymmetric The elements are quantitatively estimated by
carbons present in the molecules as these compounds elemental analyzer and atomic absorption
rotate the plane of polarized light [dextro (+) or laevo spectrophotometer to arrive at the percentage
(-) rotatary]. Optical isomers may rotate the plane of composition. Physical methods involve
polarization by equal and opposite amounts. These sophisticated instruments for the structural and
particular isomers are called enantiomers and also functional group analysis. These methods are
can be visualized as the mirror image to each other very useful in the recovery of costly compounds
with respect to their spatial arrangement. after the analysis which is not possible in the
case of chemical methods. Additional methods
and improvements are being added up with the
B advancements in the instrumental analyses.
A C B
C A

Molecular weight determination

It is done based on the physical parameters like
vapour density, boiling point elevation, freezing
point depression for simple molecules. Physical
methods like rate of diffusion, rate of sedimentation,
viscosity of the solution, osmotic pressure are use
F D D F
E for the determination of molecular weight of larger
E
molecules. X-ray (XRD) and mass spectrometry are
Enantiomer 1 mirror Enantiomer 2 also used.

367
 UV-visible spectroscopy properties. Eg. 1H1, 15N7, 2H1, 13C6. These nuclei are
having resultant spin behave as spinning magnet and
It includes the wavelength range of 200-400 nm for UV they will orient themselves in an applied magnetic
and 400-750 nm for visible spectroscopy. The presence field with possible energy levels of 2I+1, where I is the
and nature of unsaturation can be effectively detected nuclear spin quantum number. Eg. The two possible
by this method. The concentrations of compounds are orientations of the simple atom like proton are align or
also estimated from the absorption parameters. against the direction of the applied field. The energy
levels of these orientations are different and are
Infra-red (IR) spectroscopy quantized. It is possible to change the alignment of
proton to orient against the applied electromagnetic
Apart from the conventional chemical methods, radiation with definite frequency which is absorbed
functional groups, H-bonding (inter- and intra- by the proton to go from lower to higher energy
molecular), geometrical isomerism, conformational level and the proton is said to be in resonance. In
orientation of groups in both aliphatic and aromatic practice with a fixed frequency, the magnetic field
compounds can be detected by IR spectroscopy is varied to get signals depending on the magnetic
effectively for the absorption in the IR region of moment of the nucleus (proton) resulting in NMR
4000-650 cm-1. With the improved high resolution spectrum. The protons in a molecule are in different
by fourier transform mode the spectra are nowadays chemical environment ie. Shielding or deshielding
recorded. The unsymmetrical charge distribution depending on the electronic influence. Shielding
due to various vibrations in the molecule makes the causes a shift of the resonance frequency to higher
molecule to be detected in IR spectra. The various values of the applied field, (upfield). Deshielding
vibrational motions are stretching and bending causes a shift of the resonance frequency to lower
modes. Stretching regions have higher frequencies values of the applied field (downfield). The magnitude
than the deformation regions. The spectrum of a of the shift is known as chemical shift (Δ). Since these
compound can be recorded in gas, liquid (thin film), values can not be determined accurately, chemical
solid (thin film or mull) or solution in CCl4, CHCl3, shifts are measured relative to come standards like,
CS2. Identification of compound is carried out by tetramethylsilane which has protons which occur at
comparison with published spectral data. The region relatively highest upfield.
1400-650 cm-1 is the “finger-print region” as it is
having the vibrational energy changes of molecular It is usually in the range 1-10 and is quoted in parts
skeleton which will be characteristic for every per million (ppm). Eg. Ethanol. The position and
molecule. The absorption values of functional groups the intensity of the peaks give information of the
throw light on the type of compound. Characteristic type of protons present in the molecule. In the high
bands of the functional group are also recorded resolution NMR spectrum, the multiplicity of each type
together inevitably. Eg. C=O str. and C-O str. bands of proton gives the information about its environment
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at 1750-1735 cm-1 and 1250-1170 cm-1 respectively. and neighbouring protons leading to the partial
It is also the case that absence of a particular band structure of the molecule. Chemical equivalence
is not a warrant that particular functional group of the protons is deduced from the spectral study.
is not present and this situation requires chemical Chemical structure, configurations, conformations,
information about the compound along with other tautomerism, H-bonding, molecular weight can be
spectral data by UV and NMR. determined using NMR techniques.

Nuclear magnetic resonance Mass spectrometry

(NMR) spectroscopy
When a compound is bombarded with electron under
Nuclei with odd atomic and odd mass numbers, odd vacuum, it is converted into positively charges ion with
atomic and even mass number, and even atomic the loss of electron and this ion is called molecular
number and odd mass number are having magnetic ion. The excess energy present in the molecular ion

368
enables to break down further into neutral and with different spectroscopic detection methods like
positively charged fragments. Further break down is PDA, MS or NMR offers two ways of identification;
the case of excess energy fragments also. Coupling TLC/HPTLC with mass spectrometry either
by compound extraction with specific interfaces or
These positive charged ions are accelerated in an by ambient mass spectrometry significantly increased
electric field and separated by their passage through the spectral information on selected compounds.
an electric field and then magnetic field. The ions of Recently, using a TLC-MS extraction interface and
like charges of mass/charge (m/e) ratio are sorted coupling to NMR; analysing biological material
out and so the masses of ions are determined. The containing volatile constituents like essential oils,
instrument is capable of resolving adjacent beams of GC-MS analysis still represents the method of choice,
m/e and (m+1)/e and also ions of masses differing taking advantage of the unsurpassed peak capacity
in third decimal place. of capillary GC columns.

The largest peak is the most abundant and is X-ray diffraction (XRD)
called base peak with the given value of 100. All
other peaks are reported as percentages of the Complete structure of any crystalline compound is
base peak. Fragmentation pattern is characteristic possible by X-ray diffraction by the process of the data
of a compound and by this pure compound is with the help of computer. It has become a powerful
characterized. This interpretation is difficult when tool to determine the structure and molecular weight
the ion undergoes rearrangement and gives the as well without involving any chemical methods in
fragment pattern not expected from the structure some cases. Crystalline form of the compound is the
of the compound. However mass spectrometry is prerequisite for the analysis.
valuable for the determination of molecular weights,

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

molecular formula, structure elucidation, quantitative Chemical synthesis
analysis of mixtures, ionization potential, etc. From
the pattern of peaks of compounds having various The confirmation of the chemical structure deduced
functional groups, the structure of an unknown by various means be it a chemical or physical method,
compound can be proposed. List of common is done by chemical synthesis. It is the protocol of the
peaks table is also useful for this work. It is also not chemical analysis of any unknown pure compound to
necessary to identify every peak. illustrate the structure of the compound by synthesis.
So whenever a novel compound is discovered in one
With the advancement in the instrumentation in place, the synthesis of the compound is followed
mass spectrometry, it is now possible to interpret the by subsequent report elsewhere. It may not be an
structure of complex molecules like, metal complexes, economic one involving multiple steps but it is the
supramolecules, nanostructures, biopolymers (peptides, chemical confirmation of the structural entity they
proteins and nucleic acids) and other macro molecules arrived at for a particular compound.
with the help of fast atom bombardment mass
spectrometry (FABMS) and inductively coupled mass Conclusion
spectrometry (ICPMS). Matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI- As the knowledge is expanding, means of
TOF-MS) has revolutionized in situ identification of identification of an organic compound is also
microorganisms by analysing them in a short time from expanding exponentially. Modern instrumental
colonies grown on culture plates. methods involving micro of quantity of the analyte,
is indeed amazing compared to the conventional,
Hyphenated techniques such as LC-NMR, LC-MS, laborious and lengthy methods used previously
LC-PDA35, UHPLC-PDA-TOF-MS and combinations like degradative studies and various chemical
thereof compounds could be unambiguously analyses involving large quantity of samples. This
characterized. Hyphenation of HPLC separation situation is overcome with the help of modern

369
 sophisticated instruments. Good candidate Suggested readings
compounds have emerged paving way to medicinal
screening and pharmacological studies especially Silverstein R M, Bassler G C, Morrill T C, Spectrometric identification
the stereochemistry which is vital to the structure of organic compounds, 5th edn, John Wiley & sons, NY.
Finar I L, 2005 Organic chemistry Vol 1 The fundamental principles
activity relationship. 6th edn, Pearson education
Dyer J R, 1972 Organic spectral problems, Prentice-Hall, Englewood
Cliffs, N.J.
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370
General Methods of Isolation Procedures
and Separation Methods for Organic
Compounds
I. Rajendran* and P. Vijayagopal
Principal Scientist
Mandapam Research Center of CMFRI
e-mail: [email protected]

Introduction to know the complexity. Before this the mixture

is simplified by filtration through sieves to evade
Exploration of marine natural products (MNPs) from impurities. Preliminary analysis is done by thin layer
bioprospecting of marine environment is involving chromatography (TLC), gas chromatography. Using
various steps like collection of source organisms, a TLC-MS extraction interface and coupling to NMR,
preservation, processing for crude component, screening compounds can be identified and quantified as well.
of the crude extract for the targeted activity, isolation
of the compound of activity by physical methods of Isolation protocol of MNPs starts with identification
separation and final identification of the compound and collection of the biological material with the

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

by instrumentation. Inline multi analytical methods help of biologist. Extraction with different solvents
like LC-NMR or LC-MS gave the first hand information from low to higher polarity of both organic and
about the structure of the compounds prior to actual aqueous moieties follows. Prior to isolation of pure
isolation of the compound. Once this information is compounds, often by semi preparative high pressure
obtained from the given extract, actual quantitative liquid chromatography (HPLC) or liquid-liquid
isolation of the product becomes easy with appropriate chromatographic techniques, several purification
separation technique. The quantity of active ingredient steps are necessary to remove most of the non-
of the mixture may be low after a series of steps involved targeted matrix.
in the isolation process, but with the modern powerful
instrumentation, it is now comfortably possible to get Solvent extraction
the actual structural information.
The majority of isolation procedures employ simple
Types of extraction procedures with organic solvents of
different polarity, water and their mixtures. The
isolation techniques methods include maceration, percolation, Soxhlet
The relative solubility of the compounds present in extraction, ultrasound-assisted extraction and
an extract based on their polarity determines their turbo-extraction. Maceration is carried out at room
isolation/extraction in the lipophilic or lyophilic nature temperature by soaking the material with the
of the extractant. The complexity of the extract is solvent with eventual stirring. It has the advantage
thus fractionated with like group of compounds of moderate extraction conditions but suffers from
with respect to their polarity and solubility. This high solvent consumption, long extraction times and
sub fractionated compounds can easily be analyzed low extraction yields. Extraction yield is improved by
and the components can be isolated with less percolation, i.e. packing the pre-soaked plant material
difficulty. The isolation of active compound from in a container which allows the constantly controlled
the crude mixture is first of all tested analytically removal of the extract via a valve at the bottom

371
 and adding fresh solvent from the top. Soxhlet extraction technologies like liquid-liquid extraction
extraction is a popular method for extraction due (LLE), UAE, MAE or liquid-phase micro-extraction
to its reduced solvent consumption; however, heat (LPME), N,N-dimethylethanolammonium octanoate
sensitive compounds might be degraded during the (DMEA oct) and bis(2-methoxyethyl)ammonium
extraction process. For liquid samples extraction by bis(tri-fluoromethylsulfonyl)imide (BMOEA bst)
organic solvents or heterogeneous solvent mixtures showing the best performance
can be done, either simply in a separating funnel or
similar to a Soxhlet apparatus in a perforator. On a Accelerated (pressurized) solvent
smaller scale, extraction of the liquid sample absorbed
extraction (ASE)
on a porous matrix (like diatomaceous earth) packed
in a column with non-miscible solvents is an option. In ASE, sequential extraction with solvents of different
polarity and mixing of solvents is possible, ASE or
Ultrasound-assisted similar instrumentation can also be used for subcritical
water extraction (SWE) employing temperatures of
extraction (UAE)
100–280ºC. Subcritical water (superheated water,
In UAE the source material, usually in a glass container, pressurized hot water) is heated to a temperature
is covered by the extraction solvent and put into an between the boiling point at atmospheric pressure
ultrasonic bath. It decreases extraction time and (100ºC) and the critical temperature (374ºC) under
improves extraction yields due to mechanical stress pressure, thereby increasing its solution properties for
which induces cavitations and cellular breakdown, organic lipophilic compounds. For phenolic type of
and has gained increasing popularity. temperature- compounds, SWE seems to be an attractive alternative
controlled water bath connected to an ultrasound to organic solvent extraction, however, artifact
probe, showed superior extraction efficiency compared formation and degradation has to be scrutinized.
to steam distillation or superheated water extraction.
Supercritical fluid extraction (SFE)
Microwave-assisted
Replacing extraction with organic solvents by extraction
extraction (MAE)
technologies which are less detrimental to environment
Nowadays extraction employing either diffused and meet the increasing regulatory requirements
microwaves in closed systems or focused microwaves certainly can be considered as a driving force for the
in open systems are established methods.MAE has increasing application of supercritical fluid extraction,
been modified in different ways leading to vacuum above all using supercritical CO2. The utilization of
microwave assisted extraction (VMAE), nitrogen- organic solvents as modifiers for supercritical CO2 to
protected microwave assisted extraction (NPMAE), increase its solvating capabilities to medium polar
ultrasonic microwave-assisted extraction (UMAE) or and polar compounds has broadened the spectrum
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dynamic microwave-assisted extraction (DMAE). Some of NP compound classes accessible to SFE, accepting
recent examples of application of MAE to NP isolation the ecological problems related to organic solvent
employing ionic liquids are mentioned below. extractions which increase to a small extent.

Extraction with ionic liquids Chromatographic techniques

These ILs, also designated as “designer solvents”, Extraction processes which take advantage of
are organic salts in the liquid state consisting of an adsorption of the analytes or unwanted impurities
organic cation and an organic or inorganic anion. ILs on a solid phase have gained a dominant role in
are able to dissolve a wide range of polar to non-polar purification of NP extracts, not least due to its
compounds, have a low vapour pressure, show a high integration into automated sample preparation and
thermal stability and low combustibility, and some of isolation systems. Most applications utilize solid-
them are biodegradable. Ionic liquids with different phase extraction (SPE) which employs a wide range

372
of stationary phases with diverse chemistry like silica activated carbon adsorbant. Some of the common
gel, reversed-phase material, ion-exchange resins or adsorbents used are:
mixed-mode material and hydrophilic interaction
chromatography (HILIC) stationary phases in pre- Cellulose < Starch < Sucrose < Calcium carbonate <
packed glass or plastic columns. Elution of the Magnesia < Silica gel < Alumina < Activated charcoal
compounds of interest might be done stepwise by
applying a gradient with increasing eluting power, Alumina is widely used and is available in three forms
i.e. the procedure is then related to vacuum liquid – acidic, basic and neutral.
chromatography (VLC). An exciting development of
recent years was the design of molecularly imprinted Preparative planar
polymers (MIP) to be used in SPE applications for
chromatography (PPC)
selective enrichment of various compounds. Either
ionic liquid-imprinted silica particles or copolymers An attractive feature of PPC is the wide range
of acrylamide and ethylene glycol dimethacrylate of chemical detection methods characteristic
with the respective template compounds are used for compound classes which can be carried out
to create material which will have a high affinity to on a narrow section of the plate leaving most
the template structures. of the compound unchanged and available for
isolation. In bioassay-guided isolation strategies,
In a first elution step the unwanted material is planar chromatography has the advantage of
removed from the SPE column whereas target direct application of bioassays on thin layer
compounds bound to the solid phase are obtained chromatography (TLC) plates, making the rapid
in a concentrated solution usually upon elution with localization of bioactive compound zones possible.
organic solvents like methanol, though additional To overcome the disadvantage of classical TLC

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

purification steps might be necessary. A sophisticated of uncontrolled flow rates of the mobile phase,
combination of SPE columns representing strong forced-flow techniques such as centrifugal
anion and cation exchangers, a mixed-mode polymeric planar chromatography or over-pressured layer
RP-anion exchanger with a poly (divinylbenzen-co- chromatography have been developed enabling
vinylpyrrolidone) backbone and a size exclusion column elution and online detection of compounds. Rf
of a hydroxypropylated dextran gel (Sephadex LH-20) value is the ratio of the distance travelled by a
were used for explorative fractionation of extracts component to the distance travelled by the solvent
front and it is characteristic of each component.
Chromatography is the process of separation of This is the similar condition for RT value observed
components of the mixture, by distribution between in HPLC. The spots can be visualized by I2 vapour
two phases, one stationary phase and the other for most of the cases or by spraying the reagent
mobile phase. The solubilities and polarity of the solution on the plate for specialized structured
components present in the mixture determine the compounds eg. Ninhydrin for amino acid detection.
extent of elution/distribution from the matrix. To get The visualization can also be done by viewing the
systematic elution, the eluent should be either from plate under UV irradiation.
non polar to polar or polar to non polar depending on
the normal phase or reversed phase chromatography Vacuum liquid
respectively. Eg. The order of increasing eluting
chromatography (VLC).
power for silica gel: (Eluotropic series)
In contrast to other forced-flow column
PE < Cyclohexane < CCl4 < Φ < CH2Cl2 < CHCl3 < chromatographic techniques, not pressure but
Et2O < EtOAc < Acetone < PrOH < EtOH < MeOH vacuum is applied in VLC to increase flow rate and
< H2O < AcOH hence speed up the fractionation procedure. Column
beds in VLC usually consist of silica of 40–60 mm
This sequence is roughly reverse in the case of particle size or reversed-phase silica.

373
 Flash chromatography (FC). Preparative gas
chromatography (PGC)
Similarly to VLC, FC is mainly used for rapid
fractionation of crude extracts or coarsely purified For isolation of volatiles, PGC is an attractive option.
fractions. By applying nitrogen or compressed air, the
mobile phase is flushed through the stationary phase Distillation
in a tightly closed glass column or prepacked cartridges
Volatiles such as essential oils are still obtained
Low-pressure liquid mainly by distillation techniques, although working
at elevated temperatures can lead to chemical
chromatography (LPLC)
changes. Liquid compounds/mixtures are separated
Column chromatographic methods which allow flow by distillation under atmospheric pressure or vacuum
of the mobile phase at atmospheric pressure without at different boiling points which are specific to
additional forces either by vacuum or pressure are pure compounds. Distillation under vacuum also
still a major tool in the fractionation protocols for prevents the chemical changes or destruction of heat
NP isolation. sensitive compounds.

Medium-pressure liquid Electrophoresis

chromatography (MPLC).
Under the influence of applied potential difference,
MPLC is commonly used to enrich biologically active anions in the dispersion medium move to the cathode
secondary metabolites before further purification by and cations to the anode resulting in the separation
HPLC due to its lower cost, higher sample loading and of the mixture. Eg. Amino acids and peptides with
higher throughput using RP-18 and polyamide CC 6 resultant charge on the molecule which decides the
stationary materials afforded highly pure compounds. direction and extent of movement at a particular pH
of the medium.
High-performance (high-pressure)
liquid chromatography (HPLC). Conclusions
Octadecyl silica (RP-18) columns are still widely used With the development and improvement of the
for NP isolation and purification. However various existing extraction and analytical techniques, it is
laboratories have benefited from the availability now comfortable to explore the complex sample to
of high-quality modern-generation HPLC columns retrieve the components present in it. Hyphenation of
with modified matrices such as cyano, phenyl, chromatographic and spectroscopic or spectrometric
trimethylsilane, triazole, secondary and tertiary techniques thus increases the possibility of the
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amines, b-cyclodextrine and dihydroxypropane for elucidation of the structures of known as well as novel
successful isolation and purification of different compounds without the need for isolation.
groups of MNPs.
Suggested readings
Chiral chromatographic methods
Bucar F, Wube A and Schmid M 2013 Natural product isolation –
This separation technique allows separating how to get from biological material to pure compounds, Nat.
Prod. Rep, 30:525-545
enantiomers either indirectly with chiral derivatization Finar I.L 2005 Organic Chemistry, vol 2, Pearson Education
reagents or directly with chiral stationary phases or (Singapore) Pte. Ltd., Indian Branch, 482,FIE, Patparganj, Delhi.
chiral mobile-phase additives.

374
Instrumental Methods in Bioprospecting:
Chromatography and Spectroscopy
Kajal Chakraborty
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Chromatography:
a stationary phase and a mobile phase. Those
An overview components held preferentially in the stationary
Chromatography, although primarily a separation phase are retained longer in the system than those
technique, is mostly employed in chemical analysis. that are distributed selectively in the mobile phase.
Nevertheless, to a limited extent, it is also used for As a consequence, solutes are eluted from the system
preparative purposes, particularly for the isolation as local concentrations in the mobile phase in the
of relatively small amounts of materials that have order of their increasing distribution coefficients
comparatively high intrinsic value. In a single step with respect to the stationary phase; ipso facto a
process it can separate a mixture into its individual separation is achieved.
components and simultaneously provide an

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

quantitative estimate of each constituent. Samples In practice, the distribution system, (that part of the
may be gaseous, liquid or solid in nature and chromatographic apparatus where the solutes are
can range in complexity from a simple blend of distributed between the phases) can take the form
two entantiomers to a multi component mixture of a column such as a tube packed with particulate
containing widely differing chemical species. The matter on which the stationary phase is bonded or
first scientist to recognize chromatography as an coated. The mobile phase (which may be a gas or a
efficient method of separation was the Russian liquid) passes under pressure through the column
botanist Tswett, who used a simple form of liquid- to elute the sample. The column form may also
solid chromatography to separate a number of plant be a long, small-diameter open tube that has the
pigments. The colored bands he produced on the stationary phase coated or bonded to the internal
adsorbent bed evoked the term chromatography surface. Alternatively, the chromatographic system
for this type of separation (color writing). Although may take the form of a plate (usually glass) the surface
color has little to do with modern chromatography, of which is loaded with particulate matter to which
the name has persisted and, despite its irrelevance, the stationary phase is coated or bonded. The mobile
is still used for all separation techniques that employ phase (a liquid) is arranged to percolate up the plate
the essential requisites for a chromatographic (usually by surface tension forces) to elute the sample.
separation, viz. a mobile phase and a stationary The sample is injected into the mobile phase stream
phase. Today, chromatography is an extremely just before the front of the columns. The column is
versatile technique; it can separate gases, and designed to allow two processes to take place that
volatile substances by gas chromatography (GC), will produce the separation. Firstly, as a result of
in-volatile chemicals and materials of extremely different forces between each molecular type and the
high molecular weight (including biopolymers) by stationary phase, each solute is retained to a different
liquid chromatography (LC). Chromatography is a extent and, thus, the more weakly held will elute first
separation process that is achieved by distributing and the more strongly held elute last. The process is
the components of a mixture between two phases, diagramatically depicted below.

375
 Gas Liquid Chromatography
Gas-liquid chromatography (GLC) was in invented by
James and Martin and is a chromatography separation
technique in which the mobile phase is a gas (usually
helium or nitrogen) and the stationary phase is a
liquid. In the original columns used by James and
Martin, the liquid stationary phase was adsorbed
on the surface of an inert support such as Celite (a
diatomateous earth) or calcined Celite (a form of brick
dust). The support was usually deactivated before
use by acid treatment and subsequent reaction with
hexamethyldisilazane. The technique was extensively
used for the separation of a wide range of volatile
Classification substances including fatty acids.
of Chromatography
The modern gas chromatograph is a fairly complex
As all chromatographic separations are carried out instrument mostly computer controlled. The samples
using a mobile and a stationary phase, the primary are mechanically injected, the analytical results are
classification of chromatography is based on the automatically calculated and the results printed out,
physical nature of the mobile phase. The mobile together with the pertinent operating conditions
phase can be a gas or a liquid which gives rise to in a standard format. However, the instrument has
the two basic forms of chromatography, namely, gas evolved over many years although the majority of
chromatography (GC) and liquid chromatography (LC). the added devices and techniques were suggested or
describe in the first three international symposia on
Table 1 The Classification of Chromatography gas chromatography held in 1956, 1958 and 1960.
Mobile phase Stationary phase The layout of the modern gas chromatograph is
shown as a block diagram:
Gas Liquid

Gas Chromatography (GC) Liquid

Gas-liquid chromatography (GLC)
Solid
Gas Solid Chromatography (GSC)
Liquid Liquid
Liquid chromatography (LC) Liquid –liquid chromatography (LLC)
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Solid
Liquid solid chromatography (LSC)

The stationary phase can also take two forms, solid

and liquid, which provides two subgroups of GC and
LC, namely; gas–solid chromatography (GSC) and
gas–liquid chromatography (GLC), together with liquid
solid chromatography (LSC) and liquid chromatography
(LLC). The different forms of chromatography are
summarized in Table 1. Most thin layer chromatography
techniques are considered liquid-solid systems although
the solute normally interacts with a liquid-like surface
coating on the adsorbent or support or, in some cases
an actual liquid coating.

376
Different components Pressure controller
of GLC
Injectors
Gas supplies
The sample is injected by a hypodermic syringe,
Gases (carrier gas-N2 or He; and fuel gas-air and H2) through a silicone rubber septum directly into the
for use with the gas liquid chromatography were column packing or into a flash heater. An example of
originally all obtained from gas cylinders fitted with a septum injection system used for packed columns
reducing valves that are set to supply the gas to the is shown in following figure. The silicone septum is
instrument at the recommended pressure defined compressed between metal surfaces in such a manner
by the manufacturers. The reducing valves on the that a hypodermic needle can pierce it, but when
gas tanks are examples of simple pressure controllers it is withdrawn the hole is closed as a result of the
and the flow controllers that are used for detector septum compression and there is no gas leak. The
and column flow control often involve devices based glass liner prevents the sample coming in contact
on the same principles.  The pressure controller with the heated metal wall and thus, reduces the
consists essentially of two chambers separated by a chance of thermal decomposition. The glass liner can
diaphragm, in the center of which is a needle valve be fitted with a separate heater and the volatilization
that is actuated by the diaphragm. The diaphragm temperature can, thus, be controlled. By using a
is held down by a spring that is adjustable so that syringe with a long needle, the tip can be made to
the pressure in the second chamber, and thus the penetrate past the liner and discharge its contents
outlet flow, can be set at any chosen value. When directly into the column packing. This procedure is
gas enters the lower chamber, the pressure on the called ‘on-column injection’ and, as it reduces peak
lower part of the diaphragm acts against the spring dispersion on injection and thus, provides higher

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

setting, and opens the valve. Gas then passes into the column efficiencies, is often the preferred procedure.
upper chamber and pressure is built up in the upper
chamber to the value that has been set at which time The basic difference between the two types of
the diaphragm moves downward closing the valve. If injection systems is that the capillary column now
the pressure falls in the upper cylinder, the diaphragm projects into the glass liner and a portion of the carrier
again moves upward due to the pressure in the lower gas sweeps past the column inlet to waste. As the
chamber, which opens the valve and the pressure in sample passes the column opening, a small fraction
the upper chamber is brought back to its set value. is split off and flows directly into the capillary column,
ipso facto this device is called a split injector. The
split ratio is changed by regulating the portion of

Packed Column Injector

377
 can be U-shaped but columns more than a meter
long are usually coiled. Glass columns are sometimes
treated with an appropriate silanizing reagent to
eliminate the surface hydroxyl groups which can be
catalytically active or produce asymmetric peaks.

Supports for GLC

There have been a number of materials used as
supports for packed GC columns including, Celite
(a proprietary form of a diatomaceous earth),
fire-brick (calcined Celite), fire-brick coated with
Split Injection System metallic silver or gold, glass beads, Teflon chips and
polymer beads. Today however, the vast majority
the carrier gas that flows to waste which is achieved of contemporary packed GLC columns are filled
by an adjustable flow resistance in the waste flow with materials that are either based on of Celtic
line. This device is only used for small diameter or polystyrene beads as a support. There are two
capillary columns where the charge size is critical. processes used to modify Celite. One was to crush,
Consequently, quantitative analyses carried out using blend and press the Celite into the form of a brick
the high efficiency small diameter capillary columns and then calcine it at a temperature of about
may have limited accuracy and precision, depending 900˚C. Under these conditions some of the silica
on the nature of the sample. is changed into cristobalite and traces of iron and
other heavy metals interact with the silica causing
GLC Columns the material to become pink in color. This material
is sold under the trade name of Chromosorb P.
There are two types of columns in common use in GC The second process involves mixing the Celite
and they are the conventional packed column and the with sodium carbonate and fluxing the material
open tubular column. The former are usually 2 to 4 at 900˚C. This causes the structure of the Celite
mm I.D. and 1-4 m long and, packed with a suitable to be disrupted and the fragments adhere to one
adsorbent, are mostly used for gas analysis. As a another by means of glass formed from the silica
result of the simpler injection procedure and the more and the sodium carbonate. As the original Celite
precise sampling method, the packed column tends structure is disrupted, the material exhibits a wide
to give greater quantitative accuracy and precision. range of pore sizes which differs significantly from
However, despite its problems with sample injection, the material that was calcined in the absence of
the open tubular column is seen as the ‘state of the sodium carbonate. This materials is sold under the
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art’ column and is by far the most popular column name of Chromosorb W together with two similar
system in general use. The length of open tubular or materials called Chromosorb G and Chromosorb
capillary columns range from about 10-100 m and S. The residual deleterious adsorptive properties
can have internal diameters from 100-500 μm. The
stationary phase is coated on the internal wall of the
column as a film 0.2-1 μm thick.

Packed GC Column
Packed columns are usually constructed from stainless
steel or Pyrex glass. Pyrex glass is favored when
thermally labile materials are being separated such as
essential oils and flavor components. Longer columns

378
of the support are due to silanol groups on the
surface and these can be removed by silanization.
The support is treated with hexamethyldisilazane
which replaces the hydrogen of the silanol group
with a trimethylsilyl radical. The reaction proceeds
as follows,

In this way the strongly polar silanol groups are

methylated and assume dispersive characteristics
that do not produce peak tailing. Although the major
contributors to adsorption by the support are the
Open Tubular Column Types
silanol groups, a residual adsorption results from the
presence of trace quantities of heavy metals such as
iron, which can be largely removed by acid washing from 320-530 μm with a porous layer that can be
prior to silanization.  5-50 μm thick.

Capillary or Open Chiral stationary phases

Tubular Column Modern organic chemistry and pharmaceutical
Capillary columns are fabricated from stainless steel. research are becoming increasingly interested in
Metal columns provide the high efficiencies expected methods of asymmetric syntheses. This enthusiasm
from open tubular columns and were used for the has been provoked by the differing physiological
analysis of petroleum, fatty acids and fuel oils, etc. activity that has been shown to exist between

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Metal columns, however, have some disadvantages the geometric isomers of pharmaceutically active
as although easily coated with dispersive stationary compounds. A tragic example being the drug
phases (e.g., squalane, Apiezon grease etc.) they are Thalidomide, which was made available as a
not so easily coated with the more polar stationary racemic mixture of N-phthalylglutamic acid imide.
phases such as CARBOWAX®. In addition, hot metal The important physiological activity resides in the
surfaces can cause decomposition or molecular R-(+)-isomer and it was not found, until too late,
rearrangement of many thermally labile materials such that the S-enantiomer was probably tetratogenic
as the terpenes contained in essential oils. Metal can and caused serious fetal malformations. The
also react directly with some materials by chelation separation and identification of isomers can, clearly,
and adsorb polar material which results in asymmetric be very important and chromatography can be
and tailing peaks. Nevertheless, metal columns are very effective in the resolution of such mixtures.
rugged, easy to handle and easy to remove and The use of GC for the separation of asymmetric
replace in the chromatograph consequently, their isomers is not as common as LC, but nevertheless
use has persisted in many application areas despite there some very effective optically active stationary
the introduction of fused silica columns. phases that can be used in GC for the separation
of enantiomers. Some of the more useful GC
Open Tubular Column Types stationary phases are based on cyclodextrins already
described. The columns are usually 30-60 m long
Open Tubular columns are broadly split into two 0.25 mm I.D. and have an operating temperature
classes, the wall coated open tubular columns or range of 30˚C to 250˚C. In order to employ the
WCOT Columns (which have already been described cyclodextrins as stationary phases for GC the
and are by far the mot popular,) and the porous permethylated cyclodextrins are often embedded
layer open tubes or PLOT Columns. The two types of in a siloxane matrix (e.g. 35% phenyl-65% methyl
column are shown diagramatically in the following polysiloxane) which is deposited on the walls of
figure. The external diameter of PLOT columns range fused quartz capillary tubes.

379
 GC detectors
A large number of GC detectors have been developed
and made commercially available. The detectors with
the highest sensitivity tend to be specific and sense
specific types of sample (e.g., halogenated substances
by the electron capture detector). The detectors
with a catholic response are the most popular
and the majority of GC separations are monitored
by the flame ionization detector (FID). The most
commonly used specific detectors are the nitrogen
The Structure of Cyclodextrin
phosphorus detector (NPD) and the electron capture
detector (ECD).

Derivatization of the base cyclodextrin structure can Flame Ionization Detector

introduce groups to which only one enantiomer can
interact, while the other(s) are partially or wholly The FID detector employs hydrogen as the combustion
entropically hindered from interaction. This increases gas which is mixed with the column eluent (helium,
the differential interaction between the enantiomers nitrogen or other appropriate gas) and burnt at a small
and the stationary phase, thus, increasing the jet situated inside a cylindrical electrode. A potential
separation ratio and hence the resolution. of a few hundred volts is applied between the jet and
the electrode and when a carbon containing solute is
Column oven burnt in the jet, the electron/ion pairs that are formed
are collected at the jet and cylindrical electrode. The
and accessories current is amplified and fed to a recorder or to the
The column oven should operate over a fairly wide A/D converter of a computer data acquisition system.
temperature range (e.g. from 5˚C to 400˚C). In During the process of oxidation, oxidized or partially
practice, however, the maximum oven temperature oxidized fragments of the solute are formed in the
needed is usually less than 250˚C, particularly flame which is thought to generate electrons by
when synthetic stationary phases are being used, thermionic emission. The background current (ions
as many of them tend to be unstable and either and electrons from the hydrogen flame alone) is
decompose or volatilize at higher temperatures. very small (1-2 x 10-12 amperes) and consequently,
Similarly, initial temperatures below 50˚C are
also rarely needed. The oven usually has air
circulation driven by a powerful fan to ensure
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an even temperature throughout the oven. The

temperature programmer (hardware and software)
usually has a range of linear gradients from 0.5˚C/
min. to about 20˚C/min. Some programmers
include nonlinear programs such as logarithmic
and exponential, but most GC analyses can be
effectively accomplished using linear programs
only. The program rate can be changed at any
time in the chromatographic development or
intermittent isothermal periods can be inserted
where necessary in the program. The temperature
programming limits are usually the same as those
Flame Ionization Detector
of the oven (viz. 5˚C to 400˚C).

380
 can be used in two modes, either with a constant
potential applied across the cell (the DC mode) or with
a pulsed potential across the cell (the pulsed mode).
In the DC mode, hydrogen or nitrogen can be used as
the carrier gas and a small potential (usually only a few
volts) is applied across the cell that is just sufficient to
collect all the electrons available and provide a small
standing current. If an electron capturing molecule
(for example a molecule containing a halogen atom
which has only seven electrons in its outer shell) enters
Nitrogen Phosphorus Detector
the cell, the electrons are captured by the molecule
and the molecules become charged. The mobility of
the captured electrons is much smaller than the free
the noise level is also commensurably small (about electrons and the electrode current falls dramatically. In
10-14 amperes). the inactive period of the wave form, electrons having
thermal energy only will attached themselves readily to
Nitrogen any electron capturing molecules present in the cell with
the consequent production of negatively charged ions.
Phosphorus Detector The negative ions quickly recombine with the positive
The nitrogen phosphorus detector (NPD) is a highly ions (produced simultaneously with the electrons by the
sensitive but specific detector and evolved directly β particles) and thus become unavailable for collection.
from the FID. It gives a strong response to organic Consequently the standing current measured during
compounds containing nitrogen and/or phosphorus. the potential pulse will be reduced.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Although it appears to function in a very similar
manner to the FID, in fact, it operates on an entirely The basic electron capture detector consists of a small
different principle. The actual NPD sensor is a chamber one or two ml in volume enclosing two metal
rubidium or cesium bead contained inside a small
heater coil. A potential is applied between the bead
and the anode. The heated alkali bead emits electrons
by thermionic emission which is collected at the
anode and thus produces an ion current. When a
solute containing nitrogen or phosphorus is eluted,
the partially combusted nitrogen and phosphorus
materials are adsorbed on the surface of the bead.
This adsorbed material reduces the work function
of the surface and, as consequence, the emission of
electrons is increased which raises the anode current.
The sensitivity of the NPD is about 10-12 g/ml for
phosphorus and 10-11 g/ml for nitrogen). Electron Capture Detector

Electron Capture Detector electrodes. The electrodes may be concentric cylinders

or metal discs separated by an insulator. The cell
The electron capture detector contains a low energy contains the radioactive source, electrically connected
β-ray source which is used to produce electrons for to the entrance conduit and to the negative side of
capturing by appropriate atoms. Although tritium the power supply. A gauze “diffuser” is connected
adsorbed into a silver foil has been used as the β particle to the cell exit and to the positive side of the power
source, it is relatively unstable at high temperatures, the supply. The output from the sensor is processed
Ni63 source was found to be preferable. The detector by suitable electronics and the output passed to

381
 1. The preparation of the sample.
2. The development of the separation and the
production of the chromatogram
3. The processing of the data and the presentation
of the results.

Each stage is equally important and if not carried out

correctly the results will be neither precise nor accurate.
Sample preparation can be very simple involving no
more that diluting a known weight of sample with
mobile phase or be much more complex including an
extraction procedure followed by derivatization and
either a potentiometric recorder of a computer data then dilution. Liquid extraction is a clumsy procedure,
acquisition system. The electron capture detector is particularly when used on the micro scale which is
very sensitive, probably the most sensitive GC detector often necessary in sample preparation. An alternative
available (ca. 10-13 g/ml) and is widely used in the procedure is solid phase extraction. The procedure
analysis of halogenated compounds. is relatively simple and involves the use of a short
1 2 3 4 Heptachlor tube packed with an appropriate adsorbent such as
5 6 Aldrin 7 Heptachlor 8 Endosulphan silica, reversed phase silica or, for some applications,
Epox. macro porous polymer beads. The adsorbent must
9 p,p'-DDE 10 Dieldrin 11 Endrin 12 p,p'-DDD
be capable of removing the substances of interest
13 14 p,p'-DDt 15 Endin 16Endosulp.
Endosulphan Aldehyde Sulf. from the liquid medium.
11

Derivatization
Analysis of
chlorinated insecticides GC samples are usually derivatized to render highly
polar materials sufficiently volatile so that they can be
eluted at reasonable temperatures without thermal
Data acquisition and processing decomposition or molecular re-arrangement. Examples
Originally, analytical results were calculated from of such materials that need to be derivatized are the
measurements made directly on the chromatogram organic acids, amides, poly hydroxy compounds,
provided by the chart recorder. The output from amino acids etc. In order to render such materials
the detector (which is only rarely the direct output more volatile, they are either esterified, silanated or
from the detector sensor) is usually in millivolts and acetylated using one of a number of different methods
is suitable for direct connection to a potentiometric of derivatization. Acids can be esterified by treating
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recorder. The output from the detector usually passes them with an appropriate alcohol using an inorganic
directly to a scaling amplifier that modifies the signal acid to catalyze the reaction. Hydrochloric acid was
to a range that is appropriate for the analog-to- popular for this purpose because it’s strength was
digital (A/D) converter. The output can alternatively adequate and any excess could be easily removed.
pass to a potentiometric recorder and produce the Other catalysts that have been found effective are
chromatogram in real time. The computer system can trifluoroacetic acid, dichloroacetic acid, benzene
also produce a real time chromatogram but, to do so, sulphonic acid, p-toluene sulphonic acids and suphuryl
the data must be processed and the chromatogram and thionyl chlorides. A volatile acid is recommended
presented on the printer. such as hydrochloric acid or thionyl chloride. However,
the derivative must be must be sufficiently involatile
Quantitative analysis not to allow loss when removing the excess alcohol
and where appropriate the catalyst itself. The Lewis
There are three important stages in a GC analysis, acid boron trifluoride or the equivalent reagent

382
boron trichloride is also very useful for forming ester ml/minute depending on the type of LC that is
derivatives. Boron trifluoride is supplied as a 14% carried out. Modern detectors can detect solutes at
solution in methanol. Boron trifluoride catalyzed concentration levels of 1x10-9 g/ml and an analysis
reactions are very fast and can be complete in a few can be completed in a few minutes with just a few
minutes. The esters can be extracted with n-hexane micrograms of sample.
with vigorous shaking. Another popular esterifying
reagent is diazomethane. Diazomethane is a yellow Modern High Pressure Liquid
gas but is used in the form of an ethereal solution. Its
Chromatography (HPLC)
reacts with an organic acid in the following manner,
HPLC is liquid chromatography which has been
R-COOH + CH2N2 R—COO–CH3 + N2 optimized to provide rapid high resolution separations.
The basic liquid chromatograph consists of five basic
When the reaction is complete, the yellow color units as follows. A block diagram of the basic liquid
persists and thus the reagent acts as its own indicator. chromatograph is shown in the following figure.

High Pressure
Liquid Chromatography
Liquid chromatography (LC) was the first type of
chromatography to be discovered and, in the form
of liquid-solid chromatography (LSC) was originally
used in the late 1890s by the Russian botanist, Tswett
to separate and isolate various plant pigments. The

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

colored bands he produced on the adsorbent bed
evoked the term chromatography (color writing) for
this type of separation. In the late 1930s and early
1940s Martin and Synge introduced a form of liquid-
liquid chromatography by supporting the stationary
phase, in this case water, on silica gel in the form of
a packed bed and used it to separate some acetyl
amino acids. Martin and Synge suggested the use of
small particles and high pressures in LC to improve the The Basic Liquid Chromatograph
separation which proved to the critical factors that
initiated the development of high performance liquid 1. Mobile phase supply system and gradient mixers.
chromatography (HPLC). The statement made by 2. HPLC high pressure pumps and sample valves.
Martin in 1941 contains all the necessary conditions 3. HPLC columns with inert packing materials.
to realize both the high efficiencies and the high 4. High sensitivity low dispersion HPLC detectors.
resolution achieved by modern LC columns. Despite 5. High speed data acquisition systems.
his recommendations, however, it has taken nearly
fifty years to bring his concepts to fruition. The major Mobile Phase Supply System and
impediment to the development of LC was the lack
HPLC Gradient Mixers
of a high sensitive detector and it was not until the
refractive index detector was developed by Tiselius and HPLC gradient mixers provide a very precise control
Claesson in 1942 could the technique being effectively of solvent composition to maintain a reproducible
developed. The contemporary chromatograph, gradient profile. The mobile phase supply system
however, is a very complex instrument operating consists of number of reservoirs (200-1,000 ml). At
at pressures up to 10,000 PSI providing flow rates least two reservoirs would be necessary and are usually
ranging from a few microliters per minute to 10-20 constructed of glass or stainless steel and contain an

383
 exit port open to air. Each reservoir is usually fitted with
a gas diffuser through which helium can be bubbled.
Many solvents and solvent mixtures (particularly
aqueous mixtures) contain significant amounts of
dissolved nitrogen and oxygen from the air. These
gasses can form bubbles in the chromatographic
system that cause both serious detector noise and
loss of column efficiency. As helium is very insoluble
in most solvents, it purges the oxygen and nitrogen
from the solvent but does not produce bubbles in the
system itself. Applying a vacuum to the reservoir is not
a permanent solution to dissolved air as, on releasing High Pressure Gradient Programmer
the vacuum to allow the solvent to pass to the pump,
air again dissolves in the solvent. The solvent is filtered be generated either by external oscillators or, if the
through a stainless steel or sintered glass filter to chromatograph is computer controlled, directly from
remove any solid contaminants. Depending on the the computer itself.
type of solvent programmer that is employed, the
supply from each reservoir may pass either to a pump HPLC Pumps
or to a valve blending device. Solvent reservoirs are
not usually thermostatted but, when necessary, the Because of the small particles used in modern HPLC,
solvent can be brought to the column temperature LC pumps need to operate reliably and precisely at
by the use of an appropriate heat exchanger. pressures of 10,000 PSI or at least 6,000 PSI. To
operate at these pressures and remain sensibly inert
Gradient Programmer to the wide variety of solvents used HPLC pumps
usually have sapphire pistons, SS cylinders and return
valves fitted with sapphire balls and stainless steel
High Pressure seats. For analytical proposes HPLC pumps should
Gradient Programmer have flow rates that range from 0-10 ml/min.,
There are two basic types of solvent programmer. In but for preparative HPLC, flow rates in excess of
the first, the solvent mixing occurs at high pressure 100 ml/min may be required. There are a number
and in the second the solvents are premixed at low of different types of pumps that can provide the
pressure and then passed to the pump. Theoretically, necessary pressures and flow-rates required by the
there can be any number of solvents involved in a modern liquid chromatograph. In the early years of
mobile phase program, however, most LC analyses the LC renaissance, there were two types of pump
require only two solvents, nevertheless, up to four in common use; they were the pneumatic pump,
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solvents can be accommodated. The layout of a high where the necessary high pressures were achieved
pressure gradient system is shown in the following by pneumatic amplification, and the syringe pump,
figure and includes, as an example, provision for three which was simply a large, strongly constructed syringe
solvents to be mixed by appropriate programming. with a plunger that was driven by a motor. Today the
majority of modern HPLCs are fitted with reciprocating
Solvent passes from each reservoir directly to a pumps fitted with either pistons or diaphragms.
pump and then to a mixing manifold from which it
passes to the sample valve and column. The pumps Single Piston Reciprocating Pump
control the actual program and are usually driven
by stepping motors. The volume delivery of each The single piston reciprocating pump was the first of
solvent is controlled by the speed of the respective its type to be used with high efficiency LC columns
pump which is precisely determined by the frequency (columns packed with small particles) and is still very
of its power supply. The controlling frequency can popular today. It is simple in design and relatively

384
inexpensive. A diagram of the single piston pump is demands on the piston-cylinder seal are not so great.
shown in the following figure. The diaphragm has a relatively high surface area and
thus, the movement of the diaphragm is relatively
small and consequently the pump can be operated
at a fairly high frequency.

HPLC Sample Valves

Since sample valves come between the pump and
the column it follows that HPLC sample valves must
also tolerate pressures up to 10,000 PSI. For analytical
HPLC, the sample volume should be selectable from
sub- micro liter to a few micro liters, whereas in
preparative HPLC the sample volume may be even
Single Piston Reciprocating Pump
greater than 10 ml. The higher the operating pressure
the tighter the valve seating surfaces must be forced
Most pistons of modern LC pumps are made of together to eliminate any leak. It follows that any
synthetic sapphire to reduce wear and extend the abrasive material, however fine, that passes into the
working life of the pump. The cylinder is usually made valve can cause the valve seating to become scored
of stainless steel and is attached to two non-return each time it is rotated which will ultimately lead to
valves in line with the inlet and outlet connections leaks. This will cause the sample size to vary between
to the pump. The piston is driven by a stainless samples and eventually affect the accuracy of the
steel cam which forces the piston into the cylinder analysis. In LC, the sample valve contains an extra

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

expressing the solvent through the exit non-return loading port and behaves like an internal loop valve.
valve. After reaching the maximum movement, the The basic difference between this type of valve and the
piston follows the cam and returns as a result of the normal external loop sample valve is the introduction
pressure exerted by the return spring. During this of an extra port at the front of the valve. This port
movement the cylinder is loaded with more solvent allows the injection of a sample by a syringe directly
through the inlet non-return valve. The shape of the into the front of the sample loop. Position (A) shows
cam is cut to provide a linear movement of the piston the inject position. Injection in the front port causes
during expression of the solvent but a sudden return the sample to flow into the sample loop. The tip of
movement on the refill stroke. In this way the pulse the needle passes through the rotor seal and, on
effect that results from the refill action is reduced. injection, is in direct contact with the ceramic stator
face. After injection, the valve is rotated to position
Rapid Refill Pump (B) and the mobile phase flushes the sample directly
onto the column. The sample is actually forced out
In order to avoid the refill pulses resulting from a
single piston pump, a number of rapid refill systems
have been developed. The designs have ranged from
cleverly designed actuating cams to drive the piston
rapidly in the refill mode to electronically operated
piston movements.

Diaphragm Pump
The unique property of the reciprocating diaphragm
pump is that the actuating piston does not come into
direct contact with the mobile phase and thus, the External Loop Sample Valve

385
 of the beginning of the loop so it does not have to atoms by siloxane bonds (silicon-oxygen-silicon
flow through the entire length of the loop. This type bonds). On the surface of each primary particle
of injection system is ideally suited for quantitative some residual, uncondensed hydroxyl groups from
LC, and is probably by far the most popular injection the original polymeric silicic acid remain. There are
system in use. three types of hydroxyl group. The first is a single
hydroxyl group attached to a silicon atom which has
HPLC Columns three siloxane bonds joining it to the gel matrix. The
second is one of two hydroxyl groups attached to
HPLC columns are packed with very fine particles the same silicon atom which, in turn, is joined to
(usually a few microns in diameter). The very fine the matrix by only two siloxane bonds. These twin
particles are required to attain the low dispersion that hydroxyl groups are called Geminal hydroxyl groups.
give the high plate counts expected of modern HPLC. The third is one of three hydroxyl groups attached to
Plate counts in excess of 25,000 plates per column a silicon atom which is now only joined to the silica
are possible with modern columns, however, these matrix by only a single siloxane bond.
very high efficiencies are very rarely found with real
samples because of the dispersion associated with Bonded Phases
injection valves, detectors, data acquisition systems
and the dispersion due to the higher molecular Bonded phases are formed by reacting the surface
weight of real samples as opposed to the common hydroxyl groups with an appropriate reagent to
test samples. LC columns, in general, achieve their chemical link an organic moiety to the silica surface.
separation by exploiting the different intermolecular The nature of the organic moiety will determine the
forces between the solute and the stationary pahse type of interaction that will take place between the
and those between the solute and the mobile phase. solute and the surface. The most efficient bonded phase
The column will retain those substances that interact has the maximum surface coverage. It is understood,
more strongly with the stationary pahse than those that due to stearic hindrance from the bonded moiety
that interact more strongly with the mobile phase. itself, only a proportion of the silanol groups can be
In particular optically pure compounds can be used bonded and there is little that can be done to avoid
to make Chiral HPLC stationary phases. this problem. However, there are other reasons
for incomplete silanization of the silica. Incomplete
Liquid Chromatography silanization can result from the reagent molecule being
excluded from the smaller pores of the silica. Exclusion
Stationary Phases can be a particular problem when bonding relatively
Traditionally the stationary phase used in LC has been large molecular weight materials such as long chain
silica gel which separates solutes largely on the basis hydrocarbons onto the silica surface. It is therefore,
of polarity, although, due to its unique structure, silica important to choose a silica gel that has a relatively large
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gel also exhibits strong exclusion characteristics. The pore size (e.g., a mean pore diameter of 150Å) which
bonded phases were introduced to provide a material may limit the surface area to between 150 and 250
that would separate solutes by dispersive interactions sq.m per gram and thus, reduce the retentive capacity
and also to provide some semie polar stationary of the stationary phase. The solvents normally used in
phases. The bonded phases were also based on silica bonded phase synthesis are aromatic hydrocarbons
gel. More recently, polymeric stationary phases were e.g., toluene that boils at 110˚C or mixed xylenes
introduced to provide materials that were insoluble that boil 138-140˚C. The procedure varies a little
in water and that were stable at extremes of pH. depending on the size of the batch and the type of
silanizing reagent. A method of synthesis of bonded
Structure of Silica Gel phase for the alkoxysilane reagents is illustrated below.
The most reactive alkoxy reagents are the methoxy and
The matrix of the primary silica gel particle consists of ethoxysilanes and their reaction with a hydroxyl group
a core of silicon atoms joined together with oxygen is accompanied by the release of methanol or ethanol.

386
 temperature, which controls the magnitude of the
absolute retention, can be relatively small. Its effect
H
on relative retention, however, can be very significant
Si OMe + Si O and, in fact, be the determining factor in achieving a
C8-moiety satisfactory resolution. An increase in temperature will
MeOH increase the diffusivity of the solute in both phases
and thus increase the dispersion due to longitudinal
diffusion and decrease dispersion due to resistance
to mass transfer.
Si O Si

C8-bonded phase stationary phase HPLC Detectors

The final capping process is also the same as that A large number of LC detectors have been developed
employed in the method using the chlorosilanes over the past thirty years based on a variety of different
reagents, utilizing hexamethyldisilazane as the sensing principles. However, only about twelve of
capping reagent. The alkoxy-silanes are almost as them can be used effectively for LC analyses and,
readily available as the chlorosilanes and are easier of those twelve, only five are in common use. The
and more pleasant to handle. dominant detectors used in LC analysis are the UV
detector (fixed and variable wavelength), photo diode
LC Mobile Phases array detector, the electrical conductivity detector,
the fluorescence detector and the refractive index
The choice of phase system can be very complex, detector. These detectors are employed in over 95%
particularly if multicomponent mixtures are to be of all LC analytical applications.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

separated. In the first instance the type of stationary
phase needs to be chosen and this choice must be UV Detector
based on the interactive character of the solutes
to be separated. If the solutes are predominantly The UV detector is by far the most popular and useful
dispersive then the stationary phase must also be LC detector that is available to the analyst at this
dispersive (a reversed phase) to promote dispersive time. Although the UV detector has some definite
interaction with the solutes and provide adequate limitations (particularly for the detection of non polar
retention and selectivity. If the solutes are strongly solutes that do not possess a UV chromaphores) it
polar then a polarizable stationary phase (one has the best combination of sensitivity, linearity,
containing aromatic rings or cyano groups) would versatility and reliability of all the LC detectors so far
be appropriate to separate the solutes by polar and developed. Multi-Wavelength UV detectors utilize
induced polar interactions. If the solutes are weakly a single of wavelengths to detect the solute. Most
polar then a strong polar stationary phase would be multi wavelength UV detectors can also provide a UV
required (such as silica gel) to separate the solute by spectrum of the eluted solute if appropriately arranged.
polar interactions.
Electrical
Column Ovens Conductivity Detectors
The effect of temperature on LC separations is often The electrical conductivity detector can only detect
not nearly so profound as its effect in GC separations, those substances that ionize and consequently, are
but can be critical when closely similar substances frequently used in the analysis of inorganic acids,
are being separated. In LC a change in temperature bases and salts. It has also found particular use in
will change the free energy of the solute in both the detection of organic acids and bases that are
phases, (generally in a commensurate manner) and frequently required in environmental studies and in
so the net change in the free energy difference with biotechnology applications. The sensor is the simplest

387
 of all the detectors consisting of only two electrodes are incompletely resolved. The routines deconvolute
situated in a suitable flow cell. the individual peaks from the composite envelope
and calculate the area of the individual de-convoluted
Fluorescence Detector peaks. Such algorithms can be used very effectively
on peaks that are entrained in the tail of a major
The fluorescence detector is one of the most sensitive peak but are not so accurate for composite envelopes
LC detectors and for this reason is often used for trace containing many unresolved peaks.
analysis. Unfortunately, although the detector is very
sensitive, its response is only linear over a relatively Spectroscopy
limited concentration range. In fact, the response of
the detector can only be assumed to be linear over Spectroscopy is a technique that uses the interaction
a concentration range of two orders of magnitude. of energy with a sample to perform an analysis. The
Unfortunately, the majority of substances do not data that is obtained from spectroscopy is called a
naturally fluoresce which is a serious disadvantage to spectrum. A spectrum is a plot of the intensity of
this type of detector. It follows, that in many instances energy detected versus the wavelength (or mass
fluorescent derivatives must be synthesized to render or momentum or frequency, etc.) of the energy. A
the substances of interest detectable. spectrum can be used to obtain information about
atomic and molecular energy levels, molecular
Refractive Index Detector geometries, chemical bonds, interactions of
molecules, and related processes. Often, spectra
The refractive index detector is one of the least are used to identify the components of a sample
sensitive LC detectors. It is very sensitive to changes (qualitative analysis). Spectra may also be used
in ambient temperature, pressure changes, flow-rate to measure the amount of material in a sample
changes and can not be used for gradient elution. (quantitative analysis). Because the response of
Despite these many disadvantages, this detector is a compound to electromagnetic (EM) radiation
extremely useful for detecting those compounds depends on its structure, spectroscopy can be used
that are nonionic, do not adsorb in the UV, and do to educate the structure of unknown chemical
not fluoresce. products. EM radiation behaves both as a particle
of light (called a photon) and as a wave moving at
HPLC Data Acquisition the speed of light (c; c = 3 x 108m/s).

The output from the detector, usually in millivolts, is Properties of EM particles

passed to a scaling amplifier that converts the signal
to a voltage that is acceptable to the analog to digital
and waves
(A/D) converter The A/D converter changes the voltage 1. Wavelength (λ): Distance between two peaks or
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output to a binary number which is temporarily stored troughs in a light wave.

in a register. This process is continuously repeated at 2. Frequency (ν): Number of wave cycles that pass
a defined rate, called the ‘sampling rate’. The current a given point per line. Usually measured in Hertz
binary number, stored in the register is regularly (Hz; 1 Hz = 1 cycle/second).
sampled by the computer and stored (usually on hard 3. Energy of a photon: E = hν = hc/λ, where h =
disk). On completion of the analysis the computer Planck’s constant = 6.6 x 10–34 J/sec.
accesses all the data from store, calculates the
retention report, compares peak heights or peak areas Types of Spectroscopy
to provide the quantitative analysis according to the
processing program that is used and finally prints out There are several types of spectroscopy, and among
the results in tabulated form. Modern data processing all these three are important for bioprospecting.
software often includes routines that can process
chromatograms where the components of the sample • Nuclear magnetic resonance (NMR) spectroscopy:

388
 Measures interaction of radio waves with atomic NMR spectra are displayed as plots of intensity of
nuclei in a magnetic field. energy emission (due to resonance) versus the energy
• Infrared (IR) spectroscopy: Measures absorption of of the radiation applied to the sample. Peaks in the
infrared light by chemical bonds. spectrum represent resonance energies for nuclei in
• Ultraviolet/Visible (UV/Vis) spectroscopy: Measures a molecule.
absorption of ultraviolet or visible light by π bonds.
Shielding: An electron cloud circulates around each
Nuclear magnetic resonance nucleus and creates a small magnetic field opposing
the applied magnetic field. The electron cloud around
(NMR) spectroscopy each atom depends on the surrounding atoms. As a
Atomic nuclei have a “spin” associated with them (i.e., result, each nucleus experiences a slightly different
they act as if they were spinning about an axis) due to magnetic field (the sum of the applied field and
the spin associated with their protons and neutrons. the field from the electron cloud). For this reason,
Because nuclei are positively charged, their spin induces the energy at which a nucleus achieves resonance
a magnetic field. When a magnetic field is applied to depends on its surroundings.
atomic nuclei, the magnetic fields of the nuclei align
themselves either parallel or antiparallel to the applied Chemical shift (δ): The resonance energy for a
magnetic field. The nuclei have a slight preference given nucleus is reported in an NMR spectrum as the
for the parallel alignment, as it has a slightly lower difference (in parts/million) between the resonance
energy, but nuclei can flip between the two possible frequency for a given proton and the resonance
alignments. When EM radiation with energy equal to frequency for protons in a reference compound,
the energy difference between the two alignments which is usually tetramethylsilane, (CH3)4Si. Chemical
is applied to the nuclei, it induces them to flip from shifts give information about the atomic surroundings

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

parallel to antiparallel alignment. Rapid flipping of a given nucleus.
between alignments occurs. The nuclei are said to be
in resonance, and the energy they emit when flipping Peak intensity: The area under a peak in an NMR
from the high to the low energy state can be measured. spectrum is proportional to the number of nuclei
The energy at which a given nucleus achieves resonance in a given chemical environment in a molecule
depends on its chemical surroundings. NMR spectra (e.g., if the area under a peak is two times the area
are taken by applying a magnetic field to as ample, under another peak, there are twice as many nuclei
irradiating the sample with EM radiation whose energy responsible for the larger peak than for the smaller
is varied over a given range, and measuring the energy one). The intensity of an NMR peak gives information
emitted by flipping nuclei at each energy. about the relative number of a given type of nucleus
in a molecule.
1. The range of radiation energies is generally chosen
such that emission from only one type of nucleus Spin-spin splitting: In 1H NMR, a given hydrogen
(e.g., 1H) in a molecule is seen. nucleus interacts with hydrogen nuclei on neighboring
2. NMR spectroscopy does not work for nuclei that carbon atoms such that the peak from that nucleus
have an even number of protons and neutrons— is split into multiple peaks called a multiplet. Relative
these nuclei have no net spin. intensities of the peaks in a multiplet follow Pascal’s
3. NMR spectroscopy is most commonly done on triangle. Spin-spin splitting gives information
1
H and 13C. about the hydrogen atoms neighboring a given
hydrogen nucleus.
Features of an
NMR Spectrum Infrared (IR) spectroscopy
The distinguished features and terms related to NMR The principles and theory related to IR spectroscopy
spectrum are as follows: are detailed below:

389
 • Covalent bonds are similar to springs—bonded Ultraviolet/Visible (UV/Vis)
atoms vibrate (i.e., stretch and compress) and bend
about their bonds. As a consequence of quantum
spectroscopy 
mechanics, these bonded atoms can vibrate and If a molecule has π electrons, it can absorb UV or
bend only at frequencies that are integral multiples visible light to promote one of those electrons into a
of a fundamental frequency that depends on the higher-energy orbital. UV/Vis spectroscopy generally
type of bond. involves the promotion of the π electron in the
• Bonds about which vibration and bending occur highest-energy occupied orbital to the lowest-energy
can absorb light if the frequency of the light wave unoccupied orbital. A UV/Vis spectrum is taken by
is the same as the frequency of the movement irradiating a sample with UV/Vis light over a range
about the bond. The frequency of light absorbed of wavelengths and measuring the amount of light
by these bonds is generally in the infrared region absorbed at each wavelength.
of the EM spectrum.
• In IR spectroscopy, a chemical sample is irradiated Features of UV/Vis spectra
with infrared light over a wide range of frequencies,
and the light absorbed by the sample at each The distinguished features and terms related to UV/
frequency is measured. Vis spectrum are as follows:
• Bonds about which a molecule is symmetric cannot
absorb IR light and therefore cannot be detected • UV/Vis spectra are displayed as plots of absorption
by IR spectroscopy. versus wavelength. Peaks represent wavelengths
at which light was absorbed by the molecules in
Features of an IR spectrum the sample.
• The energy of the UV/Vis light absorbed by a π
The distinguished features and terms related to IR electron system in a molecule depends on the
spectrum are as follows: nature of the π system. As a result, the presence
of a particular type of π system in a molecule can
• IR spectra are displayed as plots of absorption be identified by UV/Vis spectroscopy.
versus wave number (cm-1, 1/λ), which is another • The energy gap between the highest-energy
measure of the energy of a light wave (similar to occupied orbital and the lowest-energy unoccupied
frequency). Peaks in an IR spectrum represent orbital decreases as the size of the π electron
wavelengths at which light was absorbed by the system increases. Consequently, the wavelength
molecules in the sample. of UV/Vis light absorbed by a molecule increases
• Because each functional group that is IR-active as the size of its conjugated π electron system
has a characteristic set of frequencies at which increases (because the energy of a light wave
it absorbs IR light, IR spectroscopy is useful in decreases with increasing wavelength).
Central Marine Fisheries Research Institute

detecting the presence of specific functional • UV/Vis spectroscopy is generally used on

groups in a molecule. conjugated hydrocarbon systems, but other
• Because nearly all organic molecules contain C-C molecules containing π electron systems, such as
and C-H bonds that absorb IR light in similar carbonyls, are also weak absorbers of UV/Vis light.
ways, IR spectroscopy is most useful in identifying
functional groups that contain other bonds besides Suggested readings
C-C and C-H bonds.
• Fingerprint region: Region between 1200 cm–1 and Baianu, I. C., Costescu, D., Hofmann, N. E., Korban, S. S. Infrared
1700 cm–1 in an IR spectrum; contains complicated Chemical Imaging and Fluorescence Microspectroscopy. 2004.,
q-bio/0407006 (July 2004)
absorption peaks that are characteristic of a Beesley T. E., Scott R. P. W. Chiral Chromatography, John Wiley and
specific molecule. An unknown compound can be Sons, Chichester-New York, (1998)39.
Dandenau, R. D., Zenner, E. M. J. High Res. Chromatogr. 2(1979)351.
identified with reasonable certainty if its fingerprint Desty, D. H., Goldup A., Wyman, B. F. J. Inst. Petrol., 45(1959)287.
region matches that of a known compound. Drago, R. S., Physical Methods for Chemists, Surfside

390
 Publishing, 1992. Ogan, K. L., Reese, C., Scott, R. P. W. J. Chromatogr.
Dubois, J., Sando, G., Lewis, E. N. ,G.I.T. Laboratory Journal Europe, Sci., 20(1982)425.
No. 1-2, 2007 Pasto, D. J., Johnson, C. R., Organic Structure Determination,
Harley, J., Nel, W., Pretorious, V. Nature, London, 181(1958)177. Prentice-Hall, 1969.
Harris, D. C., Bertolucci, M. D., Symmetry and Spectroscopy, Schmidt G.J., Scott R.P.W. Analyst, 110(1985)757.
Dover, 1978. Scott R. P. W. Analyst, 124(1999).
James A. T., Martin, A. J. P. Biochem. J., 50 (1952) 679. Scott R. P. W., Beesley T. E. Analyst, 124(1999)713
James, A. T. The Times Science Review, Summer (1966)8. Scott R.P.W. Chromatography Detectors: Design. Function and
Katz E., Scott R. P. W. J. Chromatogr., 253(1982)159. Operation, Marcel Dekker Inc., New York-Basle (1997).
Lide, D. R. CRC Handbook of Chemistry and Physics, 75th ed. (Boca Tiselius A., Claesson D. Ark. Kemi. Mineral. Geol. 15B(No 18)(1942).
Raton, FL: CRC Press, 1994), 9–79. Tswett M.S. Tr. Protok. Varshav. Obshch. Estestvoispyt Otd.
Luthria, D. Oil Extraction and Analysis. pp.241-273, AOCS Press., Biol. 14(1905).
Champaign, IL, 2004. Williams, D. H., Fleming, I., Spectroscopic methods in organic
Martin A. J. P., Synge R. L. M. Biochem. J. 35(1941)1358. chemistry, McGraw-Hill, 1987.
Martin A. J. P., Synge, R. L. M. Biochem. J., 35 (1941)1358. Willstatter R., Stoll A. Utersuchungenuber Chlorophy, Springer,
Nakanishi, K., Berova, N., Woody, R. W., Circular Dichroism, VCH Berlin 1913
Publishers, 1994

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

391
 Bioassays – Types and Evaluation
I. Rajendran
Principal Scientist
Mandapam Research Center of CMFRI
e-mail: [email protected]

Introduction extraction (PLE), accelerated solvent extraction (ASE),

Bioassay is a tool for the determination of a biological pressurized hot water extraction (PHWE), ultrasound-
activity, or the quantification of a target analyte assisted extraction (UAE), and microwave assisted
based on this activity, using the recognition elements extraction (MAE) techniques.
like bacteria, cells or tissues. It is the navigational
pathway to find the active component present in Types of bioassays
the crude extract of marine organisms which have
biological activity of interest for exploration. When
for screening
a particular organism is found to have activity Methods and applications represent a unique attempt
of definite interest, the extract was obtained by to describe assays to evaluate the bioactivities
conventional methods from the marine organism. of therapeutic moieties resulting from chemical,
With the bioassay guided fractionation, the extract biological, and natural processes and to explore the
was sub fractionated to the limited fractions which mechanism of action of therapeutic moieties in the
have maximum activity of interest. When the complex cells, tissues, and organs of living beings, as well as
mixture is simplified to few active fractions, it will in living beings themselves. Such assays are highly
be easy to isolate active pure component using the important in the pharmaceutical industry and are
powerful isolation techniques depending on the the prime engine for the advancement of chemistry,
quantity of the expected final product. Exploratory pharmacy, biology, and medicine.
work would be complete only if the active component
is retrieved from such extracts with the support of As a routine record of bioactivities, in any exploratory
the bio assays. The final pure product of the extract work the samples are screened from simple activity
usually happens to be in low yield when compared to the much interested bio assay. The assays are
to the total weight of the source organism. If the of different kinds and can be performed in the
compound/extract is to be screened against a wide following heads:
Central Marine Fisheries Research Institute

spectrum of activity, it is normally inadequate for

full screening experiments to find out the overall Anticancer, antiviral, antitubercular, antimicrobial
potential. The activity of drugs is dose dependent (antibacterial or antifungal), antihelminthic,
and toxic in higher doses. Eg. Pesticides at low doses antimalarial, thrombus-related anticoagulation,
act as PGR; at high dose as poison. Any prediction analgesic, antiallergic, antiarrhythmic, hypolipidaemic,
of the activity of a component can be done using hypoglycemic, hypotensive, antihypertensive, diuretic,
dose response spectra library software. immunomodulatory, choleretic and anticholestatic,
blood pressure, Parkinson’s disease and Graves’
Extraction techniques Disease, Alzheimer’s disease, antiosteoporosis,
immunomodulation, anti-inflammation, antioxidant
Some of the latest isolation techniques include activity, epilepsy, diabetes, assays for toxins from
new state-of-the art extraction techniques, such as microorganisms, hepatotoxicity and hepato protective
supercritical fluid extraction (SFE), pressurized liquid assays, cytogenetic receptor and enzyme assays.

392
The screening models for the evaluation of these Neurosuppressives and
properties on the given analyte are available.
muscle relaxants
Types of Glutamate receptor antagonist, serotonergic
receptor (SR) antagonist, IP3-inhibitor, adrenergic
screening experiments receptor antagonist, Actomyosin ATPase inhibitor
The screening for the activities is done in vitro or in (antiasthmatic, uterine relaxation), Glutamate receptor
vivo modes depending on the nature and necessity antagonist (GRA)
of testing. Appropriate models for the screening
by biological, toxicological and clinical evaluations Antiinflammatory
are now available to find out the type of activity
for the given sample. As a protocol, the extract of Antiinflammatory activity – Carrageenin-induced
a marine organism is subjected to various kinds Oedema in Mice–Volume of both the paws is
of activity tests depending on the type of activity measured plethysmographically daily for ten days.
chosen for screening. Based on this the active Phospholipase A2 (PLA2) inhibitor
component is retrieved from the extract. In vitro
assays usually consist of cell culture systems with Antiviral
neoplastic cell lines from human or other animal
tumors as targets. The capacity of test compounds The potential antiviral agents should be evaluated in
inhibiting the growth or reducing the survival of a living cell or animal host like cell culture, chicken
cancer cells in culture media and the potency of test eggs and animal models. The cells are infected with
compounds inducing structural change of cancer the virus and then exposed to the test substance. If
cells in culture media are generally correlated with the substance has antiviral activity the multiplication

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

the in vivo potency of a cancer therapeutic agent. of the virus will be inhibited which will be evident
from the morphology of the cell monolayer. Several
Anticancer tests viral targets are studied to estimate the antiviral effect
of test substance in a cell culture system. Some of
22 models used in anticancer research are described: these are viral DNA polymerase activity; ribonucleotide
3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium diphosphate reductase; mRNA polyadenylation
bromide (MTT) assay for six carcinoma cells, flow and RNA dependent RNA polymerase; terminal
cytometric assay for cell apoptosis, DNA fragmentation deoxynucleotidyl transferase; thymidine kinase; uracyl
assay, Bcl-XL/BH3 interaction assay, dissociation DNA-glycolase; d-UTPase and reverse transcriptase.
enhanced lanthanide fluoro-immunoassay (DELFIA), AV (HIV-1 integrase inhibitor), AV (herpes and polio),
Ishikawa cell and rat assay for detecting antiestrogens, AV (feline leukemia, mouse influenza, mouse corona),
adenosine triphosphate (ATP) assay for eight cells, UAG suppressor glutamine tRNA inhibitor, Interferon
alkaline phosphatase (AP) activity assay, tumor mediator, herpes simplex virus type 1 (HSV-1), HIV
endothelial cell tube formation assay, antiangiogenic reverse transcriptase (HIV RT).
assay, in vivo hollow fiber assay, VX2 rabbit lung assay,
insulin-like growth factor-I (IGF-I)-induced kinase Antibiotic
receptor activation assay, insulin-like gD. trkA-induced
kinase receptor activation assay, UV spectra-based Antibacterial, antifungal, antimalarial, antibiotic tests
calf thymus DNA intercalation assay, fluorescence are carried out involving the pathogens: gram-(+)
spectra-based calf thymus DNA intercalation assay, ve: Bacillus subtilis, Staphylococcus aureus, Candida
P-glycoprotein pump (P-gp) in MCF-7R cells assay, albicans; Fungus- Cladosporium cucumerinum, Malarial
P-gp-related efflux carrier assay, substrate transport parasite: Plasmodium falciparum. Antimicrobial testing
inhibition assay, lactate dehydrogenase release assay, involves various methods, viz. poison food technique;
functional assay of mitochondrial P-glycoprotein disc diffusion method, tube dilution method and
(P-gp), and resistance index value assay. microtitre technique. Antimalarial Activity–Against

393
 Plasmodium berghei in primary screening in infected 5 to 6 hours after dosage for toxic symptoms. If
swiss mice. Subsequent screening is conducted in death occurs during this time, the cause of death
rhesus monkey infected with P. cynomolgi. Chloroquine is recorded. The approximate LD50 is estimated
(5 mg per kg for 7 days). and the maximum tolerated dose is also recorded
for use in subsequent investigations.
Pharmacological activities tests
Clinical Trials
Antitumor and Anticancer tests – They involve the
following factors in identification of the activity of Any proven fraction/compound with promising
the given sample for screening. Protein kinase C activity from preliminary experiments on animals
(PKC) inhibitor, 3-Fucosyltransferase (FTase) inhibitor, will be extended for human trials under different
Kinesin motor protein (KMP) inhibitor, Stabilization phases. Phase I clinical trials are performed on health
of microtubules (MTS), Tubulin polymerization human volunteers, Phase II on patients and Phase III
(TP) inhibitor, Actin-depolymerisation (actin DP), on multicentric mode involving clinical trials protocol
Topoisomerase (TI) II inhibitor, Nitric oxide synthetase and duplicating trials.
(NOS) inhibitor, NKT cell activator, Reverses drug
resistance of cancer cells, v-ATPase inhibitor, Ca2+ Aquaculture
channel blocker; Immunosuppressive (IS), cytotoxic
(CTX), P-388 leukemia (P-388L) cells, Inhibitor of T-cell In aquacutrue the extract is tested for the efficient
proliferation, IL-2 inhibitor, IL-8 inhibitor, Histamine fish culture practices. Antifouling – repellent;
release (HR) inhibitor, Inhibitors of proton pump Inhibition–fertilized seaurchin egg cell division,
activity (PPAI), Inhibition of DNA replication (DNAR), Icthiotoxic–artemia, fish, feed deterrent, Nematocidal
Lipoxygenase (LP) inhibitor, Cysteine Protease (CP) – Screening of harmful worms.
Inhibitor, phosphatase activity (PA) inhibitor, Ovarian
human tumor (OHT) cell, Inhibitors of serine- Blood-Related Diseases
threonine (STI)
Serine protease inhibitor (SPI), Thrombin
Toxicity and CNS Activities receptor antagonist (TRA), VCAM-1 inhibitor,
α-glucosidase inhibitor.
LD50 is determined in mice using the method of
Horn et al. with a dose of 464 mg/kg (ip) and Anthelmintic Activity
mortality in 24 hour recorded. The samples are
evaluated against experimental animal to find out Against parasites of hookworms, ascarids, oxyurids
its toxicity. An experiment on albino rats is given. and filarids with test animal – hamsters.
The following typical tests are undertaken while
Central Marine Fisheries Research Institute

screening the samples. eg. The test substance is Analgesic activity

administered by the oral or intraperitoneal (ip)
route in two or three adult albino mice of either sex It can be divided into two categories: (a) centrally
and usually 15 to 20 g in weight. The test material acting analgesics, and (b) peripherally acting drugs.
is suspended in 0.1% agar or in 10% gum acacia
in distilled water. Concentrations are so adjusted Antiarrhythmic and
that a 20 g mouse receives a volume of 0.2 ml.
antithrombotic activities
The initial dose is at a level of 400 or 500 mg/kg
going up or down by a factor of 2. Occasionally, Ventricular arrhythmias
an interval of 1.5 is used for closer approximation.
Doses higher than 1000 mg/kg are not generally Hypolipidaemic activity
used. Control animals are administered only the
vehicle (placebo). The animals are observed for Testing lipid lowering activity

394
Hypoglycaemic activity are exposed to a variety of chemicals such as carbon
tetrachloride, D-galactosamine, thioacetamide, heavy
With test animals of Charles Foster albino rats in single metals and drugs such as paracetamol, fungal toxins
and multiple doses. A fall of more than 30% in blood and parasitic infections. Some chemicals apart from
sugar is taken as active. these on exposure also result in carcinogenesis. Extracts
from marine sources show hepatoprotective activity.
Hypotensive Activity
Conclusion
The test animal is generally anaesthetised with sodium
pentobarbiton (35–40 mg/kg, iv or ip) or a chloralose With the advancement of evaluation and screening
(70–80 mg/kg, iv). The blood pressure is recorded. techniques it is now possible to explore the range of
The test extract is given at 25 and 50 mg/kg (iv), and activities for the given sample. The characterization
the effect noted. In rats with deoxycorticosterone of the active component of the active fraction is
acetate (DOCA)-salt model is used. also simplified with low quantity of sample using
powerful instrumental methods which require only
Diuretic Activity micro level quantity. The required screening facilities
are maintained both in private and public funded
In dogs at 5 mg/kg dose by intravenous administration. institutions to enable the activity testing. The work
may be outsourced or collaborated to develop a drug.
Mechanism of drug action
Suggested readings
The drug action on a living organism may be attributed
to the counter biological functions stimulated by the

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Researching and planning bioassays: advanced applied science: GCE
host with the help of drug or by drug itself against the A2 UNITS © The Nuffield Foundation 2008, p.1-19
Comprehensive Analytical Chemistry Petrovic´ and Barcelo´ (Eds)
invading pathogens. In this way, immunomodulators are Volume 50 ISSN: 0166-526X 2007 Elsevier B.V. (DOI: 10.1016/
substances which have potential to modulate an immune S0166-526X(07)50009-7 r)
Shiqi Peng, Ming Zhao, 2009 Pharmaceutical Bioassays: Methods
response in vivo and/or in vitro. Immunostimulants and Applications, John Wiley & Sons Guillaume P, Provost
enhance antigen specific (vaccines) and non-specific D, Legrand P, Godes M, Lacroix P 2009 Chapter 5: Unit
immune response against infection and malignancy. 5.53Models of cardiovascular disease: Measurement of
antihypertensive activity in the conscious rat (SHR, DOCA-salt,
Immunotherapy of viral infection is done by potentiating and Goldblatt hypertension models) In: Curr Protoc Pharmacol.
the efficacy of drugs in immuno compromised host. DOI: 10.1002/0471141755.ph0553s44
Dhawan, B. N.; Srimal, R. C. 1992 In: The use of Pharmacological
Extracts from marine organisms show wide range of Techniques for the Evaluation of Natural Products, UNESCO-
immunomodulation during the screening expereiments. CDRI, Lucknow, India
M. Hayes (ed.), Marine Bioactive Compounds: Sources,
Characterization and Applications, DOI 10.1007/978-1-4614-
Hepatoprotective Activity 1247-2_2, © Springer Science+Business Media, LLC 2012
D.S. Bhakuni, D.S. Rawat, 2005, Bioactive Marine Natural Products,
Co-published by Springer, with Anamaya Publishers, New Delhi
It is known that the biochemical and physiological Lee et al. 2013 Marine algal natural products with anti-oxidative,
functions of liver and the activity of a wide range anti-inflammatory, and anti-cancer properties, Cancer Cell
of hepatic enzymes are altered when the animals International, 13:55

395
 Chitin, Chitosan and their Applications
K. G. Ramachandran Nair
Principal Scientist (Rtd.)
Fish Processing Division
Central Institute of Fisheries Technology
e-mail: [email protected]

Introduction is animals it is frequently present as cell wall material

in plants replacing cellulose or sometimes occurring
Chitin is a nitrogenous polysaccharide (poly N-acetyl together cellulose. This polymer in the deacetylated
Amino D- glucose) found in the outer Skeleton form, i.e. chitosan is present in various fungi eg.
of insects, crabs, shrimps and lobsters and in the Zygomycetes contain both chitin and chitosan (Austin
internal structure of other invertebrates. It is the most et al., 1981; Rudall, 1969)
abundant organic compound next to cellulose in the
earth. It was first isolated by Braconnot (1811) from Table 1: Annual availability (global) of
mushroom and was named ‘fungine’. An identical chitinaceous materials
material was isolated from insects in 1821 by Odier Resources Quantity Chitonous Chitin
(1823) and named it as “chitine”. He was the first harvested (103 waste (103 potentials (103
tonnes) tonnes) tonnes)
person to observe remarkable similarity between
Shell fish 1700 468 39
cellulose and chitin. In 1859 chitosan, a partial Krill (potential 18200 3640 56
deacetylated chitin was discovered by Rouget on landing)
boiling chitin in concentrated potassium hydroxide. Clam/Oysters 1390 521 22
It was finally named chitosan by Hoppe-Seiler (1894) Squid 660 99 01
Fungi 790 790 32
but most information available now today has been
Total 22740 5118 150
obtained since 1950. The book “The Integument
of Arthropods” by Richards (1951) gave thrust on The utilizable resources of Antarctic krill 1 x 108 to 5
chitin while Tracey (1957) reviewed the structure and x 108 tonnes ( Naczk, et al.,1981) with chitin content
detection and quantity analysis of chitin. Jeuniaux of 0.33 to 1.74% (Sikrosky, et al., 1980). Marine
(1963) published a book on chitin and its enzymatic benthic organisms yield substantial amounts of chitin
breakdown and in 1964 Brima Combe and Webber (Jeuniaux, 1978) marine surface zoo planktons are
wrote a monogram on chitin. In 1967, Rudall first valuable source of chitin with a mean production
Central Marine Fisheries Research Institute

addressed the concept of chitin-protein complex of about 1 gm/m.sq/year which is higher than krill
which opened the door for additional work on the chitin production. Chitin production is the highest in
subject (Brine, 1984). A bibliography on chitin and its eutropic fresh water ecosystems. Fresh water bryozans
derivatives was published by Pariser and Boch (1972). are able to produce colonies whose envelope is made
of chitin and protein in a non-calcified form allowing
Resources easy extraction of chitin (Jeuniaux, et al.,1989).

The major sources of chitin are shell fish, krill, clam, Structure
oysters, squid fungi and insects. Allan at al. (1978)
quantified various sources of chitin (Table 1). Kong Chitin occurs in three polymorphic forms which
(1975) and Naczk et al., (1981) estimated the chitin differ in the arrangement of molecular chain within
content of selected crustacean, insects, molluscan the crystal cell. a-chitin the tightly compacted most
organs and fungi. Though the main source of chitin crystalline polymorphic form where the chains are

396
 of mineral acids (Austin, 1975) and dimethyl
acctamide containing 5% lithium chloride (Rutherford
and Austin, 1978). On hydrolysis of chitin with
concentrated acids under drastic conditions gives
relatively pure amino sugar D-glucosamine.

On deacetylation with the strong alkali yields the

free base chitosan. Chitosan is insoluble in water but
soluble in dilute acid. Pure chitosan is not hydrolysed
by lysozyme while chitin or partially deacetylated chitin
is hydrolised. For hydrolysis to occur it is important
Chitin is a polymer of b (1-4)-N-actyl- D- glucosamine (Fig.1). that at least 6 contiguous actamido side groups
should be present in the substrate. The probability
arranged in an anti-parallel fashion, b- chitin is the of having 6 contiguous residues with the necessary
form where the chains are parallel and g- chitin is acetamido side groups decreases as the deacetylation
the form where the chains are “up” to everyone increases (Pangburn et al., 1984).
“down” (Muzzrelli, 1977a). Deacetylation of chitin
with strong alkali yields chitosan, polymer of b (1-4)-D Rutherford and Austin (1978) compared the molecular
glucosamine (Fig.2). weight derived from intrinsic viscosities of chitin from
crab and shrimp and found molecular weight from
0.4 x 106 to 1.8 x 106 for chitin. Anon (1991) obtained
a molecular weight of 1.0 to 1.50 x 105 for chitin
produced from Indian prawns.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

The acetylated amino groups form hydrogen bonds
which prevent swelling and dissolution in aqueous
media. Depending on extent of deacetylation chitin
contains 5 to 8 % nitrogen, which in chitosan in
Properties the form of primary aliphatic amino group. Chitosan
undergoes the reactions typical of amines, of which
Most commercial poly saccharides eg. Cellulose, N-acylation and schiff reactions are the most
dextran, pectin, alginc acid, agar, agarose, important. Chitosan derivatives are easily obtained
carrageenans and heparin are neutral or acidic. under mild conditions and can be considered as
Chitin and chitosan are the only abundant basic substituted glucans.
polysaccharides. Their unique properties include
solubility behavior in various media, solution The properties of chitin and chitosan vary considerably
viscosity, polyelectrolytic behavior, polyoxysalt depending on the source and production process. The
formation, ability to form films, chelate metal ions quality requirements of chitosan and its derivatives
and optical and structural characteristics (Austin et vary with the end use.
al. 1981). Although b-1-4 anhydro glucosidic bond
of chitin is also present in cellulose, characteristic Production
properties of chitin/chitosan are not shared by
cellulose (Muzzrelli, 1978). Chitin represents 14-27% and 13-15% of the
dry weight of shrimp and crab processing waste
Chitin is highly hydrophobic and insoluble in water respectively (Ashford et al., 1977). Madhavan and
and most organic solvents. It is soluble in hexafluoro Nair (1975) found that dry prawn waste contained
isopropanol, hexafluoro acetone (Capaozza 1975), 23% and dry squilla contained 15% chitin. Chitin is
Chlor-alcohols in conjunction with aqueous solution present as chitin protein complex along with minerals

397
 mainly calcium carbonate. So the process of chitin commercially available or can be made available
production consists of deproteinisation with dilute (Ravikumar, et al., 2004). The five international
alkali and demineralization with dil. acids (Fig. 3). The conferences on chitin and chitosan (1977, 1982,
chitin thus obtained is deacetylated to chitosan using 1985, 1988 and 1991) have thrown light on various
conc. alkali. Chitin and chitosan are now produced application in various fields. These applications can
commercially in Japan, USA, India, China, Poland, be classified under the following heads.Clarification
Indonesia, Norway and Australia and purification

Fig. 3: Production of chitosan from prawn/crab shells 1. Chromatography

Prawn/crab shell 2. Paper and textiles for photography
3. Food and nutrition
Dil HCl 4. Medical and pharmaceuticals
5. Agriculture
Demineralisation
Agriculture
Dil. NaOH
Chitin and its derivatives have potential applications
in agriculture for various uses such as germination
Deprotenisation and culturing to enhance self-protection against
pathogenic organisms in plants and suppress them in
Chitin soil to induce chitinase activity and proteinase inhibitor
synthesis for antivirus activity, in encapsulation of
Conc. NaOH fertilizers, in liquid fertilizers and controlled release
of herbicides.
Chitosan
A thin coating of chitosan or its derivatives in seeds
Organic acids of radish or soyabean, rice and black pine induce and
/or enhance chitinase activity which help the seeds to
germinate with little infection (Hirano et al. 1984).
The water holding capacity of soil was increased by
Milling Dissolving mixing N-methyl chitosan gels with it and found
that radish seedling could survive for 4 days after
Chitosan powder Filtration water was stopped (Hirano et al., 1984). Chitinous
materials are efficient to control parasitic nematodes
Organic acid
in ornamental plants, in cucumber and tomato
Central Marine Fisheries Research Institute

Mixing Dehydration (Brown et al., 1989). Chitosan has found application

in pesticides and herbisides because of its sustained
Chitosan acid blend chitosan salt release property (Mc Cormick and Anderson, 1984).
The chitosan is used in liquid fertilizers to reduce
(self dissolving) (water soluble)
drying rate, controlled nutrient release and to reduce
phyto- toxic affect (Strusczyk, et al., 1989). It can also
be incorporated in foliar sprays for fixing of nutrients,
slow release and to minimize moisture loss.
Applications of chitin
and chitosan Activation of plants cells with chitosan and its
derivatives may relate to (a) the induction and chitinase
Chitosan is a versatile polymer and interest in chitosan and chitosanase (b) an increase in the callus growth
is due to the large variety of useful forms that are of cabbage leaves and (c) a yield increase in the field

398
 Proc. Of third Intl. conf., Chitin and Chitosan, Italy
culture of Japanese radish plants. The growth and Braconot, H. 1811 Ann. Chi. Phy 79, 265-304. In: Distribution
chitinase activity of the callus increased in the presence and Quantative Importance of chitin in fungi in Proc. First Intl.
Conf. on Chitin/Chitosan, MIT, Combridge by Jose R H, 1978.
of low molecular weight chitosan and chitosan Brine C J, 1984 in Chitin, Chitosan and Related Enzymes. Academic
oligosaccharides and the specific activity of chitinase Pros. Inc. London.
also increased. This strongly suggests an increase Brown, L R, Brown, W., Teicherl, C, Blassingame, D J and Lander, C
M 1982. The Use of chitinous sea food wastes for the control
in the protein synthesis in the presence of chitosan of parasitic nematodes in chitin and chitosan. Japanese Society
and its derivatives. Several investigators reported of Chitosan. Tottori, Japan, 227-232
Capozza, R C 1975. Decomposable Biodegradable pharmaceutical
the induction of chitinase and chitosanase activities carrier. Ger patent, 2505 305
in entomorprphogenic fungi with the degradation Hadwiger, L. A., Kendra, D F., Fristansky, B W and Wagner, W.
products of chitin and chitosan (St. Lager et al., 1986). 1986. Chitosan both activates genes in plants and inhibits
RNA synthesis in fungi in Chitin. In: Nature and Technology,
Silverstre and Tosti (2010) reported the efficiency of Plenum Press, New York, 209-214.
chitosan in protection of plants against diseases. It has Hirano, S., Hayashi, M., Kokuko, M., Hasaya, T. and Takashi, N.,
1988. Chitosan and derivatives as activators of plant cells in
also been reported by Boonlentnirum et al., (2008) tissues and seeds. Applied Bioactive Polymeric Material. Plenum
that rice production could be increased by foliar spray Publishing Corporation, 45-59.
of chitosan which tended to show ability in disease Hirano, S., Hayashi, M., Nishide, T. and Yamamoto, T. 1989. In:
Chitin and Chitosan. Elsevier Applied Science, London, 743-747
control. (Uthairantnakij et al., 2009) found that Hoppe-Seiler, F. 1894. Bes. Dent. Chem. Gas. 27, 3329-3331.
chitosan has improved orchid production and quality. Jeuniaux, ch, 1963 Chitine et Chitinolyse, Masson, Paris.
Jeuniaux, ch, 1978. Distribution and quatitative importance of chitin
in animals. In Proc. Intl. Conf. Chitin/Chitosan. MIT, Combridge.
Conclusion Kong, N. 1975. A feasibility study of New Routes of Marine
Polymers. M S Thesis. Univ. of Washington.
Madhavan and Nair K G R 1975. Chitosan from Squilla. Fish.
Many products are marketed based on chitosan for Technol. 12, 81-82
plant protection, growth promotion, seed coating Mc Cormick, C.L. and Anderson, K.W., 1984. In: Chitin, Chitosan
and Related Enzymes. Acedamic Press, Inc. London. 41-53.
etc., but these applications have not put in to large

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

MIT, Cambridge, 54-63
scale application so far. The shelf life of vegetables Muzzarelli; R.A.A, 1978. Chitin an important Natural polymer. Proc.
and fruits also can be extended with the application of Int. Conf. Chitin/Chitosan. MIT, Combridge, 1-3.
Nacsk M. Synowiecki, J and Sikorski, Z.E. 1981. Food
chitosan coating. With the growing awareness of the Chemistry 7,175
adverse effects of hazardous chemicals in agriculture Odier, A., 1823. Mem. Soc. Hist. Nat. Paris. 1, 29-42.
Pangburn, S H, Trescony, P V and Helks, J. 1984. In; Chitin and
and popularization of organic farming, chitosan Chitosan Related Enzymes. Acedemic Press, Orlando.
products will find use. Antivirus, antibacterial, Pariser, E.R. and Boch, S., 1972. Derivatives – An annotated
nematocidal, insecticidal and pesticidal properties Bibilography of selected publications from 1965 to 1971.
142-146. MIT Cambridge, Mass.
of chitin and chitosan have to be taken to the field by Richard, A G, 1951. The integument of Arthropods. Univ. Minnesota
researchers to ensure safety of agriculture products. Press, Minneapolis.
Rudall, K M. 1969. J. Polymer Sci., 28, 83
Rudall, K. M. 1967. Conformation of Biopolymers, Academic
Suggested readings Press, London.
Rutherford, F A and Austin, P R. 1978. Marine chitin properties and
solvents. Proc. Int. Conf. Chitin/Chitosan, MIT, Cambridge. 182-
Allan, G.G., Fox. J.L. and King, N. 1987. Critical evaluation of 192.
potential sources of chitin and chitosan. In Proc. First Int. Sikrosky, Z E, Bykowsky, P and Kryszewski J 1980. Food process
Conf. Chitin/Chitosan Engineering. Vol.1. Applied Sciences Publishers, London.
Anon, 1983. Annual Report, ICAR, 1983 (of CTRL), ICAR, New Delhi. Silvester and Tosti E, 2010. Chitosan in plant protection. Marine
Anon, 1991. Annual Report, 1990-91, Central Institute of Fisheries Drugs, 23.
Technology, Cochin-29 Struszczyk, H Pospiezhy, H. and Kotlinsky, S. 1989. Chitin and
Ashford, N A, Hattis, D and Murray, A E 1977. Industrial Prospects Chitosan, Elsevier Applied Science, London, 733-742.
for chitin and chitosan from Prawn shell waste. MIT, Sea Grant Tracey, M V. 1957, Rev. Pure Appl. Chem. 7, 1-14
Report. MITSG 77-3, MIT, Cambridge. Ulthairatarakj, A, Jaine, A T and Obsuwan. 2007. Chitosan
Austin, P.R. and Sennet, S., 1985. Dry chitosan salts and complexes for improving orchid production. In: Orchid Science and
of alphatic carboxylacids in Chitin in Nature and Technology, Biotechnology. 1(1) 1-5

399
Nutrition
Nutrient Requirement of Cultivable
Brackishwater Fish and Aqua Feed
Processing Techniques
K. Ambasankar*, J. Syama Dayal, K. P. Kumaraguru Vasagam and K. P. Sandeep
Principal Scientist
Central Institute of Brackishwater Aquaculture,
75, Santhome High Road, R.A. Puram, Chennai 28
e-mail: [email protected]

Introduction and fish feeds on a variety of food items and derive

their balanced nutrition for healthy growth. When
Aquaculture farming have shown phenomenal they are cultured in confined pond they should be
growth in the last decade in India producing protein provided with a balanced diet as close to natural food
rich health food and earning valuable foreign as possible. This is the reason for understanding the
exchange. Feed is a major input in fish farming. The nutritional requirement of candidate species which
development of nutritionally balanced feed involves assumes paramount importance in developing the
understanding the dietary requirements of candidate feeds for the candidate species.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

species, selection of feed ingredients, formulation
of feeds and appropriate processing technology for Nutritional requirement
producing water stable pellet feeds. Depending upon
the type of farming, a wide range of feeds are used
of different
for feeding stocked shrimp and fish. While no feed brackishwater species
is used in traditional farming systems, supplementary
and balanced feeds are used in extensive and Protein
semiintensive aquaculture.
Protein is the most important nutrient in the diet
All animals including fish requires food to supply the of shrimp and fish. Protein requirement of aquatic
energy that they need for movement and all the other organism is higher than terrestrial animals. Fish require
activities that they engage in for growth. However, food protein in the form of essential amino acids
they are ‘cold-blooded’ and as their body temperature for maintenance of life, growth and reproduction
is the same as the water they live in, they do not and the requirement of protein depends on animal
therefore need to consume energy to maintain a characteristics i.e., species, physiological stages, size
steady body temperature and they tend to be more as well as dietary characteristics, i.e., protein quality
efficient users of food than other farm animals. The (digestibility and biological value), energy level etc.
nutrient requirement of different species of finfish Scarcity of carbohydrate and abundance of protein
vary in quantity and quality according to the nature and lipid in the natural aquatic food web is also
of the animal, its feeding habits, size, its environment probably responsible for the common trend of aquatic
and reproductive state. organisms to use protein as an energy source.

Fish diet should have adequate energy, not only to Protein is required in the diet to provide
meet the needs of body maintenance called basal indispensable amino acids and nitrogen for synthesis
metabolism, but also for growth. In nature shrimp of non-indispensable amino acids. A deficiency of

403
 indispensable amino acid creates poor utilization Lipid
of dietary protein and hence growth retardation,
poor live weight gain and feed efficiency. In severe Lipid is a complex mixture of simple fat,
cases, deficiency reduces the ability to resist diseases phospholipids, steroids, fatty acids and other fat
and lowers the effectiveness of the immune soluble substances such as pigments, vitamins A,
response mechanism. Experiments have shown that D, E and K. Apart from its major role to supply
tryptophan deficient fish become scoliotic, showing energy lipid also act as precursors to many reactive
curvature of the spine, and methionine deficiency substances. Phospholipids are responsible for the
produces lens cataracts. structure of cell membranes (lipid bi-layer). Fatty
acids are the main active components of dietary
Protein requirement vary with the age of the fish. lipids. Deficiency of essential fatty acid result in
Younger animal generally require higher levels of general reduction of growth and a number of
protein (5-10% more protein) than older animals. deficiency signs including depigmentation, fin
Carnivores require high dietary protein (40-50%) erosion, cardiac myopathy, fatty infiltration of liver
than omnivores (25-35%). Among the brackishwater and ‘shock syndrome’ (loss of consciousness for a
finfishes, requirement of protein for Asian seabass few seconds following an acute stress. Fat levels
(Lates calcarifer), milkfish (Chanos chanos) and of 6-8% are adequate in most of the fish diets.
mullet (Mugil cephalus) is 40-45%, 40% and However, the quality of fat in terms of fatty acids is
27-35%, respectively. more important. Carnivorous fish such as seabass
can utilize lipids more effectively and lipid level as
Amino acids high as 20% can be used in their diet. However,
lipid level should be adjusted in diet considering
The growth of fish is directly related to the quality of the technological problems in feed manufacture
protein in terms of amino acids. After digestion of and storage. Fish oil and soya oil are generally used
protein, amino acids are metabolized at tissue level as lipid source during feed formulation.
to form new proteins for growth, maintenance and
energy. Among 25 amino acids present in protein Fatty acids
10 amino acids must be supplied in the diet since
fish cannot synthesize them and termed as essential Fish and shrimps are unable to synthesize fatty acids
amino acids (EAA). These are arginine, histidine, of the n-3 and n-6 series and must be provided
isoleucine, leucine, lysine, methionine, phenylalanine, in their diets. Aquatic animals require higher
threonine, tryptophan and valine. A large proportion n-3 fatty acids than terrestrial animals. Among
of the amino acid consumed by a fish are catabolized aquatic animals, marine habitat requires more
for energy and fish are well adapted to using an excess HUFA than freshwater counterparts. Among the
energy in this way. It is found that if the amino acid long chain fatty acids polyunsaturated fatty acids
Central Marine Fisheries Research Institute

composition of the protein in the feed matches with (PUFA) such as linoleic acid (18:2n6), linolenic acid
the amino acid composition of shrimp body tissue, (18:3n3), eicosapentaenoic acid (20:5n3) (EPA) and
such feed promotes good growth. docosahexaenoic acid (22:6n3) (DHA) are essential
for growth, survival and good feed conversion ratio.
Table 1. Nutrient requirement of different brackishwater fishes The n3 fatty acids are more essential than the n6
Nutrient L. calcarifer Mugil Etroplus
cephalus/ Liza suratensis
acids. The fatty acids, EPA and DHA, which are
tade known as highly unsaturated fatty acids (HUFA) of n3
Energy (Kcal/ 4000-4500 4000-4500 4000-4500 series, are particularly important. Quantitatively EPA
kg)
and DHA are needed at 0.5% and 1.0% in the diet
Protein % 45-55 27-35 30-32
of larvae and fry of brackishwater fish. Fresh water
Lipid % 6-18 6-9 6-8 fish show requirement for n6 and n3 essential fatty
Carbohydrate 10-20 30-40 30-40 acids (EFA), whereas marine fish show requirement
%
of n3 and also HUFA.

404
Phospholipids largely dependent on the mineral concentration of the
aquatic environment. About 20 inorganic elements
Fish require phospholipids for growth, metamorphosis (macro and micro) are required to meet the metabolic
and maturation. Lipids of squid, clam, shrimp, fish and structural functions in the body of animals. The
and polychaetes are excellent natural source of aquatic organisms regulate the mineral needs through
phospholipids. The phospholipid phosphatidylcholine dietary source and also through internal regulatory
(lecithin) is essentially required in the diet of larval and mechanisms in the kidneys and gills. In saline water,
fry stages of fish for fast growth and good survival. calcium (Ca) is abundant, which is absorbed by most
Soya lecithin is a good source of phospholipid. It is aquatic animals. Since the availability of phosphorus
required at 1-2% level in the diet. The development (P) through water medium is poor, P should be made
and survival of larvae is significantly improved when available through diet. Usually the preferred Ca:P
the diet contains lecithin. ratio is 1:1 in feeds of aquatic species. Mono and
dicalcium phosphate contain more available P than
Carbohydrate tricalcium phosphate. Incorporation of P should be
very discrete in fish feeds, as most of it gets excreted
Carbohydrate is an inexpensive source of energy in leading to eutrophication. The dietary requirement
fish diet. Among the different types of carbohydrates of P ranges from 0.5-0.9% in fishes. The requirement
available, fish are found to utilize disaccharide of magnesium (Mg) in fish ranges between 0.04-
and polysaccharide better than monosaccharide. 0.3%. The requirement of zinc (Zn) ranges from 15-30
Omnivorous fishes have enzymes to digest mg/kg diet for fishes. The requirement of iron (Fe)
carbohydrates while carnivorous fishes have poor ranges from 150-200 mg/kg diet for fishes. Major
ability to digest carbohydrate. Polysaccharides are deficiency symptoms of manganese (Mn) in fishes are
better utilized than monosaccharide. Generally cataracts and abnormal curvature of the backbone

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

carbohydrate utilization by fish is found to be lower and malformation of tail. A dietary supplementation
than that of terrestrial animals. Fish can utilize of 11-13 mg/kg restores normal growth in fishes.
dietary carbohydrate up to 40%. For carnivorous fish
carbohydrate level in the diet may be in the range of Trace minerals like copper (Cu), cobalt (Co), selenium
10-20 %. Depending upon the total energy content (Se), iodine (I) and chromium (Cr) have some role
required in the diet, carbohydrate can be used from in general upkeep of the organism. Their dietary
10-40% level. Using starch as source of carbohydrate incorporation enhances growth and survival.
in diet has dual advantage. Besides being energy
source, it can act as binder if gelatinized by cooking Vitamin requirement
with moisture and hence improve water stability of
diet. Corn flour, wheat flour, tapioca flour and other Micronutrient such as vitamins and mineral
grain flours are good source of starch in shrimp significantly influence the growth and survival of fish
and fish diet. Another polysaccharide, cellulose is and this cannot be synthesized by these organisms.
required in the diet as roughage for improving the Even though, some vitamin such as niacin can be
feed efficiency in fish. synthesized by number of animals but are typically
insufficient to meet physiological demand. Hence,
Mineral requirement supplementation of vitamins in feed becomes
necessary for most of the aquatic organisms.
Micronutrient such as vitamins and minerals Unlike domestic higher animals, the recommended
significantly influence the growth and survival of fish doses of vitamin for aquatic animals are higher, as
and these cannot be synthesized by these organisms. many vitamins are lost during the process of feed
manufacture and also due to leaching. Destruction
Fish can absorb minerals directly from aquatic of vitamin - C due to oxidation is one of the biggest
environment through gills and body surfaces or by problem during feed manufacture. Many fishes
drinking. Hence, dietary requirement of minerals is cannot synthesize Vitamin - C from glucose due to

405
 absence of enzyme L-gulonolactone oxidase. Major of final feed. All the solid ingredients are procured
role of vit C is in the formation and maintenance of in dry form with moisture levels preferably below
intracellular material having collagen or related basal 10%; otherwise the materials may be subjected to
drying before they are processed.
Table 3. Vitamin requirement of fish
Vitamin (mg/kg) Fish
Thiamin 10 Grinding
Riboflavin 20
Pyridoxine 10 Pregrinding of solid ingredients to uniform particle
Pantothenic acid 40 size is essential for making homogenous mixture of
Niacin 150 a compounded feed. Fine powdering of materials
Folic acid 5
increases the surface area and improves the digestibility
Vit B12 0.1
besides helping in making compact pellets. Materials
Choline 3000
Inositol 400 such as dry fish, prawn head waste, squilla and squid
Vit C 100 are subjected to two stage grinding process. First, size
Vit E 30 reduction, by passing through a hammer mill. In this
Vit A (IU) 2500 the materials are roughly powdered so that they can
Vit D (IU) 2400 be further powdered to finer particles. Subsequently
Vit K 10
these coarse materials are further powdered to fine
constituents in bones and in soft tissues. Among the particle size in a micropulverizer. Different kinds of
11 water soluble vitamins, three (vit C, inositol and grinding machines such as hammer mill, pulverizer,
choline) are required in large quantities. Sources of flour mill and impact pulverizers are employed for
choline include cottonseed meal, fish meal, shrimp grinding feed ingredients.
meal, soyabean meal and yeast. Stable form of vit C
is available commercially. Sieving

Feed Processing The powdered ingredients are passed through

a standard mesh sieve for obtaining the desired
One of the important factors that determine the particle size. In case the grinding equipment does
final quality of feed is the adoption of appropriate not have an inbuilt sieving mechanism, the materials
processing technology. With the best of machinery should be subjected to sieving. Feed materials that
at the disposal, working out right combination of are commercially available in fine powder form may
various factors in processing and standardizing them also be sieved to screen the presence of extraneous
would only lead to production of feed of desired materials and metal pieces, which might otherwise
water stability. The following are the steps involved inadvertently enter the pelleting equipment and
in processing of aquaculture feeds. cause damage. Sieving the ingredients helps in
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preparing feed pellets with uniform and attractive

Processing of feed ingredients physical appearance.

The quality of feed ingredients has an important Vibrating or gyratory type of sieve assemblies are
bearing on the quality of final feed. Feed ingredients available which are generally employed for sieving
should be fresh and confirm to the nutrient quality. feed materials.
Contamination with foreign matter, especially,
sand, stones and earthen materials will affect the Mixing
quality of the materials. Old stock of oil cakes
may contain aflatoxin, while PUFA rich fish oils The powdered ingredients after weighing
are oxidized leading to rancidity. Quality control according to the formulation are mixed together
of the raw materials should be done at the time and homogenized into a feed mixture. The liquid
of their procurement itself, to ensure the quality materials such as fish oil may be added at the

406
end and further homogenized. Materials, which water stability to the resultant pellets. However, the
are heat sensitive and get destroyed, may not be pellets expand as they come out of the die due to
added in the feed mix at this stage. Water required sudden drop of pressure and air gaps develop inside
for increasing the moisture may also be added. the pellet, which makes them float or sink very
Binders, which need mixing with water, should slowly. This is an excellent process for producing
also be incorporated at this stage. Horizontal or floating pellets for finfish culture. By adjusting the
vertical types of batch mixtures are employed for pressure in the barrel and moisture in the feed, it
mixing feeds. For proper mixing of different feed is possible to prepare sinking pellets by extruder.
ingredients into a homogeneous mass, the mixing The new generation extruders are made with twin
time will vary based on the homogeneity of the screwbarrel arrangement, which are more suited
material and formulations. for sinking pellet feed manufacture. The resultant
pellets are dried as the moisture will be high.
Feed Production
Pelletizer technology
The final form of the feed is produced in the form
of pellets. For shrimp compact sinking pellets are Pelletizer is primarily used for making sinking pellets.
produced. For finfish floating pellet feeds are preferred The basic principle of pelletizer is that the finely
even though sinking pellets are equally good for ground feed mixture is pelleted by compression
fish. However, for fish species such as Asian sea bass process. The main components are a pair of rollers
that feed the moving or live prey floating pellets are and a die, which are driven by a high-speed motor.
more desirable. The following technologies are for The pelletizer works with a combination of high
commercial production of pellet feeds. pressure (42-1800kg/cm 2) between rollers and
the die, steam (0.5-3.5kg/cm 2) and moderate

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Pelletization temperature (75-95ºC). Moisture is the limiting
factor in the pelletizer. It works satisfactorily at 15%
Pelletization is a process in which the feed mixture moisture and higher moisture levels choke the die.
is compacted into predesigned cylindrical pellets. Because of this reason starch present in feed cannot
Pelletization is done mainly using two types of be fully gelatinized for binding. Hence, additional
machines namely, extruder and pelletizer. binder, which works on the principle of thermo
plasticity, has to be used. Conditions for proper
Extruder technology reaction between binder and feed ingredients during
pelleting should be standardized. Several steam
The basic components in an extruder are a barrel conditioners in series are used to prolong contact
fitted with a die plate and a screw shaft conveyer, between steam and ingredients for producing pellets
which is connected to a high-speed motor. The with good water stability.
feed mixture is fed into an extruder by proper
arrangement of water/steam injection facility. Wet pelletizer
The extruder operates at high pressure (1498kg/
cm2) and steam (Pressure 5-7kg/cm2) injection. For small and laboratory scale production of feed
Depending upon the characteristics of the feed pellets, wet pelletizer which can work with high
mixture and moisture content, the pressure moisture levels (30%) is used. This is similar to noodle
develops before the material passes through the or spagatti making machine. Moist pellet can be
die. Because of this the temperature rises and the successfully produced in this machine. Feed mixture
material is forced through the die and the pressure is wetted with water (30%) and steam cooked in
suddenly drops. The temperature of the material batches and passed through the wet pelletizer. Starch
rises to 110-130ºC for a short spell of time and is well gelatinized and acts as an effective binder in
cooks the food, gelatinizing the starch present in this process. Because of higher moisture content, the
the feed mixture. This imparts good binding and pellets should be dried for longer period.

407
 Drying Conclusion
After pelleting, the feed should be dried to reduce Nutrient requirements are the basic points on which
the moisture content below 10%. This is essential the feed formulations are drawn and hence to develop
for good shelflife of the feed. Different types of a feed for any species understanding the nutrient
dryers are used for drying feed pellets. There are requirement is of prime significance. The final feed
horizontal conveyer type, vertical hopper type and quality is based on all the processing steps and
fluid bed dryers. Dry steam or hot air (heated hence all the machineries has to be suitably selected
either electrically or otherwise) is used for drying and the processing has to be carried out as per the
feed at temperatures 7080ºC. Higher temperature requirement of finished feed. CIBA has developed
is not desirable. Feed pellets with low moisture feed processing technology for shrimp and fish and
obtained through a pelletiser should be dried in commercialized these technologies. The knowhow
cooler dryer. of the technology for commercial production can be
obtained from CIBA on agreed terms and conditions.
Central Marine Fisheries Research Institute

408
Shrimp Nutrition
J. Syama Dayal*, K. Ambasankar and K. P. Kumaraguru Vasagam
Principal Scientist
Central Institute of Brackishwater Aquaculture,
75, Santhome High Road, R.A. Puram, Chennai 28
e-mail: [email protected]

Introduction Scientists, farmers, feed manufacturers, as well as

suppliers of feed ingredients, all contributed to the
Aquaculture have shown phenomenal growth in the last better understanding of feeding shrimps raised under
three decades in India producing protein rich healthy semi-intensive or intensive conditions.
food and earning valuable foreign exchange. Shrimp
farming is a major component of aquaculture in coastal Feeding behavior of shrimp
states of India. In exports of seafood’s from India, farmed
shrimp continued to be the major value item. Feed is a L. vannamei is an omnivorous scavenger, feeds
major input in shrimp farming which accounts about voraciously on organic detritus and benthos
50 to 60% of the production cost. The development of (copepods, polychaeta, ostracods, nematodes and
nutritionally balanced feed involves understanding the insects). Since, it can graze/nibble on microbial flocs,
dietary requirements of candidate species, selection of plankton sediments and faeces very effectively than

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

feed ingredients, formulation of feeds and appropriate Penaeus monodon, gives better feed conversion
processing technology for producing water stable pellet efficiency, improved water quality parameters and
feeds. Depending upon the type of farming, a wide minimized organic load. Nutrients inputs and stocking
range of feeds are being used for feeding shrimp and densities are directly proportional. During high
fish. While no feed is used in traditional farming systems, stocking density maintaining good water quality is
supplementary and balanced feeds are used in extensive real challenge; it requires feeds with high cohesive
and semiintensive aquaculture. property, high digestible protein, optimal essential
amino acids and fatty acids.
Whiteleg shrimp (Litopenaeus vannamei, formerly
Penaeus vannamei), also known as Pacific white shrimp, Significance of formulated
is a variety of shrimp of the eastern Pacific Ocean
native to the eastern Pacific, from Sonora in Mexico
feed for white shrimp
to northern Peru. The main sources of whiteleg shrimp
are Thailand, Indonesia, Vietnam, Ecuador, Mexico,
Brazil and India. Shrimp farming is now synonyms with
L. vannamei culture. Since, it is endowed with many
positive traits for current farming, it can readily accept
low protein and low fishmeal diet, trouble-free seed
production, suitable for intensive and super intensive
farming, resistant to wide range soil and water quality
fluctuation, increased disease resistance through SPF
and good customer acceptance.
Figure: 1. Schematic demonstration of significance
Nutrition and feeding of white shrimp have received of formulated feeds with stocking density and
a great deal of attention over the last 20 years. natural productivity

409
 In Indian conditions, different levels of stocking energy. Using protein as an energy source is relatively
densities are being practiced 20 to 60 pcs/m2, intended inefficient as compared with lipids and would reduce
at 10-40t/ha/yr biomass production. Feed represents the amount of protein available for tissue deposition.
40 to 60% of total production cost; hence, a range
of feed formulations must be aimed to produce Among all penaeid shrimps, white shrimps utilize
most economical and nutritionally rational as well. plant protein very effectively and nearly 2/3rd of dietary
Currently essential nutrients and their digestibility protein comes from plant by products. Plant proteins
and utilization are best known and with the recent includes soya bean meal, rapeseed meal, ground
advancement in analytical and experimental facilities nut/peanut meal, wheat gluten and corn gluten are
in nutrition, feeds can be more precisely formulated widely used as source of protein in India. Protein that
to expand the industry further. is utilized for energy and not deposited for growth
contributes to the release of nitrogen metabolites
Protein nutrition into the culture medium. According to Wu (1995),
Nitrogenous wastes are dietary in origin with estimates
This is the most expensive and major nutrient in shrimp of up to 52-95% of feed nitrogen being excreted as
feed. Proteins are primary nutrient for the structure waste, depending on the species and the diet; thereby
and function of shrimp growth and survival as in all high protein and imbalanced amino acid diet leads to
living organisms. Since proteins are continually being environmental deterioration. Adequate dietary intake
used by the animal for growth and repair of tissues, of high-quality protein is required to support rapid
a continuous supply of proteins or its constituent growth of fish and crustaceans, but shrimp do not
amino acids is considered obligatory. Protein is a have a dietary requirement for protein per se, but
major and the expensive component of formulated rather for amino acids.
aqua feeds. Hence, nutritional studies of shrimp often
start with investigating the optimal dietary protein Table: 2. Commonly used ingredients in white
level. As a consequence, the most researched nutrient shrimps feed
in terms of the number of penaeid shrimp species Protein sources Inclusion levels (%)
being studied is proteins. Protein requirement varies Fish meal 10-25
Squid meal 5-10
from 18-50% under different stocking densities and
Crustacean meal (Acetes spp.) 5-10
primary productivity. Ideally, 32% crude protein in
Soya bean meal 25-50
diets found to very effect for white shrimps at varied Peanut meal 5-10
stocking densities. 32% protein diet was found to Rapeseed meal 5-10
induce superior growth in juvenile and sub adult L. Corn gluten meal 5-8
vannamei as compared to 16% and 48% protein Wheat gluten 3-5
diets. However, the 48% protein diet had higher Feed Yeast biomass 3-5
Energy sources
Efficiency (FE) on an isonitrogenous basis feeding.
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Wheat 15-35
The lower weight gain, which resulted from feeding Broken Rice 10-20
the diet containing 48% protein, is possibly due to Wheat bran 3-5
the low energy to protein ratio of the diet, which Rice bran 5-10
would cause shrimp to utilize protein as a source of Lipid sources
Fish oil 1-3
Table: 1. Protein requirement for white shrimp Squid oil 1-3
Dietary crude protein Reference Soya lecithin 1-3
15 Aranyakananda, 1993 Cholesterol 0.15
25 Velasco et al., 2000 Plant oil 1-3
30 Cousin et al. 1993; Colvin and
Brand, 1977
32 Kureshy and Davis 2000 Amino acid requirements
36 Smith et al. 1985
40-55 Sturmer et al. 1992; Treece and Optimal dietary amino acid profile will depend
Fox, 1993

410
Table 3: List of potential feed ingredients for use in commercial shrimp feeds in near future
Moisture Crude Protein Crude Fat Crude Fibre Crude Ash
Protein sources of plant origin

Mustard meal 10.15 32.2 8.9 8.1 9.2

Coconut meal 8.7 21.5 3.5 14.8 7.1
Cottonseed meal 10.0 32.9 1.7 21.8 6.0
Soy protein concentrate 7 59 5.4 1.5 7.9
Corn protein concentrate 10 76.2 4.5 1 1.3
Distillers dried grains with solubles (DDGS) 8.2 28.4 8.5 9.4 4.9

Corn gluten meal 8.6 56.1 4 2.9 2.1

Animal meal by-products of marine origin

Fish soluble 7.4 55.9 6.5 3.2 12.6

Shrimp head meal 8.8 46.6 6.4 11.1 26.5
Crab meal 7.1 33.9 2.8 10.7 41.9
Squid viscera meal 10.3 50.3 18.6 1.5 9.8
Animal meals by-products of terrestrial origin

Blood meal 9.0 85.5 1.4 0.9 5.3

Feather meal 8.4 84.0 4.2 1.0 3.6
Meat and bone meal 7.5 50.1 10.6 2.4 28.8
Poultry by product meal 7.4 59.0 12.4 2.6 15.3

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Terrestrial invertebrate meals

Silkworm pupae meal 11.1 55.1 23.2 5.5 3.8

Maggot meal 3.0 43.2 23.0 1.0 16.2
Earthworm meal 7.4 56.4 7.8 1.6 8.8
Polychaete worm meal 8.0 55.0 15.0 1.0 12.0
Protein sources of microbial origin

Brewer’s yeast 7.6 46.1 1.3 2.9 34.0

Fungal biomass 8.5 44.4 9.4 16.9 16.1
Spirulina 6.4 62.1 4.8 0.5 17.3
Chlorella meal (micro algae) 5.7 47.2 7.4 8.3 20.8
Methanol Substrate 6.4 73.1 5.7 0.4 2.7

on the amino acid requirement of an animal for with lysine alone, could be further substituted
protein synthesis and the use of individual amino up to 75% level supplementation with arginine,
acids as energy substrates or for other purposes. methionine, and phenylalanine.
The amino acid composition of the food should
be very similar to that of the animal’s proteins. Lipid nutrition
Mente et al., (2002) found notable differences
between the FAA concentrations in whole animal Lipids constitute a broad group of naturally occurring
and in tail-muscle tissue. Tryptophan is a candidate molecules which include fats, waxes, sterols, fat-
amino acid that limits the rate of protein synthesis, soluble vitamins (such as vitamins A, D, E and K),
Forster et al. (2002) reported that 50% of the fish monoglycerides, diglycerides, phospholipids, and
meal could be replaced in the diet of L. vannamei others. Dietary lipids are a highly digestible and
with soy protein concentrate supplementation concentrated source of energy which supply 9 kcal/g,

411
 about double of that supplied by either carbohydrate and modulators of transmembrane signaling. PL
or protein. The main biological functions of lipids act as emulsifiers and facilitate the digestion and
include energy storage, as structural components absorption of FA, bile salts and other lipid-soluble
of cell membranes, and as important signaling matters. They also have a role in the transport of
molecules. Although penaeid shrimp, like other lipids, not only in the transport of absorbed lipids
animals use various biosynthetic pathways to both from the gut epithelium into the hemolymph,
break down and synthesize lipids, some essential but also in the transport of lipids between
lipids cannot be made this way and must be obtained tissues and organs since they are constituents of
from the diet. lipoproteins. Shrimp growth increased with PL levels
up to 3-5% of diet.
It has been demonstrated that shrimp have a limited
ability to synthesize de novo the n-6 and n-3 families Carbohydrate nutrition
of fatty acids (FA), including the polyunsaturated
linoleic (18:2n-6, LOA) and linolenic (18:3n-3, LNA) Carbohydrates are classified among simple (glucose,
acids. They also have a limited ability to elongate and trehalose) and complex (starch, glycogen, chitin,
desaturate these polyunsaturated fatty acids (PUFA) cellulose) and the bulk of organic matter in the
to highly unsaturated fatty acids (HUFA) such as environment is provided by carbohydrates. With
arachidonic (20:4n-6, AA), eicosapentaenoic (20:5n- disaccharides, the α 1-4 bond is broken down
3, EPA) and docosahexaenoic (22:6n-3, DHA) acids. by amylase, and glycosidic bond is much more
Consequently, these FA are considered EFA. For F. resistant. Most animals do not have enzymes for α
chinensis, Xu et al., (1993) observed that n-3 or n-6 1-4 bonds. Within polysaccharides, starch presents
HUFA had greater value than PUFA of the same family a combination of α 1-4 (amylose) and branched
following this trend: DHA>AA>LNA>LOA. chains (α 1-6 amylopectin), glycogen, cellulose and
chitin, all are formed from monosaccharide chain
Phospholipid (PL) units. Carbohydrate is the cheapest source of energy.
Digestibility will be moderate if it is processed well.
The importance of phospholipids (PL) in penaeid Critical component of feed processing because, only
shrimp nutrition, including L. vannamei (Boone), has under optimal moisture and temperature it gets
been demonstrated by researchersGonzález-Félix et gelatinized completely which gives natural binding
al., 2002a). Phospholipids are a class of lipids and to the pellets that makes pellets more durable and
are a major component of all cell membranes as they water stable.
can form lipid bilayers. Most phospholipids contain a
diglyceride, a phosphate group, and a simple organic Carbohydrates utilization by shrimp varies with
molecule such as choline; one exception to this rule carbohydrate complexity and processing procedures.
is sphingomyelin, which is derived from sphingosine These differences are represented by the range of
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instead of glycerol. The first phospholipid identified digestible energy values reported for Pacific white
as such in biological tissues was lecithin, or shrimp, L. vannamei, for typical ingredients processed
phosphatidylcholine, in the egg yolk, by Theodore under different conditions: whole wheat 3,571-3,857
Nicolas Gobley, a French chemist and pharmacist, in kcal/kg, corn flour 3,037-3,917 kcal/kg, rice flour
1847. They maintain cell structure and function, and 3,093-4173 kcal/kg, and milo/sorghum 2,821-3,785
have regulatory activities within the membrane and kcal/kg. Although these values are considerably lower
outside the cell. For instance, they serve as second than those of lipids, which contain about twice the
messengers in cell signaling, an essential process in energy of carbohydrates and proteins, the cost per
regulating cell growth, proliferation, differentiation, energy unit is much lower for carbohydrate sources.
metabolism, nutrient uptake, ion transport, and Among botanical origin we can cite potato, corn
even programmed cell death. In addition, there is (several forms), wheat, cassava, sago palm, rice
evidence that PL containing choline, sphingomyelin, basically. Digestibility of starch from various botanical
and their metabolites are important mediators origins is given in Table 7. Starch is rich in amylose

412
and is poorly digestible compared to starch rich in decaying organic matter suspended in the water
amylopectin. Native potato starch is less digestible column of the rearing system. But with fishes and
than pre-cooked one. Wheat native starch is other species shrimp, a wide variety of factors affect
well digestible (92%). Corn starch with 76-99% the quantity, quality, and accessibility of natural
amylopectin is equally digestible. foods. The decision to reduce feed costs through
the utilization of nutritionally incomplete feeds is
Chitin a viable strategy. However, it is difficult to predict
and manage the availability and nutritional quality
It is the major structural component of the exoskeleton of highly dynamic natural foods. Consequently,
of shrimps and one of the carbohydrates that shrimp if nutritionally incomplete feed is used, farmers
meet in natural environment might be chitin. Minimum increase the risk of suboptimal production due to
of 0.5 % chitin is recommended in shrimp feeds nutritional inadequacies.
because dietary chitin has growth promoting effect
(Akiyama et al. 1992). Glucosamine, a monomer of Vitamin-C
chitin, has inhibitory effect on growth by diminishing
the growth-promoting effect of cholesterol. The vitamin C is essentially required in the diet to
preventing the development of the black death
Vitamin and syndrome in penaeid shrimp. Recommended dietary
ascorbic acid (AA) levels for shrimp using ascorbic
Mineral nutrition acid-polyphosphate (ApP) are 20 and 120–130 mg/kg
Vitamins are organic compounds required by for the postlarvae of tiger shrimp, Penaeus monodon
shrimp in very small quantities to maintain essential and white shrimp, L. vannamei (Boone), respectively. A
functions. Quite often they function as coenzymes significant effect was shown to be produced on stress

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

in chemical reactions and hence are described as the sensitivity for both the species by high dietary AA
“spark plugs” of cells. The qualitative and quantitative levels, and moreover, P. vannamei displayed a higher
vitamin requirements of shrimp have not received resistance to Vibrio harveyi infection.
major attention. The difficulties in determining the
vitamin requirements are exemplified by the wide Minerals
variations in vitamin supplementation utilized by
researchers and the commercial industry. As there A variety of studies with shrimp illustrate the
is considerable debate regarding the absolute nutritional importance of the inorganic components
requirements for shrimp, vitamin values can be of the diet. The quantitative mineral requirements
tempered with those established with other species. of penaeid shrimp have not been established, but
Shrimp feeds are generally supplemented with a are reasonably well studied. Practical diets generally
vitamin premix in a sufficient quantity to exceed the contain a substantial amount of endogenous
estimated vitamin requirements, including losses due minerals. Consequently, complete mineral
to feed processing. Levels above those required for premixes are not necessary. With the exception
maximum growth may facilitate the ability of shrimp of phosphorus, macro minerals are generally not
to respond to stress. Feed processing and storage supplemented to commercial feeds, as there is no
can degrade many of the vitamins, and due to the evidence these minerals are required in feed under
slow feeding habits of shrimp, considerable leaching normal production conditions. Since many of the
can occur prior to consumption. As exemplified trace minerals can have low biological availability
in studies, the use of vitamin premixes may not or are found at relatively low levels, a number
be required under some culture conditions if the of trace minerals are commonly supplemented.
animals have access to natural foods and there Phosphorus is the most costly and problematic
are no environmental stresses. This stands more mineral. Its biological availability varies with the
appropriate for L. vannamei, which prefers to feed source, with the water-soluble forms having higher
on bioflocs, an assemblage of microorganisms and availability to shrimp.

413
 Feed additives introduction of post larval shrimps. During first
30 days of culture (DOC), feeding based on
Free amino acids: used individually or in assumption and majority of the feed goes as
combinations to reduce dietary protein levels and nutrient for planktons rather for shrimps; which
nitrogen excretion, to overcome dietary amino acid in turn feed and shelter for post larval shrimps.
deficiencies resulting from fish meal replacements, Initially, culturing adequate planktons is critical
or as feeding stimulants for better survival, good health and also reducing
size variations of the post larvae. Second month
Feed enzymes: used individually or in combinations onwards daily feeding must be controlled by
to increase carbohydrate and mineral digestibility introducing feed trays/check trays since 85% of
and reduce nutrient (e.g. phosphorus) loss to the total feeding left. Minimum of 4 feed trays for one
aquatic environment. acre pond is recommended. The dimension of the
feed trays is 80cmX80cmX10cm, and made up of
Chemoattractants and/or feeding stimulants: Table: 4. The recommended physical dimension for white shrimps
used to increase feed palatability and stimulate feed
Name Dimension Shrimp size % feed
intake (especially in plant-based feeds with low (g) required
levels of marine protein sources), increase growth Starter I 0.3-0.6 mm 0.02-0.5 1
(by minimizing the time the feed remains uneaten in Starter II 0.5-1.0 mm 0.5-2.0 3
water and thereby minimizing nutrient loss through
Starter III 0.8-1.2 mm 2.0-5.0 8
leaching), and reduce feed wastage
Pregrower 4s 1.6 x 3-4 mm 5.0-15.0 30

Probiotics: live micro-organisms and/or their Grower 4 1.8/2.0 x 4-5 15.0-25.0 30

mm
processed products are used as dietary supplements
Finisher 4L 2.0/2.2 x 4-5 25.0-harvest 28
or added directly to the water to stabilize or enhance mm
a healthful and appropriate microbial community
in the gastrointestinal tract of the cultured shrimp steel frame with nylon mesh. Physical size of feed
and/or within the culture system, so as to improve also very important in controlling feed conversion
growth, survival, and/or disease resistance. However, efficiency and reducing pollution.
it is important to mention here that although good
scientific evidence exists concerning the beneficial The role of feed boy is very important for white shrimp
effects of probiotics on shrimp performance and farming. He should have adequate knowledge of
health under clearwater hatchery conditions, this has demand feeding, indication of over/under feeding,
not always been the case under practical pond grow- moulting on feed consumption, effect of cold weather
out conditions, where resident aquatic/sediment
microbial flora already exists. Table: 5. The recommended protein and lipid levels for whiteleg
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shrimp
Immunostimulants: used to stimulate the Shrimp Protein Lipid Energy E:P
size
nonspecific immune system mechanisms of shrimp 0.02-3.0 38-36 7-8 3550- 3650 9.5
and thus increase disease resistance 3.0-15.0 36-34 6-7 3450-3550 10.0
15.0-40 34-32 5-6 3350-3450 10.5
Miscellaneous: Other additives that can be used in
shrimp feeds include antioxidants, mold inhibiting
compounds, pigments, and to a lesser extent and other climatic conditions. He should not follow
chemotherapeutants and hormones feeding guides all the time since, it is based on a
rough theoretical calculations. Actual feeding may
Feeding Management vary greatly with temperature, dissolved oxygen,
plankton bloom (natural productivity), pond bottom
Feed management starts immediately after the and pollution.

414
Table: 6. Feeding guide

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

* Feed table is for 100000pcs, assuming stocking at 10pcs/m2

Suggested readings González-Félix, M. L., Perez-Velazquez, M., 2002. Current status of

lipid nutrition of Pacific white shrimp, Litopenaeus vannamei.
In: Cruz-Suárez, L. E., Ricque-Marie, D., Tapia-Salazar, M.,
Akiyama, D. M., Dominy, W.G., Lawrence, A. L., 1992. Penaeid Gaxiola-Cortés, M. G., Simoes, N. (Eds.). Avances en Nutrición
shrimp nutrition. In: Fast, A.W., Lester, L.J. (Eds.), Marine Acuícola VI. Memorias del VI Simposium Internacional de
Shrimp Culture: Principles and Practices. Elsevier, Amsterdam, Nutrición Acuícola. 3 al 6 de Septiembre del 2002. Cancún,
The Netherlands, pp.535-568. Quintana Roo, México.
Cuzon, G., Rosas, C., Gaxiola, G., Taboada, G. and Van Wormhoudt, Kuresh, N., Davis, D.A., 2000. Metabolic requirement for protein by
A. 2000. Utilization of Carbohydrates By Shrimp. In: Cruz– pacific white shrimp Litopenaeus vannamei. In: Cruz -Suárez,
Suárez, L.E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, L.E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa, M.A.
M.A. y Civera-Cerecedo, R., (Eds.). Avances en Nutrición y Civera-Cerecedo, R., (Eds.). Avances en Nutrición Acuícola
Acuícola V. Memorias del V Simposium Internacional de V. Memorias del V Simposium Internacional de Nutrición
Nutrición Acuícola. 19-22 Noviembre, 2000. Mérida, Yucatán. Acuícola. 19-22 Noviembre, 2000. Mérida, Yucatán, Mexico.

415
 Broodstock Feeds and Nutrition
D. Linga Prabu* and S. Chandrasekar
Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction RNA, maternal deposited nutrients, hormones

In many cultured fish species the knowledge on which all may affect the egg quality. The larvae
nutritional requirement of broodstock is very scanty. with high quantity of yolk sac are considered as
This is mainly because the broodstock nutrition study good yolk sac larvae.
requires huge facilities to keep considerably extensive
group of large brood fish and also requires high Feeding period on
cost for construction and maintenance of these
facilities. And also, it is important to carry out long
broodstock performance
term feeding experiments on brood fish. This is The feeding period plays an important role in
the underlying reality in the broodstock nutrition reproductive function of brood fish. The length
studies. Improvement in the nutritional condition of the vitellogenic periods, the feeding history
of broodstock diet have significant effects on the during reproduction, the time period needed to
time of first maturity, number of eggs produced, change the nutrient composition of gonads are
egg size and egg quality as measured by chemical the important factors that affects the reproductive
composition, hatchability and larval survival. Hence, success. For example, gilthead sea bream, Sparus
the dietary supplementation of broodstock with aurata is batch spawners with group synchronous
essential amino acids, fatty acids, vitamins, minerals ovaries and have short vitellogenesis period where is
and carotenoids will produce desirable effects of egg possible to change or improve the spawning quality
and larval quality. by modifying the nutritional quality of the diet even
for few weeks of feeding. In sea bass, Dicentrarchus
Preferred egg and labrax vitellogenisis is slightly larger than sea bream
and hence relatively extended period is needed for
larval quality obtaining appropriate levels of n-3 HUFA in the
The broodstock performance is assessed in terms of eggs. In case of Lutjanus campechanus to obtain
Central Marine Fisheries Research Institute

quality and quantity of eggs and larvae produced by sufficient quantity of n-3 HUFA, will take at least 2
the brood fish. In a hatchery operation, the brood months before the spawning period commences.
fish that produce more number of eggs per kilogram Salmonids are synchronous spawners and the
of body weight during the spawning cycle is most vitellogenesis period extends up to 6 months.
preferred. Good quality eggs are those exhibit low Hence, the brood fish should be fed with good
levels of mortality at fertilization, hatch and first quality diet for several months before spawning
feeding and produce fastest growing healthiest fry period. Only then, the fatty acid profile of muscle
and adult fish. Egg quality in nutritional studies and developing eggs will started reflecting the
are indicated by fertilization rate, survival during dietary fatty acid profile.
egg stage, cleavage symmetry during 8 and 32 cell
stage, blastomere morphology and hatching rate Many fish species tend to decrease their feed
and later rearing success. The ovulated egg contains intake during sexual maturation. The swelling
genetic code for new individual and also maternal of ovaries due to egg hydration may restrict

416
the space in the body cavity and hence the Specific
feed volume ingested is reduced. Endocrine
nutrient requirement
changes in connection with sexual maturation
and spawning may also interfere with appetite
regulation. As a consequence, the energy and Protein and amino
nutrient required for gonadal development may acid requirement
be obtained from body reserves. Rainbow trout
mobilizes its lipid reserves from carcass and viscera The quality protein level in the broodstock diet
where as African catfish utilizes their abdominal affects the reproductive performance. The protein is
fat as major energy source for sexual maturation. the most abundant nutrient in fish eggs and is main
Atlantic salmon uses its muscle protein and lipid energy source during embryonic development. The
where as cod uses the lipid reserve of the liver growth of embryos occurs through the deposition
for sexual maturation. Gilthead sea bream brood of protein. Hence, protein have important role in
fish continues to feed during sexual maturation fertilization and embryonic development. A positive
and spawning period, and therefore producing correlation exists between size at first spawning and
an egg biomass equivalent to its body mass. In protein content of the broodstock diet. Therefore,
these circumstances, nutrients deposited in the less protein (lower than optimum level) fed fishes
ovaries may come from broodstock diets and the spawn at smaller size, spawn less frequently and had
nutritional composition of eggs can be modified higher relative fecundity (number of eggs per kg body
during spawning season. weight), induce female to mature earlier and spawn
over longer period of time. It is advisable to feed the
Feed intake on broodstock with low protein-high energy diets than
the high protein low energy diets. The need for correct

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

spawning performance protein requirement in the diets of brood fish has not
Feed availability is the important factor determines been studied in detail in most of the fish species. But
the fecundity in the wild fish. The food deprivation in in general when the dietary protein level is optimal
case of long lived species may delay in reproduction most of the fish species will spawns successfully.
by favoring somatic growth over ovarian growth
where as in case of short living species the ovarian Protein covering the yolk is significant for fertilization
growth is preferred than the somatic growth at times and their amino acid composition generally rich in
of inadequate feed supply. In cultured fish fecundity proline and glutamic acid and low in cystine. The yolk
and other spawning quality parameters are affected protein precursors and vitellogenins are lipoproteins
by feed ration size and feeding rate. Inadequate and their amino acid composition contains high level
feeding rate may inhibit gonadal development in of alanine, glutamic acid and leucine but low in serine
many fishes. level. Free amino acids are available in high levels
in eggs of pelagic fishes. The abundant free amino
The sea bass fed with half the actual feed ration acids are leucine, lysine, valine, isoleucine, alanine
showed decreased growth rate, delayed spawning and serine. Hence while formulating the diet for the
period, smaller eggs and newly hatched larvae than broodstock these points should be kept in mind for
the fish fed with full feed ration. The restricted feed better broodstock performance.
ration to the brood fishes showed decreased plasma
estradiol level and reduced fecundity. In tilapia feed The sufficient amount of protein with balanced
intake influence the fecundity but did not affect the composition of amino acids is essential for the
hatching percentage and egg diameter. High feed emerging embryo which should be obtained from
intake not only affects the fecundity increment but yolk. Hence, the broodstock diet should be balanced
also egg size and female body size. The length of the with required protein and amino acid composition
time between successive spawns was lower in fish which improve the synthesis of vitellogenin. In most
with high feed intake. of the carnivorous marine fishes the broodstock diet

417
 should contain at least 45-50% of protein level for affected by fatty acid composition of broodstock
better reproductive performance. This will increase diets. However, fatty acid composition of lipids in
fecundity and reduced the rate of deformed larvae. fish eggs may vary with the species and even different
During spawning period if the diet is provided with batches of same species. In general ovarian lipids
insufficient level of protein that may altered the contain higher proportions of n3 HUFA, particularly
secretion of gonadotrophin releasing hormone 20:5 n3 and 22:6 n3 fatty acids. Fatty acids are
(GnRH) and leutinizing hormone (LH) which play an essential sources of energy during early embryonic
essential role in the regulation of oocyte maturation development, important in phospholipid synthesis
and ovulation. The squid meal and cuttlefish and hence in formation of bio-membranes. The ratio
meal incorporation in place of fish meal in the between the saturated and unsaturated fatty acids
diet of red seabream showed significantly better regulates the fluidity of cell and organelle membranes
reproductive performance. and their functions.

The amino acids tryptophan and taurine are also Fatty acids, Arachidonic acid (ARA) and
very essential for fish reproduction. Tryptophan is Eicosapentaenoic acid (EPA) are precursors of
a precursor of serotonin and can affect increase in eicosanoids as well as production of prostaglandins
the testosterone levels which favoring spermiation and leukotrienes. Prostaglandins are involved in
in males and induced female gonad maturation. numerous reproductive processes such as production
Taurine being most abundant free amino acid in of steroid hormones, gonad development and
tissues of fish, the supplementation of taurine can ovulation. Optimal arachidonic acid may increase
improve fecundity, percentage of viable eggs and the fecundity, egg viability, hatching rate and larval
fertilization rate. survival. ARA will stimulate the release of testosterone
through its conversion into prostaglandin E2 which
Lipids and Fatty acids in turn increase the fertilization rate. The ARA, EPA
and DHA will compete with each other for eicosanoid
The broodstock required great amount of energy production. Hence, the ratio of ARA/EPA and EPA/DHA
for production of primary sex products, secondary will have the effect on the reproductive performance.
sexual characters development and for reproductive The diet with higher lower EPA will have higher DHA/
behaviors. The relative proportion of energy required EPA that may affect the reproductive performance. In
between growth and reproduction varies according gilthead seabream broodstock dietary EPA and ARA
to type of breeding, sex and age. The energy required levels show a correlation with fertilization rates. There
for reproduction is around 15-30% in most of the should be a balance between n3 and n6 fatty acid
fishes. During the somatic growth phase of life around ratios in broodstock diet and high n-3 to n-6 ratio will
40% of energy may be diverted for growth but after provide better maturation effect. The optimal n-3 to
maturation hardly 10% of energy will be allotted for n-6 fatty acid ratio should be 2:1 to 3:1 for efficient
Central Marine Fisheries Research Institute

growth. When the energy supply from the feed is not performance of broodstock.
sufficient most of the fishes produce the gonads at the
expense of somatic growth and mobilize their body Diets deficient in essential fatty acids cause an
reserve for gonad development. Increased dietary increase in the number of droplets in fish eggs.
energy in the form of fat is responsible for increase Fusion of fat droplets into a single lipid droplet leads
in gonadosomatic index. This will result in increased to increased percentage of hatching and normal
fecundity or increased egg size. The increased fat larvae. Excess or deficiency of n-3 HUFA also causes
content in the diet will influence larval length, weight negative effect on egg and larval quality. Very high
and survival rate. It is difficult to differentiate the levels of n-3 fatty acid caused a decrease in total
beneficial effects obtained are solely through lipid or number of eggs produced and egg quality. Low n-3
may be because of fatty acids not clear. HUFA lowered the fertilization rate due to decreased
sperm motility. The positive effects of n3 fatty acids
Fatty acid composition of fish egg and sperm is directly on spawning performance may be by increasing

418
the fluidity of biological membranes. High levels decrease in number and motility of spermatozoids
of linoleic acid (18:2; n6) in the broodstock diet as well as sperm plasma tocopherol reduction and
create problem on the larval quality such as body sperm viability. Elevated level of vitamin E up to 2000
deformation and inability to inflate air bladder. The mg/kg improved the percentage of buoyant eggs,
broodstock diet should possess at least 1.5% of ratio of larvae with normal development and larval
n-3 HUFA with minimum of 50-60% DHA will be survival. Optimum requirement of vitamin E in most
required for better larval survival and swim bladder of the broodstock diet is around 200-250 mg/kg.
inflation rate. The requirement of n-3 fatty acid in
sea bream broodstock is between 1.5-2.5% being Vitamin C plays a vital role in sexual maturation through
higher than the juveniles’ requirement (0.5-0.8%) the biosynthesis of steroid hormones. It generally
of the same species. affects the steroidogenesis and vitellogenesis. Also
affects the level of vitamin C in seminal fluid which is
Among the phospholipids lecithin and cephalin are directly related to sperm motility. In most of the fishes
predominant in ovaries. The phospholipid requirement dietary vitamin C level reflects in the level of vitamin
in broodstock diet is around 1.5-2%. Cholesterol is C in eggs. The level at least 1200 mg/kg in the diet
known to fulfill several endocrinological functions improves the egg hatching percentage and rate of
and its mobilization during maturation. Generally normal larvae. Fecundity and egg quality is not only
the cholesterol is supplied in the form of fresh feed by dietary vitamin C or E but also by the interaction
such as squid, clam and mussel. The low membrane of them. In rainbow trout vitamin C requirement is 8
cholesterol–phospholipid ratios are correlated with a times higher than the requirement of juveniles.
better sperm freezing resistance in cryopreservation.
Vitamin A is required for reproduction and embryonic
Carbohydrates development. Vitamin A increase fecundity, viable

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

egg percentage and normal larval percentage. The
It is an inexpensive source of energy with protein- optimum level varies with species and is in the range
sparing and lipid-sparing effects. It acts as basic source of 10000-20000 IU/kg.
of energy in some of the tissues. Broodstock diets with
low level of carbohydrates cause decrease in fecundity There is no much information is available on the
where as the increased level of carbohydrates reduce effect of B complex vitamins on fish reproduction.
the spawning quality. In the broodstock diet thiamine is importance for
normal embryo and larval development. Thiamine is
Vitamins supplied through injection into female Atlantic salmon
shows reduction in mortality of progeny. Thiamine
Vitamin C and E are antioxidant enzymes and are concentration in the egg and yolksac larvae is related
degraded as they fulfill their function. Broodstocks to reduction of early mortality syndrome. Pyridoxine is
fed with trash fishes which are often low in C an essential compound in the biosynthesis of steroid
and E and also low in thiamin because of activity hormones and other vitamin, folic acid hence the
of thiaminase. And it is advisable to avoid trash deficiency may cause reduced cell division due to
fish in the broodstock diet. Vitamin E content is impaired DNA and RNA synthesis that plays a vital
generally high in fish eggs and low in broodstock role in hatchability of eggs.
tissues after the spawning period. Vitamin E
requirement is depend on the dietary content of Minerals
HUFA, an increase in n-3 HUFA in the diet requires
the vitamin E requirement increase also. Vitamin There is scarce information available on the mineral
E deficiency inhibits the gonad maturation, loss requirement of broodstocks. Phosphorous deficiency
of sexual coloration and decreased hatching and in sea breams is related to decrease in fecundity,
survival rate of larvae. Also cause the reduction in percentage of viable eggs and hatching rate, increase
the percentage of fertilized eggs which is due to in number of abnormal larvae and lower fecundity. The

419
 content of manganese, zinc and iron in the eggs found Nucleotides
lower when the broodstock is not supplemented with
sufficient level of these trace minerals. Broodstock diets enriched with nucleotides showed
an improvement in larval survival of haddock after
Carotenoids 10 days of hatching. This may caused by better
development of intestine of nucleotide supplemented
Carotenoids are being antioxidants and their larvae and as a consequence better utilization of
inclusions improve the percentage of floating first exogenous feed. In addition, broodstock fish
eggs, egg hatchability and percentage of normal fed with nucleotide enriched diet have also recorded
larvae. Combined increase in the level of n3 HUFA higher fecundity.
and carotenoids significantly improve spawning
quality such as fecundity, percentage of viable eggs, Broodstock feeding practice
hatching rate and larval survival. Canthaxanthin and
astaxanthin in the broodstock diets of red sea bream Generally, in most of the countries there are no specific
were incorporated into the egg without converted feeds commercially available for marine broodstocks.
into beta carotene. During early maturation And hence in most of the marine fish hatcheries the
carotenoids accumulate in the liver and during broodstock is mainly feed on fresh marine by-products
secondary vitellogenesis it is mobilized from liver or in combination with commercial diets. The fresh diet
through blood to ovaries. Astaxanthin improve the includes squid, cuttlefish, mussels, clam, shrimp and
larval survival this is because of the antioxidant other small crustaceans. The use of these fresh and
properties which scavenges singlet oxygen and unprocessed fish products do not provide required levels
other free radicals and prevent the egg membrane of nutrients to broodstock fish and it also increases the
deterioration. Carotenoid reserves in the embryo risk of disease transmission and parasitic, bacterial and
and prefeeding larvae will help in the development viral pathogens to the parents and offspring.
of chromatopores and eyes.
Central Marine Fisheries Research Institute

420
Nutritional Pathology
S. R. Krupesha Sharma* and P. Vijayagopal
Principal Scientist
Marine Biotechnology Division
Central Marine Fisheries Research Institute
Kochi- 682018, Kerala, India
e-mail: [email protected]

Introduction which must be supplied in the fish diet. Functions

of proteins include maintenance, growth and
Due to rapid expansion of aquaculture industry, more reproduction. In addition to this some amino acids
research interest is being focussed on fish nutrition. are readily converted to glucose to provide energy
Due to the lack of sufficient information on the nutrient source for some tissues such as brain and red blood
requirements of cultured marine fishes, fish nutrition cells. Wild fishes especially carnivores depend largely
has not made much progress when compared to on amino acids as precursors to glucose since their
animal nutrition. Stress induced outbreak of diseases normal diet lack carbohydrates. Hence, in case of
can be attributed to nutritional factors in addition to fish, some fraction of the dietary protein is used as
various chemical, physical and biological stressors. an energy source.
Hence, understanding the nutrient requirement of

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

cultivable fishes is rapidly being recognized so as Deficiency of essential amino acids may lead to
to develop proper feed for a particular fish species improper utilization of dietary Protein resulting
being cultured. Nutritional diseases of cultured in growth retardation, lowered weight gain, and
marine fish may develop consequent to deficiency, increased feed conversion ratio. In severe cases,
excess, or imbalance of nutrients like protein, lipid deficiency of amino acid leads to poor resistance to
and fat present in the feed. Nutritional diseases do diseases due to alteration in the immune response
not generally develop all of a sudden instead they of the fish. Deficiency of individual amino acid
develop gradually since the fish have body reserves of like tryptophan may lead to scoliosis resulting in
nutrients that make up for nutritional deficiency up to spinal curvatures and also renal calcinosis, cataract,
a certain extent. Clinical signs of nutritional deficiency caudal fin erosion, decreased carcass lipid content.
develop only when supply of any diet component Methionine deficiency affects the vision of the fish.
falls below critical level. Further, when fish consumes Lysine deficiency leads to dorsal/caudal fin erosion.
excess food, whatever in excess would be converted Lysozyme activity and C-reactive protein values are
to fat which will be deposited in body tissues and reduced in case of protein deficiency in fish.
organs. The deposited fat affects the physiological
functions of the fish. Pathological features
of fatty acids and
Pathological features of lipids malnutrition
protein malnutrition
Dietary proteins are the source of essential amino Fatty acids form the nutritionally active components
acids for the fish. These proteins also supply nitrogen of dietary lipids. Fish are unable to synthesize
for the synthesis of non-essential amino acids. A total fatty acids that are unsaturated in the ω-3 or
of 23 amino acids make up the protein present in ω-6 positions unless a suitable precursor is
the tissues of which 10 are essential amino acids supplemented in their diet. Unlike mammals, which

421
 have a major requirement for ω-6 fatty acids, most requirement of vitamins in case of fish depends
of the marine fishes require ω-3 fatty acids. Hence, on the size of the fish, uptake of other nutrients
adequate quantity of essential fatty acids must be and environmental stresses. Deficiency of vitamin
included in the dietary lipids. in fish may arise by their low content in feeds,
environmental or physiological stressors and by
When fish are affected with lipoid liver disease, they diseases especially those, which occur in the early
exhibit severe anemia, bronzed, rounded heart and stages of development. Clinical signs of most of the
swollen liver with rounded edges. Microscopically, vitamin deficiency disorders are non-specific in nature.
the conspicuous feature is extensive lipoid infiltration It is rather complicated to identify them. Deficiency
of hepatocytes which appear as clear, round or oval disorders caused by vitamin deficiency adversely
vacuoles in H and E stained sections since the lipid affects the utilization of other nutrients and reduces
is dissolved during routine tissue processing. Excess the resistance to diseases.
carbohydrates are known to induce damage to the
liver and destructively affect the natural microbial Some of the deficiency disorders caused by vitamin
flora in the gut of the fish. deficiency in fish include:

Pathological features of Pathological features of

vitamin deficiency trace elements deficiency
Vitamins are micro-nutrients that are required for In higher vertebrates, trace elements like zinc, iron,
growth, reproduction, and disease resistance. The copper and selenium are required for the immune

Vitamin Clinical signs of deficiency

Ascorbic acid (Vitamin C) Reduced growth, anaemia impaired collagen formation, scoliosis, lordosis, internal and fin haemorrhage,
distorted gill filaments, slow wound repair, mortality, reduced hatchability, exophthalmia, caudal fin
erosion, renal granuloma, club-shaped gill lamellae, fatty degeneration of liver, muscle degeneration, skin
haemorrhage,  swollen abdomen, changes in head bones, blindness, surface swimming, lower lip ulceration,
scale loss, emaciation.

Pyridoxine (Vitamin B6) Nervous disorders, hyperirritability, anorexia, ataxia, oedema of peritoneal cavity, erratic and rapid swimming,
greenish-blue colouration of skin, anaemia, rapid breathing, poor growth, muscular spasms, abnormal
pigmentation, convulsions, reduced food conversion ratio,  lesions of lower lip, avoidance of schooling

Cyanocobalamin (Vitamin Anorexia, reduced growth, microcytic hypochromic anaemia, fragmented erythrocytes, low FCR, dark
B12) pigmentation, distended abdomen, skin and fin haemorrhage, loss of skin mucosa , grey-white intestine.

Choline Reduced growth, fatty liver, low FCR, haemorrhagic kidney and intestine, exophthalmia, clouding and thickening
of corneal epithelium, degeneration of the retina, elevated liver/muscle lipid content. 
Central Marine Fisheries Research Institute

Vitamin E (Tocopherol) Reduced growth, exophthalmia, ascites, anaemia, clubbed gills, mortality, erythrocyte fragility, muscle
degeneration, reduced hatching rate/spawning efficiency, reduced antibody response, ceroid deposition in liver,
spleen and blood vessels, splenic haemosiderosis.

Vitamin K3 Increased blood clotting time, anaemia, haemorrhage in gills, eyes, skin and vascular tissue.

Thiamine

(Vitamin B1) Anorexia, poor growth, nervous disorders, increased sensitivity to shock, fin haemorrhage,

Folic acid Macrocytic normochromic anaemia, poor growth, anorexia, lethargy, dark colouration, pale gills, exophthalmia,
distended abdomen with ascites fluid 

Pantothenic acid Anorexia, reduced growth, high mortality, clubbed gills, haemorrhage under the skin, fragile fins, oedema, rapid
breathing, swelling at base of pectoral fins, pale gills, sluggish activity.

Riboflavin (Vitamin B2) Anorexia, poor growth, corneal vascularisation, cloudy lens, snout erosion, spinal deformities, mortality, fin
erosion, fin haemorrhage, muscular weakness, light or dark pigmentation, photophobia, in coordination,
lethargy, anaemia.

422
system to function. However, in fish not much is anemia in many fish species. Copper deficiency rarely
known about the effects of these trace elements occurs in fish since water is abundant in copper. In
on the immune system. In case of fish, minerals case of copper toxicity, susceptibility to bacterial
execute important functions in osmoregulation, infections like vibriosis increases. Manganese
metabolism and in formation of the skeleton and deficiency occurs in fishes when the diet contains
scales. The minerals required in finfish diets include high concentration of calcium or ash. Manganese
calcium, zinc, manganese, cobalt, selenium, iodine deficiency in fish results in cataract. Deficiency of
and fluorine. Iron is an essential requirement in zinc is also associated with occurrence of cataract
fish diet. Low concentrations of free iron in mucus especially in young fish. Absorption of zinc will be
membranes and in other tissues are reported to hindered when feed contains excess amount of ash,
be one of the non-specific host defences against calcium or phytate. Zinc deficiency also results in
bacterial infections. Iron deficiency causes microcytic poor growth and darkening.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

423
 Feed Ingredients and Database
S. Chandrasekar* and D. Linga Prabu
Scientist
Marine Biotechnology Division,
Mandapam Regional Centre of CMFRI
e-mail: [email protected]

Introduction commercially referred as protein rich feed ingredients.

These ingredients may be animal or plant origin
The rapid world-wide expansion of aquaculture and
livestock production strongly indicates that a crisis Classification Based on
will be predicted in the livestock and aquaculture
feed industries in the near future. Nutrient content
Commonness of Usage
of individual feed ingredients varies widely from The feed ingredients are broadly divided into two
country to country or region to region depending types based on its commonness of usage as:
on local climate, post harvest processing methods and
storage. Some generalization can be made regarding • Conventional feed ingredients
the chemical composition of individual feed stuffs. • Unconventional feed ingredients
Hence, the nutrient profiling of each ingredient
should be done and database need to be developed Conventional feed ingredients: Those feed
for the better feed formulation. Ingredients used in ingredients which are commonly used in the
commercial fish diets can be classified different means commercial aqua feed preparation is known
such as sources of protein (amino acids), energy, as Conventional feed ingredients. For example,
essential fatty acid content, vitamins and minerals. Groundnut oil cake, Mustard oil cake, Soy bean meal,
Special ingredients may be used as additives to Sunflower oil cake, rice bran, Rice polish, Wheat bran,
enhance growth, pigmentation or sexual development Fish meal, Meat meal, Blood meal etc.
and to prepare diets having the required physical
quality, palatability and preservation properties. Unconventional feed ingredients:Some of the
ingredients which are not commonly used may be
Classification of Feed used in the aqua-feed after removal of some of the
anti-nutritional factor present in the same ingredients.
Ingredients Based on
Central Marine Fisheries Research Institute

These ingredients are referred as unconventional feed.

Protein Content Some of the common examples are Guar meal, Squilla
meal, Feather meal, Frog meal, Rubber seed cake,
The ingredients used for fish feed based on protein Neem seed cake, Karanja cake, Niger cake, Coconut
content can be broadly classified into two major meal, Tapioca flour, Dried whey, Crocodile meal, etc.
groups such as:
Commonly used ingredients
• Energy rich feed ingredients- Energy rich feed
ingredients belongs to those groups which contain
in aquafeed
<20% protein and <18% crude fibres. These
ingredients mostly plant origin.
Cereals and cereal by-products
• Protein rich feed ingredients- Those feed ingredients Cereals: Carbohydrates are the primary nutritional
which contain more than 20% crude protein are contribution of cereals/grains. Whole grains contain

424
62 to 72 % starch, which is 60 to 70 % digestible extraction. Corn germ meal contains high level of
by warm-water fish but markedly less digestible by hemicelluloses fibres, which delivers good hydration
carnivores fishes. Starch in grains is an important and pelleting characteristics.
binding agent in steam-pelleted and extruded fish
feeds. It also contributes significantly to the protein Rice Bran with Germ: Rice bran with germ is primarily
and lipid content of the diet. Though deficient in some the pericarp or bran layer and the germ portions of
of AA (e.g. Lysine) they can be used to balance high the rice kernel. The crude protein level is approximately
protein animal and vegetable ingredients. Some of the 13.3%, crude fat 15%, and crude fibre 11%.
common cereals used in aqua feed are sorghum grains,
wheat, rice, sorghum/maize/wheat gluten meals. Tapioca flour: Tapioca- In the strictest sense, starch
from cassava is to be referred as tapioca. Tapioca
Cereal by-products flour is rich in carbohydrates (starch), is mainly
used as binder Tapioca flour have a considerably
Wheat flour: This ingredient is mainly used as a higher amount of amylopectin compared to other
binder for shrimp and prawn feeds. Primarily Consists starch sourced, which helps in better expansion and
of wheat flour together with fine particles of wheat functional properties of aquafeeds.
bran, wheat germ and the offal from the “tail of the
mill”. This product usually obtained from commercial Corn Gluten Meal: Corn gluten is an excellent protein
milling process and must contain less than 1.5 percent source, with minimum of 60 % protein. Corn gluten
crude fibre. India is one of the world’s largest wheat meal is commercial produced from dried residue of
producers and processors. Second-grade wheat flour, corn after the larger part of the starch and germ
which is only marginally fit for human consumption, have been removed and the bran separated by the
is used in animal feeds. Indian wheat has a low wet process employed in the wet milling manufacture of

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

gluten index (<28 percent) relative to the ideal index corn starch or syrup, or by enzymatic treatment. Corn
for shrimp feed pelleting (32 percent). gluten meal may contain fermented corn extractives
and/or corn germ meal. White corn gluten meal will
Wheat Bran: Wheat bran is the coarse outer covering likely be priced at a premium to regular (yellow) corn
of the wheat berry as separated from cleaned and gluten meal, but protein levels are at least 10% higher,
scoured wheat in the usual process of commercial which justifies the increased cost.
milling.Wheat bran contributes bulk to ruminant
feeds and is a source of carbohydrates, protein, Wheat gluten meal: Wheat gluten meal is a good
minerals and vitamins. Too much wheat bran in a natural protein binder, containing 70-80% of protein.
formulation results in pellets with poor water stability Wheat gluten meal having rich source of cystine and
due to the water absorption characteristics of fibre. glutamine, but is very deficient in lysine. It is produced
from wheat flour after starch extraction. Due to high
Wheat Germ Meal: Wheat germ meal consists level of yellow pigments, its use in aquaculture is
chiefly of the germs of the wheat berry, with some restricted to only 10 %.
bran and middlings. A rather wide variety of wheat
germ grades are produced, depending on the area Oil Extractives/Oil
and regional demand. The quality of wheat germ
meal is influenced by the presence of screenings and
Seed Cakes
the level, of other wheat products, which can lower Oil extractive or Oil seedcakes are by-products from
the fat or protein level. Excessive storage time may vegetable oil extraction industry. The protein-rich
result in rancidity, due to the oxidation of the rather residues left after removal of oil represent an immense
high level of fat. resource upon which the world’s production of animal
protein for human consumption largely depends. Oil
Corn germ meal: Corn germ meal is produced from extraction from seed or fruit is carried out by two
corn germ by complete removal of oil through solvent methods: by pressing, or with chemical solvents. The

425
 product obtained by pressing is termed oilcake and Sunflower seed meal: Sunflower meal is obtained
that by solvent extraction, oil meal. by grinding the residue remaining after extraction of
most of the oil from dehulled sunflower seed by a
Soybean Meal: Use of soybean products in the solvent extraction process. Sunflower meal caontains
aquaculture industry has become the focus of protein crude protein level upto 46%. Sunflower seed meal
substitution in fish food around the world. The high is a rich source of tryptophan and arginine, but it is
protein level makes it a key ingredient for aquaculture deficient in lysine and to a lesser extent tyrosine. In
feeds since soybean meal is considerably less expensive addition, sunflower meal may contain anti-nutritional
than traditionally used marine animal meals. Soybean factors, including protease inhibitors, tannins and
meal is universally available and has one of the best arginase inhibitor.
amino acid profiles of all protein-rich plant feedstuffs for
meeting most of the essential amino acid requirements Rapeseed meal/Canola meal: Meal from canola
of fish. Some fish, such as young salmon, find soybean seed (low-glucosinolate rapeseed) has been used in
meal unpalatable while others, such as channel catfish, experimental feeds for salmonids with success. It has
readily consume diets containing up to 50 % soybean an amino acid profile comparable to soybean meal,
meal. Soybeans contain several anti-nutritional factors but it is lower in protein and higher in fat, sulphur
but heating during commercial oil extraction destroys containing aminoacids particularly methionine, fiber
much of the activity. Soybean meal is produced in two and tanins.
major protein levels by different processes Forty-four
% soybean meal is usually mechanically extracted to Linseed cake: Linseed (flax) cake is the product
produce a meal of 44 % crude protein; crude fat, 4.7 obtained by grinding the flakes which remain after
%; and crude fibre, 6.0 %. Forty-eight percent soybean removal of most of the oil from flaxseed by a solvent
meal is dehulled and solvent extracted to yield meal extraction process. It must contain not more than
with a crude protein I level of 48 %; crude fat, 0.9 %; 10 percent fibre. The oilcake has protein content
and crude fibre, 2.8 %. Soybean meal must be heated comparable to cottonseed oilcake but is considerably
(toasted) sufficiently to destroy, the trypsin inhibitor. richer in methionine.
Soybean meal generally classified according to their
protein level. The limiting amino acid (lysine and Animal by-products
methionine) content of soybean meal is high, and is
not on the level of Whole fish meal and especially egg. Fish Meal: Fish meal is the clean, dried, ground tissue
of un-decomposed whole fish or fish cuttings, either
Cottonseed Meal: Solvent extracted cottonseed or both, with or without the extraction of part of
cake is used for fish feed preparation. Cottonseed the oil. Fish meal provides a balanced amount of all
meal is a rich source of arginine (and to a lesser extent essential amino acids, phospholipids, and fatty acids
tryptophan and cystine), but is deficient in methionine for optimum development, growth, and reproduction,
Central Marine Fisheries Research Institute

and lysine. Most cottonseed meal contains free especially of larvae and brood stock. The nutrients
gossypol, which is moderately toxic to monogastric in “Whole Fish Meal” or “White Fish Meal” (such
animals and limits its use in fish feeds. as Omega 3 fatty acids DHA or docosahexaenoic
acid and EPA or eicosapentaenoic acid) also aid
Groundnut Meal/Oil cake (GNOC): Groundnut meal in disease resistance by boosting and helping to
has been traditionally used as principal protein source maintain a healthy functional immune system. Fish
for fish feed preparation. GNOC is obtained by grinding meals are manufactured by cooking fish, pressing to
the cake, chips or flakes obtained by removal of most of remove water and oil, and then drying. Fish meals
the oil from peanut kernels by a mechanical or solvent are often made from a single species of fish, e.g.,
extraction process. GNOC is known to be very poor in herring meal, Fishmeal made from fish parts, such
lysine and methionine, so the oilcake should be used as waste from fish processing and canning plants,
with lysine and methionine supplements to achieve a has a lower percentage of high-quality protein than
proper balance of essential amino acids in the diet. that of meal from whole fish. Whole Fish Meal

426
averages between 17% and 25% ash content. More the material comes from bone and other non
ash indicates a higher mineral content, especially muscle tissue.
calcium, phosphorus, and magnesium. Calcium and
phosphorus constitute the majority of the ash found Squid Meal: Squid Meal is made from squid viscera
in fishmeal. This makes Whole Fishmeal an important portions from cannery plants including egg and testis.
source of very essential minerals that fish need for Squid Meal is high digestibility of protein source,
osmoregulation. As it has high in ash content should which provides a full range of amino acids for fish.
be used prudently in fish diets because it can produce It provides various kinds of vitamins and minerals and
mineral imbalances. Over-all protein digestibility also 1.0-1.5% of cholesterol that is suitable for fish
values for fishmeal are consistently above 95%. In fry and young fish.
comparison protein digestibility for many plant-
based proteins varies greatly, for example, from 77% Acetes meal: A small size shrimp called the Acetes
to 96%, depending on the species of plant. Whole shrimp is caught in large quantities seasonally in
Fish Meal is an excellent source of DL-methionine. Karnataka, Maharashtra, and Gujarat. The sun dried
Fishmeal also contains certain compounds that make and pulverized meal of Acetes shrimp is one of the
the fish food more acceptable and agreeable to the good protein sources having 60% protein.
taste. It is thought that the non-essential amino acid
glutamic acid is one of the compounds that impart Blood Meal: Blood meal is produced from clean fresh
to fishmeal its palatability. This property allows for animal blood, exclusive of all extraneous material such
the feed to be consumed rapidly, and hence reduce as hair, stomach contents, etc. Blood meal may be
the nutrient leaching. dried by several processes, but most often by spray
drying. Spray dried blood meal has approximately a
Shrimp meal: Shrimp meal can be made from either crude protein level of 85 %; crude fat, 0.5-3 %; crude

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

cull shrimp that are being processed before freezing fibre, 2.5 %; ash, 6 %; and lysine, 9-11 %, with an
or from whole shrimp that is not of suitable quality availability of 80-90 %. Flash or spray-dried blood meal
for human consumption. The material to be made is rich in protein (80 to 86 %) but low in methionine
into shrimp meal is dried (sun or using a dryer) and and unbalanced in branched-chain amino acids.
then ground. Shrimp meal has been used in trout
and salmon diets as a source of pigments to impart Hydrolyzed Poultry Feathers: Feathers from poultry
the desirable color in the tissues. Shrimp either whole are collected, ground and hydrolyzed under pressure.
or as a part of another prepared food is an excellent During processing the cystine linkage is broken,
source for fats needed for the growth of hump heads increasing the value of the meal. Not less than 75
in Flowerhorn Cichlids. Shrimp meal has been found % of the crude protein must be pepsin digestible.
to be an acceptable supplemental protein source for Generally, hydrolyzed poultry feathers will range in
fish, but inferior to whole fish meal. crude protein from 80 to 85 %; crude fat, 2.5 %;
crude fibre, 1.5 %; phosphorus, 0.75 %; and ash
Meat and Bone Meal: Meat and bone meal is about 3 %. Feather meal is high in crude protein (80
the dry rendered product derived from mammalian %) but, unless the feathers are thoroughly hydrolyzed
tissue, exclusive of hair, hoof, horn, manure and during processing, digestibility is low.
paunch contents. The relatively high fat content of
meat and bone meal apparently helps protect the Miscellaneous
lysine content during rendering. Meat and bone
meal generally contain a crude protein of 45-50
%; fat, 8-11 %; crude fibre, 3 % phosphorus,
Dried Distillers Grains
4.4-5 %; calcium, 8.8-10 %; and ash, about 30 %. with Solubles
The quality of the protein in this product is less This product is obtained by removal of ethyl alcohol
than that of whole fishmeal, and the ash content by distillation from the yeast fermentation of corn or
is usually high because a significant amount of corn mixture by condensing and drying at least 75 % of

427
 the solids of the resultant whole stillage. Corn distillers raw protein and seven major vitamins: A1, B1, B2,
dried grain with solubles has a crude protein level of B6, B12, C and E. Spirulina naturally contain beta-
26-27 %; crude fat, 7-8 %; and crude fibre, 8.5-9.5 %. carotene color enhancing pigments (1500 mg/kg.
Carotenoids; Orange/ Red pigment enhancers),
Brewers Yeast: Brewers dried yeast is the dried, and high amount of minerals. In addition, it
sterilized, unextracted yeast (Saccharomyces sp.,) contains all essential fatty acids and eight amino
resulting as a by-product from the brewing of beer acids required for complete nutrition. Spirulina is
and ale. Brewers dried yeast has a crude protein level similar to cyanobacteria in structure (spiral shape),
of 45 %; crude fat, 1 %; and crude fibre, 2.7 %. which can be toxic. Spirulina is recognized by the
body (fish in particular) as a bacterium, causing
Brewers Dried Grains: Brewers dried grain is the an increase in antibodies, which in turn increases
extracted dried residue of barley malt alone or in mixture disease resistance. Spirulina is also high in usable or
with other cereal grain or grain products resulting from digestible amino acids. Spirulina is probably one of
the manufacture of beer and may contain pulverized the best fish food ingredients available, including for
spent hops in an amount not more than 3 %. The carnivores (usually fed via gut loading). Any staple
protein content may be around 25-30 %. fish food diet for community fish is improved by the
addition of Spirulina Algae.
Dried Whey: Dried whey is the residue obtained by
drying whey, a by-product of cheese manufacturing. Silkworm pupae: It contains 72.5% crude protein.
It contains at least 65 % lactose; crude protein, 13 It is oil extracted, sun dried, powered and used as
%; about 0.8 % crude fat; and no crude fibre. a source of animal protein in fish feed. It is widely
utilized in carnivorous fish diets.
Spirulina: Spirulina is blue–green algae rich in

Table 1: Proximate composition of selected aquafeed ingredients

Ingredients Moisture (%) Crude Protein Ether Extract (%) Crude Fibre (%) Total Ash (%) Nitrogen Free
(%) Extract (%)
Plant Origin
Rice Polish 8.4-12.6 11.4-14.5 15.3-17.3 7.5-11 6-12.9 41- 46.8
Rice bran 7.8-10.1 2.9-12.6 4.2-11.3 5.3-19.3 3.1-20.5 36.5-37.5
Deoiled rice bran 7.2-8.1 12.1-14.3 1.3-1.8 15.2-16.7 23.8-29.1 40.4-43.3
Wheat bran 9-13 8.2-15.8 2.6-6.6 4-13.5 0.2-4.2 34.5-37.6
Wheat flour 12.6-12.9 14.5-15.6 3.7-3.9 2.7-2.9 2.3-2.8 64.2-64.6
Groundnut cake 7-10 42-48 7.3-13.8 13-13.2 2.5-13.4 25.2-29.9
Sunflower cake 8-10 31-32.6 2.1-2.9 18.4-24.7 1.5-6.2 39-40.1
Central Marine Fisheries Research Institute

Coconut cake 8.9-9.1 12.2-13.7 4.9-5.1 25.6-26.5 2.6-2.8 45.8-46.4

Soybean meal - 26-32.8 2.1-2.9 18.4-24.7 1.5-6.5 39-40.1
Soybean meal, defatted, undehulled - 42-44 - 6.5-7.0 .5.5-6.0 30-32
Soybean meal, defatted, dehulled - 48-50 - 3.0 5.5- 6.0 30-31
Safflower cake 11-12.1 35.9-36.8 0.9-1.6 11.5-13.2 6.9-7.1 32.1-34.3
Cotton Seed cake 7-8.2 37-42.7 7-10 12.6-13 6.5-8.2 27.3-35.3
Sorghum 10-11.6 9-10.2 2.8-3.6 3-3.6 0.1-0.9 75.1-78.1
Lucaena meal 11.8-12.3 35.9-36.8 0.9-1.6 11.5-13.2 6.9-7.1 32.1-34.3
Coffee husk 12.3-14.1 14-15.2 1.2-1.7 20.8-23.1 8.2-9.2 43.5-45.4
Mulberry leaves 8.9-9.4 27.7-35.6 2.4-4.6 11.5-12.5 8.1-9.1 41.4-43.2
Salvinia leaves 2.6-2.8 16.2-17.1 1.1-1.8 18.5-18.6 22-23.5 39.6-40.4
Pistia meal 4.9-5.3 19.5-20.6 1.3-1.8 11.7-11.9 25.6-27.2 37.9-39.8
Colocasia meal 5.8-15.6 24.6-28.2 4.5-4.8 8.2-9.6 9.9-11.2 47-47.1

428
Sesame seed cake 8.3-10 29-42.7 6.9-12.9 5.7-18.3 10-14.8 19.8-21.8
Eichornia meal 3.3-4.1 19.5-20.2 2.3-2.6 18.3-18.4 9.3-9.6 47.3-48.5
Sal seed cake 8.6-8.9 8.2-9.1 2.9-3.5 1.7-1.8 10.2-11.6 68.4-69.7
Spirulina 8.7-10.1 50.5-51.3 1-1.8 2.1-2.6 11-11.7 26.7-27.5
Mustard oil cake 8.5-9.2 23.6-30.8 9.3-9.6 6.2-6.3 10.3-10.4 34.9-40.9
Gingely cake 7.9-9 34-40 2-7.8 7.9-9.6 2.9-3.1 38.2-38.4
Gingely extract 7-9 34-40 2-7.8 9.6-9.7 2.9-3.1 38.2-38.4
Corn / Maize 10.4-10.6 4.6-5 7.8-8 3.5-4 1-2 72.7-75
Corn gluten meal - 60-64 - 8.0 45-50 -
Maize meal 10.4-13.5 4.6-9.5 4-7.8 3.5-4 1-1.5 67.5-72.7
Tapioca flour 8-11.5 1.8-3.1 1.3-2.3 1.8-2 0.2-2.3 78.8-86.9
Barley grains 10-12 8-10 2-3 4-6 2-3 70-80
Rice broken 10-10.5 12-12.6 4.2-4.8 5.3-5.9 3.1-3.6 65.4-69.1
Wheat broken 9-10 11.5-12 1.9-2 4-4.5 0.2-1 73.4-75.2
Palm kernel cake 8.9-10.2 12.2-12.5 4.9-5.1 25.6-26 2.6-2.9 45.8-48.2
Rapeseed cake 11-11.5 35.9-36.3 0.9-1.5 13.2-13.6 6.9-7.5 32.1-33.8
Niger extract 7-7.5 35-35.8 2-2.5 19-19.9 3.5-4 33.5-34.2
Copra cake 8.4-12 20.3-22 6-11.4 12-16.2 2.1-6.2 37.5-42.1
Tobacco seed ext 7.7-8.3 30.6-32.5 0.3-1 - 13.7-14 47.7-48.3
Coffee pulp 12.3-12.5 14-14.5 1.2-1.5 20.8-22.1 8.2-8.9 43.5-45.2
Animal origin:
Fish meal 9-14.6 44.4-72 2.5-10.3 0.3-30 2.5-20.9 7-29.0

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Shrimp meal 3.6-15.6 22.5-34.2 1.1-8 7.1-35.3 18.6-31.6 11-16.3
Squilla meal 14.1-14.9 46-47.3 2.6-3.3 13.5-15.2 18-20.1 5.8-6
Squid meal 8-8.5 75-76.9 6.5-7.1 4-5.3 - -
Clam meal 7-8.1 50.7-52 8.9-11.6 3.9-5.5 6.4-6.9 22-23.1
Silk worm pupae 7.1-7.5 43.9-45.5 25.7-26.1 4.2-4.3 15.8-16.4 3.3-4
Defatted silk worm pupae 8.1-9 68-69.2 2.6-3.1 1.3-1.9 7.2-8.1 12.8-13.7
Blood meal 10-12.9 65.3-76.6 0.5-1.1 1-1.9 3.8-4.3 4.6-5.1
Meat meal 8-10 50-71.2 4.4-13.3 0.7-6.8 5-5.6 25.8-26
Liver meal 7-7.5 65-68.3 3.4-4.2 1.2-2 2.4-3.1 21-22.3
Earth worm pupae 5-6.5 51.7-55.1 3.4-4.1 12.8-13.6 12.5-13 14.6-15

Table 2: Nutritional composition of commercially important feed ingredients

Fish meal Poultry Squid meal Krill meal Soybean Sunflower Rapeseed Wheat Corn
meal meal meal meal bran meal
Moisture (%) 6-9 6-8 8-10 7-9 7-9 6-8 7-9 10-12 8-12

Crude protein(%) 57-64 66 78-80 58-62 45-48 26-29 34-36 15 8

Lipid(%) 7-9 10-14 2-3 16-20 2-4 2-3 2-4 3-4 3-4

Crude fibre(%) 5-7 24-26 10-13 9 2.2

Ash(%) 15-25 10-15 4-8 9-11 6-7 6-7 7 4.9 1.2

Gross energy 19-20 22-23 21-22 22-23 17-18 17-18 17-18 16.5 16

Essential Amino Acid

Arginine(%) 3.7 3.4 7.9 3.8 3 2.1 1.8 0.9 1.55
Histidine (%) 1.4 0.9 2.0 1.4 1 0.6 0.8 0.4 0.4
Isoleucine (%) 2.5 2.0 4.1 2.8 1.8 1.1 1.2 0.4 0.8
Leucine(%) 4.3 3.6 6.9 4.7 3 1.6 2 0.8 1.3
Lysine(%) 4.5 2.3 8.2 4.9 2.4 0.9 1.7 0.5 1.3
Threonine(%) 2.5 2.0 4.0 2.7 1.6 0.9 1.3 0.4 0.7
429
 Tryptophan(%) 0.6 0.4 0.9 0.5 0.5 0.3 0.4 0.2 0.2
Valine(%) 2.9 2.8 4.2 3.4 1.9 1.3 1.5 0.6 0.9
Methionine(%) 1.6 0.7 2.7 1.6 0.6 0.6 0.6 0.2 0.2
Cysteine(%) 0.5 1.3 0.4 0.7 0.6 0.4 0.7 0.3 0.3
Phenylalanine(%) 2.3 2.0 3.7 2.5 2 1.1 1.2 0.5 0.8
Tyrosine(%) 1.9 1.3 2.3 2.7 1.4 0.6 0.9 0.4 0.6
Non essential Amino Acid
Alanine(%) 3.9 2.8 4.6 3.5 1.8 1.1 1.3 0.6 0.8
Aspartic acid(%) 5.6 3.5 8.1 5.7 4.5 2.3 2.1 0.9 2.1
Glutamic acid(%) 7.6 5.6 12.6 7.6 7.1 4.9 5 2.5 3.0
Glycine(%) 3.8 4.5 4.4 2.9 1.7 1.4 1.5 0.7 0.8
Proline(%) 2.5 4.1 3.6 2.4 2 1.1 1.8 0.8 0.8
Serine(%) 2.3 3.2 4.0 2.7 2 1.1 1.3 0.5 0.8
(Source : ARRAINA, Feed ingredients in aquaculture, Technical booklet)

Table 3: Vitamin content of selected commercially important feed ingredients

Biotin Choline Folic acid Niacin Pantothenic acid Pyridoxine Riboflavin Thiamin Vitamin B12 Vit. E
As fed basis mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
Fish meal 0.2 4400 0.2 100 15.0 4.6 7.1 0.1 352 5
Squid meal 650 194 - 1.3 8 0.1 - -
Krill meal 9000 - 1.6 - 0.1 0.1 - 2.6
poultry meal 0.3 1896 0.7 178 46 6.5 9.1 5.7 2.9
Corn gluten 0.2 352 0.3 60 3.5 7 2 0.3 - 23.4
Soybean meal 0.3 2731 1.4 22 15 6.4 3.1 3.2 - 2.3
Rapseed meal 1.0 6700 0.8 160 9.5 7.2 5.8 5.2 - 13.4
Sunflower meal 0.9 3700 2.3 242 40 13.7 3.5 3.1 - 11.1
Wheat bran 0.4 1232 1.8 2 28 8.5 3.6 8.4 - 14.3
Corn meal 0.1 504 0.3 23 5.1 4.7 1.1 3.7 - 21

(Source : ARRAINA, Feed ingredients in aquaculture, Technical booklet)

Table 4: Mineral content of selected commercially important feed ingredients

Calcium Phosphorus Sodium Potassium Magnesium Copper Iron Manganese Selenium Zinc
Unit % % % % % % % % % %
Fish meal 5.5 4.1 1.1 0.8 0.2 6 320 14 1.2 100
Squid meal 0.1 1.4 0.1 0.3 0.0 19 7 0 0.5 15
Central Marine Fisheries Research Institute

Krill meal 1.8 1.2 1.5 0.1 0.7 78 395 6 12 60

poultry meal 0.3 1.9 0.6 0.5 0.1 12 220 16 60
Corn gluten 0 0.4 0.1 0.1 0.1 12 100 8 0.8 34
Soybean meal 0.3 0.6 0 2.1 0.3 16 304 40 0.3 47
Rapseed meal 0.8 1.1 0 1.2 0.5 9 131 58 1.1 71
Sunflower meal 0.4 1 0 1.5 0.5 28 241 34 0.5 85
Wheat bran 0.1 0 0 1.2 0.4 12 137 98 - 77
Corn meal 0 0 0 0.3 0.1 2 32 4 - 18
(Source : ARRAINA, Feed ingredients in aquaculture, Technical booklet)

Table 5: Additives and other components used in the aquafeed industry in India
Additive/ Microingredients Composition/details
Cholesterol 95 % purity, used in shrimp feeds
Mould inhibitor Mixture of propionic acid and other organic acids and salts

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Antioxidant Mixture of BHT, BHA, ethoxyquin, etc
Pellet binder Modified urea formaldehyde
Monocalcium phosphate 21-23% phosphorus
Dicalcium phosphate 18% phosphorus
Limestone powder Mostly used as filler
Mineral premix Typical micro-mineral premix for fish feeds
Salt Feed grade
Potassium chloride KCl 50%
Magnesium sulphate MnSO4
Vitamin premix Typical premix for fish feeds, without Vitamin C
Choline chloride 60%; Feed Attractant
Vitamin C, coated 35 % active ascorbic Acid
Inosital Feed attractant
(Source: FAO Fisheries technical paper, 2007)

Processing Technology, Central Institute of Fisheries Education,

Suggested readings Kolkata Centre, Kolkata, pp2-27.
Hardy, R.W. and Barrows, F.T., 1989. Diet formulation and
Albert G.J. Tacon, Marc Metian, Mohammad R. Hasan, 2007, Feed Manufacture. In : Halver, J.E and Hardy, R. w., (Eds.), 2002. Fish
ingredients and fertilizers for farmed aquatic animals, FAO nutrition 3rd Edn, Academic Press, Amsterdam, pp 515-538.
fisheries and aquaculture technical paper. Nutrient requirements of fish, 1993. Committee on Animal
Feed ingredients in aquaculture -Technical booklet: A database Nutrition, Board on Agriculture, National Research Council,
of aquaculture Feed ingredients, 2013. ARRAINA Project, National Academy press, Washington, pp 80.
EU commission.
Sardar, P., 2007. Training Manual on Fish Feed Formulation and

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

431
 Feed Production Techniques
D. Linga Prabu* and S. Chandrasekar
Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction Fry feed: Once the fish fry reaches the weight of 0.5
Fish feed manufacturing technology will differ based to 0.75 g they can be shifted to fry feeds. Generally
on the types of feeds produced. In aquaculture the fry feeds are crumbles, flakes or small diameter
operation different types of feeds were used according pellet feeds. The nutritional composition of this feed
to the life stage and needs of the fish. The aim of is slightly lesser than the starter feed.
the feed production technology is to produce a high
performance feed with lowest cost possible. The Fingerlings feed: The fingerlings are the fishes that
feed should have better water stability, less nutrient metamorphosis into the size of about 10-20 g in
leaching, highly palatable, good feed acceptance and weight. The nutritional composition of fingerling diet
better efficiency. is lesser than the fry feed. For most of the species the
fingerling diet is not available. They are directly fed
Types of feeds to grow out diets of small diameter pellets.

Grow-out feeds: The major portion of commercial

Based on life stage of fish diet (around 80%) is produced for grow-out stages.
According to life stage of fish the feed can be classified The diet will be available in 2 or 3 different dimensions
as starter feed, fry feed, fingerling feed, grow-out feed and nutritional compositions for different phase of
and broodstock feed. These feeds are generally called grow-out stages. It is most necessary to ensure that
as phase feeds. In most case, fingerling feed will not the protein content of the grow-out diet should be
be commonly available. used for the growth and not for the metabolic activity.
Hence, faster body growth will be achieved in less
Larval feed: The feed size is less than 400 µm and feed which will reduce the production cost.
intended for the fish larvae which is not aggressive
in their first feeding and with partially developed Broodstock diet: The broodstock diet should be
Central Marine Fisheries Research Institute

digestive system. The micro encapsulated diets, enriched with essential fatty acids (EPA, DHA and
micro bound diets and micro coated diets falls in ARA), essential amino acids, higher fat content
this category. to compensate the higher energy requirement,
essential vitamins (A, E and C), trace minerals (Zinc,
Starter feed: The starter feeds are larger than larval Magnesium, Iron and Selenium) for getting maximum
feeds and the size is above 400 µm. Starter feeds fecundity, viable eggs, fertilization rate, hatchability
are produced for the fish species which are fully rate and larval survival ship.
aggressive and have developed digestive system
during first feeding. The feeds should be highly Based on moisture content
digestible and water stable and readily acceptable
by the fish. The diet should contain, high protein, Wet feed: The wet feed comprises fresh ingredients
fat and balanced amino acid and essential fatty such as fish flesh, salmon viscera, chicken viscera, beef
acid content. liver, yeast and 2% salt added to congeal the mixture.

432
The moisture content of the feed will be 45-60%. Generally the antibiotic is added by top dressing with
Generally the wet feeds are used in hatcheries mainly oil or 5% of gelatin. Immunostimulant incorporated
for broodstock feeding served as wet balls or served diets also come under medicated feeds.
as the texture similar to saw dust. FCR is ranged from
3 to 8. It requires frozen storage. Disease causing Pigmented feeds: The inclusion of carotenoid
organisms also transmitted through unpasteurized pigments in the diets of salmonids for the production
wet feed. The thiaminase present in the wet fish meat of pinkish red colour flesh is commonly practiced.
may cause thiamin deficiency in fishes. Feed manufacturers are allowed to only 100 mg/kg
either astaxanthin or canthaxanthin in salmon diets
Semi moist feed and moist feeds: The semi which is allowable to feed the salmon and trout fishes
moist feed are made by mixing the fresh ingredients after 6 months of the age. The pigmented feeds also
and dry ingredients. The wet meat is pasteurized used in ornamental fish feeds.
before it is mixed into the feed mixture. This feeds
contains binding materials such as CMC, gelatin or Based on nature of the
other hydrocolloidal binding agents. The semi moist
ingredients used
diet is made as pellets through cold extrusion. The
moisture content of the diet is 20-40%. It can be Purified feeds: The purified diets are generally made
store in refrigerator for certain period. by the selection of different individual nutrients such
as all amino acids, fatty acids, vitamins, minerals
Dry feed: These feeds are made from dry ingredients and binders. This diet is applicable for experimental
through the addition of water. The feed contains purpose and the palatability is very poor.
less than 10% moisture. The feed is produced by
either extrusion cooking or compressed pelleting or Semipurified feeds: This diet is used for the

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

steam pelleting. The extrusion cooking is employed experimental purposes. The diets are made by the
to produce floating feeds where as steam pelleting combination of purified ingredients such as casein,
can produce compressed sinking pellets. The dry gelatin, sucrose, glucose, cellulose, dextrin, refined
feeds based on shape may be divisible into pellet fish oil, cholesterol, carboxy methyl cellulose, vitamins
feed and crumbles. and minerals, Casein to gelatin in should be in the
ratio of 4:1. These diets are not much palatable.
Based on contents of ingredients
Practical feeds: The diet made with all the
and nutrients
commercially available ingredients comes in
High energy feeds: Contains high level of crude fat this category.
15-36% which increases the total energy content of
the diet and spare dietary protein for growth. It is Feed
mainly used in salmonid culture.
manufacture techniques
Low pollution feeds: The use of high quality raw Aquafeed manufacturing is often a challenging
materials with high bioavailability and high digestibility task that involves in the production of feed with
will reduce the faecal output and aquatic pollution. good water stability, manufacturing difficulties
The feeds produced by extruder will increase the due to high fat content, good keeping quality
carbohydrate digestibility and inclusion of phosphorus due to high unsaturated fatty acids and protein
with high digestibility and low solubility will greatly content and protecting the water soluble vitamins
helpful in pollution reduction. and minerals from leaching. Though several kinds
of feeds are available in the market basically two
Medicated feeds: The feeds produced by the methods are employed for the production of most
incorporation of antibiotics are generally known as of the commercial feeds. They are steam pelleting
medicated feeds for the control of bacterial diseases. and extrusion pelleting techniques. For both of the

433
 techniques most of the preparatory procedures and different feed ingredients from its respective storage
equipments are common except the pelletizer and bins as per the feed formula and weighing in the
extruder respectively in steam pelleting and extrusion weighing scale will be done mechanically by providing
pelleting. The basic steps in feed manufacture: comments in the computer. The batching size may
be one or two tones as per the capacity of the feed
Grinding/particle mill. Each batch will take 3 to 4 minutes to complete
the operation.
size reduction
The particle size of the different feed ingredients Mixing
varies while arrives the feed mill. The grinding process
reduces the particle size of the feed ingredients and Finely ground ingredients mixed together properly in
increases the surface area to facilitate uniform mixing, a desired proportion to form a homogenous blend.
pelleting and digestibility of finished feed as well as The uniform size particles of blend ingredients reduce
prevent blockages of die. Grinding is an expensive the segregation of particles and produce pellets of
process in feed manufacture and hence over-grinding uniform formulation. The mixing time should be
significantly increase the feed production cost. The optimum and insufficient or over mixing may cause
ingredients with high fat contents mainly meat meal particle segregation and non-homogenous mixing.
and dry fish meal are most difficult to grind than the The micronutrients should be mixed separately with
low fat ingredients. There are different grinding mills fillers (rice bran or wheat bran) to make considerable
available and the most popular size reduction system volume before mix with other formula ingredients
used in aquafeed manufacture is hammer mills. The to reduce the non-homogeneity in the feed. At the
other grinding mills are plate mills, attrition mills and time of batching, the dry ingredients should be mixed
air-swept pulverizers. The plate mills are not suitable thoroughly first followed by the liquid ingredients
for aquafeed production because they are incapable addition should be done as the mixing continues.
of producing fine particles. Air-swept pulverizers are The mixer efficiency should be checked periodically by
common in aquafeed production when the required collecting the samples from the mixer for analyzing
particle size is 100 µ or less. Also air-swept pulverizers the known markers such as salt in the feed formula.
are desirable for grinding high fat ingredients where Mixing can be done by mixers which are either batch
air pressure is used for control the grinding rather or continuous mixture. The batch mixers (paddles,
screen. When the hammer mill is alone used for augers and ribbons) only useful in small scale
grinding high fat ingredients it is advisable to use feed production units where as in feed industries
along with low fat ingredients such as de-oiled rice continuous mixers should be in place. Generally,
bran and soy bean meal to avoid screen clogging in the feed industry horizontal and vertical ribbon
with fatty materials. While using air-swept pulverizers, mixers are used for uniform mixing. Nanta mixers and
hammer mills are required for initial particle size turbine mixers are also used less frequently. Twin-shell
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reduction. In hammer mills the grinding chamber blenders are used in mixing the micronutrients with
composed of moving or fixed hammers attached the carrier materials.
to the rotors which breakup the incoming material
and forced through the different size of steel screen Pre-processing or Pre-
depending on the required particle size. There are lot
of ferrous materials may present in the ingredients
conditioning
and hence magnetic protection should be given to In a feed mill different kinds of diets are produced
remove the iron particles time to time before feeding for same species or for different species. The diet
the raw material into hammer mill. production specifications may vary among the
diets. Therefore, proper attention must be taken to
Batching ensure high quality feeds are produced consistently.
Conditioning is the term used to prepare a feed
Batching is the process in which the drawing of mixture for pelleting which includes thermal and

434
mechanical processing. The preprocessing condition cut into desired length. The pellet quality is influenced
will vary for steam pelleting and extrusion pelleting. mainly by fat and moisture content of the feed. The
The conditioning is done at conditioning chamber fat content below 2 % makes the pellet too hard and
that may be a same diameter cylinder or differential increase dustiness whereas the fat content beyond
Diameter Cylinder (DDC), which is common in 8-10% makes pelleting more difficult due to increased
extrusion operation. This chamber contains agitators lubrication and insufficient compression. Hence
that mix the incoming feed mixture and conditions optimum fat content is more advisable. The moisture
through the thermal processing which are achieved content of the pellet affects the hardness of the pellet.
by addition of steam. For compressed pellets, the The pellets with high moisture content results in softy
feed mixtures remain in the condition chamber for pellets due to insufficient compression where are
about 30 seconds where as for extrusion pellets it may insufficient moisture content produce crumbly and
be prolonged to about 2 minutes. Besides thermal dry pellets. The lubricating power of moisture and
processing, the heat generated by the shear force fat will reduce electric power consumption and also
(pressure) applied along the barrel and frictional increase the life span of the die. The feed produce by
energy generated as the feed mixture forcedly pass the compressed pelleting will be a sinking feed with
through the narrow gap created by the presence great water stability.
of cone in the tapered outlet in the chamber that
helps the starchy materials to get gelatinized for the Extrusion dry pelleting by extruders
production of extrusion pellets. A phase transition
analyzer is used to determine the phase transition The extruded pellets also produced in the same way
temperature of individual ingredients or complete as compressed pellets produced but involves the use
feed. Generally for extrusion cooking the temperature of different physical conditions and different type
is maintained around 120-150ºC in the DDC. of dies. The process allows the feed mill operator to

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

have a control over addition of large quantity of lipid
Pelleting as top dressing and buoyancy of pellet by managing
the bulk density. There are two kinds of extruders,
Pelleting process converts the feed mixture into single screw and twin screw extruders are used for
durable forms of physical characteristics of feed that is extruded feed production. As the name spells the
suitable for feeding. The feed mash is fed into the die single screw extruder contains single shaft in the
and comes out the die whole where cutter assembly DDC whereas in twin screw extruder, co-rotating
cut the pellets into defined length according to the twin shafts are present. The capital equipment cost
speed of the cutter. The pellets may be a compressed of twin screw extruder is one and half to two times
one or expanded one according to the conditioning higher than single screw extruder with comparable
in the conditioning chamber. The different types of hourly production capacity. The advantages of twin
pellets are as follows: screw extruders are,

Compressed Pelleting by pelletizer 1. It can be used for the production of feed with
very high internal fat content above 17% fat
Compressed pellet process forces the feed mixture into 2. Products with high levels of fresh meat
the conditioning chamber where dry steam is applied (above 35%)
for about 30 seconds to increase the temperature of 3. Ultra small sized products (0.5-2.0 mm
feed mixture to about maximum of 90ºC together dia products)
with the moisture content around 15-16% through 4. Co-extruded products.
the die holes in the metal die by the action of rollers in
the ring die. Here the combination of heat, moisture In a single screw extruder it is very difficult to produce
and pressure helps to gelatinize the feed mixture into a product with more than 7% of fat content because
compressed pellet with the bulk density beyond 0.5 g/ the higher fat actually provides lubricity and reduce
cm3 while it is coming out from the ring die; they are friction with in the extruder barrel. The extruder

435
 works by increasing the temperature to 120-150ºC 18-20 mt/h is achieved in UP/C by accelerating
in a pressurized DDC through shear force and the speed of the rotor in the extrusion barrel 2-3
increasing the moisture content to about 22-26% times faster than conventional extruder which can
which makes the starch ingredients to get gelatinized. produce 12-14 mt/h. Due to faster rotor rotation
The preconditioned mixture becomes dough like along with lower moisture content increase the
consistency in the extrusion barrel and the pressure frictional energy over the feed mixture which
increases as the dough move towards the die which is enhances the gelatinization up to 80%. The bulk
sufficient to convert the water vapour into liquid. As density of UP/C produced pellets in the range of
the pellet leaves the die hole, the sudden reduction in 0.5-0.65 g/cm3. Due to lower moisture content the
pressure take place that cause the instant expansion pellets can be dried in cooler rather in drier. The
of water vapor to evaporate and allow the pellet to pellets appear without glaze unlike compressed
form an air pockets. Because of the high gelatinization pellets hence, the top dressing of oil can be go up
the durability and water stability of pellet will be to 30%. By the faster knife speed, crumbles can
excellent and the lower bulk density of 0.3-0.4 g/ be produced without much fines in UP/C method.
cm3 makes the pellets to float.
Drying, cooling and crumbling of
In the extruder, post extrusion pressure chamber is
compressed pellets
also available which is known as External Density
Management System (EDMS) where desired pressures The compressed pellets are passed through cooler-
are maintained in the knife enclosure by a special drier assembly where it spread thinly in a moving belt
airlock through which the product discharges. The in horizontal cooler and cool air is blown through the
special airlock system is created by compressed air pellets. In case of vertical cooler the hot pellets are drop
steam which can be used to generate the required down from a cooling tower due to its temperature
pressure in the chamber, as the pressure increases its moisture content gets dried and cooled due to air
in the chamber the water vapour point increases flow. The whole operation takes around 10-15 min
which reduce product flash off expansion and thus and the final product reaches the moisture content
reduce product bulk density. This technique used of 10%. This product goes to storage bins or passed
for the production of shrimp feed in the twin screw through the corrugated rollers of crumbler to make
extruder with sinking capacity. The advantages of them crumbles of required size. Then the crushed
extrusion cooking are that increase the digestibility crumbles separated according to the size in a shaking
of carbohydrates and there by the energy supplied sieve. The fine particles produced send back to feed
by carbohydrates and eliminates the need for non mixture for further feed pellet production.
nutrient binders. The extruded feed reduce the fine
production during transportation and water stability Drying, oil coating and cooling of
is better than steam pellets that is at least 10 h which
extruded pellets
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can be very much suitable for slow feeding fishes.

Extruder pellets contains more moisture than the
Pellets from Universal Pellet/ compressed pellets and hence required to heated at
70ºC by hot air/steam to reduce the moisture content
Cooker (UP/C)
to 10-12%. In the extruded pellet drier the product
The universal pellet/cooker is combination of has to move into 4 layers of beds and hot air need to
compressed pelleting and extrusion cooking pass over the pellets in a crosscurrent fashion which
and hence having the combined benefits of will take at least 30-45 minutes based on the diameter
both systems. In UP/C process the preprocessing of the pellet. After drying the dried extruded pellets
is around 3 minutes that results in 40-50 % of are shifted and sieved in a shaking, vibrating sieve
starch gelatinization. The feed mixture moisture assembly to remove the over sized and under sized
is achieved about 16-18% due to less steam and pellets and the correct sized pellets are transferred
moisture addition. The higher feed production of to vacuum oil coater for oil coating at 60ºC. Then

436
the pellet comes to cooler, where it gets cooled by electronic balance and transferred to polyethylene
blowing cooling air in a vertical cooler. Once the pellet bags with inner liner and get stitched. After that
is completely cooled sieved in a shaking sieve assembly stitched bags are sent through conveyer to the
to remove fines and then goes to storage bins. warehouse for storage where the bags will be shifted
manually by labours and stored in wooden raised
Bagging and storage platform for proper ventilation. The stacked height
of feed bags should be optimum to avoid physical
The pellet feed is weighed automatically in a damages to pellets.
predetermined quantity in a computer assisted

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

437
 Bomb Calorimetry
D. Linga Prabu* and S. Chandrasekar
Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction withstand pressures of minimum 3000 psig.

Calorimetry is the science of measuring quantities
of heat and the instrument that is used for heat Bucket: The bucket or container is provided for
measurement is known as calorimeter. Bomb holding the bomb in a measured quantity of water
calorimeter is one such instrument used to measure along with a probe to read temperature and a
the calorific values of solid or liquid ingredients. Heats stirrer to promote rapid thermal equilibrium without
of formation for organic compounds are generally allowing excessive heat in the form of mechanical
determined by measurements of heats of combustion energy. Buckets are made with a highly polished outer
or calorific values. Heats of combustion or calorific finish to minimize the absorption and emission of
value is defined as number of heat units liberated by radiant heat.
a unit mass of a sample when burned with oxygen
in an enclosure of constant volume that is bomb or Insulating Jacket: The calorimeter jacket is holds
in other words, the heat liberated by the combustion the bomb and bucket that serves as a thermal shield
of all carbon, hydrogen and even other elements by controlling any heat transfer between the bucket
such as sulfur, nitrogen with oxygen to form carbon and surroundings. The jacket will minimize the effects
dioxide and water. Heat energy measured in the bomb of radiant energy and changes in room temperature
calorimeter is expressed either in calorie (cal), British during a test. Anyone of the 3 major types of jacket
thermal unit (Btu) or Joule (J). 1 cal= 4.18 J; 1 Btu systems are employed in bomb calorimeter namely
= 251.99 cal. uncontrolled or plain insulating jacket, adiabatic
system and isoperibol mode.
Bomb calorimeter
Thermometer: Precise measurement of temperature
The bomb calorimeter consists of 4 important is very essential in bomb calorimeter which is achieved
parts. They are bomb, bucket, insulating jacket by any one of the thermometers like mercury-in-glass
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and thermometer. thermometers, platinum resistance thermometers,

quartz oscillators and thermistor systems.
Bomb: The bomb or vessel in which the feed
materials are burned hence, it must be a strong, thick- Standardization
walled corrosion free chromium-nickel alloy and can
be opened for inserting the sample, for removing
of calorimeter
the products of combustion. The bomb should be Before analyzing a material with an unknown heat
provided with valves for filling the bomb with oxygen of combustion in a bomb calorimeter, the energy
under pressure and for releasing residual gases after equivalent or heat capacity or water equivalent
completion of a test. It also provide with electrodes of the calorimeter must first be determined. This is the
to carry an ignition current to a fuse wire. At times sum of the heat capacities of the components, the
of combustion internal pressure may reach up to metal bomb, the bucket and the water in the bucket.
1500 psig, hence, oxygen bombs are constructed to Since, the exact amount of each of the metals used

438
in the bomb and bucket is difficult to determine and Energy estimation from
it is changing continually with use. Hence, energy
equivalents are determined empirically at regular
test samples
intervals by burning a sample of a standard material, After the energy equivalent of the calorimeter has
benzoic acid with a known heat of combustion (6319 been estimated, the calorimeter is ready for testing
cal/g) under controlled and reproducible operating feed samples. Samples of known weight are burned
conditions. Benzoic acid is used as a reference and the resultant temperature rise is measured and
material because it burns completely in oxygen and recorded. The amount of heat of combustion or
it is not hygroscopic and readily available in very calorific value of each sample is determined by
pure form. The amount of heat generated by the multiplying the observed temperature rise by the
reference sample is determined by multiplying the energy equivalent of the calorimeter and divide
heat of combustion of the reference material by the by the weight of the sample. The calorimetric
weight of the sample burned which is divided by corrections need to be considered for acid and
the temperature rise generated in the test, resultant fuse wire.
energy equivalent for this particular calorimeter.
(T× W - (C1+C2))
Water equivalent (W) =(M× H + C1+ C2 )⁄(Temperature rise) Gross calorific value =

(Weight of sample)
Where, M–Weight of benzoic acid; H- Heat of combustion of benzoic acid;
C1- Heat of combustion of fuse wire (1400 cal/g); C2-Heat of combustion Where, T- Temperature rise; W- Water equivalent of the calorimeter
of acids

Estimation of gross energy

Calorimetric corrections

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

value from feed stuff
The factors which cannot be held constant will require
corrections to compensate for their effects. The burning Principle
fuse wire in the bomb contributes additional heat to
the bomb combustion. As the amount of fuse wire The gross energy is the measure of amount of heat
consumed in each test may vary, the energy contributed produced from unit volume of feed in a bomb
by the fuse must be determined for each test and a calorimeter when it is completely burnt down to its
correction applied to compensate for this variance. ultimate oxidation products, CO2 and H2O.

In normal combustion all sulfur in a feed material is Procedure

oxidized to sulfur dioxide and discharged with the
stack gases. Whereas if the same material is burned 1. Bomb assembly and filling the bomb: Weigh the
in an oxygen bomb, the oxidation is carried further to feed material in the range of 0.1 – 0.2 g and
trioxide which then reacts with moisture in the bomb place it in the metal crucible. Connect a 7 cm
to form sulfuric acid. Similarly, in normal combustion platinum wire to the two electrodes of the bomb.
nitrogen in the air is not affected. But when a feed Keep the crucible over the spring stand in such a
sample is burned in bomb, some of the molecular way that it should have contact with ignition wire
nitrogen trapped in the bomb is oxidized and loop. Close the bomb tightly without shaking and
combined with water vapor to form nitric acid. Hence fill oxygen into the bomb through its inlet value
corrections required for the heat energy contributed up to 30-lbs pressure per square inch.
by these acids. American Society for Testing and
Materials (ASTM) and ISO test methods contain 2. Fill exactly 400 ml of water in the bucket
procedures for calculating the correction which must and temperature of water should be 2-3ºC
be applied to account for the heat liberated in the lower than the jacket temperature (jacket-
formation of these acids. constant temperature).

439
 3. Place the bomb in the holder fitted in the bucket 6. The stirrer will start functioning and after 15
and connects with electrodes. Then, place the min the result will be obtained in the monitor
stir, thermometer and lid of the calorimeter in of the bomb calorimeter. The gross energy value
their proper position. is represented as cal/g.

4. Keep the bomb calorimeter in determination 7. Remove the connecting wires, thermometer,
mode for test samples and for benzoic stirrer and covering lids. Take out the bomb and
acid standardization, it should be in release slowly the gas pressure inside the bomb.
standardization mode. Open the bomb clean the interior surfaces with a
jet of distilled water and wipe with tissue paper
5. Press start menu and enter sample weight, before store it.
sample id and specify the bomb number.
Central Marine Fisheries Research Institute

440
Atomic absorption spectroscopy: Analysis
of minerals
Kajal Chakraborty
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
high quality protein they are so well known for.
The minerals present in fish include iron, calcium, Fish should therefore be an integral component
zinc, iodine, phosphorus, and fluorine. These of the diet, preventing malnutrition by making
minerals in the fishes are highly ‘bioavailable’ these macro and micronutrients readily available
meaning that they are easily absorbed by the body. to the body. The contents of K, Na, Cl, Mg, P and
Iron is important in the synthesis of hemoglobin in Ca are up to 1 mg/100 g, whereas those of Fe,
red blood cells, which is important for transporting Zn, Cu and I are less than 1 mg/100g However,
oxygen to all parts of the body. Iron deficiency is it is difficult to generalize and to establish the
associated with anemia, impaired brain function mean mineral values, because they depend on
and in infants is associated with poor learning several factors such as species, sex, biological

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

ability. Due to its role in the immune system, its cycle, and on the portion of fish analyzed and
deficiency may also be associated with increased also on ecological factors, such as season, place of
risk of infection. Calcium is required for strong development, nutrient availability, temperature and
bones (formation and mineralization) and for the salinity of the water. Fish flesh is a good mineral
normal functioning of muscles and the nervous source of Ca and P however, it is interesting to
system. It is also important in the blood clotting take into account that the bony fish skeleton are
process. Vitamin D is required for its proper richer in these elements. Because bone tissue, in
absorption. The intake of calcium, phosphorus and its stage of highest crystallization, is constituted
fluorine is higher when small fish are eaten with by the hydroxyapatite salt, which has Ca and P in
their bones rather than when the fish bones are a 2.15/1 ratio (w/w), its addition to the flesh could
discarded. Deficiency of calcium may be associated contribute to high Ca and P contents. Fish cannot
with rickets in young children and osteomalacia be eaten with bone in its natural state, as it cannot
(softening of bones) in adults and older people. be chewed or digested. However, it is technically
Fluorine is also important for strong bones and possible to process some fish with bone by careful
teeth. Zinc is required for most body processes prior homogenization, which could be incorporated
as it occurs together with proteins in essential in some manufactured foods, increasing the Ca
enzymes required for metabolism. Zinc plays an and P contents and the Ca/P ratio of the meal.
important role in growth and development as well Ca and P are necessary to maintain an optimal
in the proper functioning of the immune system bone development more of both minerals being
and for a healthy skin. Zinc deficiency is associated required during childhood and growing stages to
with poor growth, skin problems and loss of hair prevent rickets and osteomalacia. Although Ca,
among other problems. A deficiency of iodine Mg and P are important in bone metabolism and
may lead to goiter (enlarged thyroid gland) and development, other minerals such as Fe, Cu, Zn
mental retardation in children. It is evident that and Mn are considered to be essential for normal
fish contribute more to people’s diets than just the growth and for avoiding several pathologies.

441
 Mineral Composition • Cool the solution and filter through Whatman
No. 1 filter paper. Dilue the filtrate with distilled
Analyses by atomic water (50 ml) to be injected in atomic absorption
absorption spectroscopy spectrophotometer for determination of minerals.
Estimation of minerals needs to be carried out by atomic • The analyses of Ca, Mg, Na, K, Mn, Cu, Fe, and
absorption spectrophotometer following the di-acid Zn to be performed by flame atomic absorption
(HNO3/HClO4) digestion method (Astorga et al., 2005). spectrophotometry equipped with a hollow
cathode lamp containing D2 lamp background
Apparatus/ Reagents correction system.
• For Se, continuous flow hydride generator
Atomic absorption spectrophotometer, sand bath, coupled with atomic absorption spectrometer
fume hood, concentrated HNO3, HClO4, Whatman should be used (AOAC, 2005).
No. 1 filter paper.
Suggested readings
Procedure
AOAC., 2005. Metals. In: Official methods of analyses of AOAC
• Place the samples (2 g) in digestion tubes, to International, 18th edition, 2005 Chapter 9, (Editor) Dr William
Horwitz; Published by AOAC International, Suite 500, 481
which add concentrated HNO3 (7 ml), and the North Frederick Avenue, USA, pp. 22-46 (chapter 9), Official
content to be kept for overnight digestion in methods of Analyses, 18th edition, Association of Official
Analytical Chemists, Washington D. C.
a fume hood until no brown fumes appeared. Astorga Espana, M.S., Rodrıguez Rodrıguez, E.M., Dıaz Romero,
• Continue the digestion over the sand bath with C., 2005. Sodium, K, Ca, Mg, Fe, Cu and Zn concentrations in
HClO4 (6 ml) until the color of the solution molluscs from the Strait ofMagellan (Chile): their contribution
to dietary intake. International Journal of Food Sciences and
became pale yellow to colourless. Nutrition 56, 337–347.
Central Marine Fisheries Research Institute

442
Amino acids from marine fish and their
implications in health and diseases
Kajal Chakraborty
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
An amino acid is any molecule that contains both Carboxyl group
amino and carboxylic acid functional groups. Amino Amino group
acid is any one of a class of simple organic compounds COO­
containing carbon, hydrogen, oxygen, nitrogen, and in I Alpha Carbon
certain cases sulfur. These compounds are the building +
H3N - C�
blocks of proteins. Amino acids are the building blocks
(monomers) of protein, and are utilized by every cell in
the body for a variety of crucial functions. The shape ---- R Group
and other properties of each protein is dictated by

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

the precise sequence of amino acids in it. Normally,
we obtain them from our food sources, particularly bond, and a chain of amino acids is known as a
those high in protein; the body breaks these proteins polypeptide. Each type of protein differs in its amino
down into their constituent parts, and then our cells acid sequence. Thus the sequential position of the
use these to build the specific types of protein each of chemically distinct side chains gives each protein
them needs. Amino acids form short polymer chains its individual properties. The two ends of each
called peptides or polypeptides which in turn form polypeptide chain are chemically different: the end
structures called proteins. Each amino acid has at least that carries the free amino group (NH3+, also written
one carboxyl (COOH) group, which is acidic, and one NH2) is called the amino, or N-, terminus; and the end
amino (NH2) group, which is basic. carrying the free carboxyl group (COO–, also written
COOH) is the carboxyl, or C-, terminus. The amino
Each amino acid consists of an alpha acid sequence of a protein is always presented in the
carbon atom to which is attached N to C direction, reading from left to right.
• A hydrogen atom
• An amino group (hence “amino” acid) There are two types of amino acids: essential and
• A carboxyl group (-COOH). This gives up a proton nonessential. Essential ones are defined as those
and is thus an acid (hence amino “acid”) which the body cannot manufacture on its own and
• One of 20 different “R” groups. It is the structure must obtain from food sources (or supplements);
of the R group that determines which of the 20 it nonessential ones, on the other hand, can be
is and its special properties. The amino acid shown produced by our own bodies from an available source
here is alanine. of nitrogen and a carbon skeleton, but can also be
consumed as supplements. The essential amino
Amino acids join together in long chains, the amino acids are isoleucine, leucine, lysine, methionine,
group of one amino acid linking with the carboxyl phenylalanine, threonine, tryptophan, and valine.
group of another. The linkage is known as a peptide The nonessential amino acids are arginine, alanine,

443
 asparagine, aspartic acid, cysteine, glutamine, Essential amino acids and
glutamic acid, glycine, proline, serine, and tyrosine. their importance
However, cysteine can partially meet the need for
methionine (they both contain sulfur), and tyrosine Isoleucine: Isoleucine belongs to a special group
can partially substitute for phenylalanine. Semi- of amino acids called branched-chain amino acids
essential amino acids are ones that can sometimes (BCAAs), which are needed to help maintain and
be made internally if conditions are right. Histidine repair muscle tissue. Leucine and valine are other two
is considered semi-essential because the body does branched-chain amino acids. Isoleucine is an essential
not always require dietary sources of it. Other amino acid that is not synthesized by mammalian
amino acids, such as carnitine, are used by the body tissues. Isoleucine is needed for hemoglobin formation
in ways other than protein-building and are often and also helps to maintain regular energy levels.
used therapeutically. Isoleucine is important for stabilizing and regulating
blood sugar and energy levels and is required through
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Leucine, isoleucine, and valine are called branched-chain the diet as it cannot be produced by our bodies.
amino acid (BCAAs) because human beings cannot
survive unless these amino acids are present in the diet. Leucine: Leucine is a member of the branched-chain
The combination of these three amino acids makes up amino acid family, along with valine and isoleucine.
approximately one-third of skeletal muscle in the human The branched-chain amino acids (BCAAs) are found
body. In addition to their role in protein and enzyme in proteins of all life forms. Leucine ties glycine for
synthesis, amino acids are extremely crucial for good the position of second most common amino acid
health. Amino acids contribute significantly to the health found in proteins with a concentration of 7.5 percent
of the nervous system, muscular structure, hormone on a molar basis compared to the other amino
production, vital organs and cellular structure. Some acids. Leucine is necessary for the optimal growth
affects low levels of the essential amino acids result in of infants and for the nitrogen balance in adults. It
hormonal imbalances, irritability, low concentration, functions on balancing blood sugar level in the body.
and depression. It also promotes in the development of the muscle

444
The molecular structures of the major amino acids are listed below:

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

tissue. It modulates the level of hormone production in the formation of collagen.
and energy regulation. It also provides support by
preventing the breakdown of muscles. Deficiency in Methionine: Methionine is an important amino
leucine may include dizziness, irritation, headache, acid that helps to initiate translation of messenger
fatigue, etc. RNA by being the first amino acid incorporated into
the N-terminal position of all proteins. Methionine
Lysine: Lysine is an essential amino acid that has a net supplies sulfur and other compounds required by the
positive charge at physiological pH values making it body for normal metabolism and growth. Methionine
one of the three basic (with respect to charge) amino reacts with adenosine triphosphate to form S-adenosyl
acids. Lysine is an essential amino acid because it methionine. S-adenosyl methionine is the principal
cannot be synthesized in the body and its breakdown methyl donor in the body and contributes to the
is irreversible. It is an essential building block for all synthesis of many important substances, including
protein, and is needed for proper growth and bone epinephrine and choline. It helps in breaking down
development in children. Lysine helps the body absorb of fatty acids, and hence it prevents in building up
and conserve calcium and it plays an important role fatty elements on the artery walls. It also works

445
 significantly in the normal detoxification of liver. It is to pH) amino acids due to its aromatic nitrogen-
essential in promoting energy production as well as heterocyclic imidazole side chain. Histidine is the direct
in muscle building. Methionine, one of the essential precursor of histamine; it is also an important source
amino acids, functions like an effective anti-oxidant of carbon atoms in the synthesis of purines. Histidine
by supplying sulfur for inactivating free radicals. is needed to help grow and repair body tissues, and to
maintain the myelin sheaths that protect nerve cells. It
Phenylalanine: Phenylalanine is an essential amino also helps manufacture red and white blood cells, and
acid that is also one of the aromatic amino acids helps to protect the body from heavy metal toxicity.
that exhibit ultraviolet radiation absorption properties Histamine stimulates the secretion of the digestive
with a large extinction coefficient. Phenylalanine is enzyme gastrin.
part of the composition of aspartame, a common
sweetener found in prepared foods (particularly Non-essential amino acids
soft drinks, and gum). Phenylalanine plays a key
role in the biosynthesis of other amino acids and Alanine: Alanine is one of the simplest of the
some neurotransmitters. amino acids and is involved in the energy-producing
breakdown of glucose. L-alanine is created in
Threonine: This amino acid is perfect in assisting muscle cells from glutamate in a process called
protein balance in the body. Additionally, it helps transamination. Alanine comes from the breakdown
in the development of collagen and maintaining of DNA or the dipeptides, anserine and carnosine,
elasticity in the skin. It also functions of liver. It and the conversion of pyruvate, a compound in
functions well in reducing liver fat. In addition to carbohydrate metabolism. Alanine is used by the body
other essential amino acids, threonine promotes well to build proteins. Alanine is vital for the production
balancing of immune system in terms of antibody of protein, essential for proper function of the central
production and thymus growth. nervous system and helps form neurotransmitters.
Alanine is necessary for the promotion of proper
Tryptophan: Tryptophan is an essential amino acid blood glucose levels from dietary protein.
formed from proteins during digestion by the action
of proteolytic enzymes. Tryptophan is also a precursor Arginine: Arginine is a complex amino acid that
for serotonin (a neurotransmitter) and melatonin (a is often found at the active (or catalytic) site in
neurohormone). This is an essential ingredient for proteins and enzymes due to its amine-containing
the formation of vitamin B3. It is responsible for side chain. Arginine is involved in multiple areas of
the production of serotonin which is exclusively human physiology and metabolism. Arginine plays an
important for balancing nerve and brain functioning. important role in cell division, the healing of wounds,
It is beneficial for controlling hyperactivity among removing ammonia from the body, immune function,
children. It aids in alleviating stress. It works effectively and the release of hormones. Arginine has a number
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as an appetite suppressant. It also promotes in of functions in the body such as assisting in wound
reducing weight. healing, hormone production, immune function and
removal of excess ammonia.
Valine: Valine is a branched-chain amino acid (BCAA)
that is closely related to leucine and isoleucine both in Asparagine: Asparagine is the ß-amide of aspartic
structure and function. Valine is a constituent of fibrous acid synthesized from aspartic acid and ATP
protein in the body. As a branched-chain amino acid (adenosine triphosphate). Asparagine is one of the
(BCAA), valine has been found useful in treatments principal and frequently the most abundant amino
involving muscle, mental, and emotional upsets, and acids involved in the transport of nitrogen. Asparagine
for insomnia and nervousness. Valine may help treat is very active in converting one amino acid into
malnutrition associated with drug addiction. another (amination and transamination) when the
need arises. Asparagine serves as an amino donor in
Histidine: Histidine is one of the basic (with reference liver transamination processes.

446
Aspartic acid: Aspartic acid is alanine with one of important amino acid glutamine. Because it has a
the β hydrogens replaced by a carboxylic acid group. carboxylic acid moiety on the side chain, glutamic acid
Aspartic acid is a part of organic molecules containing is one of only two amino acids (the other being aspartic
an amino group, which can combine in linear arrays to acid) that has a net negative charge at physiological
form proteins in living organisms. Although aspartic pH. This negative charge makes glutamic acid a very
acid is considered a non-essential amino acid, it plays polar molecule and it is usually found on the outside of
a paramount role in metabolism during construction proteins and enzymes where it is free to interact with
of other amino acids and biochemicals in the citric acid the aqueous intracellular surroundings. On a molar
cycle. Among the biochemicals that are synthesized from basis, glutamic acid is incorporated into proteins at a
aspartic acid are asparagine, arginine, lysine, methionine, rate of 6.2 percent compared to the other amino acids.
threonine, isoleucine, and several nucleotides.
Glycine: Glycine is the simplest amino acid and is
Cysteine: Cysteine is a naturally occurring the only amino acid that is not optically active (it
hydrophobic amino acid which has a sulfhydryl group has no stereoisomers). The body uses it to help the
and is found in most proteins. Cysteine is one of the liver in detoxification of compounds and for helping
key components in all living things. N-acetyl cysteine the synthesis of bile acids. It has a sweet taste and
(which contains cysteine) is the most frequently used is used for that purpose. Glycine is essential for
form of cysteine. N-acetyl-L-cysteine (NAC) helps the synthesis of nucleic acids, bile acids, proteins,
break down mucus and detoxify harmful substances peptides, purines, adenosine triphosphate (ATP),
in the body. Both cysteine and NAC have been shown porphyrins, hemoglobin, glutathione, creatine, bile
to increase levels of the antioxidant glutathione. salts, one-carbon fragments, glucose, glycogen, and
l-serine and other amino acids.
Cystine: Cystine is the product of an oxidation between

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

the thiol side chains of two cysteine amino acids. As Proline: Proline is a non-essential amino acid
such, cystine is not considered one of the 20 amino that is involved in the production of collagen
acids. This oxidation product is found in abundance and in wound healing. Proline is the precursor
in a variety of proteins such as hair keratin, insulin, the for hydroxyproline, which the body incorporates
digestive enzymes chromotrypsinogen A, papain, and into collagen, tendons, ligaments, and the heart
trypsinogen where it is heavily involved in stabilizing muscle. Proline plays important roles in molecular
the tertiary structure of these macromolecules. recognition, particularly in intracellular signalling.
Proline is an important component in certain medical
Glutamine: Glutamine is one of the twenty amino wound dressings that use collagen fragments to
acids generally present in animal proteins. Glutamine stimulate wound healing.
is the most abundant amino acid in the body. Over
61% of skeletal muscle tissue is glutamine. It contains Serine: The methyl side chain of serine contains
two ammonia groups, one from its precursor, a hydroxy group making this one of two amino
glutamate, and the other from free ammonia in the acids that are also alcohols. Serine plays a major
bloodstream. Glutamine is involved in more metabolic role in a variety of biosynthetic pathways including
processes than any other amino acid. Glutamine is those involving pyrimidines, purines, creatine, and
converted to glucose when more glucose is required porphyrins. Serine has sugar-producing qualities, and
by the body as an energy source. Glutamine assists is very reactive in the body. It is highly concentrated
in maintaining the proper acid/alkaline balance in the in all cell membranes, aiding in the production of
body, and is the basis of the building blocks for the immunoglobulins and antibodies.
synthesis of RNA and DNA.
Tyrosine: Tyrosine is metabolically synthesized
Glutamic acid: Glutamic acid is biosynthesized from from phenylalanine to become the para-hydroxy
a number of amino acids including ornithine and derivative of that important amino acid. Tyrosine is
arginine. When aminated, glutamic acid forms the a precursor of the adrenal hormones epinephrine,

447
 norepinephrine, and the thyroid hormones, Creatine: Creatine is a natural derivate of an amino
including thyroxine. L-tyrosine, through its effect acid and is synthesized in the liver, kidneys and
on neurotransmitters, is used to treat conditions pancreas out of arginine, methionine and glycine.
including mood enhancement, appetite suppression, Creatine functions to increase the availability of cellular
and growth hormone (HGH) stimulation. ATP, adenosine triphosphate. Creatine works by acting
on mechanisms of ATP by donating a phosphate ion
Hydroxyproline: Hydroxyproline is derived from the to increase the availability of ATP. Creatine is stored
amino acid proline and is used almost exclusively in in muscle cells as phosphocreatine and is used to
structural proteins including collagen, connective help generate cellular energy for muscle contractions.
tissue in mammals, and in plant cell walls. An
unusual feature of this amino acid is that it is not Citrulline: Citrulline is a precursor to arginine and is
incorporated into collagen during biosynthesis involved in the formation of urea in the liver. Arginine
at the ribosome, but is formed from proline by is a contributing member of the various amino acids
a posttranslational modification by an enzymatic found in the urea cycle, which is responsible for
hydroxylation reaction. Non-hydroxylated collagen detoxifying ammonia. Citrulline supports the body
is commonly termed pro-collagen. in optimizing blood flow through its conversion to
l-arginine and then nitric oxide (NO).
Non protein amino acids
Gamma-aminobutyric acid: Gamma-aminobutyric
In humans, non-protein amino acids also have acid (GABA) is a non-essential amino acid formed
important roles as metabolic intermediates, such as from glutamic acid with the help of vitamin B6. GABA
in the biosynthesis of the neurotransmitter gamma- (gamma-aminobutyric acid) is found in almost every
aminobutyric acid. These class of amino acids are region of brain, and is formed through the activity of
described in detail. the enzyme glutamic acid decarboxylase (GAD). GABA
serves as a inhibitory neurotransmitter to block the
Carnitine: Carnitine is a non-essential amino acid transmission of an impulse from one cell to another
produced in the liver, brain and the kidneys from the in the central nervous system.
essential amino acids methionine and lysine. Carnitine
is a nutrient responsible for the transport of long- Glutathione: Glutathione (GSH) is a tripeptide
chain fatty acids into the energy-producing centers composed of three different amino acids: glutamate,
of the cells (known as the mitochondria). Carnitine is cysteine and glycine that have numerous important
recommended as a daily supplement to help maintain functions within cells. Glutathione plays a role in
blood lipid profile and promote fatty acid utilization such diverse biological processes as protein synthesis,
within heart muscle. enzyme catalysis, transmembrane transport, receptor
action, intermediary metabolism, and cell maturation.
Central Marine Fisheries Research Institute

Carnosine: Carnosine is a dipeptide composed Glutathione acts as an antioxidant used to prevent

of the covalently bonded amino acids alanine oxidative stress in most cells and help to trap free
and histidine and is found in the brain, heart, radicals that can damage DNA and RNA.
skin, muscles, kidneys and stomach. Carnosine
is one of the most important and potent natural Ornithine: Ornithine plays an important role in the
antioxidant agents which act as universal urea cycle and is the precursor of the amino acids
antioxidants both in the lipid phase of cellular citrulline, glutamic acid, and proline. Another primary
and biological membranes and in the aqueous role of ornithine is being an intermediate in arginine
environment protecting lipids and water-soluble biosynthesis, although this is due to its participation
molecules like proteins (including enzymes), DNA in the urea cycle (responsible for the production of
and other essential macromolecules from oxidative urea). Ornithine is not directly incorporated into
damage mediated by reactive oxygen species and proteins and enzymes and does not have a codon
lipid peroxides. in the genetic code.

448
Taurine: Taurine is a non-essential sulfur-containing often involves moving its amino group to alpha-
amino acid that functions with glycine and ketoglutarate, forming glutamate. In many
gamma-aminobutyric acid as a neuroinhibitory vertebrates, the amino group is then removed
transmitter. Taurine is the body’s water soluble through the urea cycle and is excreted in the
anti-oxidant, and inhibitory neurotransmitter. The form of urea. However, amino acid degradation
major antioxidant activity of taurine derives from can produce uric acid or ammonia instead. For
its ability to scavenge the reactive oxygen species example, serine dehydratase converts serine to
hypochlorite. Taurine plays an important role in pyruvate and ammonia. Nonstandard amino
numerous physiological functions. acids are usually formed through modifications to
standard amino acids. For example, homocysteine
Metabolism of amino acids is formed through the transsulfuration pathway
or by the demethylation of methionine via the
In plants, nitrogen is first assimilated into organic intermediate metabolite S-adenosyl methionine,
compounds in the form of glutamate, formed from while hydroxyproline is made by a posttranslational
alpha-ketoglutarate and ammonia in the mitochondrion. modification of proline. Microorganisms and plants
In order to form other amino acids, the plant uses can synthesize many uncommon amino acids. For
transaminases to move the amino group to another example, some microbes make 2-aminoisobutyric
alpha-keto carboxylic acid. For example, aspartate acid and lanthionine, which is a sulfide-bridged
aminotransferase converts glutamate and oxaloacetate derivative of alanine. Both of these amino acids are
to alpha-ketoglutarate and aspartate. Other organisms found in peptidicl antibiotics such as alamethicin.
use transaminases for amino acid synthesis too. While in plants, 1-aminocyclopropane-1-carboxylic
acid is a small disubstituted cyclic amino acid that
Transaminases are also involved in breaking is a key intermediate in the production of the plant

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

down amino acids. Degrading an amino acid hormone ethylene.

449
 The health benefits of depression, rheumatoid arthritis and osteoarthritis,
menstrual cramps, Parkinson’s disease, vitiligo, and
amino acids cancer. Proline is an important component in certain
Amino acids are needed to build the various proteins medical wound dressings that use collagen fragments
used in the growth, repair, and maintenance of to stimulate wound healing. Serine is needed for the
body tissues. Amino acids play innumerable roles in metabolism of fats and fatty acids, muscle growth, and
human health and disease. Alanine is necessary for the a healthy immune system. Taurine helps regulate the
promotion of proper blood glucose levels from dietary contraction and pumping action of the heart muscle
protein. Alanine stimulates lymphocyte production and it helps regulate blood pressure and platelet
and may help people who have immune suppression. aggregation. Threonine may enhance immunity by
Alanine strengthens the immune system by producing assisting in the production of agents that fight viral
antibodies. L-arginine is used by the immune system to infections. L-theanine reduces stress and anxiety
help regulate the activity of the thymus gland, which without the tranquilizing effects found in many other
is responsible for manufacturing T lymphocytes. The calming supplements. Tryptophan is important for the
body uses arginine to produce nitric oxide. Nitric oxide production of serotonin. Increasing tryptophan may
is an endogenous messenger molecule involved in a help to normalize sleep patterns. Tyrosine may act as
variety of endothelium-dependent physiological effects an adaptogen, helping the body adapt to and cope
in the cardiovascular system. In the central nervous with the effects of physical or psychological stress
system, asparagine is needed to maintain a balance, by minimizing the symptoms brought on by stress.
preventing over nervousness or being overly calm. As a branched-chain amino acid (BCAA), valine has
Aspartic acid can help protect the liver from some drug been found useful in treatments involving muscle,
toxicity and the body from radiation. Carnosine is the mental, and emotional upsets, and for insomnia and
water-soluble counterpart to vitamin E in protecting nervousness. Creatine supplements fuels and enhances
cell membranes from oxidative damage. L-carnosine short bursts of high-energy exercise. Creatine prevents
supports healthy aging and cellular rejuvenation by the body from relying solely on the process of glycolysis.
its effects on two mechanisms: glycosylation and free Citrulline supports the body in optimizing blood flow
radical damage. Cysteine strengthens the protective through its conversion to l-arginine and then nitric
lining of the stomach and intestines, which may help oxide (NO). GABA has been used in the treatment
prevent damage caused by aspirin and similar drugs. of depression, manic-depressive (bipolar) disorder,
The health benefits of glutamine include immune seizures, premenstrual dysphoric (feeling depressed)
system regulation, nitrogen shuttling, oxidative stress, disorder, and anxiety. Glutathione are necessary for
muscle preservation, intestinal health, injuries, and supporting the immune system, glutathione is required
much more. Glycine is an inhibitory amino acid with for replication of the lymphocyte immune cells.
important functions centrally and peripherally. Glycine
may be indicated to help alleviate the symptoms Suggested readings
Central Marine Fisheries Research Institute

of spasticity. Histidine is known to be vital in the

maintenance of the myelin sheaths surrounding Heinrikson L, Meredith SC (1984). Analytical Biochemistry
nerves, particularly the auditory nerve and is used 136, 65-74.
Anfinsen CB, Edsall JT, Richards FM (1972). Advances in Protein
to treat some forms of hearing disability. Isoleucine Chemistry. New York: Academic Press. pp. 99, 103.
is necessary for the optimal growth of infants and Fennema OR (1996). Food Chemistry (3rd ed.). CRC Press.
pp. 327–8.
for nitrogen balance in adults. Leucine is used as a Sakami W, Harrington H (1963). Annual Review of Biochemistry
source for the synthesis of blood sugar in the liver 32: 355–98.
during starvation, stress, and infection to aid in Young VR (1994). The Journal of Nutrition 124 (8
Suppl): 1517S–1523S.
healing. Lysine is used in managing and preventing Imura K, Okada A (January 1998). Nutrition 14 (1): 143–8.
painful and unsightly herpes sores caused by the Lourenço R, Camilo ME (2002). Nutrición Hospitalaria 17 (6): 262–
70.
herpes simplex virus (HSV). Methionine is both an Fürst P, Stehle P (June 2004). The Journal of Nutrition 134 (6
antioxidant and lipotrope, meaning it helps remove Suppl): 1558S–1565S.
fat from the liver. Phenylalanine is used to treated Reeds PJ (July 2000). The Journal of Nutrition 130 (7): 1835S–40S.

450
Fatty acids from marine fish and their
implications in health and diseases
Kajal Chakraborty
Senior Scientist
Marine Biotechnology Division, CMFRI, Kochi
e-mail: [email protected]

Introduction
families’ viz., ω-3 and ω-6 PUFAs (otherwise termed
Fatty acids are carboxylic acids with long hydrocarbon as n-3 and n-6 PUFAs). However, ω-3 fatty acids are
chains (usually C12-22). Dietary fats are used to build found to be abundantly available in marine sources
every cell in the body and cell membranes are made particularly fish and phytoplanktons. These fatty acids
of a variety of individual fatty acids. The essential fatty affect many physiological processes including cognitive
acids from marine fish have protective mechanisms function, visual acuity, immunosuppressive, and anti-
against coronary heart disease, which became thrombic activities along with having major role on
apparent in the investigations of the health status of glucose and lipid metabolism. Table 1 illustrates the
Greenland Eskimos who consumed diets very high in details regarding the differential changes of fatty
fat from seals, whales, and fish, and yet had a low acids and their structures including their abbreviated

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

rate of coronary heart disease events. This paradox formulae, molecular formulae, and molecular weight.
was explained by the fact that Eskimos consumed
contained large quantities of the very-long-chain Biosynthetic route of fatty acids
and highly polyunsaturated fatty acids with C20-22
carbons and 5-6 olefinic bonds, which are abundant Fatty acid synthesis is a metabolic process to
in marine fish, and are scarce or absent in land combine eight C2 – moieties (-CH3C(=O) group from
animals and plants. Classification of fatty acids is CH3COSCoA) to synthesize saturated fatty acid with
based on to denote hydrocarbon chain length and C16-moiety (C16H32O2), which thereafter modified
number and positions of olefinic bonds. However, to form homologous fatty acid analogues. These
the most accepted system of classification is based on modifications include: elongase-catalyzed chain
the number of olefinic bonds. Saturated fatty acids elongation to synthesize fatty acids with longer
(SFAs) donot possess olefinic bonds in hydrocarbon hydrocarbon chain, e.g., stearic acid (C18H36O2),
chain. Examples of SFAs are lauric acid, myristic acid, arachidic acid (C20H40O2), and so on. These SFAs, on
palmitic acid, stearic acid, arachidic acid, behenic desaturation yield unsaturated fatty acid analogues.
acid, and lignoceric acid (Table 1). Monounsaturated In general, fatty acid synthesis takes place in
fatty acids (MUFAs) possess one double bond, the cytoplasm of liver, adipose, central nervous system,
typical examples being myristoleic acid, palmitoleic and lactating mammary gland tissues of human.
acid, elaidic acid, oleic acid, erucic acid, and nervonic Glycolytic breakdown of glucose yields acetyl CoA
acid. Fatty acids with ≥ 2 double bonds are termed as through pyruvate (CH3COCOOH) by aerobic glycolysis
polyunsaturated fatty acids (PUFAs). The tetrahedral that is starting material for fatty acid synthesis.
bond angles on carbon results in a molecular geometry Acetyl CoA serves as substrate to synthesize citrate
for saturated fatty acids that is relatively linear. Olefinic that transported out of mitochondria to cytosol
bonds in hydrocarbon chain of unsaturated fatty and generates acetyl CoA. The overall reaction of
acids results in kinks in their structure results in weak anabolism of fatty acids to form unsaturated faty
stacking. PUFAs are broadly divided into two major acids is as follows:

451
 Table 1. Nomenclature of fatty acids
Central Marine Fisheries Research Institute

O
O O O
HO C O

H3 C S
CoA
HO S
CoA Fatty acids are stored in adipocytes as triacylglycerol
Acetyl CoA carboxylase
Acetyl CoA Malonyl CoA that must be hydrolyzed to release free fatty acids.
O

ATP ADP + Pi R

Polyunsaturated fatty acids and

2NADP+ + CO2 + H2O SCoA

Fatty acyl CoA

Fatty acid elongase

NADPH + H+ + O2
2NADPH + 2H+
CoA-SH
their importance in health and
R
SCoA
Desaturase
Coenzyme A
SCoA
disease
R

To prevent cancer
O
Unsaturated fatty acid NADP+ + 2H2O Elongated fatty acyl CoA (extra C2 moiety)

Among dietary factors postulated to influence cancer

Figure 1. Synthesis of unsaturated fatty acids from acetyl CoA development are long chain polyunsaturated ω-3 fatty
acids, found in fish. Earlier studies revealed inverse

452
relation between marine fatty acid consumption and that the polyunsaturated fat may affect these
mortality rates of prostate (Hebert et al., 1998) and pathological processes through other mechanisms.
breast cancer (Hebert et al., 1996). The mechanisms There is evidence from epidemiology that marine n-3
proposed how the intake of marine fatty acids might PUFA is associated with a reduced risk of coronary
lower the risk of cancer is the inhibition of eicosanoid heart disease. This was originally found in Greenland
biosynthesis from AA, a ω-6 fatty acid. Prostaglandins Eskimos with an extremely high intake of n-3 PUFA
converted from AA by the cyclooxygenase-2 enzyme, (10–14 g/day) and later also reported in several other
notably PGE2, have been linked to carcinogenesis populations (Schmidt et al., 2005; Kris-Etherton et
viz., mammary tumor development, proliferation of al., 2002) including Western populations with an
breast and prostate cancer (Erickson, 1986). Tumor average intake of marine n-3 PUFA below 0.2–0.4
cells typically produce large amounts of AA-derived g/day. Recently, a meta-analysis was published on
PGE2, which may impede immune system function, fish consumption and CHD mortality from 13 cohort
possibly through their role in the generation of studies including a total of 222,364 individuals
suppressor T cells (Erickson, 1986). Marine fatty acids with an average of 11.8 years of follow-up (He et
were reported to inhibit cyclooxygenase-2 and the al., 2004). Fish consumption was inversely related
oxidative metabolism of AA to PGE2. EPA and DHA also with fatal CHD and sudden cardiac death (He et
inhibit lipoxygenases which metabolize AA to HETEs al., 2004).
and leukotrienes. 12-HETE has been linked to the
suppression of apoptosis, stimulation of angiogenesis, Estimation of fatty acids
stimulation of tumor cell adhesion, and expression
in laboratory
of the invasive phenotype. It is apparent that both
EPA and DHA can inhibit the biological activity of Broadly fatty acid estimation is divided under the
eicosanoids and androgens (Liang et al., 1992), which broad categories, viz., (1) lipid extraction and acid-

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

are known to have a stimulating effect on cell growth catalyzed transesterification of fatty acid to methyl
and uncontrolled cell proliferation (Ghosh & Myers, esters (FAMEs) and N-acyl pyrrolidides; and (3) gas-
1997). It is well established that in animal models and liquid chromatography and gas chromatography-mass
in human cancer cell lines, EPA and DHA were found spectrometry (GC/MS) analysis of FAMEs. Below are
to suppress cell growth. However, because intakes of illustrated the details under each head.
fish and marine fatty acids are highly correlated, it is
difficult to disentangle the effect of fatty acids from Lipid extraction
the effect of fish per se.
Lipid from the crude sardine oil was extracted by using
To combat atherosclerosis and CHCl3-CH3OH-H2O (Bligh, & Dyer, 1959). In brief, about
cardiovascular diseases 10 g tissue together with chloroform methanol mixture
(2:1) ratio is homogenized, and CHCl3-CH3OH mixture
Eating ω-3 fatty acids abundantly available in (15 times) was added and mixed (to 1/3rd of the total
marine fish were reported to protect human volume). The resulting solution was filtered, and the
beings from heart failure (European Heart Journal. filtrate was collected. The process was repeated two
doi:10.1093/eurheartj/ehp111). Researchers in the more times with rest of the CHCl3-CH3OH mixture.
USA and Sweden followed 39,367 Swedish men, To the filtrate, add distilled water (20% of the total
aged between 45-79, from 1998 to 2004. They volume of the filtrate) and leave overnight. The water-
recorded details of the men’s diet and tracked the soluble residue diffuses away from the solvent and
men’s outcome through Swedish inpatient and occupies the top position in the separating funnel.
cause-of-death registers. PUFAs in the diet have Solvent containing lipid (bottom layer) is collected
long been considered essential to the growth and by filtering through anhydrous Na2SO4. Evaporate to
proper nutrition of humans and other vertebrates. dryness and make up the volume using CHCl3. On
It was reported that atherosclerosis and thrombosis extraction with CHCl3-CH3OH, lipid (bottom layer) is
represent essential fatty acid deficiencies, but rather separated from the sample and is collected by filtering

453
 through anhydrous Na2SO4. After saponification Mass Spectroscopic Analyses of
of the dried extract PUFA is determined using gas FAME Derivatives
chromatograph as illustrated below.
The following are the mass spectrometric data
Extraction and derivatization of FAME derivatives.

of fatty acids to fatty acid

Methyl Palmitate. EI-MS m/z (relative intensity, %):
methyl esters (FAME) and 270 (M+, 61.11), 239 (15.74), 227 (31.48), 213
N-acyl pyrrolidides (7.41), 199 (14.81), 185 (12.96), 171 (12.96), 157
(7.41), 143 (31.48), 129 (11.11), 87 (74.07), 74
The lipid extract thus obtained was saponified (100), 55 (18.52).
with 0.5 N KOH in CH3OH. After removal of the
nonsaponifiable material with n-hexane and Methyl Oleate. EI-MS m/z (relative intensity, %): 296
acidification with 1 N HCl, the saponifiable materials (M+, 20.00), 111 (76.67), 264 (33.33), 222 (26.67),
were extracted with petroleum etherdiethyl ether (1:1 180 (18.33), 166 (23.33), 152 (23.33), 123 (23.33),
v/v) and transesterified to furnish fatty acid methyl 110 (38.33), 97 (75.00), 83 (70.00), 74 (66.67), 69
esters (FAME) by reaction (30 min under reflux) with (78.33), 55 (100).
a methylating mixture (14% BF3/ CH3OH, 5 mL) in
a boiling water bath under an inert atmosphere Methyl Linoleate. EI-MS m/z (relative intensity, %):
of N2 (Metcalf, Schimtz, & Pleka, 1966). The FAME 294 (M+, 52.46), 263 (24.59), 220 (8.20), 178
thus obtained was cooled to ambient temperature, (13.11), 164 (19.67), 150 (21.31), 136 (18.03),
and distilled water (20 mL) was added. The solution 123 (18.85), 109 (37.70), 95 (70.49), 81 (100), 67
was extracted with n-hexane (10 mL X 6), and the (91.80), 55 (50.82).
upper n-hexane layer was removed and concentrated
under an inert atmosphere of N2. The resulting FAME Methyl Linolenate. EI-MS m/z (relative intensity, %):
concentrate was reconstituted in petroleum ether, 292 (M+, 16.67), 261 (5.00), 236 (6.67), 173 (6.67),
flushed with N2 in glass vials, and stored in deep 163 (6.67), 149 (20.00), 135 (20.00), 121 (25.00),
freeze (-20ºC) until required for GC/GC-MS analyses. 108 (56.67), 95 (58.33), 79 (100), 67 (56.67),
Analysis was performed in triplicate. 55 (35.00).

Gas-liquid chromatography Methyl Arachidonate. EI-MS m/z (relative intensity, %):

318 (M+, 1.82), 290 (1.82), 264 (1.82), 175 (5.45),
and gas chromatography-mass
150 (7.27), 133 (7.27), 105 (30.91), 91 (70.91), 79
spectrometry (GC/MS) analysis of (100), 67 (80.00), 55 (49.09).
fatty acid derivatives
Central Marine Fisheries Research Institute

Methyl Eicosapentaenoate. EI-MS m/z (relative

Quantitative and qualitative analyses of FAME intensity, %): 315 (M+, 1.67), 175 (6.67), 161 (8.33),
obtained by transesterification were performed on 145 (11.67), 131 (18.33), 119 (31.67), 108 (31.67),
gas chromatograph using a flame ionization detector 91 (70.00), 79 (100), 67 (68.33), 55 (48.33).
(FID). FAMEs were identified by comparison of
retention times with the known standards. In another Methyl Docosahexaenoate. EI-MS m/z (relative
process, FAMEs were derivatized to N-acyl pyrrolidides intensity, %): 342 (M+, 0.60), 145 (4.20), 131 (6.60),
by condensation of fatty acid methyl ester with a 119 (10.80), 108 (11.40), 91 (28.20), 79 (100), 67
mixture of pyrrolidine (1 mL) and acetic acid (0.1 (20.40). (Chakraborty et al., 2010).
mL) at 100ºC under reflux (2 h) for GC-MS analyses
(Andersson, 1978). The GC-MS analyses need to be Mass Spectroscopic Analyses of
performed by GC interfaced with mass spectrometer
N-Acyl Pyrrolidide Derivatives
for confirmation of fatty acid identification.

454
The following are the mass spectrometric data as essential and must be supplied in diet. The
of N-acyl pyrrolidide derivatives. importance of PUFAs in human nutrition has been
extensively investigated during the past 20 years.
1-(Pyrrolidin-1-yl)hexadecan-1-one/ DHA is one of the important PUFAs, which maintains
Palmitoylpyrrolidine. EI-MS m/z (relative intensity, structural and functional integrity in larval cell
%): 309 (M+, 16.00), 294 (2.00), 168 (8.00), 140 membranes in addition to the neural development
(10.00), 126 (16.00), 113 (100), 98 (8.00), 70 and function, while AA and EPA are involved in,
(12.00), 55 (14.00). respectively, the production and modulation of
eicosanoids. Docosahexaenoic acid (22:6ω-3), which
1-(Pyrrolidin-1-yl)octadec-9-en-1-one. EI-MS m/z is a vital component of the phospholipids of cellular
(relative intensity, %): 335 (M+, 27.56), 250 (8.62), membranes, especially in the brain and retina, is
236 (10.34), 208 (6.90), 196 (5.17), 182 (12.07), necessary for their proper functioning. The ω-3
126 (53.45), 113 (100), 98 (18.97), 85 (8.62), 72 fatty acids favorably affect atherosclerosis, coronary
(20.69), 55 (27.59). heart disease, inflammatory disease, and perhaps
even behavioral disorders. Membrane fluidity is
1-(Pyrrolidin-1-yl)octadeca-9,12-dien-1-one. EI-MS essential for proper functioning of these tissues.
m/z (relative intensity, %): 333 (M+, 77.97), 290 In the retina, where ω-3 fatty acids are especially
(10.17), 236 (15.25), 222 (20.34), 182 (16.95), 168 important, deficiency can result in decreased vision
(15.25), 140 (22.03), 126 (44.07), 113 (100), 98 and abnormal electroretinogram results. The ω-3
(25.42), 70 (42.37), 55 (49.15). fatty acids are essential fatty acids, necessary from
conception through pregnancy and infancy and,
1-(Pyrrolidin-1-yl)octadeca-9,12,15-trien-1-one. EI-MS undoubtedly, throughout life. AA has been an
m/z (relative intensity, %): 331 (M+, 44.00), 182 essential function of producing eicosanoids, making

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

(22.00), 168 (24.00), 140 (26.00), 126 (60.00), 113 it an essential fatty acid because prostaglandins
(100), 98 (30.00), 72 (64.00), 55 (42.00). (PGF2R) are produced from 20:4n6, and has roles
in reproduction. AA is the basis for cyclo-oxygenase
1-(Pyrrolidin-1-yl)icosa-5,8,11,14-tetraen-1-one. (COX) action to produce PGF2R. AA, being a major
EI-MS m/z (relative intensity, %): 357 (M+, 18.97), component of phosphoinositol, was reported to have
232 (10.34), 180 (10.34), 126 (13.79), 113 (100), a vital role in the transduction signal mechanism. An
85 (17.24), 70 (22.41), 55 (27.59). imbalance in ω-3/ω-6 ratio can accentuate ω-3 fatty
acid deficiency state, as shown by earlier studies. The
1-(Pyrrolidin-1-yl)icosa-5,8,11,14,17-pentaen-1-one. ratio may have increased in industrialized societies
EI-MS m/z (relative intensity, %): 355 (M+, 3.85), because of increased consumption of vegetable oils
286 (7.69), 232 (7.69), 126 (13.46), 113 (100), 85 rich in ω-6 fatty acids, ie, linoleic acid (18:2n26),
(17.31), 72 (26.92), 55 (21.15). and reduced consumption of foods rich in ω-3 fatty
acids. Another important feature of ω-3 fatty acids
1-(Pyrrolidin-1-yl)octadeca-9,12-dien-1-one. EI-MS is their role in the prevention and modulation of
m/z (relative intensity, %): 381 (M+, 3.91), 312 certain diseases that are common (Importance of
(7.05), 272 (7.29), 232 (16.22), 218 (15.76), 192 n23 fatty acids in health and disease (W. E. Connor
(8.24), 166 (23.67), 153 (22.85), 113 (100), 98 Am J Clin Nutr 2000;71(suppl):171S–5S). Below
(46.62), 72 (21.98) (Chakraborty et al., 2010). is appended a partial list of diseases that may be
prevented or ameliorated with ω-3 fatty acids:
Conclusions
1. Coronary heart disease and stroke
Research on exploring sources long-chain PUFAs, 2. Cancers of the breast, colon, and prostate
viz., DHA, EPA, and AA for use in nutrition have 3. Retinal and brain development);
received considerable attention. These PUFAs, which 4. Immunostimulant
are usually low in abundance in human, are regarded 5. Hypertension

455
 Chakraborty, K.; Paulraj, R. (2007) Eicosapentanoic acid enrichment
The first two functions are extremely important and from sardine oil by argentation chromatography. J. Agric. Food
are related directly or indirectly with other diseases Chem. 55 7586-7595.
Connor W. E. (2000) Am J Clin Nutr 71(suppl):171S–5S
as listed earlier. Erickson, K. L. (1986) Prog. Clin. Biol. Res. 222: 555–586
Ghosh, J., and Myers, C. E. Biochem. Biophys. Res. Commun.,
235: 418–423, 1997
Suggested readings He, K.; Song, Y. Daviglus, M.L. (2004). Circulation 109: 2705–2711
Hebert, J. R. & Rosen, A. (1996) Cancer Detect. Prev. 20: 234–244
Bligh, E.G., Dyer, W.J. (1959). Canadian Journal of Biochemistry Hebert, J. R., Hurley, T. G., Olendzki, B. C., Teas, J., Ma, Y. & Hampl,
and Physiology, 37, 911-917. J. S. (1998) J. Natl. Cancer Inst. 90: 1637–1647
Chakraborty, K; Vijayagopal, P., Chakraborty, R.D., Vijayan. K.K. Kris-Etherton, P.M.; Harris, W.S., Appel, L.J. (2002). Circulation
(2010) Enrichment of eicosapentaenoic acid concentrates from 106: 2747–2757
sardine oil by bacterial (Bacillus circulans) lipase isolated from Liang, T., and Liao, S. (1992) Biochem. J. 285: 285–562, 1992
seaweed Turbinaria conoides. Food Chemistry 120: 433-442 Metcalf, L. D., Schimtz, A. A., & Pleka, J. R. (1966). Analytical
Chakraborty R D., Chakraborty, K.; Radhakrishnan E.V.R. Variation Chemistry, 38, 514–515.
in fatty acid composition of Artemia salina nauplii enriched Schmidt, E.B. (1997). Dan. Med. Bull. 44: 1–22
with microalgae and baker’s yeast for use in larviculture J. Schmidt, E.B., Arnesen, H., Caterina, R. De, Rasmussen, H.,
Agric. Food Chem. 55 (2007) 4043-4051. Kristensen, S.D. (2005). Thromb. Res. 115: 163–17.
Central Marine Fisheries Research Institute

456
Nutrigenomics
S. Chandrasekar* and D. Linga Prabu
Scientist
Marine Biotechnology Division,
Mandapam Regional Centre of CMFRI
e-mail: [email protected]

Introduction of an organism reflected by dietary bio-molecules.

Nutrition research has the long tradition for In nutrition research studying the Gene Expression
recommending the species specific diets to farmed Profiling (GEP) may be used for three distinct
fishes for more production. Over last two decades, purposes such as to assist in the identification and
nutritional research has also made a progress in characterization of basic molecular pathways that may
the application of molecular biological tools to be impacted by nutrients, to provide insights upon
understand the basics of gene regulation that specific mechanisms that trigger either beneficial or
affected by dietary factors. Nutrigenomics (Nutritional negative effects and to identify specific genes altered
genomics) refers to the research that investigates the by nutrients that might prove valuable as molecular
interaction between nutrition and the genome. From biomarkers or nutrient sensors.
the view point of nutrigenomics, nutrients are act as

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

dietary signaling molecules and detected by cellular Genomics
sensing mechanism, that influence the gene, protein
and metabolite expression. Nutrients may act directly Generally the genomic study covers the development
as ligands for transcription factor receptors; may of molecular markers, linkage mapping, quantitative
be metabolized by primary or secondary metabolic trait loci (QTL) analysis and characterization of
pathways, thereby altering concentrations of genomic resources for understanding all physiological
substrates or intermediates involved in gene regulation functions. The development of genomic resources
or cell signaling; or alter signal transduction pathways that is cDNA libraries is the first step in any genomic
and signaling. Nutrigenomics is the collective terms, studies which represents the maximum of expressed
includes genomics, transcriptomics, proteomics and RNA and the expressed sequence tag (EST) resources
metabolomics. Application of these modern research getting increased as well. The EST analysis is the
tools may yield new knowledge in molecular responses most useful method for gene identification, gene
expression profiling and cataloguing. The EST
analysis is also used for the development of cDNA
or oligonucleotide microarrays. The information on
the fish genes responsible for metabolic pathways is
helpful in understanding the nutritional metabolism
in molecular level.

Transcriptome
and Transcriptomics
The genome is only a source of information and it
must be expressed to understand the function. The
Figure: Fate and activities of nutrients in the cell
(Adopted from: Kaput and Rodriguez, 2004) transcription of genes to produce RNA is the first stage

457
 of gene expression. Transcriptome is the complete set hepatic transcriptome of fish using cDNA microarray
of RNA transcripts produced by the genome at any techniques. The rainbow trout fed with low protein
one time and is extremely dynamics and varies with levels showed that the over expression of one gene
differing circumstances due to different patterns of that is responsible for the inhibitor of growth that is
gene expression. And hence, transcriptomics refers cell multiplication or division than the high protein fed
to the study of the complete set of RNAs encoded fishes. Polyunsaturated fatty acid is being an essential
by the genome of a specific cell or organism at a component in the central nervous system, the dietary
specific time or under a specific set of conditions. PUFA leads to the brain gene expression through
Transcriptomics, also referred as gene expression the transcriptional modulators. Hence the functional
profiling to examines the expression level of mRNAs in a genome studies with high density microarrays useful in
given cell population. Understanding the transcriptome the discovery of regulatory pathways linked to dietary
is essential for interpreting the functional elements components in fish.
of the genome and understanding the functions
such as growth, health, physiological well-being and Nutritional regulation of
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flesh quality. Transcriptomics uses DNA microarray

technology for studying the gene profiling expression.
candidate genes in fish
In fishes the variations of transcriptome expression
analysis is used to study the stocking stress, handling Larval Nutrition
stress, chemical contaminants, salinity, temperature
and hypoxia conditions and its mitigation through The production of marine fish larvae in hatcheries
dietary intervention. The effects of dietary nutrients on depends on supply of live feed and hence substituting
the hepatic transcriptome expression will helps to learn the live feed with inert compound diet reduce the cost
the nutrient requirement and its effects in fish species. of production of juveniles. Hence, it is necessary to
The protein nutrition in fish affects the transcription determine the time of initiation of compounds in fish
of number of genes through the regulation of larvae. The digestive enzyme of seabass larvae is altered
transcription regulators expression. The protein by dietary composition of diet which was detected
requirement study can be effectively studied by the by its gene expression. During larval development of

458
seabass larvae specific activity and mRNA levels of to species. In fish different types of glucose receptors
amylase enzyme is regulated transcriptionally based such as Glut 1, Glut 2 and Glut 4 are available for
on the dietary carbohydrate level. transport and utilization of glucose. The nutritional
control of these genes and its expression for their
In larval nutrition, physical abnormalities particularly important function was started studied in few fishes.
skeletal abnormalities observed is due to malnutrition. The carnivorous fish fed with high level of dietary
In Japanese flounder larvae high levels of dietary carbohydrates expresses the lower level of its utilization
retinoic acid results in the development of bone due to the atypical hepatic regulatory mechanism.
deformities which occurs due to retinoic acid In rainbow trout and gilthead sea bream, dietary
dependent depression of a transcriptional factor, carbohydrate feeding induce the synthesis of first
sonic hedgehog (shh). phosphorylation enzyme, glucokinase which is related
to higher levels of glucokinase gene expression. In
Lipid metabolism case of endogenous glucose production the enzymes
such as glucose 6 phosphatase, fructose 1,6 bis-
The substitution of fish oil with plant based oil phosphatase and phosphoenolpyruvate carboxykinase
is the prevailing scenario in aquafeed industry. are very much essential. These are expressed based
The knowledge on molecular level of fatty acid on the dietary levels of carbohydrates in most of
biosynthesis particularly C20 and C22 from C18 will the fishes but in some of the fishes especially in
give the information on the manipulation of fatty rainbow trout, the exogenous glucose production
acid bioconversion pathways through the series of is not affected by dietary glucose level. The reason
fatty acid desaturation and elongation reactions for for absence of the hepatic gluconeogenic enzymes
efficient utilization of plant based oils in aquafeeds. regulation may be due to high levels dietary fatty
In general freshwater fishes can express almost all the acids and gluconeogenic amino acids. The need of

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

enzymes responsible for long chain highly unsaturated the hour is to study the gene level expression on the
fatty acid but marine fishes are inefficient in the utilization of dietary carbohydrates.
production of sufficient level of DHA due to the
deficiencies of one or more enzymes of the pathway. Phosphorous regulation
The expression of ∆6 desaturase by the dietary lipids
and carbohydrate sources was noticed in marine Dietary phosphorous utilization in most of the fish
fishes in recent studies. But, the ∆5 desaturase gene species is around 50% of the original level. This can
was not characterized in marine fish in contrast to be tackled by the understanding the mechanism
freshwater fish. of absorption of dietary phosphorous by fish. The
genes associated with phosphorous metabolism
Dietary lipid content influences the body particularly such as type II sodium phosphate co-transporter,
the muscle lipid content in fish. The key enzymes intestinal meprin 1A and cysteine sulphinic acid
mainly the lipoprotein lipase is necessary for lipid decarboxlase are essential bio-indicators for the
transport and storage. In rainbow trout and red sea dietary phosphorous optimization in addition to the
bream, this enzyme expression studies revealed that traditional phosphorous estimation methods.
this enzymes works similar to the lipase enzymes of
higher vertebrates. In some fishes, lipoprotein lipase Proteomics
gene expressed in a tissue specific manner according
to the nutritional status of the fish that indicates the Proteomics is the study of all the proteins (proteome)
lipid storage based on the tissue. in a particular cell, tissue or an organism. The
proteome represents the protein equivalent of the
Glucose metabolism genome, which is determined by the sequence, the
type and number of its nucleotides. In contrast to
The carnivorous fishes generally do not utilize dietary static nature of the genome, the proteome represents
carbohydrates and the level of utilization varies species a tremendously dynamic object, which is influenced

459
 Reproductive endocrinology in
Aquaculture with special reference to
captive maturation of penaeid shrimps
C. P. Balasubramanian* and K. K. Vijayan
Principal Scientist
Central Institute of Brackishwater Aquaculture,
75, Santhome High Road, R.A. Puram, Chennai 28
e-mail: [email protected]

Introduction food production system. During 1980s there was an

upsurge for the export oriented agricultural crops,
Fish and fisheries rank top among all the global and shrimp farming in India is paradigmatic example
natural resources and fish has been considered to for this. In aquaculture, there are certain areas
be the last wild food of humans (Walsh 2011). It where science can play crucial roles. For example:
contributes about US $ 100 billion annually to reproduction, early development, nutrition, health
global trade, which is greater than the GDP of 70 and genetics (Donaldson, 1996). The most essential
world nations (McClanahan et al. 2015). About 520 characteristic of an aquaculture species is the ability
million people, i.e. 8% of world population, directly to control reproduction and produce viable offspring
or indirectly depend on marine fisheries (Sumaila et from captive broodstock. Many aquaculture species,
al. 2011). Fish has been acknowledged as the major however, faces some form of reproductive dysfunction
nutrient dense animal source protein for a significant in captivity. This dysfunction is largely due to dramatic
proportion of nutritionally vulnerable people. It difference in the environmental conditions faced
overshadows most terrestrial generated animal foods, by animal in natural environmental conditions and
and fish production was double that of poultry and captivity. The captive environmental conditions, which
three times of cattle. The unique long chain poly lack the natural stimuli, fail to induce appropriate
unsaturated fatty acids and highly bioavailable endogenous response in fish. It is almost impossible
essential micronutrients, which are not readily to simulate the natural conditions for breeding under
available in most terrestrial food resources, make it the captivity for many aquacultured species.
as unique food which is highly essential for the adult
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health and cognitive development for the children. Complete control over the reproduction is the most
With all these widely acknowledged importance, there elusive goals of almost all form of aquaculture. It
has been a growing concern about the sustainability is crucial for seed production, selective breeding,
of fisheries. Sustainable harvest of wild fisheries has growth rate, feed efficiency, meat quality and
been central theme of many international fora, media biosecurity (Weber, 2009). Among all, seed
headlines, scientific publications and environmental production is the primary need of development
campaigns as most wild fisheries are under crisis of any form of aquaculture. Sustainability of any
due to overfishing. Aquaculture has often been aquaculture, therefore, largely depends on how
considered to be the best option to meet the deficit efficiently aquaculturists can manage reproduction
of the capture fisheries. under captivity. Some species reproduce more easily
than others. For example, Indian white shrimp,
Although aquaculture has been vogue in Asia, the Penaeus indicus, is relatively easy to breed species
modern scientific aquaculture is youngest of all when compare to tiger shrimp, Penaeus monodon.

460
Table: Selected candidate genes in fish nutrigenomic study:

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

(Source: Panserat & Kaushik, 2010)

by a variety of parameters. However, arraying of Metabolomics

proteins is more difficult than the arraying of DNA,
because they have to maintain their correctly folded The study of metabolites present in a cell, tissue
conformations. The major tools of proteomics study or organism is known as metabolomics. The
are two dimensional gel electrophoresis (2D) for metabolomic study includes the essential steps such
separation of complex protein mixtures and mass as separation of analytes, detection, identification
spectrometry (MS) for identification and structure and quantification of the analytes/metabolites. The
analysis of protein. In a typical proteomic study, separation of analyte is done by chromatographic
protein extraction followed by high resolution 2D techniques such as Gas Chromatography (GC),
electrophoresis along with gel image analysis permit Capillary Electrophoresis (CE), High Performance
the identification and expression of n-numbers of Liquid Chromatography (HPLC) and Ultra Performance
protein molecules. The hepatic proteomes of fishes Liquid Chromatography (UPLC). The detection and
is used to study the effect of quality protein, plant identification of metabolite is carry out by Nuclear
based protein in fish meal replacement and fasting on Magnetic Resonance Spectroscopy (NMR) and Mass
the transport and primary energy generation, cellular Spectrometry (MS). In fish nutritional research the
protein degradation, bile acid biosynthesis etc. metabolomic study is still in its infancy stage.

461
 However, this relative easiness in breeding may not and external environment. These two systems have been
be a desirable quality in grow out production system, evolved in animals to initiate and coordinate appropriate
where reproductive growth adversely affect the responses. These stimuli may either be an active danger
somatic growth of the animal. Conversely, delayed posed by the predator or may be a gradual threat
maturation in P monodon is a desirable characteristic posed by seasonal environmental change. Although
in grow out production system. However, delayed these two systems are morphologically different, both
maturation of a species hampers the seed stock the system will operate fundamentally in similar ways.
production and selective breeding. These diverse needs The nervous system is organized as cellular network,
illustrate that each species require different levels where axons direct information via chemical messengers
of control over reproduction, and even within the called neurotransmitters, and these neurotransmitters
same segment of the industry. Whatever the goal of are secreted into synapses and act on target located on
aquaculturists to control or manipulate reproduction, the other side. Neurotransmitters can also diffuse into
the progress in gaining control over reproduction circulatory system and act on distantly located target
process is dependent on our greater understanding of tissue as hormones do. These neurotransmitters that act
mechanism controlling these processes. This chapter outside the synaptic cleft are called neurohormones. In
summarizes the present understanding on control of addition to endocrine and neurohormones, hormones
reproduction in aquaculture with special reference to have a broader usage in recent years (Table 1).
crustacean reproductive control. The first part of this
chapter is a preamble to the general endocrinology Method of hormone delivery: The specific
providing the general concepts and principles in receptors in the target cells determine the selectivity
endocrinology. The second part deals with history hormone action. Receptors are protein present in the
of endocrinological applications of aquaculture, and target cells and bind a particular hormone and initiate
third part provides present understanding of penaeid a response. There are two types of receptors: cell
endocrinology, endocrine methodologies and future surface receptors and intra cellular receptors (Fig 1).
perspective for the application of endocrinology Protein/peptide hormones use cell surface receptors
whereas steroid hormone uses intracellular receptors.
Endocrinology: a preamble
When the life began as a unicellular organism, about
three billion years ago, the time was simpler and
communication between cells (organisms) were modest
than that those required to maintain multicellular
organisms. During the course of evolution the
multicellular organisms developed a communication
system through chemical signals that coordinate
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multiple organic functions. The substances that provide

a chemical basis for communication between cells are
Figure 1 Action of peptide hormone on target tissue
called hormones. The investigation of these chemicals
in early years of twentieth century gave birth to a new How do hormones function: Hormones modulate
discipline, the endocrinology (Fingerman, 1997). In the activity of target tissues, and effects of hormones
strict sense, endocrine glands are specialized organ are found even long after the levels of hormones
that delivers their products, the hormones, directly into returned to the basal levels. They are usually present
the interstitial spaces and enter circulatory system. In in tracer levels ranging from 10-6 g/ml to 10-9 g/
early years, the endocrinology is confined to the study ml. Combined effect of more than one hormone on
of these specialized cells. Later, it was demonstrated a biological response will be in number of different
that nervous system is also closely associated with the ways (Fig 2). The action of these hormones may
functioning of endocrine system in order to respond to be additive, if they cause same response. Thus the
varieties of stimulus and contingencies in their internal combined effect is simply sum of the response of

462
 Training manual in Molecular Biology and Biotechnology for Fisheries Professionals
Table 1 Terminology of different hormone producing organ

for another hormone to have an effect

Hormones and aquaculture

Husbandry of aquatic organisms is an ancient enterprise.
However, the modern science and technology applied
to it is only in the past quarter of the century. Fish
endocrinology has been developed as a basic science
with little applied interest. The past fish endocrinologists,
largely confined the physiological and evolutionary
aspects and not with fish production. Only recently,
Figure 2 Various forms action of hormones on target tissue results
the endocrinologists have become concerned on
different types of responses. aquaculture. Now aquaculture and ranching of salmon
two individual hormones. Sometimes two hormones are considered to be the domain of fish endocrinologists.
have same biological effect, but the combined
effect may be non- additive. In still different way Initial studies on endocrinological applications in
the combined effect will be more than the additive biology largely confines to describe the effect of
effect of individual hormone effect, and it is known a hormone at organismal level. For example: how
as synergistic. In some cases some hormones will does hormonal therapy influences viable spawning?
not have effect on their own but must be present Although target and applicability of this approach

463
 is obvious; this approach fails to investigate the extract are another threat of the use of pituitary
fundamental biology of a particular hormone. extract. Although not totally reliable, hypophysation
was not abandoned.
Hormonal application in induced breeding started in
Brazil, when fish pituitary glands were injected to induce This first generation technology paved the way for
maturation. This is originally described by Argentinean the second generation technology, which involve
physiologist, B. A. Houssay who recorded precocious the purified human chorionic gonadotropin (hCG).
spawning in viviparous fishes when they administered Although it has been proved to be success in many
with crude extract of the pituitary gland. The role species, owing to its species specificity it did not work
of pituitary gland (hypophysis) in the reproduction in many species (Zohar, 1989). This hormone has high
of vertebrates may considered to have commenced molecular weight and when injected these hormones
three years before, when S. Aschheim and B. Zondek stimulate the immune system of fish and adversely
found that pituitary implants accelerated the sexual affect the health of the animals. The discovery of
development and cycle of female mice, and P. E Smith for the portal (closed) circulation between pituitary and
the first time unequivocally demonstrated the effect of brain initiated the discovery of the gonadotropin
hypophysectomy and subsequent replacement therapy releasing hormone (GnRH) (Bowers et al. 1970). These
on the gonads of the laboratory rats (Allen,1939). This discoveries radically changed our thoughts on the
new finding came to the attention of R. von Ihering, organization of the hormonal system, but some time
who was the Director of the Comissao Tecnica de passed before they affected aquaculture practices.
Piscicultura do Nordeste at Ceara, Brazil, who had Bretton and Well (1973) used the brain hormone for
been facing the problems of failure in spawning in the first time to release gonadotropic hormones from
native fresh water food fish. Even when caught in the pituitary in fresh water carps. Later many workers
nearly ripe condition these species rarely completed the used the GnRH for inducement of spawning in fishes.
maturation. In nature, spawning precipitously followed The advantages of use of brain hormones are obvious:
the first storms of rainy season, but these environmental pituitary gonadotropins differ in species used for
cues could not be duplicated in the captivity. By 1934, aquaculture in their molecular structure and success
von Ihering had developed successful techniques to rate is unpredictable in many cases. Brain hormones
induce ovulation using fish pituitary- this is generally have undergone little change during evolution
known as hypophysation. During almost similar period, (McCeery et al. 1982), and thus same peptide molecule
Russians were also discovered successful spawning and may well activate the gonadotrophs of pituitary of
breeding of sturgeon using freshly removed pituitary any vertebrates. Further, being low molecular weight
gland extract. These findings were in fact a fortunate compound, the immunogenitic potential of GnRH is
discovery as hydroelectric stations and dams were built in far less than the pituitary hormones. GnRH molecules
rivers and it prevents the upstream spawning migration can be modified by replacing amino acids, and the
of sturgeon. resulted products in vivo degraded far slower than
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the native forms. The use of GnRH in the induced

Although hypophysation revolutionized fish breeding of finfish is the third generation technology
culture, and contributed enormously towards the and it has attracted significant interest in recent
development of finfish aquaculture, it suffers from years especially: the discovery of new natural forms
number of limitations. Crude pituitary extract vary of GnRH, the development and testing of potent
in their content of gonadotropin depending upon GnRH analogue and the development of novel
state of maturity of donor fish, and in addition to the means of administration of hormones (Donaldson,
gonadotropin it contain a range of other hormones 1996). In some teleosts gonadotrophs of pituitary are
which may have a synergistic and antagonistic effect innervated by nerve fibers producing the dopamine,
on reproductive process. It is extremely difficult to the neurotransmitter inhibits the production of
quantify the gonadotropin content of pituitary extract gonadotropin 2. A combination of therapy with
and standardize the dosage (Zohar, 1989). The dopamine antagonists have been found successful
chances to the transmission of pathogen via pituitary in some cases, where GnRH alone does not provide

464
successful spawning. The most recent development in of reproductive maturation is successfully conducted
induced maturation of fin fishes is the incorporation by Aquaculture team of Centre Océanologique du
of GnRH analogue into a polymeric sustained-release Pacifique (Aquacop). Alain Michael, the head of the
Aquacop, serendipitously found that marine shrimp,
Penaeus aztecus, who had lost the eye, matured and
spawned in the tank. This observation, he connected
with the work of Panouse, and developed the most
revolutionized technology in the captive breeding
of shrimp. It had far reaching impact on crustacean
aquaculture in general and penaeid shrimp farming
in particular. The first successful maturation and
spawning of P. monodon was achieved by Santiago
(1977), although Alikunhi et al. (1975) achieved
maturation with unviable spawning. The great
majority of the captive maturation has been from
ablated females, although few workers have reported
maturation in unablated females (Santiago, 1977,
Primavera, 1978. Emmerson, 1983), only Emmerson
(1983) was successful in obtaining viable spawning
Figure 3 Schematic representation of hypothalamus-pituitary-gonad-
(16.7 to 82% hatch rate). Although eyestalk ablation
liver axis in finfish
started as a stop- gap procedure to induce maturation
delivery system, which releases the hormone over a and spawning in penaeid shrimp in early 1970s, this
period of days. The current model for the control of procedure has been continuing in commercial seed

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

reproduction of fin fishes is presented in the figure 3. production industry across the world.

Reproductive endocrinology The most acknowledged consensus of crustacean

reproductive endocrinology is that reproduction
of penaeid shrimp and
is controlled by two antagonistic hormones, one
shrimp aquaculture inhibits (Vitellogenin inhibiting hormone or Gonad
Endocrinological/neuro-endocrinological studies inhibiting hormone, V/GIH) and other stimulates
on crustaceans started in first half of the 20th (Vitellogenin stimulating hormone, Gonad stimulating
century. Generally, in invertebrates, oogenesis can be hormone, V/GSH). This simple endocrine axis has
stimulated by a release from inhibition or by a secretion been questioned by many recent researchers and
of a stimulator (Schuetz, 1969). Ovarian maturation postulated a multi hormonal system involving several
(oogenesis) in Crustacea is said to be stimulated by neuroenodcrine and endocrine pathways, involving
the gonad stimulating hormones secreted by the neurotransmittes (serotonin or 5 hydroxytreptamine),
brain and thoracic ganglia and inhibited by Gonad steroids (progensterone, estradiol), terpenoids(methyl
Inhibiting hormones (GIH) of the eyestalk (Adiyodi farnesoate) and vertebrate peptide hormones (GnRH).
and Adiyodi, 1970). The antagonism of eyestalk may Additionally the role of other neurohormones in
be reduced by a decline in the titre of the GIH as the the CHH family (i.e. MIH and CHH) in reproduction
shrimp grows and moves into an environment suitable in penaeid shrimps has also been reported. Thus
for spawning. Final spawning act may, in fact, be crustacean reproduction is an end result of multiple
triggered by a stimulus, either visual or hormonal vitellogenic related endocrine cascades (Figure 4).
originating in the eyestalk (Muthu and Laxmynarayan, Nevertheless the bi hormonal axis is still central to
1982). Panouse (1943) demonstrated that the removal the shrimp reproductive endocrinology.
of the eyestalk of palaemonid shrimp would lead to
ovarian development and spawning. However, the The neural portion of the decapod eyestalk is an
first use of eyestalk ablation procedure for inducement extension of brain (supra-esophageal ganglion). A

465
 peptides are recognized type 1 and type 11; type
1 peptides are cHH sensu stricto are with typically
72 amino acids. Their protein precursor contains
cryptic peptide, cHH precursor related peptide (CPRP),
between signal peptide and cHH progenitor sequence.
This type 1 cHH is named because upon injection it
elicit hyperglycemia in animals. Type 11 peptides are
not with CPRP and it contains three neuro-hormones:
Gonad/vitellogenesis inhibiting hormone (G/VIH),
Molt inhibiting hormone (MIH) and Mandibular organ
Figure 4 Schematic representation of shrimp reproduction and various inhibiting hormone (MOIH).
factors controlling reproduction. CNS: central nervous system; GIH:
Gonad inhibiting hormone; GnRH: Gonadotropic releasing hormone;
GSH: Gonad stimulating hormone; 5HT: 5 hydroxy tryptamine; HP:
hepatopancreas; LG: Lamia ganglioris, MF: methyl farnesoate; MT:
Gonad/vitellogenesis
Medulla terminalis; ME: Medulla externa: MI: Medulla interna; MIH:
Molt inhibiting hormone, MOIH: Mandibular organ inhibiting hormone;
inhibiting hormone:
Vg: vitellogenin
G/VIH actively participate in ovarian development and
group of cell bodies usually found as faint blue white is a key hormone for the reproduction in crustacea.
in live specimens is located in the middle portion It is believed that GIH is more intense than any other
of the medulla terminalis is termed as X organ. At hormone in crustacea. Gene coding for GIH has been
least eight neuro-hormones appear to be synthesized characterized and cloned from several crustaceans: For
in the X organ and it contains about 150 -200 example, terrestrial isopod (Armadillidium vulgarae),
neurosecretory cells. The neuro hormones produced lobsters (Homarus americanus H. gammarus and
in these cells are transported via axon and ends in Nephropse norvegicus) and shrimps (P. monodon,
the blood sinus called sinus gland in the medulla Metapenaeus ensis, Litopenaeus vannamei), prawn
externa (Figure 5). These hormones regulate several (Macrobrachium nipponnese) and deep sea shrimp
physiological functions for example, gonad activity, (Rimicaris Kairei). GIH is expressed in both male and
molting, and blood-sugar level. CHH or crustacean female; it has been expressed in tissues such as eyestalk
hyperglycemic hormone family are structurally related and brain. The presence of GIH has also found in larval
neuro-hormones of X-organ. Two sub types of cHH crustacean as well. The expression of GIH mRNA is
found to be lower in immature stage, however it was
found to be higher in previtellogenic stage.

Gonad stimulating hormones/

gonadotropins
Central Marine Fisheries Research Institute

It is generally accepted that identification of gonad

stimulating hormones (GSH) in crustaceans would
greatly expedite domestication and closing of life cycle
of commercially important aquacultured crustaceans
that do not reproduce readily in captivity. Research on
GSH has been started in 1960s. A substance known
to stimulate vitellogenesis has been found in brain
and thoracic ganglion of several crustacean species
(Otsu, 1963; Gomez, 1965). But the chemistry of
this species is not yet elucidated. De Kleijn and Van
Herp (1998) suggested that this hormone might be
Figure 5 Schematic diagram of eyestalk showing X organ, sinus gland a crustacean hyper glycemic hormone.
and optic nerve

466
Methyl Farnesoate: Parallel to the search for (MOIH). The inhibition is artificially reversed in vivo by
GSH, studies on other gonad stimulating hormones eyestalk ablation. However, MF can be applied directly,
(gonadotropins) have been carried out by several and in many cases successful ovarian maturation and
researchers. These investigations results in the spawning have been achieved as well. Table 2 shows
discovery of a crustacean hormone, Methyl farnesoate that experimental results of MF treatment to study the
(MF) (Laufer, 1987). MF is an intermediate compound effect of this hormone on reproduction of females.
produced during the juvenile hormone biosynthetic
pathway (Fig 6) juvenile hormone is well known and Ecdysteroid: Ecdysteroid is a polyhydrated keto
extensively studied in insects where they play several steroid, found in most arthropods and have a
regulatory roles both as gonadotropin in adults and primary function as molting hormone. In crustacea
morphogens during development. Juvenile hormone ecdysteroids are synthesized in ‘Y’ organ (=ecdysal
as such is not found in crustaceans, but MF is isolated gland), and the alternative sources of Ecdysteroid is
from mandibular organs of crustaceans. Mandibular the epidermis, and ovary. It has been reported that
organ actively synthesize MF during vitellogenesis Ecdysteroid has a possible role in reproduction and
and become less active during non-reproductive maturation as in the case of insects (Subramoniam
periods. The secretion of MF is tissue specific and 2000). It has been suggested that crustacean
circulated through the blood, and circulating levels hormones are multifunctional in nature, a single
are positively correlated with the reproductive state hormone can mediate different functions. Ecdysteroid
of females. It has also been shown that mandibular mediate as hormone that promotes protective
organ is negatively regulated by an eyestalk neuro membrane in embryos, then they function as molting
hormone, mandibular organ inhibiting hormones hormone from larvae to adult life. In adult they
function as gonadotropin. Chang (2001) called it as
an ‘amazing economy of nature’

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Vertebrate-like steroids: Several decapod crustaceans
have the ability to synthesize the vertebrate-type steroids
(e. g. progesterone, 17-β- estradiol, and testosterone).
Some of them fluctuate during the reproductive cycles,
and therefore, indicates their role in the reproduction.
Many crustaceans have been found to be responding
to the injections of these steroids. In additions to the
steroids, biogenic amines such as, 5 hydroxy tryptamine
(serotonin) are also found to have ovarian stimulatory
Figure 6 Schematic diagram of eyestalk showing X organ, sinus gland effect. Recently Wongprasert (2006) reported that
and optic nerve serotonin injected P. monodon had ovarian maturation
Table 2 Effect of methyl farnesoate on crustacean reproduction
Species Mode of study Nature of response Reference
Triops longicudatus In vitro No effect Riley and Tsukimura 1998
Triops longicudatus In vivo Inhibition Tsukimura et al. 2006
Litopenaeus vannamei In vitro Reproductive stimulation Tsukimura and Kamemoto 1991
Litopenaeus vannamei In vivo Improvement of reproductive Laufer 1992
performance
Sicyonia ingentis In vivo Reproductive stimulation Balasubramanian et al. 2010
Penaeus monodon In vivo Improvement of reproductive Hall et al. 1999
performance
Penaeus monodon In vivo Reproductive inhibition Marsden et al. 2008
Macrobrachium rosenbergii In vivo No effect Wilder et al. 1994
Homarus americanus No effect Tsukimura et al. 1993
Procambarus clarkii In vivo Reproductive stimulation Laufer et al. 1998

467
 and spawning similar to that of unilateral eyestalk analytical methods (Table 3). Today, assay of peptide
ablated females. Although the mode of action of hormones take advantages of powerful recombinant
these hormones/neuro transmitters is not properly DNA and antibody technologies. If aminoacid
understood, there seems to be a tremendous potential in sequence of a hormone is known, it is possible to
using these hormones to stimulate gonadal maturation synthesis nucleic acid probes that can recognize gene
in the aquacultured species. that codes for the hormone. The knowledge gene
sequence also helps to understand the temporal and
In summary, the crustacean egg production is spatial pattern of expression of mRNA
controlled by a cascade of hormonal activities which
is triggered by environmental and nutrional factors Future Directions of reproductive
There fore, all these factors should be considered
physiological research
when any reproductive technology is developed.
Basic research on reproductive physiology/endocrinology
Endocrine methodologies inevitably leads to the advances in applied research and
eventually the practices of aquaculture. Achievements
Like most other discipline progress in endocrinology is in the fin fish reproductive physiology is an excellent
largely depends on the analytical techniques. The most example for how basic endocrinological findings can
important step in the endocrinological study is the be effectively integrated into broodstock management
characterization of endocrine system, and understand (Zohar and Mylonas, 2001). On the contrary, we are
how different organs function. This knowledge can
Table 3 Conserved hierarchy of endocrine methodologies.
be used to improve the reproductive performance of Sl no Study level Techniques
the animals. The information needed to characterize 1 Gene mRNA expression pathways (RT PCR, qRT
the hormone are: chemical structure of the hormone, PCR, microarrayn, Northern Blot
biosynthesis and storage, and pathway of metabolisms. 2 Protein PAGE, western blot, ELISA
3 Functional Histology Histochemistry
In most cases the fist assay when dealing with an cell Immunohistochemistry,In-situte hybridization
unknown hormone is a bioassay. It is based on the
measurement of physiological response caused by a still far from achieving adequate understanding on
hormone. In a bioassay, the hormone is injected directly control of reproduction in penaeid shrimp. The growth
into the animal to observe the phenotypic effect, or of penaeid shrimp aquaculture has been spectacular
it is dissolved in a medium in which an appropriately and regarded as a remarkable success story of modern
responsive tissue can be cultured. An ideal bioassay is aquaculture. Twenty five years ago, Primavera (1985)
one that produces a graded response to the hormone, identified the replacement of eyestalk ablation and
rather than an all or none response. The response of the development of low cost maturation diet as key goals
assay is then transferred to numerical scale, by which to increase spawner yield and reduce the production
the relative activity of the hormone can be quantified. cost of shrimp culture industry. Unfortunately, even
Central Marine Fisheries Research Institute

Hormone concentrations are generally expressed in after twenty five years later, endocrine replacement for
titers because they are usually determined indirectly eyestalk ablation yet to be achieved. When we look into
by progressive dilution (titration) of the hormone the global scenario of farmed shrimp production, there
sample being tested until its activity in the bioassay is an obvious shift at production level. While P. monodon
is no longer detectable. The goal of the bioassay is production remains at constant level, there is a drastic
to detect the approximate concentration of hormone hike in the production of Litopenaeus vannamei. The
normally occurs in the animal. When excessive poor performance of P. monodon is essentially due
hormone is used in bioassay a non-specific response to the non-availability of specific pathogen free (SPF)
may occur. This abnormal physiological response is broodstock. The most significant hindrance for the
called pharmacological response development of SPF broodstock is the poor reproductive
performance of captive P. monodon. Although findings
Chemical structure, biosynthesis and pathways on reproductive physiology of penaeid shrimps have
of hormones can be studied by various chemical been increased during the recent years, findings are

468
 Breteon, B., and Well, C. 1973. Endocrinologie compare-effets
still inadequate to resolve sufficient background for du LH/FSH-synthetiqeu et d’extrats hypothalmiques de carpe
successful broodstock management. Information on sur la secretion d’hormone gonadotropique in vivo chez la
carpe (Cyprinus caprio L). C. R. Hebd. Seances Acad. Sci.
the key hormones involved in reproduction is being 277: 2061-064
just published. Bondad-Reantaso, M. G. and Subasinghe, R.P. 2005. Aquatic
animal diseases and their economic importance: a global
perspective. Aquaculture Health International 1: 4-5
Many of the research work in crustacean physiology are Durate, C. M. Marba, N. and Holmer, M. 2007. Rapid domestication
criticized as semi scientific (Adiyodi and Subramoniam, of marine species. Science, 316: 382-383
Donaldson, E. M. 1996. Manipulation of reproduction in farmed
1985) or lack sound experimental design (Benzie, fish. Animal Reproduction Science. 42: 381-392
1997). Further, most endocrinological studies in Emmerson W. D. 1983. Maturation and growth of ablated and
aquaculture are based on non-model wild caught unablated Penaeus monodon Fabricius. Aquaculture 32: 235-
241.
animals, and, therefore, the likelihood for the genetic FAO 2007. Fishereis Report No 819, 262p
and physiological heterogeneity is extremely high. The Fingerman, M. 1997. Roles of neurotransmitters in regulating
reproductive hormone release and gonadal maturation
data generated by these studies does not have much I decapod crustaceans. Invertebrates Reproduction and
value to develop a model for reproductive control. Development, 31: 47-54
Gomez, R. 1965. Acceleration of development of gonads by
implantation of brain in the Paratelphusa hydrodromus.
The endocrine manipulation for induced maturation Naturwissenschaften 9-216
and spawning should be made at different Kirk, A. 1987. A history of marine fish culture in Europe and North
America, Farnham, Fishing news Ltd.
physiological command levels. The choice at which Laufer et al. 1987. Identification of a juvenile hormone-like
level these interventions to be made are largely compound in crustacean. Sceince 235: 202-205
determined by at which level reproductive cycle McCreery, B. R. et al. 1982. Action of agonist and antagonistic
analogs of gonadotropin releasing hormone (GnRH) in
disruption occurs due to the impact of captivity. These the bull frog, Rana catesbeiana. General and comparative
disruptions vary from simple inhibition of spawning endocrinology. 46: 511-520
Muthu, M. S. and Laxminarayana A. 1977. Induced maturation
to complete lack of gonad development. The success

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

and spawning of Indian penaeid prawns. Indian Journal of
of endocrine manipulation largely depends upon Fisheries 24: 172-180
the physiological stage of female. If female is not Overturf, K. 2009. Convergence of Aquaculture and Molecular
Biology. In Molecular Research in Aquaculture (ed. Overturf
competent for oocyte maturation, it is less likely for K.), Wiley Blackwell, 408 p
having responded for hormonal therapy. Panouse, J. B. 1943. Inflence de I’ablation de pedoncule oculaire
sur la croissance de I’ovarie chez la crevette Leander serratus.
Comptes Rendus Hebdomadaies des SeancesdeI’ Academie
Modern technology has shifted endocrine research des Sciences, Paris, 217: 553-555
from traditional reductionist approach (part by part Primavera J. H. 1985. A review of maturation and reproduction in
closed thelycum penaeids. In: Y. Tak, J. H. Primavera and J. A.
study) to an integrative approach. Although traditional Liobreara (eds. ), Proc. Ist international Conf. of Cult. of penaeid
approach progressed the science of endocrinology, it prawns/shrimps. SEAFDEC, Illoilo City, Philippines, pp. 47-64
Primaveral J. H. and Boriongan E. 1978. Ovarian rematuration of
has limitations. The integrative approach investigates ablated Sugpo prawn Penaeus monodon Fabricius. Ann Biol.
the complex system at a time. The modern molecular Anim. Biochem. Biophys. 18: 1067-1072
techniques such as microarray techniques would assist Santiago, Jr., A.C. 1977. Successful spawning of cultured Penaeus
monodon Fabricius after eyestalk ablation. Aquaculture
to unravel the many complexities of endocrinology. 11: 185-196
The selective breeding for the high reproductive trait Smith, M. D., Roheim, C.A. Crowder, L.B., Hapern, B.S., Turnipseed,
M., Anderson, J. L., Asche, F., Bourillon, L, Guttormsen, A. G.,
is found to have tremendous scope in improving the Khan, A., Liuori, L. A. McNevi, A., O’Connor, M.I., Squires, D.,
captive maturation of penaeid shrimps. Tyedmers, P., Brownstein, C., Carden, K., Klinger, D.H., Sagarin,
R. and Selkoe, K. A 2010. Sustainability and Global Seafood.
Science, 327: 784-786.
Suggested readings Soyez, D. Van Deijnen, J. E., and Martin, M. 1987. Isolation and
characterization of a vitellogenesis inhibiting factor from
sinus glands of the lobster Homarus americanus, Journal of
Alikunhi, K. H. and Poernomo, A., Adisukresno, S, Budiono, M., Experimental Zoology 244: 479-484
and Busman S. 1975. Preliminary observations on inducton Zohar 1989. Endocrinology and fish farming: aspects in
of maturation and spawning in P. monodon Fabricius and reproduction, growth, and smoltification. Fish Physiology and
Penaeus merguinesis de Man by eyestalk extirpation. Bull. Shr. Biochemistry, 7: 395-405
Cult.Res. Cent. 1: 1-1 Zohar, Y. and Mylonas, C.C. 2001. Endocrine manipulation
Allen, 1939. Sex and internal secretions. Williams and of spawning in cultured fish: from hormone to genes.
Wickins, Baltimore, Aquacultlure 197: 99-136

469
Protocols
Basic Tools in the Biotechnology
Laboratory

A training in Molecular Biology can’t be initiated single drop and as large as a few milliliters.
without imparting a basic knowledge about the They are not graduated and are not used to
common lab equipment pieces and describe measure volumes.
their function. b. Beral pipettes: (transfer pipettes) are plastic
pipettes with a bulb at one end used for transfer
Measurement of Volume of liquids. Sometimes they have calibration marks,
which have a low level of accuracy. They are often
Erlenmeyer flasks: are used primarily to prepare disposable, sterile and individually wrapped.
solutions prior to an accurate volume adjustment. c. Serological: or “blowout,” pipettes are
Although there are volumetric markings on these graduated glass tubes used to measure anywhere
flasks, they are not calibrated and should not be relied from 0.1 to 50 mL. When the liquid has drained

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

upon for exact volume measurements. from this pipette, the final drop in the tip is
transferred with a puff of air.
Beakers: are also used for preparing solutions, d. Mohr: or “to deliver,” pipettes are similar to
especially when a pH adjustment requires access to blowout pipettes, but do not require a puff of
the solution by a pH probe. The volumetric markings air to accurately deliver the desired volume. They
on beakers are also not reliable. can be identified by the label ‘TD’ on the top.
e. Volumetric pipet: s are not graduated, but
Graduated cylinders: are calibrated with sufficient are carefully calibrated to deliver a single, highly
accuracy for most volume measurements when accurate volume, and are used for the transfer
preparing solutions. For example, the calibration of of exact volumes.
most 100 mL graduated cylinder can be relied upon f. Micropipettes: are calibrated to deliver highly
to accurately measure to within +/-0.6mL. accurate volumes generally less than 1.0 mL, and
as little as 0.1 microliter. They are often adjustable
Volumetric flasks: are used to measure a specific for measuring different volumes and they always
volume with the highest degree of accuracy, and are use dispensable plastic tips to actually transfer
used to make standard solutions for analytical assays. the liquids.
For example, the calibration of a 100 mL volumetric g. Multichannel micropipettes: can deliver
flask can have an accuracy of +/-0.1 mL. the same volume from as many as 12 tips
simultaneously. All micropipettes need regular
pipettes: are glass or plastic devices that are routinely maintenance, calibration, and validation.
used to measure and transfer liquids by drawing the
liquid into the tube with a bulb or mechanical pump. Measurement of Weight
a. Pasteur pipettes: are small glass tubes used Instruments for weighing materials are called
with a bulb to transfer volumes as small as a balances, and most laboratories have more than one

473
 type of balance, depending on the amount of material plastic or glass and which usually has an internal
being measured and the degree of accuracy required. diameter of 1.0 cm, is filled with the solution and
placed in the spectrophotometer for measurements.
a. Mechanical balances: weigh an object on a pan
hanging from a beam that has a counterbalanced • An ultraviolet/visible (UV/VIS) spectrophotometer
weight. We do not use mechanical balances in can also measure absorbance of light in the
our lab. ultraviolet region of the spectrum (about 100-
b. Electronic balances: have replaced most 400nm). These spectrophotemeters require a
mechanical balances due to their greater accuracy halogen light bulb that emits ultraviolet light and
and ease of operation. They are easier to use require special cuvettes that don‘t absorb UV light.
because they usually have a digital readout, and
weighing dishes can be tared to read zero mass • A scanning spectrophotometer can measure
before using. Most balances used for preparation the absorbance of a solution over a range of
of solutions have a sensitivity of +/–0.01 g, but wavelengths, creating an absorbance spectrum that
electronic analytical balances can be sensitive to can be used to identify substances in a solution.
+/–0.1 mg or less. Electronic balances require
routine maintenance and recalibration • A NanoDrop spectrophotometer is a brand of
scanning UV/VIS spectrophotometer that allows
Measurement of pH the user to measure the absorbance of a very small
sample of liquid (1-2 uL). This instrument makes it
Most solutions prepared in the biological laboratory easy to quickly evaluate the quality and quantity
must have a carefully controlled pH. Buffers are of nucleic acids or proteins in a small sample prep.
prepared by adjustment to a specific pH with strong
acid and base solutions, using a meter to monitor • A microplate reader is a spectrophotometer that
the pH. A pH meter is a volt meter that measures can measure the absorbance in the individual wells
the electrical potential between two electrodes. One of a plate. Usually the plates are 96 wells, but other
electrode is in contact with your solution, and the formats are available, such as 48 wells and 384 wells.
other is in contact with a reference solution. Usually This allows the user to prepare and read many small
both of these electrodes are combined in a single pH samples at once, saving time and money. Microplate
probe that you place in your solution. These meters can readers may also be capable of reading fluorescence
read to the nearest 0.1 pH unit, but require frequent and chemiluminescence, which are two types of light
calibration with reference buffers of known pH. emission that are frequently used in biologic al research.

Measurement of light Solution Preparation

Central Marine Fisheries Research Institute

Solutions are often analyzed in the biotechnology lab Solution preparation involves mixing liquids and
by measuring how the solutes interact with light. A dissolving solids in liquids. There are many specialized
spectrophotometer measures the amount of light that devices in addition to balances, volume measuring
is absorbed by a solution at a specific wavelength or over devices, and pH meters involved in these processes.
a range of wavelengths. If you know a wavelength at
which a specific substance absorbs light, you can calculate • Magnetic stirrers come in the form of a box with a
the amount of that substance in a solution from the magnet inside attached to a motor that spins the
measured absorbance of that solution at that wavelength. magnet. When a vessel containing a magnetic stir
bar is on top of the magnetic stirrer, the stir bar
• A visible (VIS) spectrophotometer measures spins and stirs the contents of the vessel.
absorbance of light in the visible region of the • A vortex mixer rotates the bottom of a tube rapidly;
spectrum (wavelength of about 400-700 nm). A setting up a vortex in the liquid that rapidly mixes
small vessel called a cuvette, which is generally the contents.

474
Microbiological techniques • A microcentrifuge holds microcentrifuge tubes
that can hold about 1.5 mL of liquid. These
Specialized equipment is required to isolate, transfer, microcentrifuges can also spin at high speeds and
and grow up cultures of microbes and tissues in are sometimes refrigerated.
the laboratory. • A sonicator emits ultrasonic waves that can be
used to disrupt cells, allowing their contents to
• Autoclaves are machines that achieve a high be released into the surrounding buffer in -grind
internal temperature and pressure and are used and find strategies.
to sterilize solutions and glassware. The kitchen
pressure cooker achieves the same results and can Separation
be used instead of an autoclave.
• A biological safety or cell culture hood filters
of macromolecules
small particles out of the air in order to avoid Since there are thousands of different macromolecules
contamination of cultures or sterile media. The in each cell, purification of a specific one from all
filters are similar to those used to decontaminate the others requires powerful separation techniques,
air for operating rooms in hospitals or clean rooms such as chromatography and electrophoresis. Both
used in the semiconductor industry. of these approaches take advantage of physical
• Fermenters are used to grow up a large quantity and chemical properties that differ between the
of cells with automatically controlled pH and levels individual macromolecules.
of oxygen and other nutrients.
• Since most cells are generally too small to be In gel electrophoresis, the macromolecules are
seen with the naked eye, microscopes are placed in a solid matrix, called a gel, which is
used to magnify their images. Light or Bright under a liquid buffer. An electric field is applied to

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

field microscopes and inverted microscopes this system, and since biological macromolecules
are the most common types found in carry ionic charges, they will be attracted towards
biotechnology laboratories. one pole of the electric field and repelled by the
opposite. Thus, macromolecules characteristically
Preparation of biological migrate in either direction in the field. The migration
speed is determined by the charge to mass ratio of
samples for analysis the macromolecule.
There are many pieces of equipment that are used to
prepare biological samples for analysis. • In a flat gel, also called a horizontal or submarine
gel, electrophoresis system, an agarose gel lies
• A Sorvall type centrifuge, or preparative centrifuge, horizontally below the electrophoresis buffer. This
has a balanced rotor that holds vessels and spins technique is mainly used to separate large nucleic
them at high speed, up to 20,000 rpm. This will acids (DNA and RNA).
cause most insoluble particles such as cells and • A vertical electrophoresis system holds a
many subcellular components to rapidly form polyacrylamide gel in the vertical position, and
a pellet at the bottom of the vessel. Rotors are is mainly used to separate proteins or small sized
available that hold vessels as small as a few milliliters nucleic acids.
to as large as a liter. These centrifuges are often
refrigerated so that heat sensitive compounds are Chromatography is a family of methods used to
not damaged during centrifugation. separate macromolecules through their relative
• A tabletop, or clinical, centrifuge is generally not affinity to a stationary phase (generally, solid
refrigerated and spins at a much slower speed chromatography beads) and a mobile phase
than a preparative centrifuge. Rotors for clinical (generally, an aqueous buffer). The chromatography
centrifuges generally hold tubes with a capacity be ads are loaded into a tube, called a chromatography
of 15 mL or less. column, and buffer is dripped, or pumped, through

475
 the column to carry the macromolecules along. molecular biology procedures will include:
The macromolecules separate on their affinity for
the mobile front. Some chromatography beads • A thermal cycler is a machine that is used for
separate by charge (ion exchange chromatography), amplification of a specific section of DNA by PCR
by hydrophobicity (hydrophobic interaction (polymerase chain reaction). The machine cycles
chromatography), or by a specific property of that through several temperatures, which allows an
protein (affinity chromatography). Macromolecules enzyme called DNA polymerase to use chemicals
can also be separated by size otherwise known as in solution to build DNA molecules identical to a
size exclusion or gel filtration chromatography. template provided.

• To overcome this limitation, high performance (or • An electroporator is used to discharge a high
high pressure) chromatography (HPLC) uses high voltage, high amperage pulse of electricity of
pressure pumps and metal jacketed columns to very short duration through a cuvette containing
operate at high pressures and speed up the process. suspended cells to disrupt their plasma membranes,
• A fraction collector collects the released mobile allowing DNA to be introduced.
phase (eluent) of a chromatography column. It
automatically measures a programmed volume • A real time PCR machine amplifies and measures
(sometimes by the number of drops of liquid) into the production of amplicons in one step. It is a
a line of test tubes or microcentrifuge tubes. thermal cycler and fluorescent analyzer in one
instrument and is usually computer controlled.
Manipulation of You do not have to load your product onto a gel
to determine if it was made; the machine measures
Nucleic Acids its production photometrically.
Some of the specialized pieces of equipment used
in the isolation, transfer, and analyze DNA in the
Central Marine Fisheries Research Institute

476
Nucleic acid Isolation

Research in biotechnology field largely depends on the carefully carried out under optimum conditions. Even
genome analysis and recombinant DNA technology. rapid stirring can break high molecular weight DNA into
Good quality nucleic acid is an essential prerequisite for shorter fragments. If physical disruption is necessary,
consistent results in most of the downstream applications as in the case with certain tissues, it should be kept to
in the genome analysis and recombinant DNA technology. the minimum, and should involve cutting or squashing
of cells, rather than the use of shear forces. Ultrasonic
The general principle underlying the isolation of nucleic sound is used to disrupt cell wall of certain bacteria.
acids is common with few modifications depending
on the type of nucleic acid being isolated. Firstly the Care has to be taken to prevent degradationof DNA by
nucleic acid is to be made free form the other biological deoxyribonucleases (DNase). These enzymes are found
macromolecules and cell debris. This is achieved by in most cells, and may also be present in dust, which
properly lysing the cell wall/cell membrane and then could contaminate laboratory glassware. Hence all the
by selectively denaturing the other macromolecules glassware, plastic wares and the buffers are to be made
like proteins. Nucleic acids thus recovered in its native sterile by autoclaving. These enzymes activity can also
form is precipitated by alcohol and suspended in sterile be inhibited by using EDTA in lysisbuffer, which will

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

buffer or distilled water. Finally the qualitative integrity chelate the Mg2+ ions needed for the DNase activity
of the isolatednucleic acid is checked by agarose and also essential for preserving the overall structure of
gel electrophoresis and ethidium bromide staining. the cell membrane thus helps in cell lysis. Cell disruption
Quantitative estimation of nucleic acid is carried out and most of the subsequent steps should be performed
by spectrophotometric methods. at 4ºC. The cell wall could be lysed enzymatically as
well. The bacterial cell wall is usually lysed by the
The types of nucleic acids usually isolated on a routine enzyme Lysozyme. The cell membrane on the other
basis are, total genomic DNA (gDNA), total RNA, extra hand are solubilized by including suitable detergent
chromosomal DNA (Plasmid, Mitochondrial DNA, like Sodium dodecyl Sulfate (SDS) in the lysis buffer.
Chloroplast DNA etc.). Earlier DNA was one of the SDS aids in disrupting the cell membranes by removing
most difficult molecules to analyze. But now, DNA the lipids of the cell membranes.
has become the easiest macromolecule of the cell to
study. DNA can be easily purified from the cells and Upon lysis the entire content of the cell including
once isolated it is much more stable than any other nucleic acids (DNA, RNA), cytoplasm, organelles etc.
macromolecule. It can be manipulated precisely and will be released in to the lysis buffer and now the
reproducibly with various molecular tools like restriction target molecule DNA is to be made free from RNA
enzymes, PCR, cloning etc. enabling genome analysis and other associated proteins. The RNA molecules
and recombinant DNA technology with much ease. can be selectively denatured by enzymatic treatment
with RNase.The other major contaminant, protein, is
removed by enzymatic treatment with proteinase K
Isolation of total genomic followed by treatment with Tris saturated Phenol(pH
DNA (gDNA) 8.0) or Phenol: Chloroform mix (1:1), either of
which will denature proteins. Centrifugation of the
Breaking of the bacterial and plant cell wall as well as emulsion formed by this mixing produces a lower,
solubilizing the cell membrane of animal cells are to be organic phase, separated from the upper aqueous

477
 phase by an interface of denatured proteins.The upper • Micro centrifuge tubes (1.5ml)
aqueous phase was recovered and deproteinised • Micropipettes
repeatedly until no material is seen at the interface
and washed with chloroform: isoamyl alcohol (24:1), Procedure/Protocol
to remove traces of phenol. Finally by the addition • 50 mg of tissue (muscle/fin clips) was homogenized
of two volumes of ice-cold absolute alcohol in the with 400µl of lysis buffer in a sterile 1.5 ml micro
presence of salt (3M Sodium Acetate, pH 5.2) the centrifuge tube.
high molecular weight DNA can then be efficiently • 100ul of 10% SDS solution and 10 µl of Proteinase
precipitated out of the solution in a -20ºC freezer. K was added to the homogenate
• The above mixture is mixed well and incubated
After centrifugation, the DNA pellet is dissolved in a at 55ºC for an hour in an incubator / water bath.
buffer containing EDTA for protection against DNases, • 500 µl of phenol-chloroform (1:1) mixture is added
and this solution can be stored. DNA solutions canbe to the contents, mixed well by inverting several
stored frozen, but repeated freezing and thawing tends to times and incubated at room temperature for
damage long molecules by shearing and hence the DNA 5 minutes.
preparations in frequent use are normally stored at 4ºC. • The contents are centrifuged at 10,000 rpm for
10 minutes at 4ºC.
Quantitative estimation of • The supernatant is collected using cut/wide bore
tips and is transferred to a fresh tube.
nucleic acid. • 10µl of RNase A was added to the supernatant
DNA can be spectrophotometrically estimated by taking and incubated at 37ºC for an hour in an incubator
optical density (OD) at 260nm, 1 OD corresponds to 50 • The phenol: chloroform extraction is repeated and
microgram of DNA. Purity of the DNA can be checked after centrifugation at 10,000 rpm for 10 minutes
spectrophotometrically by taking OD at 260 & 280 at 4ºC the supernatant is collected in a fresh tube.
nanometers (nm). The ratio of 260 and 280 will result • Equal volume chloroform-isoamyl alcohol (24:1
a value of 1.8 with pure DNA preparations. v/v) is added to the supernatant, mixed well by
gently inverting several times.
Protocol for the isolation • The contents are centrifuged at 10,000 rpm for
10 minutes at 4ºC.
of DNA from animal • The supernatant is collected using cut/wide bore
tissue using Phenol tips and is transferred to a fresh sterile micro
Chloroform method. centrifuge tube.
• 1/10 volume of 3M sodium acetate (pH 5.2) is
Equipment and reagents required added to the contents and is mixed gently.
• 1 ml of ice cold ethanol (100%) is added and
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• Lysis buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0; 1 mixed gently by inversion till white strands of DNA
mM EDTA, pH 8.0, 400mM NaCl) precipitates out. If no precipitate is observed the
• Sodium dodecyl sulfate (SDS) solution (10%) tube is incubated overnight at -20ºC.
• Proteinase K (20mg/ml) • After incubation the tube is centrifuged at 10000
• RNase A (10mg/ml) rpm for 10 min to pellet the DNA.
• Tris saturated Phenol (pH 8.0): Chloroform (1:1 v/v) • The pellet was washed in 70% ethanol by
• 24:1 (v/v) chloroform-isoamyl alcohol centrifugation at 1000 rpm for 10 min at 4ºC
• 3 M sodium acetate, pH 5.2 • Decant the ethanol and air dry the pellet
• Ice cold Ethanol (100%) • Dissolve the pellet in 50 µl to 100µl of TE buffer
• Ethanol (70%) or sterile dH2O.
• TE buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0; 1 mM • The stock of the DNA samples is then stored at
EDTA, pH 8.0) -20ºC and diluted working solution can be stored
• Micro centrifuge at 4ºC.

478
Isolation of DNA from animal tissue using
salting out procedure

Reagents required: Solution 1:

Tris-HCl (pH8.0) - 50 mM
Stock solutions:
EDTA (pH8.0) - 20 mM
SDS - 2 %
0.5M Tris Cl (pH-8.0) Prepared in double distilled water. Autoclave and
Tris base - 3.028 g store at 4ºC
Distilled water - 40 ml
Adjust pH to 8.0 using HCl. Solution 2:
Make up the volume to 50 ml, autoclave and store
at 4ºC. NaCl solution (saturated)–(6 M)

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Prepared in double distilled water.
0.5M EDTA (pH-8.0) Autoclave and store at 4ºC

EDTA - 9.31 g Proteinase K

Distilled water - 40 ml
Adjust pH to 8.0 using NaOH. Proteinase K – 20 mg/ml
Make up the volume to 50 ml, autoclaved and Prepared in autoclave double distilled water and
stored at 4ºC. store at -20ºC.

10mM Tris Cl (pH-7.5) TE buffer

Tris base - 0.030 g Tris Cl (pH-8.0) - 10 mM
Distilled water - 20 ml EDTA (pH-8.0) - 1 mM
Adjust pH to 7.5 using HCl. Prepared in double distilled water. Autoclave and
Make up the volume to 25 ml, autoclaved and store at 4ºC
stored at 4ºC
RNAase
RNAase buffer
RNAase–10 mg/ml of RNAase buffer (autoclaved)
10mM Tris Cl (pH 7.5)- 10 μl
15mM NaCl - 30 μl Protocol
Distilled water - 960 μl
Autoclaved and stored at 4ºC. Tissue stored in alcohol was washed with TRIS buffer
(pH 8.0) by spinning and then followed below
Working Solutions listed steps.

479
 • Placed tissue sample in 1.5 ml tube & added 500 incubated at 37ºC on heating block for 15 minutes.
μl Solution 1. • Added twice the volume (~1 ml) of ice cold 100
• Homogenized tissue sample with sterile % molecular biology grade Ethanol to precipitate
homogeniser or melted filter tip. the DNA.
• Added 5μl of Proteinase K (20 mg/ml) • Incubated overnight at -20ºC.
• Incubated at 55ºC in water bath for 2 hours (with • Next day, centrifuged at 11000 rpm for 15 minutes
occasional mixing). and removed supernatant.
• Chilled on ice for 10 minutes. • Rinsed DNA pellet in 250 μl of ice-cold 70% ethanol.
• Added 250 μl Solution 2 and inverted several times • Centrifuged at 11000 rpm for 5 minutes.
for through mixing. • Carefully removed supernatant and partially dry
• Chilled on ice for 5 minutes. with lid off at room temperature.
• Centrifuged at 8000 rpm for 15 minutes. • Resuspended partially dried DNA in 50-200 μl
• Carefully collected clear supernatant (~500 μl) (depending on size of pellet) of TE buffer (pH-8)
with wide-bore filter tip into a newly labeled 1.5 by gently pipetting the sample with wide-bore filter
ml tube. tip until dissolved.
• Added 1.5 μl RNase (final conc.20 μg/ml) and
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480
Isolation of DNA from animal tissue
using Kits

QIAGEN DNeasy Blood & Tissue Kit Discard flow-through and collection tube.

1. Cut up to 25 mg tissue (up to 10 mg spleen) into 5. Place the DNeasy Mini spin column in a new
small pieces, and place in a 1.5ml microcentrifuge 2 ml collection tube (provided), add 500 μl
tube. For rodent tails, place one (rat) or two Buffer AW1, and centrifuge for 1 min at ≥ 6000
(mouse) 0.4–0.6cm lengths of tail into a 1.5 ml x g (8000 rpm). Discard flow-through and
microcentrifuge tube. Add 180 μl Buffer ATL. collection tube.

2. Add 20 μl proteinase K. Mix thoroughly by 6. Place the DNeasy Mini spin column in a new 2
vortexing, and incubate at 56ºC until the tissue ml collection tube (provided), add 500 μl Buffer

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

is completely lysed. Vortex occasionally during AW2, and centrifuge for 3 min at 20,000 x g
incubation to disperse the sample, or place in (14,000 rpm) to dry the DNeasy membrane.
a thermomixer, shaking water bath, or on a Discard flow-through and collection tube.
rocking platform.
7. Place the DNeasy Mini spin column in a clean 1.5
3. Vortex for 15 s. Add 200 μl Buffer AL to the ml or 2 ml microcentrifuge tube, and pipet 200
sample, and mix thoroughly by vortexing. Then μl Buffer AE directly onto the DNeasy membrane.
add 200 μl ethanol (96–100%), and mix again
thoroughly by vortexing. 8. Incubate at room temperature for 1 min, and
then centrifuge for 1 min at 6000 x g(8000 rpm)
4. Pipet the mixture from step 3 (including any to elute.
precipitate) into the DNeasy Mini spin column
placed in a 2 ml collection tube (provided). 9. Recommended: For maximum DNA yield, repeat
Centrifuge at ≥ 6000 x g(8000 rpm) for 1 min. elution once as described in step 7.

481
 Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a technique the reaction to 94-98ºC for 30 seconds. It causes
widely used in molecular biology. It derives its name melting of DNA template and primers by disrupting
from one of its key components. A thermostable DNA the hydrogen bonds between complementary
polymerase isolated from a thermophilic bacterium bases of the DNA strands, yielding single strands
Thermus aquaticus (Taq polymerase) is most often of DNA.
used and since it is heat stable, it does not have to be • Annealing step: The reaction temperature is
replaced after each cycle. Developed in 1984 by Kary lowered to 50-65ºC for 30 seconds allowing
Mullis. PCR is now a common and often indispensable annealing of the primers to the single-stranded
technique used in medical and biological research DNA template. Typically the annealing temperature
labs for a variety of applications. PCR is a technique is about 3-5 degrees Celsius below the Tm of the
that employs in-vitro enzymatic amplification of a primers used. Stable DNA-DNA hydrogen bonds
particular or desired DNA fragment of up to several are only formed when the primer sequence very
kilobases (kb) in size from a complex genome. PCR closely matches the template sequence. The
allows the production of more than 10 million copies polymerase binds to the primer-template hybrid
of a target DNA sequence from only a few molecules. and begins DNA synthesis.
• Extension/elongation step: At this step the
• Basic PCR set up requires several components and DNA polymerase synthesizes a new DNA strand
reagents. These components include: complementary to the DNA template strand by
• DNA template that contains the DNA region to adding dNTPs that are complementary to the
be amplified. template in 5’ to 3’ direction.
• Two primers, which are complementary to the DNA • Final elongation: This single step is occasionally
regions at the 5’ or 3’ ends of the DNA region. performed at a temperature of 70-74ºC for 5-15
• Taq polymerase to amplify the DNA minutes after the last PCR cycle to ensure that any
• Deoxynucleoside triphosphates (dNTPs); the remaining single-stranded DNA is fully extended.
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building blocks from which the DNA polymerases • Final hold: This step at 4ºC for an indefinite
synthesizes a new DNA strand. time may be employed for short-term storage of
• Buffer solution, providing a suitable chemical the reaction.
environment for optimum activity and stability of
the DNA polymerase. In PCR, the double stranded DNA is denatured by
heat and then the temperature is lowered to allow
The PCR usually consists of a series of 20 to 40 annealing of two specific primers by complementary
repeated temperature changes called cycles; each base pairing on the opposite strands of the DNA. Taq
cycle typically consists of 2-3 discrete temperature polymerase directs the synthesis of the new strand
steps. Most commonly PCR is carried out with cycles from the primed sites in both directions that results in
that have three temperature steps double stranded DNA–and the procedure is repeated.
In each cycle, the target DNA is replicated by a factor
• Denaturation step: This step consists of heating of 2 so that, after 30 cycles millions of copies of DNA

482
are available for subsequent manipulation. and DNA templates, the optimal concentration of
MgCl2 has to be selected for each experiment. Too few
PCR based techniques require only a small amount Mg2+ ions result in a low yield of PCR product, and
of template DNA of relatively inferior quality and too many increase the yield of non-specific products
therefore, the amount of material required for analysis and promote mis-incorporation. The recommended
is greatly reduced in comparison to all other methods. range of MgCl2 concentration is 1-4 mM under the
PCR can be used to identify a target organism even standard reaction conditions specified.
in small numbers in mixed infections. For methods
involving PCR, the major potential drawback is the dNTPs
risk of contamination of the sample with material
from another sources, especially previously amplified The final concentration of each dNTP in the reaction
DNA. A control reaction, omitting template DNA, mixture is usually 200 µM. It is very important to
should always be performed, to confirm the absence have equal concentrations of each dNTP (dATP, dCTP,
of contamination dGTP & dTTP), as inaccuracy in the concentration
of even a single dNTP dramatically increases the
Materials required misincorporation level.

Thermocycler, micro centrifuge, vortex mixer, thin- Taq DNA polymerase

walled PCR tubes, Micropipettes and tips.
Usually 1-1.5-units of Taq DNA polymerase are
Reagents required used in 50µl of reaction mix. Higher Taq DNA
polymerase concentrations may cause synthesis of
Template DNA, Primers, 10mM dNTP mix, 25mM nonspecific products.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

MgCl2, Taq DNA polymerase with 10x buffer
Preparation of
Components of the reaction mixture
reaction mixture
Reagent Final Conc. Volume for 50µl
Template DNA reaction mix
10x buffer 1x 5.0
Usually the amount of template DNA is in the range dNTP mix (10mM each) 200 µM 1.0
of 0.01-1ng for plasmid or phage DNA and 0.1-1 µg MgCl2 (25mM) 1mM 2.0
for genomic DNA, for a total reaction mixture of 50 Forward primer (10pmol/µl) 10pmol 1.0
Reverse primer (10pmol/µl) 10pmol 1.0
µl. Higher amounts of template DNA usually increase
Taq polymerase enzyme(2U/ 1U 0.5
the yield of nonspecific PCR products. µl)
Template DNA 50ng 1.0

Primers Sterile H2O 38.5

PCR primers are usually 15 -30 nucleotides in length. To perform several parallel reactions, prepare a master
The GC content should be 40-60 %. More than three G mix containing water, buffer, dNTPs, primers, Taq
or C nucleotides at the 3’-end of the primer should be DNA polymerase and MgCl2 in a single tube, which
avoided, as nonspecific priming may occur. The primer can then be aliquoted into individual tubes. This
should not be self-complementary or complementary method of setting reactions minimizes the possibility
to any other primer in the reaction mixture, in order of pipetting errors and saves time by reducing the
to avoid primer-dimer and hairpin formation. number of reagent transfers. Template DNA solution
is added after aliquoting into tubes.
MgCl2 concentration
Gently vortex and briefly centrifuge all solutions
Since Mg 2+
ions form complexes with dNTPs, primers after thawing.

483
 Add the following in a thin-walled PCR tube placed Tm = 4 (G + C) + 2 (A + T)
on ice:
Annealing temperature (ºC) = Tm–5ºC
A Negative control can be maintained by replacing
the Template DNA with sterile water. Where,
Tm = Melting temperature; G, C, A, T =number of
Gently vortex the sample and briefly centrifuge to respective nucleotides in the primer.
collect all drops from walls of tube.
Extension
Place samples in a thermocycler and start PCR.
Usually the extending step is performed at 72ºC.
Thermocycling conditions The rate of DNA synthesis by Taq DNA Polymerase is
highest at this temperature. Recommended extension
time is 1 m for the synthesis of PCR fragments up
Initial Denaturation
to 2 kb.
The complete denaturation of the DNA template at
the start of the PCR reaction is of key importance. Number of cycles
The initial denaturation should be performed over
an interval of 1-3 min at 95ºC if the GC content is The number of PCR cycles depends on the amount
50% or less. This interval should be extended up to of template DNA in the reaction mix and on the
10 min for GC rich templates. expected yield of the PCR product. 25-35 cycles are
usually sufficient.
Denaturation
Final extension
Usually denaturation for 30–120 sec at 94-95ºC is
sufficient, since the PCR product synthesized in the After the last cycle, the samples are usually incubated
first amplification cycle is significantly shorter than at 72ºC for 5-15 min to fill in the protruding ends of
the template DNA and is completely denatured under newly synthesized PCR products.
these conditions
Visualization of PCR products
Annealing
The PCR product was visualized by electrophoresis in
Annealing temperature of the primers is calculated agarose gel stained with 0.5 µg/ml ethidium bromide.
using the following formula
Central Marine Fisheries Research Institute

484
RNA Isolation Protocol – Using TRIZOL

Reagents required • Following centrifugation, the mixture separates

into lower red, phenol-chloroform phase, an
• DEPC-treated water interphase, and a colorless upper aqueous phase.
• TRIzol Reagent (Invitrogen) RNA remains exclusively in the aqueous phase.
• 75% ethanol Transfer upper aqueous phase carefully without
• Isopropyl alcohol disturbing the interphase into fresh tube. Measure
the volume of the aqueous phase (The volume of
Equipment and supplies the aqueous phase will about 60% of the volume
of TRIZOL Reagent used for homogenization).
• Refrigerated Microcentrifuge
• Micropipettors RNA Precipitation
• Aerosol-barrier tips

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

• Vortex mixer • Precipitate the RNA from the aqueous phase by
• Powder-free gloves adding equal volume ice-cold isopropyl alcohol
• Centrifuge tubes (100%). Incubate samples at -20ºC for 10 minutes
and centrifuge at 10,000 rpm for 10 minutes at
Homogenization 4ºC. The RNA precipitate, often invisible before
centrifugation, forms a white gel-like pellet on the
• Homogenize 50 to 100 mg of tissue samples in side and bottom of the tube.
1 ml of TRIZOL reagent using a glass-Teflon or
power homogenizer. The sample volume should RNA Wash
not exceed 10% of the volume of TRIZOL Reagent
used for the homogenization. • Remove the supernatant completely. Wash the RNA
pellet once with 75% ethanol, adding at least 1 ml of
• Incubate the homogenized sample for 5 minutes at 75% ethanol per 1 ml of TRIZOL Reagent used for the
room temperature to permit the complete dissociation initial homogenization. Mix the samples by vortexing
of nucleoprotein complexes. Centrifuge to remove cell and centrifuge at 10000 rpm for 5 minutes at 4ºC.
debris. Transfer the supernatant to new tube.
Redissolving RNA
Phase Separation
• Air-dry or vacuum dry RNA pellet for 5-10 minutes
• Add 0.2 ml of chloroform per 1 ml of TRIZOL to remove all leftover ethanol. It is important not
Reagent. Cap sample tubes securely. Vortex to let the RNA pellet dry completely as this will
samples vigorously for 15 seconds and incubate greatly decrease its solubility. Partially dissolved
them at room temperature for 2 to 3 minutes. RNA samples have an A260/A280 ratio < 1.6.
Centrifuge the samples at no more than 10,000 Dissolve RNA in DEPC-treated water by passing
rpm for 15 minutes at 4ºC. solution a few times through a pipette tip.

485
 Spectrophotometric Analysis and proteoglycan-rich sources. Biotechniques
• Dilute 1 μl of RNA with 39 μl of DEPC-treated water 19(6): 942-5.
(1:40 dilution). Take OD at 260 nm and 280 nm to
determine sample concentration and purity. The TRIZOL Reagent technical insert (Invitrogen).
A260/A280 ratio should be above 1.9. Apply the
convention that 1 OD at 260 equals 40 μg /ml RNA
to calculate the exact conc. of RNA.

Suggested readings
Chomczynski P, Mackey K. (1995) Short technical
report. Modification of the TRIZOL reagent
procedure for isolation of RNA from Polysaccharide-
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486
Isolation of total RNA by modified
guanidine thiocyanate method

Preparation of glassware DEPC treated water and made upto 1 liter and
and reagents sterilized by autoclaving.

Glasswares and plasticwares Solution D

All glass wares used for RNA isolation were treated 4 M Guanidium thiocyanate
with 0.1 % Diethyl pyrocarbonate (DEPC) solution 25 mM Sodium citrate
prepared in Milli-Q water for 4 h at 37ºC and 0.5 % w/v N-lauryl sarcosine
then kept at 150ºC in hot air oven for 3 h. Sterile 0.1 M β-mercapto ethanol*

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

disposable RNase free plastic wares were used for the Filter sterilized through 0.2 µm filter and store at
preparation and storage of RNA. Utmost care was room temperature
taken to prevent the action of RNase during the entire *Add β-mercaptoethanol just before use. (3.6μl into
isolation process by ensuring the biochemical and 500μl of sol D)
microbiological sterility of the workbench by alcoholic
sterilisation. Disposable gloves were used during the Phenol
preparation of reagents, isolation and analysis of RNA.
Saturated with 0.1 M citrate buffer, pH 4.3
Reagents Chloroform
Isopropanol
All reagents were prepared using molecular grade 75% Ethanol
chemicals (SIGMA Inc, USA) in 0.1 % DEPC treated
sterilized Milli-Q water. The autoclavable reagents Protocol
were made sterile at 121ºC for 15 min at 15
lbs pressure.
Tissue homogenization:
Phosphate Bufered • A single pair of eyestalk neural tissue was
homogenized in 500 μl PBS in a 1.5 ml micro
saline (PBS) centrifuge tube using a sterile pestle.
137 mM NaCl • Homogenized samples were incubated on ice
2.7 mM KCl for 5 min to ensure the dissociation of cell wall
4.3 mM Na2HPO4 and pigments.
1.47 mM KH2PO4 • The homogenate was then centrifuged at 10,000
Adjust to a final pH of 7.4. rpm for 5 min at 4ºC and the upper aqueous phase
Dissolved the chemicals in minimum volume of was saved and the pellet discarded.

487
 ➢Phase separation • The resulting pellet was saved carefully and the
ethanol was decanted .
• The aqueous phase (0.4 ml) was transferred to a
fresh 1.5 ml micro centrifuge tube. Dissolving RNA
• Solution D (0.4 ml) was added to the preparation
and the tube was vortexed vigorously for 15 sec • The RNA pellet was air dried and dissolved in the 20
followed by incubation on ice for 3 min. μl sterile RNA storage solution (Ambion Biosciences)
• Saturated phenol (0.4 ml) and chloroform (0.1
ml) was added and vortexed vigorously for 30 sec Quantification of RNA
followed by incubation on ice for 15 min.
• The preparation was centrifuged at 12,000 rpm • Dilute 1 μl of RNA with 39 μl of DEPC-treated water
for 15 min at 4ºC (1:40 dilution). Take OD at 260 nm and 280 nm to
determine sample concentration and purity. The
RNA precipitation A260/A280 ratio should be above 1.9. Apply the
convention that 1 OD at 260 equals 40 μg /ml RNA
• The upper aqueous phase was transferred to a fresh to calculate the exact conc. of RNA.
1.5 ml micro centrifuge tube and equal volume of
isopropyl alcohol was added to precipitate the RNA Suggested readings
and kept at-80 oC for 10 min .
• Precipitated RNA was recovered by centrifugation Chomczynski P, Sacchi N (1987) Single-step
at 12,000 rpm for 10 min at 4ºC. method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal
RNA wash Biochem 162:156–159.

• Precipitated RNA pellet was washed once with 1

ml of 75 % ethanol and centrifuged at 12,000 rpm
for 5 min at 4ºC.
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488
Reverse transcriptase PCR (RT-PCR) for
First strand cDNA synthesis

The ability to synthesize DNA from an RNA template, Alternatively, RT-PCR can be done in two steps, first
via reverse transcription, enables researchers to study with the reverse transcription and then the PCR.
RNA with the same molecular approaches used for DNA The two-step protocol is usually more sensitive than
investigations. cDNA generated by reverse transcription the one-step method; yields of rare targets may be
can be amplified using polymerase chain reaction (PCR). improved by using the two-step procedure.
The combination of reverse transcription and PCR (RT-
PCR) allows the detection of low abundance RNAs Two-step RT-PCR
in a sample. In the first step of the PCR process, the
cDNA is denatured by heating to 95ºC, which disrupts • Saves RT reagents. One RT reaction will provide
the hydrogen bonds between complementary strands, templates for multiple PCR’s
yielding single-stranded molecules. The temperature is • Can be more sensitive than one-step RT-PCR
then lowered in order to allow primers complementary to
the sequence(s) of interest to anneal. The DNA polymerase

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

included in the reaction will then begin DNA synthesis. At
this point, the temperature is raised to the optimal activity
temperature of the DNA polymerase (usually 72ºC) to
synthesize a new strand complementary to the template. Two-step Protocol
The process of denaturing, annealing, and extension can
be repeated multiple times, with a two-fold increase in RT-PCR involves use of reverse transcriptase enzyme
the amount of DNA molecules with each cycle. Because to synthesize cDNA from mRNA. The two-step RT-PCR
PCR can selectively amplify a template, it is an important reaction mainly involves two major steps, 1) Synthesis
method for detecting specific nucleic acid molecules in a of the first strand cDNA and 2) Synthesis of second
particular cell or small populations of cells. PCR Products strand by PCR amplification.
can be used in many downstream applications, such as
cloning into plasmid vectors and sequencing using next Step One: Reverse Transcription
generation sequencing platforms.
After ensuring the qualitative integrity by
In the one-step protocol, the components of RT and gel electrophoresis and quantification by
PCR are mixed in a single tube at the same time. The spectrophotometer, total RNA isolated was reverse
one-step protocol generally works well for amplifying transcribed with First Strand cDNA Kit using
targets that are reasonably abundant. Oligo(dT)24 primer following the instructions given
by the manufacturer.
One-step RT-PCR
Reagents
Convenient

489
 • First Strand cDNA synthesis Kit Step Two: PCR
• Reverse Transcriptase MMLV-RT (100 units/μl)
• 10X RT Buffer (500 mM Tris-HCl, pH 8.3, 750 mM Setting up PCR reactions
KCl, 30mM MgCl2 50 mM DTT)
• Oligo-dT primer [d(T)24] (50μM) Negative Controls
• dNTP (2.5mM each dNTP)
• RNase Inhibitor (10 units/μl) Use two negative controls among the PCRs.
• PCR Thermocycler
I. The minus-RT control from the previous step,
Place RNase Inhibitor and Reverse Transcriptase ON or alternatively, untreated RNA can simply be
ICE directly from the box. subjected to PCR.
II. A minus-template PCR, it should have all the PCR
Component Stock Final Experiment Control (-RT) components, but use water as template instead
amount (+RT)
of an aliquot of the cDNA (RT reaction). This
Total RNA ~1-2 μg ~1-2 μg
control will verify that none of the PCR reagents
Oligo dT primer 50μM 5μM 2 μl 2 μl
is contaminated with DNA.
10X RT Buffer 10X 1X 2 μl 2 μl
dNTP mix 2.5mM 0.5mM 4 μl 4 μl
RNase Inhibitor 10 U/μl 10U 1 μl 1 μl Positive Control
Reverse 100 U/ 100U 1 μl 0 μl
Transcriptase μl I. Perform PCR to amplify a cDNA that corresponds
Nuclease-free x μl (to total x μl (to total to a basal/housekeeping gene transcript
water of 20 μl) of 20 μl II. Use genomic DNA isolated from cells as template.
If your primers span intron(s), note the size of
Thaw 10x reaction buffer, random decamers, and the expected PCR product and if necessary, adjust
dNTP mix quickly in your hands and place ON ICE; annealing temperature of the PCR program.
Use small 0.25ml PCR tubes.
Reagents:
1) Assemble your reaction as follows on ice. Add the
enzyme last. Taq DNA polymerase with 10X Buffer (-MgCl2) and
2) Mix gently, spin briefly. 50mM MgCl2

Component Working Stock Final Conc. Experiment Control 1 (-RT in Control 2 (no
step 1) template)
RT reaction 1-2 μl 1-2 μl 0 μl
10X PCR Buffer 10X 1X 2.5 μl 2.5 μl 2.5 μl
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Forward primer 5μM 0.25μM 1.25 μl 1.25 μl 1.25 μl

Reverse primer 5μM 0.25μM 1.25 μl 1.25 μl 1.25 μl
dNTP mix 2.5mM 0.125mM 1.25 μl 1.25 μl 1.25 μl
Taq Polymerase 5U/μl 1U 0.2 μl 0.2 μl 0.2 μl
dH2O Up to 25 μl Up to 25 μl Up to 25 μl

3) Incubate in the thermocycler at: dNTP mix

• a. 42ºC for 1 hr.
• b. 95ºC for 10 min to inactivate the The following table outlines the components needed
reverse transcriptase. for PCR.

4) Store reaction at –20ºC or proceed to the PCR. Making Master Mixes:

490
Component Experiment Neg Control 1 (-RT in step 1) Neg Control 2 (no template)
RT reaction ~1-2*(n+1) μg ~1-2*(n+1) μg 0
10X PCR Buffer 5 *(n+1) μl 5*(n+1) μl 5 *(n+1) μl
dNTP mix 1.25*(n+1) μl 1.25*(n+1) μl 1.25*(n+1) μl
MgCl2 *(n+1) μl *(n+1) μl *(n+1) μl
Taq polymerase 0.2*(n+1) μl 0.2*(n+1) μl 0.2*(n+1) μl
Nuclease-free water Variable Variable variable
Total Volume 100 *(n+1) μl 100*(n+1) μl 100*(n+1) μl

Consider making master mixes if you are testing multiple Anneal at 55ºC for 20-30 sec**
sets of primers at once. A master mix will contain Extend at 72ºC for 45 sec ***
everything except the PCR primers. If you are testing n c. Final extension: 72ºC for 5 min
sets of primers, make a master mix enough for n+1 tests. **Start with the annealing temperature suggested by your primer
design software. An annealing temperature of ~55ºC used with
Mix the components gently but thoroughly. Aliquot the cycling times shown is often a reasonable starting point, but
22.5μl of your master mix to each tube. Add 1.25μl the optimal temperature and cycling times for your primer and
of each of the appropriate primer at 5μM working template combination may need to be determined empirically.
stock concentration.
***The rule of thumb is to use an extension time of 1 min per
kilobase of target.
Assemble reactions on ice. Incubate
in Thermocycler: Run 6 μl of the PCR reaction product on 1-1.5%
a. Initial denaturation: 94ºC for 4 min Agarose gel to check for the presence of
b. 30 cycles: Denature at 94ºC for 30 sec amplified products.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

491
 Agarose Gel Electrophoresis

Agarose gel electrophoresis is used to analyze and Rate of migration of nucleic acids in agarose gels
quantitate nucleic acid. The agarose, for agarose gel depends mainly on several factors.
electrophoresis is purified from agar. Agarose is a
linear polysaccharide made up of repeating units of a. Agarose concentrations
agarobiose which comprises of alternating units of
galactose and 3, 6 anhydrogalactose. Agarose has Higher concentration of gels are used for the
an average MW of 12,000 and contains about 35-40 separation of lower molecular weight DNA and RNA
agarobiose units. Agarose in solution exist as a left fragments and vice-versa
handed double helices. About 7 to 11 such helices
form bundles which extend as long rods and appear b. Molecular weight
to intertwine with one another, further strengthening
the frame work of the gel. The cross links are held Duplex DNA fragments migrates at rates inversely
together by hydrogen and hydrophobic bonds. The proportional to the log Molecular weight. A plot of
pore size of the gel is controlled by the concentration log M.W vs Mobility gives a straight line
of the agarose. Higher the concentration, smaller the
pore size of the gel and vice versa. Because of large c. Conformation
pore size even at low concentration, agarose gels are
widely used for separation of DNA and RNA. Supercoiled DNA moves fastest followed by linear
forms and relaxed open circular forms.
Effect of agarose concentration on
d. Applied Voltage
separation ranges
The following table describes the relationship At low voltage (<5V/cm) the rate of migration is
between agarose concentration and separation range directly proportional to the applied voltage. However,
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of nucleic acid if the voltage is increased, mobility of high molecular

Agarose Concentration (%) Separation range (Kb) weight DNA fragments increased differentially
0.3 5 to 60
0.6 1-20 e. Presence of Ethidium Bromide (EtBr)
0.8 0.8 to 10
1.0 0.4 to 8
The presence if ethidium bromide in the gel causes
1.2 0.3 to 6
DNA to run slower, as EtBr intercalates and uncoils
1.5 0.2 to 4
the DNA.

Factors which affect the rate

Reagents
of migration of nucleic acids in
agarose gels 10X TBE/50X TBE, EtBr, distilled water, gel loading
buffer, molecular weight marker.

492
Buffers buffer of choice (TBE/TAE) and dissolved completely
by heating the flasks using a microwave oven. (Usually
There are a number of buffers used for agarose gel the percentage of agarose ranges between 0.7- 2.0
electrophoresis. The most common being Tris acetate depending on the size of DNA to be electrophoresed).
EDTA (TAE) and Tris borate EDTA (TBE) buffer. TAE has Allow the gel solution to cool down to 60ºC at room
the lowest buffering capacity but provides the best temperature and 1.5 μl of EtBr stock solution was
resolution for larger DNA fragments. This means a added to it and mixed by gentle swirling, without
lower voltage and more time, but a better product. forming any air bubbles.
On the other hand TBE will give a faster run time with
higher voltage and a good resolving power. Assemble to gel tray and place the comb at about
1 cm from the top of the tray and pour the melted
TBE buffer agarose without making any bubbles, keeping the
Tris borate (1X TBE) thickness of the gel around 0.5 to 0.9 cm. Allow
89mm Tris base the gel to set for 20 mins and take off the combs to
89mm Boric acid uncover the wells. Place the gel tray in the running
25mm Na2-EDTA unit and pour the 1x buffer over it. Make sure the
TAE buffer gel is completely covered with the buffer.
Tris acetate (1X TAE)
50mm Tris base The DNA sample (100 to 200 ng) is mixed with the
25mm Glacial acetic acid loading dye (for 5 μl of DNA sample 1μl of 6x dye is
1mm Na2-EDTA used) and loaded in to the well carefully, using a micro
Sterilize the stock solutions by autoclaving pipette. The maximum volume that can be loaded on
to a well formed from a 1.5 mm thickness tooth of

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Ethidium bromide (stock solution) the comb is 30 μl. Load one well with a DNA ladder.

Weigh 10 mg ethidium bromide into a sterile tube Once the sample is loaded in to the well, the cathode
and dissolve in 10 ml sterile distilled water. The stock (Black negative terminal) is connected towards the top
is stored at 4ºC end of the gel and the anode (Red positive terminal
is connected towards the bottom end of the gel.
Sample loading dye Glycerol &
The electrophoresis is started by switching on the
bromophenol blue (6x)
DC power pack. The gel is run at 5v/cm till the
3ml glycerol (30%), 25mg bromophenol blue (0.25%) bromophenol blue (the tracking dye) has moved
dH2O to 10mL 1 cm above the bottom end. Then the current is
switched off, the power supply is disconnected and
Preparation of agarose solution the gel is photographed under UV light on a gel
documentation system.
for casting the gel
Weigh the required quantity of agarose on to 1x

493
 Preparation of competent E. coli cells

Competence is the ability of a cell to take up extra Mix 500 mL of LB media with 7 g of Agar (Autoclave).
cellular DNA from its environment. Competency can Cool to ~55-65ºC prior to pouring. The addition of
be artificially induced by treating the cells with CaCl2 antibiotics should be made before pouring and at a
prior to adding DNA. The calcium destabilizes the cell temperature not higher than 55ºC.
membrane and adheres to the cell surface favoring
the formation of the pores for the entry of DNA. Procedure
Solutions 1. Streak E. coli cells on an LB plate
2. Allow cells to grow at 37ºC overnight
Luria-Bertani (LB) media (1 L): 3. Place one colony in 10 mL LB media (+antibiotic
selection if necessary), grow overnight at 37ºC
Mix 10 g of Bacto-tryptone, 5 of Yeast extract, and 4. Take 2 ml LB media and save for blank. Transfer
10 g of NaCl. 5 mL overnight culture into 500 mL LB media in
pH to 7.5 w/ NaOH and dH2O to 1 L (Autoclave) 1 L conical flask
5. Allow cell to grow at 37ºC (250 rpm), until
1M CaCl2 (1 L): OD600= 0.4 (~2-3 hours)
6. Transfer cells to 2 centrifuge bottles (250 mL),
Mix 111 g of CaCl2 (anhydrous) and 1 L of dH2O. and place cells on ice for 20 mins
Filter sterilize through a 0.22μ filter 7. Centrifuge cells at 4ºC for 10 mins at 3,000 g
8. Subsequent resuspensions may be done in the
0.1M CaCl2 (1 L): same bottle. Cells must remain cold for the rest
of the procedure: Transport tubes on ice and
Mix 100 mL of 1M CaCl2 with 900 mL of dH2O. resuspend on ice in the cold room
Filter sterilize through a 0.22 μ filter 9. Pour off media and resuspend cells in 30 ml of
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cold 0.1 M CaCl2. Transfer the suspended cells

50% Glycerol (500 mL): into 50 ml polypropylene falcon tubes, and
incubate on ice for 30 mins.
Mix 50 mL of Glycerol with 50 mL of dH2O (Autoclave) 10. Centrifuge cells at 4ºC for 10 mins at 3,000 g
11. Pour supernatant and resuspend cells (by
0.1M CaCl2 + 15% glycerol: pipetting) in 8 mL cold 0.1M CaCl2 containing
15% glycerol. Transfer 140 μl into (1.5 mL)
Mix 100 mL of 1M CaCl2, 300 mL of 50% Glycerol, Ependorff tubes placed on ice. Freeze the cells
and 600 mL of dH2O in liquid nitrogen. Cells stored at -80ºC can be
used for transformation for up to ~6 months
LB plates:

494
Transformation of competent E. coli Cells

Changing the genotype of a cell or organism by • 42ºC water bath

transferring foreign DNA is called transformation. • Isopropyl thiogalactoside (IPTG) 100mm, Sterilize
The transferred DNA may be maintained as extra- by filtration.
chromosomal elements or integrated into the genome. • 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
(X-gal) 20mg/ml dissolved in DMSO.
eg: ampicillin susceptible genotype of E. coli strain
TOP10 can be changed to ampicillin resistant Procedure
genotype by transferring pUC18 plasmid that carries
a gene for ampicillin resistance and the selection in 1. Competent cells were taken out from -80ºC freezer
done in a selection medium with ampicillin. and thawed in the ice.
2. To transform the CaCl2-treated cells transfer 50μl
pUC18 vectors have a short segment of E. coli DNA, suspension of competent cells to a sterile chilled

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

which contains the regulatory and coding sequences microfuge tube using a chilled micropipette tip.
of Lac Z gene that codes for β-galactosidase enzyme. Add DNA (no more than 12.5 ng in a volume of
Isopropyl thiogalactoside (IPTG) is an inducer of Lac Z gene 2.5 μl or less) to each tube. Mix the contents of
expression. β -galactosidase reacts with the chromogenic the tubes by swirling gently. Store the tubes on
substrate 5-bromo-4-chloro- β-D-Galactoside (X-gal) and ice for 30 minutes.
yields a blue colored product. A multiple cloning site 3. Transfer the tubes to a rack placed in a preheated
(MCS) is engineered inside the coding region of the Lac 42ºC circulating water bath. Store the tubes in the
Z gene. The MCS as such does not disrupt the reading rack for exactly 90 seconds. Do not shake the tubes.
frame and results only in insertion of a few amino acids 4. Rapidly transfer the tubes to an ice bath. Allow
in the amino terminal fragment of the β-galactosidase. the cells to chill for 1-2 minutes.
Therefore, the colonies appear blue in color in the 5. Add 200 μl of SOC medium to each tube. Incubate
presence of IPTG and X-gal. However, when a insert is the cultures for 45 minutes in a water bath set at 37ºC
cloned in the MCS, that becomes a harmful insertion to to allow the bacteria to recover and to express the
the functional properties of β-galactosidase and it can antibiotic resistance marker encoded by the plasmid.
no longer react with X-gal, and therefore, the colonies 6. About 100ul of the inoculum of transformed
appear white in color. This is a simple visual color test that competent cells were plated onto LB agar plate
can be used to screen thousands of colonies to identify containing appropriate antibiotic, IPTG and X-gal
the presence of recombinant plasmids. 7. About 100ul of untransformed competent cells were
plated on a LB agar plates containing appropriate
Materials antibiotic, IPTG and X-gal as a negative control.
8. Store the plates at room temperature until the
• Competent TOP10 cells liquid has been absorbed.
• Foreign DNA /plasmid 9. Invert the plates and incubate at 37ºC for overnight.
• LB medium Transformed colonies should appear in 12-16 hour.
• LB plates with 100mg/L Amp

495
 Cloning of the PCR amplified products

Bacterial Strain used

expresses a lethal restriction enzyme in the host after
for cloning transformation and hence blue/white screening is
TOP10 (Invitrogen, USA) E. coli cloning host cells not required.
were made chemically competent and used for
transformation with the recombinant plasmid. Ligation of cDNA
CloneJET™ PCR Cloning Kit The ligation reaction of the PCR amplified cDNA with
the cloning vector was carried out as described in the
pJET 1.2 Blunt-end vector (50 ng/μl) manufacturer’s protocol (Fermentas, Germany). The
ligation protocol in brief was as follows:
In order to clone PCR amplified products, the pJET1.2/ 2X reaction buffer 10.0 µl
blunt vector (Fermentas, Germany), was used. PCR product x µl
PJET1.2/blunt is a linearized cloning vector, which pJET1.2/blunt end cloning vector (50ng/µl) 1.0 µl
accepts inserts from 6 bp to 10 kb. Milli-Q water x µl
T4 DNA ligase 1.0 µl
Total 20.0 µl
Blunt-end PCR products generated by proofreading
DNA polymerases (eg. Pfu) can be directly ligated After giving a short spin, the ligation mixture was
with the pJET1.2/blunt cloning vector. All common incubated at 22ºC for 10 min and directly used
laboratory E. coli strains can be directly transformed for transformation.
with the ligation product. Only recombinant clones
containing the insert appear on culture plates as the Transformation protocol
re-circularized or self-ligated pJET1.2/blunt vector
An aliquot of 5 µl of ligation mixture was added to
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50 µl of competent cells and mixed gently by flicking.

The mixture was incubated on ice for 30 min.

The tubes were then transferred to water bath

maintained at 42ºC and held for exactly 90 sec. The
tubes were immediately transferred back on ice and
allowed to chill for 1 min.

To the above mix, 50 µl of SOC media was added and

mixed. The tubes were incubated at 37ºC for 1 h in
a rotary shaker to allow the bacteria to recover and
develop the antibiotic resistance. After incubation, 75
Schematic diagram of PCR cloning vector pJET1.2 µl of transformed mixture was spread evenly on LB

496
agar plates with ampicillin (100 µg/ml) and incubated μM of each primers (pJET1.2F and pJET1.2R) and 0.5
at 37ºC for 16-20 h. U Pfu DNA polymerase. The PCR reaction conditions
were 95ºC for 5 min then 35 cycles of 95ºC for 30
Confirmation of the cloned sec, 58ºC for 30 sec and 72ºC for 60 sec followed by
a final extension at 72ºC for 10 min. Electrophoresis
genes by Colony PCR was performed on 1 % agarose gel prepared in 1x
The transformants obtained on LB agar plates with TBE buffer and stained with ethidium bromide.
ampicillin (100 µg/ml) were screened using colony PCR
with vector specific primers pJET1.2F (5’-CGA CTC ACT Colonies with expected sized products were inoculated
ATA GGG AGA GCG GC-3’) and pJET1.2R (5’-AAG into LB broth containing ampicillin (100 µg/ml) and
AAC ATC GAT TTT CCA TGG CAG-3’) to confirm the incubated in shaking incubator at 37ºC for 12-16
presence of the insert DNA. h after which the cells were pelleted and used for
plasmid isolation.
A small portion of selected colonies picked up from
the transformed plate using sterile toothpicks were
dispensed into the PCR reaction mix composed of 1x
PCR buffer with, 2 mM MgSO4, 0.2 mM each dNTP, 0.5

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

497
 Minipreparation of Plasmid DNA by
Alkaline Lysis with SDS

Plasmid is an extra chromosomal DNA present in most • Ethanol

of the bacteria and in some yeast. Most of them are • Phenol:chloroform (1:1, v/v)
circular and they vary in size and in numbers. The • TE (pH 8.0) containing 20 μg/ml RNase A
plasmids are modified and used as vectors (carrier)
in rDNA technology. In this experiment one such Media
modified plasmid pUC18 is isolated form an E.coli
culture by alkaline lysis method LB broth

This method is based on the principle that exposure Method

of bacterial suspensions to the strongly anionic
detergent at high pH opens the cell wall, denatures 1. Inoculate 2 ml of rich medium (LB Broth)
chromosomal DNA and proteins and release plasmid containing the appropriate antibiotic with a single
DNA into the supernatant. Although the alkaline colony of transformed bacteria. Incubate the
solution completely disturbs chromosomal DNA, culture overnight at 37ºC with vigorous shaking
the circular plasmids DNA are unable to separate at 200-300 rpm.
from each other because they are topologically 2. Pour 1.5 ml of the culture into a microfuge tube.
intertwined. During lysis, bacterial proteins, broken Centrifuge at maximum speed for 30 seconds
cell wall and denatured chromosomal DNA become at 4ºC in a microcentrifuge. Store the unused
enmeshed in large complexes that are coated with portion of the original culture at 4ºC.
dodecyl sulphate. These complexes are efficiently 3. Remove the medium by aspiration, leaving the
precipitated when solution with sodium ions are bacterial pellet as dry as possible.
replaced by potassium ions. After the denatured 4. Resuspend the bacterial pellet in 100 μl of ice-
materials have been removed by centrifugation native cold Alkaline lysis solution I by vigorous vortexing.
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DNA plasmid can be recovered from the supernatant 5. Add 200 μl of freshly prepared Alkaline lysis
by precipitation. solution II to each bacterial suspension. Close
the tube tightly, and mix the contents by inverting
Alkaline lysis is a flexible method that works well with the tube rapidly five times. Do not vortex! Store
all strains of E. coli and with bacterial culture ranging the tube on ice.
in size from 1ml to 500ml. The plasmid DNA obtained 6. Add 150 μl of ice-cold Alkaline lysis solution III.
by this method is devoid of nuclear DNA. Close the tube and disperse Alkaline lysis solution
III through the viscous bacterial lysate by inverting
Buffers and Solutions the tube several times. Store the tube on ice for
3-5 minutes.
• Alkaline lysis solution I 7. Centrifuge the bacterial lysate at maximum speed
• Alkaline lysis solution II for 5 minutes at 4ºC in a microcentrifuge. Transfer
• Alkaline lysis solution III the supernatant to a fresh tube.

498
8. (Optional) Add an equal volume of Alkaline Lysis Solution II
phenol:chloroform. Mix the organic and aqueous
phases by vortexing and then centrifuge the 0.2 N NaOH (freshly diluted from a 10 N stock)
emulsion at maximum speed for 2 minutes at 1% (w/v) SDS
4ºC in a microcentrifuge. Transfer the aqueous Prepare Solution II fresh and use at room
upper layer to a fresh tube. temperature.
9. Precipitate nucleic acids from the supernatant
by adding 2 volumes of 100% ethanol at room Alkaline Lysis Solution III
temperature. Mix the solution by vortexing and
then allow the mixture to stand for 2 minutes at 5 M potassium acetate, 60.0 ml
room temperature. Glacial acetic acid, 11.5 ml
10. Collect the precipitated nucleic acids by H2O, 28.5 ml
centrifugation at maximum speed for 5 minutes The resulting solution is 3 M with respect to
at 4ºC in a microcentrifuge. potassium and 5 M with respect to acetate.
11. Remove the supernatant by gentle aspiration as Store the solution at 4ºC and transfer it to an ice
described in Step 3 above. Stand the tube in an bucket just before use.
inverted position on a paper towel to allow all
of the fluid to drain away. Use a tissue paper or EDTA
disposable pipette tip to remove any drops of
fluid adhering to the walls of the tube. To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of
12. Add 1 ml of 70% ethanol to the pellet and disodium EDTA•2H2O to 800 ml of H2O. Stir vigorously
invert the closed tube several times. Recover the on a magnetic stirrer. Adjust the pH to 8.0 with NaOH
pellet by centrifugation at maximum speed for 2 (approx. 20 g of NaOH pellets). Dispense into aliquots

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

minutes at 4ºC in a microcentrifuge. and sterilize by autoclaving. The disodium salt of EDTA
13. Remove all of the supernatant by gentle aspiration will not go into solution until the pH of the solution
as described in Step 3.Take care with this step, is adjusted to approx. 8.0 by the addition of NaOH.
as the pellet sometimes does not adhere tightly
to the tube. Glycerol
14. Remove any beads of ethanol that form on the
sides of the tube. Store the open tube at room To prepare a 10% (v/v) solution: Dilute 1 volume of
temperature until the ethanol has evaporated molecular-biology grade glycerol in 9 volumes of
and no fluid is visible in the tube (5-10 minutes). sterile pure H2O. Sterilize the solution by passing it
15. Dissolve the pellet in 50 μl of TE (pH 8.0) through a pre rinsed 0.22-μm filter. Store in 200-ml
containing 20 μg/ml DNase-free RNaseA. Vortex aliquots at 4ºC.
the solution gently for a few seconds. Store the
plasmid solution at -20ºC. NaOH

Reagents The preparation of 10 N NaOH involves a highly

exothermic reaction, which can cause breakage of
glass containers. Prepare this solution with extreme
Alkaline Lysis Solution I care in plastic beakers. To 800 ml of H2O, slowly add
50 mM glucose 400g of NaOH pellets, stirring continuously. As an
25 mM Tris-Cl (pH 8.0) added precaution, place the beaker on ice. When
10 mM EDTA (pH 8.0) the pellets have dissolved completely, adjust the
Prepare Solution I from standard stocks in batches volume to 1 liter with H2O. Store the solution in a
of approx. 100 ml, autoclave for 15 minutes at 15 plastic container at room temperature. Sterilization
psi on liquid cycle, and store at 4ºC. is not necessary.

499
 Potassium Acetate 20 minutes at 15 psi on liquid cycle. Store the buffer
at room temperature.
5 M potassium acetate, 60 ml
glacial acetic acid, 11.5 ml Tris-Cl
H2O, 28.5 ml
The resulting solution is 3 M with respect to Dissolve 121.1 g of Tris base in 800 ml of H2O.
potassium and 5 M with respect to acetate. Store Adjust the pH to the desired value by adding
the buffer at room temperature concentrated HCl.

SDS pH HCl
7.4 70 ml
Also called sodium lauryl sulfate. To prepare a 20% 7.6 60 ml
(w/v) solution, dissolve 200 g of electrophoresis-grade 8.0 42 ml
SDS in 900 ml of H2O. Heat to 68ºC and stir with
a magnetic stirrer to assist dissolution. If necessary, (1 M) Allow the solution to cool to room temperature
adjust the pH to 7.2 by adding a few drops of before making final adjustments to the pH. Adjust the
concentrated HCl. Adjust the volume to 1 liter with volume of the solution to 1 liter with H2O. Dispense
H2O. Store at room temperature. Sterilization is not into aliquots and sterilize by autoclaving. If the 1 M
necessary. Do not autoclave. solution has a yellow color, discard it and obtain Tris of
better quality. The pH of Tris solutions is temperature-
TE dependent and decreases approx. 0.03 pH units for
each 1ºC increase in temperature. For example, a
100 mM Tris-Cl (desired pH) 0.05 M solution has pH values of 9.5, 8.9, and 8.6
10 mM EDTA (pH 8.0) at 5ºC, 25ºC, and 37ºC, respectively.
(10x Tris EDTA) Sterilize solutions by autoclaving for
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500
Restriction Enzyme digestion

Restriction enzymes form part of the restriction- The unit definition of restriction enzyme activity
modification system of bacterial cells that provides is based on the amount of enzyme required
protection against invasion of the cell by foreign to cut 1µg of bacteriophage lambda DAN to
DNA – especially bacteriophage DNA. Restriction completion in one hour’s time in a reaction volume
enzymes are Nucleases which can cleave the sugar- of 50 µl under optimal concentration of salt, pH
phosphate backbone of DNA, found in bacteria. As and temperature.
they cut within the molecule, they are commonly
called restriction endonucleases. They specifically Nomenclature
cleave the nucleic acids at specific nucleotide
sequence called Restriction sites to generate a  The first three letters of the restriction enzyme refer
set of smaller fragments. Most of the restriction to the organism from which the restriction enzyme
recognition sequences are palindromic and vary in was originally isolated, the fourth letter (if present)

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

lengths between 4 and 8 nucleotide. Restriction refers to the strain, and the Roman numerals serve as
enzyme makes two incisions, once through each indices if the same organism contains several different
sugar-phosphate backbone (i.e. each strand) of the restriction enzymes.
DNA double helix. Some restriction enzymes cut
the double stranded DNA in two different positions e.g. EcoR I and EcoR V are both from Escherichia
and generate ends that are staggered, with 5’ or 3’ coli, strain R; I and V are the order in which they
protruding terminal nucleotides; others cut at the were discovered.
same position and produce blunt ends. Discovery
of restriction enzymes lead to the development of Table 1. Restriction Enzyme Nomenclature
recombinant DNA technology and are routinely Enzyme  Enzyme Source
used for DNA modification and manipulation EcoRI Escherichia coli, strain R, Ist enzyme
in laboratories.
HindIII Haemophilus influenzae, strain d, 3rd
enzyme
Types of restriction enzymes BamHI Bacillus amyloliquefaciens, strain H, Ist
enzyme
Four different classes of restriction endonucleases
SmaI Serratia marcescens, Ist enzyme
are recognized, Type I, Type II, Type III and Type IV,
each distinguished by the difference in their mode HaeIII Haemophilus aegyptius, 3rd enzyme
of action. Type I and III are complex and have a
limited role in genetic engineering since the sites Restriction Enzyme cleavage:
of actual cleavage are at variable distance from
these recognition sites, and can be hundreds of Class II restriction enzymes generate three types
bases away. Type II restriction enzymes target only of DNA ends, all possessing 5´-phosphate and
methylated DNA. 3´-hydroxyl groups:

501
 All the enzymes are usually supplied with a 10x
buffer. Most companies have different kinds of buffer
giving various levels of efficiency and occasionally a
unique buffer for a particular enzyme optimized for
a)    Cohesive 5´ ends:- For example, ends generated 100% efficiency. The enzyme storage buffer contains
by EcoR I: antifreeze (glycerol) to allow it to be preserved at
-20ºC, but it will inhibit the digestion if present in
more quantities. So the volume of enzyme in the
reaction mix should not be more than 10% of the
final reaction volume.

b)    Cohesive 3´ ends:- For example, ends generated Reagents

by Pst I:
Restriction enzymes, DNA, 10x buffer

Procedure
1. Combine the following in a PCR tube for a single
c)    Blunt ends:- For example, ends generated by reaction of 10 ml
Hae III
Components Volume
Sticky ends (Blunt ends) are produced by cutting the 10X RE buffer 1.0 ml
DNA in a staggered manner within the recognition Milli-Q water 6.5 ml
site producing single stranded DNA ends. These ends DNA 2.0 ml (depending on the concentration)
Enzyme 0.5 ml (1 to 5U/mg of DNA. Maximum
have identical nucleotide sequence and are sticky volume to be added is 1/10th of the
because they can bind to complementary tails of other totoal reaction volume)
DNA fragments cut by the same Restriction enzyme. Total volume 10 ml

Some enzymes require special conditions. Some

requires BSA (bovine serum albumin) added in to the 2. It is always recommended to prepare a premix if
mixture. This is usually provided with the enzymes at you are doing many digestions at a time with the
100x concentration. BSA stabilizes the enzymes, binds same enzyme.
to some impurities and prevent enzyme adsorption 3. Incubate the tubes in a water bath or thermal cycler
to the surface of the tubes. Some require weak at 37ºC for 1-2 h.
detergents (eg.Triton X-100) to reduce surface tension. 4. Incubate the tubes at 65ºC for 10 minutes to
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Some require to be incubated at temperatures other inactivate the enzyme.

than the standard temperature 37ºC. 5. Run a 1.5% agarose gel to see the restriction pattern.

502
Ligation

Ligation of a segment of insert DNA to a • T4 DNA Ligase

linearized plasmid vector involves the formation of • 5X Ligase buffer
phosphodiester bonds between DNA molecules. • 0.2ml PCR tube
Ligase catalyze the formation of phosphodiester • Incubator
bonds between the directly adjacent 3’ hydroxyl
and 5’phosphoryl termini of nucleic acid molecule. Procedure
The ligation process consumes ATP as energy source.
When cohesive ends are present the ligation occurs The following components were added in a 0.2ml
efficiently, but when blunt-end fragments have to be PCR tube.
ligated the efficiency is very low. Salt and phosphate Linearized Vector (100ng/μl) 9 μl
concentration is very important for the efficiency of Insert DNA (100ng/μl) 3 μl
ligation. Incubation times and temperatures vary a

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

5x ligase buffer 4 μl
great deal in the literature but the following seem to T4 DNA Ligase (0.1U/μl) 1 μl
work well in most cases. Sterile distilled water 4 μl
Total 20μl
For “sticky ends”: incubate 2-4 hrs. at 16ºC; for blunt
ends incubate overnight at 4ºC. Mixed gently and centrifuged briefly

Materials and Reagents Incubate the tubes in thermal cycler at 16ºC for 2
hrs and after incubation the samples were stored at
• Linearized vector -20ºC for future use
• Insert DNA

503
 Sodium Dodecyl Sulphate-Polyacrylamide
gel electrophoresis (SDS PAGE)

SDS-PAGE is widely used to analyze the proteins in leading edge of the proteins being separated on the
complex extracts. The most commonly used methods gel. The sample buffer also contains glycerol, which
are derived from the discontinuous SDS-PAGE system allows the protein samples to settle into the bottom
first described by Laemmli (1970). The system actually of the gel wells. The gel is vertically positioned in the
consists of two gels–a resolving (aka running) gel electrophoresis apparatus and covered with chamber
in which proteins are resolved on the basis of their buffer containing glycine.
molecular weights (MWs) and a stacking gel in
which proteins are concentrated prior to entering the Once a voltage is applied, the chloride ions in the
resolving gel. Differences in the compositions of the sample buffer and stacking gel move rapidly toward
stacking gel, resolving gel and electrophoresis buffer the positive pole, forming the leading edge of a
produce a system that is capable of finely resolving moving ion front. Glycine molecules have very little
proteins according to their MWs. charge in the stacking gel, so they migrate at the rear
of the moving ion front. This difference in chloride
The Laemmli (1970) SDS-PAGE system can be and glycine mobility sets up a steep voltage gradient
considered a 3-component system. The stacking in the stacking gel that sweeps along the negatively
and running (resolving) gels have different pore charged protein-SDS complexes. The large pores of
sizes, ionic strengths and pHs. The third component the stacking gel present very little resistance to the
is the electrophoresis buffer (25 mM Tris, 192 mM movement of protein-SDS complexes, which then
glycine, 0.1% SDS, pH ~8.3), which contains large “stack up” into a very concentrated region at the
amounts of glycine. The ionization state of the glycine interface between the running and stacking gels
is critical to the separation. At neutral pH, glycine is a (right). Protein-SDS complexes remain concentrated
zwitterion, with a negatively charged carboxyl group at the interface until the slowly migrating glycine
and a positively charged amino group. The pKa of molecules reach the boundary between the two gels.
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the amino group is 9.6, considerably higher than the

pH of the chamber buffer. Consequently, very little Dramatic changes occur as the glycine ions enter the
glycine has a negative charge in the chamber buffer running gel. The pH of the running gel is closer to
or stacking gel, and significant ionization does not the pKa of the glycine amino groups, so a significant
occur until the glycine enters the more alkaline pH fraction of the glycine molecules assume a negative
8.8 environment of the running gel. charge. Negatively charged glycine molecules begin
to move at the same rate as the chloride ions, thereby
Let’s follow the progress of protein samples during eliminating the voltage difference that controlled
SDS-PAGE to see how differences in the composition protein mobility through the stacking gel. The pores
of these three components generate the high in the running gel are much smaller than those of the
resolving power of SDS-PAGE gels. The sample stacking gel, so the pores present frictional resistance
buffer used for SDS-PAGE contains a tracking dye, to the migration of proteins. Proteins begin to migrate
bromophenol blue (BPB), which will migrate with the at different rates, because of the sieving properties of

504
the gel. Smaller protein-SDS complexes migrate more and adjusted the pH to 6.8 with HCl and
quickly than larger protein- SDS complexes (right). upto 100mL with water.
Within a certain range determined by the porosity 4. 10% SDS solution: 1g of SDS in 10mL of
of the gel, the migration rate of a protein in the distilled water.
running gel is inversely proportional to the logarithm 5. N,N,N’N’-Tetra methylene diammine(TEMED)
of its MW. 6. 10% Ammonium per sulphate (APS): 1g of APS
in 10mL of distilled water.
Proteins are visualized 7. Electrophoresis Buffer:
a) Tris: 25mM, pH 8.3
with stains. b) glycine: 250mM, pH 8.3
To visualize the positions of proteins after c) SDS: 0.1%: Dissolved in minimum amount
electrophoresis is complete, we stain the gels with of water (500mL) and then added SDS.
various dyes that bind noncovalently and with very Allowed to settle and dissolved. This was
little specificity to proteins. During the staining finally made upto 2.5liters.
process, proteins are also “fixed” in the gel, meaning 8. Sample buffer 4x: 5.0mL
that proteins become insoluble and unable to a) Tris (1M, pH 6.8): 2.1mL
diffuse out of the gel. In our experiments, we will b) 2% SDS: 100mg
use a colloidal suspension of Coomassie Brilliant c) Glycerol (100%): 1.0mL
Blue G-250. Brilliant Blue G-250 binds proteins d) b-mercaptoethanol: 0.5mL
nonspecifically through a large number of ionic and e) Bromophenol blue: 2.5mg
Van der Waals interactions. In this procedure, gels are f) Distilled water: 0.4mL
rinsed with water to remove the buffer salts used for 9. Staining solution (100mL):
electrophoresis and then treated with the colloidal a) Alcohol: 40%

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

G-250 suspension. Protein bands appear rapidly, and b) Acetic acid: 10%
when necessary, the gels can be destained to lower c) Commassie Brilliant Blue (CBB): 259mg
the gel background. Brilliant Blue staining intensity is d) Distilled water: 50%
considered to be a quantitative procedure, because 10. Destaining solution (100mL)
with some exceptions, the intensity of a stained band a) Alcohol: 50%
is directly proportional to the amount of protein in b) Acetic acid: 10%
a band. c) Distilled water: 40%

Reagents Casting SDS-PAGE gels

Preparation of stock solution and buffers: These instructions are designed for constructing two
1. 30% acrylamide 12% SDS-PAGE gels with the BioRad Mini Protean
a) Acrylamide: 29.2g system. The plates were washed in warm detergent
b) N, N-methelyne–bis–acrylamide: 0.8g solution, rinsed subsequently in tap water, deionised
Added water, dissolved and made upto water and ethanol and dried.
100mL and filtered with Whatman no.1
filter paper. Assemble the gel
2. Separating gel buffer:
casting apparatus
a) Tris-HCl: 1.5M, pH 8.8
18.171g of Tris was dissolved in 60mL of 1. Assemble the components that you will need for
water and adjusted the pH to 8.8 with HCl casting the gel: a tall glass plate with attached 1
and finally made upto 100mL with water. mm spacers, a small glass plate, a green casting
3. Stacking gel buffer: frame and a casting stand.
a) Tris-HCl: 1M, pH 6.8
6.057g of Tris was dissolved in 60mL water 2. Place the green casting frame on the bench with

505
 the green “feet” resting firmly against the bench Reagent Resolving gel Stacking gel
and the clamps open (perpendicular to the frame) Deionized water 3.5 mL 2.1 mL
30% acrylamide:bis-acrylamide 4.0 mL 0.63 mL
and facing you.
(29:1)
1.5 M Tris-HCl, 0.4% SDS, pH 8.8 2.5 mL ———
3. Place the two gel plates in the frame. Insert the 0.5 M Tris-HCl, 0.4% SDS, pH 6.8 ——— 1.0 mL
taller spacer plate with the “UP” arrows up and 0% ammonium persulfate (catalyst) 100 μL 30 μL
the spacers facing toward you into the casting TEMED (catalyst) 10 μL 7.5 μL
frame (the BioRad logo should be facing you). Polymerization occurs rapidly, so be sure to follow
Insert the short glass plate in the front of the the step-by-step instructions below.
casting frame. There should be a space between
the plates. NOTE: catalysts should NOT be included into the
mixture until you are ready to pour the gels!!
4. Secure the plates in the casting frame by pushing 1. Label two 15 mL beakers “Resolving gel” and
the two gates of the frame out to the sides. “Stacking gel”.
IMPORTANT: the bottom edges of the two plates 2. Prepare ONLY the resolving gels at this time. Mix the
should be flush with the lab bench before you acrylamide solution, pH 8.8 Tris buffer and water,
clamp the frame closed to ensure a watertight seal. as shown in the chart above. Mix the ingredients
To do this, rest the frame vertically on the bench gently, trying not to introduce air. Oxygen inhibits
BEFORE closing the gates. polymerization of acrylamide gels.
3. To the resolving gel mixture, add 100 μL of a 10%
5. Clamp the casting frame with glass plates into ammonium persulfate (APS) solution. Gently mix
the casting stand, with the gates of the casting the solution, trying not to introduce air. Oxygen
frame facing you. inhibits acrylamide polymerization.
4. Add 10 μL of TEMED catalyst. Once again, gently
6. Repeat steps 1-5 to prepare a second gel in the mix in the catalyst trying not to introduce air
casting frame. bubbles. CAUTION: TEMED has an unpleasant
odor. Cover the tube immediately after you aliquot
7. Check to see if the assembled plates in the casting this reagent.
stand are sealed properly by pipetting a small 5. Working quickly, use a plastic transfer pipette to
amount of deionized water into the gap between fill the space between the two plates until the
the plates. If the glass plates hold water and don’t resolving gel solution reaches a height just above
leak, you are ready to make the gels. Pour the water the green clamps on the gel casting frame. Draw
out by holding the entire casting platform over a up any remaining acrylamide into the transfer
liquid waste container or sink. Use paper towels pipet. (You will know that the acrylamide has
or tissues to absorb any residual water. If the gel polymerized when you can no longer push the
Central Marine Fisheries Research Institute

leaks, disassemble the frame, dry the plates and acrylamide out of the pipet.)
go back to step 3 6. Using a transfer pipet, add deionized water so
that it gently flows across the surface of the
Prepare two resolving gels. polyacrylamide mixture. The water layer ensures
that the polyacrylamide gel will have a level surface
Safety Note: Acrylamide and bisacrylamide once it polymerizes.
monomers are weak neurotoxins. Gloves and goggles 7. Allow the gel to polymerize, which takes ~15-20
should be used when working with acrylamide. minutes. You will note that the interface between
the polyacrylamide and water overlay disappears
Assemble the chemicals that you will need to pour temporarily while the gel polymerizes. A sharp
the gels. The table below shows the quantities of new interface then forms between the two layers,
each chemical that you will need to pour two gels indicating that polymerization is complete. (You
with the Mini-Protean system. can also check the remaining polyacrylamide in

506
 the transfer pipette to see if it has polymerized.) 5. Lower the chamber into the electrophoresis tank.
8. When polymerization is complete, remove the
water from the top of the resolving gel by tilting 6. Fill the space between the two gels with Tris-glycine
the gel to the side and using a paper towel or running buffer. This forms the upper chamber
Kimwipe to wick out the water. for electrophoresis.

Prepare the stacking gels 7. Add Tris-glycine running buffer to the outer (lower)
chamber until the level is high enough to cover the
1. Prepare the stacking gels. Mix the acrylamide platinum wire in the electrode assembly.
solution, pH 6.8 Tris buffer and water, as shown
in the chart above. Load and run samples on the SDS-
PAGE gel
2. Add 30 μL 10% APS and 7.5 μL TEMED to the
stacking gel acrylamide mixture. Mix the contents 1. Retrieve the cell extracts and mix with gel loading
by gently inverting the tube twice. dye, vortex vigorously for ~ 10 seconds to
thoroughly mix the contents and boil at 100ºC
3. Use a transfer pipette to pipette the stacking gel immediately for 3-5 minutes.
on top of the resolving gel between the two glass
plates. Add enough stacking solution until it just 2. Using gel loading micropipette tips (tips have very
reaches the top of the small plate. long, thin points and fit P20s or P200s), load up
to 15 μL of sample into each well. Load 5 μL of
4. Carefully, but quickly, lower the comb into a molecular weight standard into one lane of the
position, being careful not to introduce air gel. Load samples slowly and allow the samples to

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

bubbles. (The Bio Rad logo on the comb should settle evenly on the bottom of the well.
be facing you.) Adding the comb will force
some solution out of the gel, but this is fine. If NOTE: Be sure to record the order of samples loaded
air bubbles become trapped below the comb, onto the gel.
remove the comb and reposition it. 3. Connect the tank to the power supply. Fit the tank
cover onto the electrodes protruding up from the
Running SDS-PAGE gels electrode assembly. Insert the electrical leads into
the power supply outlets (connect black to black
Set up the electrophoresis apparatus and red to red).

1. Carefully remove the gels from the casting stand 4. Turn on the power supply. Run the gel at a constant
and then from their green frames. voltage of 120-150 V. Run the gel until the blue
dye front nearly reaches the bottom of the gel.
2. Carefully remove the comb from the spacer gel. This may take between 45-60 min.

3. Remove the casting frame from the gel cassette Staining SDS-PAGE gels
sandwich and place the sandwich against the
gasket on one side of the electrode assembly, with 1. After the run is complete, turn off the power supply.
the short plate facing inward. Place a second gel
cassette or a buffer dam against the gasket in the 2. Remove the gel apparatus from the tank. Open
other side of the electrode assembly. the clamping frame and remove the gel cassette
sandwich. Carefully, pry the two plates apart with
4. Clamp the green clamps on the sides of the a spatula. With the spatula, remove the lower right
electrode assembly (below). or left corner of the gel to serve as an orientation
marker. Be sure to indicate in your lab notebook

507
 whether the notched corner corresponds to lane appropriately labeled waste container.
1 or lane 10 of the gel. You may also remove the
stacking gel with the spatula, if you desire. 6. Destain the gel by filling the container about half
full with destaining solution. Shake the gel in
3. Place the gel in a small plastic tray and gently free the destaining solution for 30 minutes. Pour off
the gel from the glass plate, allowing it to slide the destaining solution and add new destaining
into the water. The gel should move freely in the solution. Repeat, if necessary, until protein bands
water. Place the gel and tray on a rocking platform. become visible.
Rock the gel for ~2 minutes.
7. When individual bands are detectable, record
4. Drain the water from the gel and add enough your data. You may photograph the gel with
staining solution to cover the gel, while allowing the gel documentation system against a white
the gel to move freely when the tray is rocked. background. Alternatively, place the gel in a clear
Cover the gel container with saran‐wrap and rock plastic page protector and scan the gel.
for 30 min. Make sure that the gel does not stick
to the bottom of the tray. 8. After recording the data, dispose of the gel in the
Biohazard waste container.
5. After that, drain the staining solution into an
Central Marine Fisheries Research Institute

508
Tricine–SDS Poly Acrylamide Gel
Electrophoresis (Tricine–SDS-PAGE)

Tricine–SDS-PAGE is used to separate proteins in 10x Anode Running Buffer (0.2 M Tris-Cl, pH 8.9)
the mass range 1–100 kDa and is the preferred
electrophoresis system for the resolution of proteins Dissolved 24.22 g Tris base in 50ml Milli-Q water. pH
smaller than 30 kDa. was adjust to 8.9 with 6 N HCl and final volume was
made to 100 ml with Milli-Q waterand store at 4ºC.
Composition of stock solutions for use in Tricine SDS-
PAGE: 1 M Tris-HCl, pH 6.8

Acrylamide / bis acrylamide (37.5% T, 1% C) 30% Dissolve 12 g Tris base in 50ml Milli-Q water. The
pH was adjusted with 6 N HCl and final volume was
made to 100 ml with Milli-Q water and stored at 4ºC.

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

2x Tricine sample buffer

Mixed 1 ml 1 M Tris-Cl ph 6.8, 2.4 ml glycerol, 0.8 g

SDS, 2 mg Coomassie blue G-250, 1 ml β-mercapto
Dissolved 30 g Acrylamide and 0.8 g bis–acrylamide ethanol and final volume was made to 10 ml with
in 50 ml Milli-Q water and final volume was made Milli-Q water and stored at 4ºC.
to 100 ml with Milli-Q water, and stored at 4ºC in
an Amber coloured bottle. Ammonium persulfate(APS) 10%

3x Gel Buffer (3 M Tris-Cl, 0.3 % SDS, pH 8.45) Tetramethylethylenediamine (TEMED)

Dissolved 36.4 g Tris base in 50 ml Milli-Q water. 15% Separating gel Preparation
The pH was adjusted to 8.45 with 6 N HCl and final Milli-Q water 0.516 ml
volume was made to 100 ml with Milli-Q water and
Glycerol 1.00ml
finally 0.3 g SDS was added and stored at 4ºC.
3X gel buffer 3.33ml

10x Cathode Running Buffer (0.1 M Tris, 0.1 M Acrylamide / Bis (30% stock) 5ml
Tricine, and 0.1 % SDS) 10% APS 150μl

TEMED 4 μl
Dissolved 12.11 g Tris base, 17.92 g Tricine and 1g
Total Volume 10ml
SDS in 50 ml Milli-Q water, and with out adjusting
the pH the final volume was made to 100ml with 5% Stalking Gel Preparation
Milli-Q water and stored at 4ºC Milli-Q water 2.4ml

509
 3X gel buffer 1.67ml
increased to 20 mA/gel. After the electrophoresis the
Acrylamide / Bis (30% stock) 840μl gel was removed and proteins were fixed by staining
10% APS 75μl in the Coomassie stain solution.
TEMED 5 μl

Total Volume 5ml

Coomassie Blue staining
and Destaining
Tricine SDS-PAGE Electrophoresis Fixing and staining was carried out simultaneously in
a solution containing 0.1 % Coomassie blue R-250
The gel was cast in a Mini-PROTEAN®Tetra Cell in fixative (40 % Methanol and 10 % glacial Acetic
(10x8cm) (Bio-Rad Laboratories, Inc, USA). After the acid). Afterwards the destaining of the gel was
polymerization, the gel assembly was transferred in to carried out by several changes of 40 % methanol
a tank containing the 1x running buffers. The cathode + 10 % glacial acetic acid mixture to remove the
and anode tanks were filled and electrophoresis was background stain. The gels were documented under
carried at constant current of 10 mA per gel till the a gel documentation system.
dye reaches separating gel, then the current was
Central Marine Fisheries Research Institute

510
Separation of DNA in Polyacrylamide Gels

Polyacrylamide gels can separate DNA that differs by

0.2% in length, well beyond the resolving capabilities Reagents required
of agarose (2% difference in DNA length). Another 1. Acrylamide- Bis acrylamide Solution
advantage to using polyacrylamide gels is that they Acrylamide (19:1) 30% stock - 4 ml
Double distilled water - 6 ml
can accommodate large amounts of DNA (up to 10 x TBE - 1.25 ml
10 μg) without any loss in resolution. Depending 10% Ammonium persulphate - 80 ml
upon the application, TBE gels can be prepared as (freshly prepared)
TEMED - 8 ml
denaturing or nondenaturing gels. 2 TBE buffer 10X (pH-8.0)
Tris base - 10.8 g

Applications Boric acid - 5.5 g

EDTA - 0.75 g
Make up the solution to 100 ml with double distilled water.
Autoclaved and stored at 4ºC

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Denaturing gels: concentrations 3. Gel loading buffer
range from 8–20% Bromophenol blue - 0.5%
Glycerol (mol. grade) - 30%
• Oligonucleotide purification Prepared in 1X TBE
Store at 4ºC.
• Separation of single-stranded DNA
4. 1X TBE buffer
• Isolate radiolabeled DNA probes 10X TBE - 10 ml
• S1 nuclease assay Distilled Water - 90 ml
• DNA footprinting
• RNase protection assays
Protocol
1. Clean the glass plates and spacers thoroughly.
Nondenaturing gels: Hold the plates by the edges or wear gloves, so
that oils from the hands do not become deposited
concentrations range from 3–20%
on the working surfaces of the plates. Rinse the
• Separation of di-nucleotide repeats plates with deionized water and ethanol and set
• Separation of DNA ranging from 20 bp–2000 bp them aside to dry. The glass plates must be free of
in length grease spots to prevent air bubbles from forming
• Study DNA-Protein interactions (Gel Shift Assays) in the gel.

Buffers for Electrophoresis 2. Assemble the glass plates with spacers in gel caster.

To ensure adequate buffering power during vertical 3. Prepare the gel solution with the desired
electrophoresis, TBE Buffer is used for polyacrylamide polyacrylamide percentage according to the table
gel electrophoresis at a working strength of 1X. Lower below, which gives the amount of each component
dilutions of the buffer or the use of TAE Buffer may required to make 12 ml (sufficient for 2 Hoefer
cause gels to overheat and result in band smiling minigels of 1 mm thickness):
throughout the gel.

511
 Volume of Reagents Used to Cast Polyacrylamide Gels 8. When ready to proceed with electrophoresis,
remove gels from gel caster, carefully clean spilled
30% 10%
Gel % Acrylamide H2O (ml) 5x TBE (ml) APS TEMED (μl) gel from back of white plates and insert gels into
(29:1) (μl) Hoefer gelbox. Add running buffer and carefully
8% 3.2 ml 6.4 2.4 200 10 pull the combs from the polymerized gel.
10% 4.0 ml 5.6 2.4 200 10
12% 4.8 ml 4.8 2.4 200 10
9. It is important to use the same batch of
Stock solutions other than 29:1 (% w/v) acrylamide:bisacrylamide
can be used to cast polyacrylamide gels. However, it is then electrophoresis buffer in both of the reservoirs
necessary to recalculate the appropriate amount of stock solution and in the gel. Small differences in ionic strength
to use. Gels can be cast with acrylamide solutions containing or pH produce buffer fronts that can greatly
different acrylamide:bisacrylamide (cross-link) ratios, such as 19:1
distort the migration of DNA.
and 37.5:1, in place of the 29:1 ratio recommended here. The
mobility of DNA and dyes in such gels will be different from those
given in this protocol. 10. Use a Pasteur pipette or a syringe to flush out
the wells once more with 1x TBE. Mix the DNA
4. Wear gloves. Work quickly after addition samples with the appropriate amount of gel-
of TEMED to complete the gel before the loading buffer. Load the mixture into the wells
acrylamide polymerizes. using a micropipette equipped with a drawn-out
plastic tip.
5. Immediately insert the appropriate comb into
the gel, being careful not to allow air bubbles 11. Connect the electrodes to a power pack, turn on
to become trapped under the teeth. The tops the power, and begin the electrophoresis run.
of the teeth should be slightly higher than
the top of the glass. Clamp the comb in place 12. Run the gel until the marker dyes have migrated
with bulldog paper clips. If necessary, use the the desired distance. Turn off the electric
remaining acrylamide gel solution to fill the gel power, disconnect the leads, and discard the
mold completely. Make sure that no acrylamide electrophoresis buffer from the reservoirs.
solution is leaking from the gel mold.
13. Detach the glass plates. Lay the glass plates on
6. Allow the acrylamide to polymerize for 30-60 the bench. Use a spacer or plastic wedge to lift
minutes at room temperature. a corner of the upper glass plate. Check that the
gel remains attached to the lower (white) plate.
7. After polymerization is complete, surround the Pull the upper plate smoothly away. Remove
comb and the top of the gel with paper towels the spacers.
that have been soaked in 1x TBE. Then seal the
entire gel in Saran Wrap or plastic bag and store 14. Stain gels with silver nitrate to visualize the bands.
Central Marine Fisheries Research Institute

it at 4ºC until needed.

512
Visualization of DNA in Polyacrylamide
gels using silver staining

Polyacrylamide gels with were stained to visualize Protocol

the DNA using the silver staining kit supplied by
Amersham Pharmacia. 50 ml of fixing solution were used for 20 minutes
to fix the gels (diluted five times with 30.4 ml
Reagents double distilled water and 9.6 ml ethanol) and
silver-impregnated (with 1X staining solution) for
Solution Composition 20 minutes. This followed by washing the gels
1 Fixing solution (5X)3.0% Benzene sulphonic acid (w/v) in with double distilled water for another 1 minute.
24% ethanol (v/v)
This followed by keeping gels in the 1X developing
2 Staining solution 1.0% Silver nitrate (w/v),
(5X) 0.35% Benzene sulphonic acid (w/v). solution in darkness for 10 minutes. When the bands
were dark enough, developing solution was poured

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

3 Developing solution 12.5% Sodium carbonate (w/v).
(5 X) 37% Formaldehyde (w/v) in water out. Then stopping and preserving solution (1X) was
2% Sodium thiosulphate (w/v) in water
immediately added and the gel was documented.
4 Stopping and 5% Acetic acid (v/v)
Preserving solution, 25% Sodium acetate (w/v)
5X 50% Glycerol (v/v)

513
 Production of Antibodies

Principle
• Mix 0.5 ml of soluble antigen to 0.5 ml of adjuvant.
A substance that can induce a detectable immune The mixture is forcibly passed through needle until
response in heterologous species in known as an water in oil emulsion is achieved.
antigen. When foreign proteins (antigen) are injected
into rabbits, they induce immune response and • Clean skin of rabbit (preferably on thigh region)
produce antibodies to that particular antigen. These with 70% alcohol and hold a fold of skin between
antibodies can be separated from serum of the rabbits thumb and finger.
injected with antigen.
• Insert the needle into the skin lying behind the
Materials skin fold and inject the desired volume of toxin
adjuvant mixture.
Antigen: Soluble antigen: Proteins, virus, etc
• Four to five injections of the same dose are given
• Particulate antigen: Bacterial cell at weekly intervals.
• Freund’s adjuvant
• 2 ml syringe • In case of particulate antigen, inject the antigen
• 20 gauge needle intravenously on 0, 3, 5, 9, 13 and 15 day
• Millipore filter without adjuvant.

Procedure • Bleed the rabbit from marginal ear vein, separate

the serum and test for antibody formation by agar
• Pass the antigen through 0.45 mm millipore filter gel precipitation test for soluble antigen and by
in case of soluble antigen and pure bacterial cell agglutination test for particulate antigen.
preparation in case of particulate antigen.
Central Marine Fisheries Research Institute

514
Author Index

Ambasankar K.������������������������������������������������������������������� 4,03,409 Prabhakaran M. P.������������������������������������������������������������������������ 104

Anusree V. Nair������������������������������������������������������������������������������ 308 Pradeep M. A.�������������������������������������� 167, 180, 267, 274, 278
Balasubramanian C. P.����������������������������������������������������������������� 466 Raja Swaminathan T.������������������������������������������������������������������� 213
Basdeo Kushwaha������������������������������������������������������������������������ 148 Rajendran I.������������������������������������������ 353, 361, 366, 371, 392
Basheer V. S.������������������������������������������������������������������������ 144, 213 Rajendran K. V.������������������������������������������������������������������������������ 252
Chandrasekar S.���������������������������������� 416, 424, 432, 443, 462 Ramachandran Nair K. G.���������������������������������������������������������� 396
Dinesh Kumar S.������������������������������������������������������������������������������ 35 Rameshkumar P.���������������������������������������������������������������������������� 259
Esha Arshad 167, 180 Ravindra Kumar����������������������������������������������������������������������������� 148
Gajendragad M. R.����������������������������������������������������������������������� 245 Rekha J. Nair������������������������������������������������������������������������������������ 35
Geetha Sasikumar��������������������������������������������������������������������������� 76 Rema Madhu����������������������������������������������������������������������������������� 29
Gopakumar G.��������������������������������������������������������������������������������� 21 Reshma K. J.����������������������������������������������������������������������������������� 291
Gopalakrishnan A.������������������������������������������������������������������������ 144 Reynold Peter������������������������������������������������������������ 155, 202, 233
Jeena N. S.�������������������������������������������������������������������������� 160, 202 Sajeela K. A.����������������������������������������������������������������������������������� 228
Joshi K. K.������������������������������������������������������������������������������������������ 11 Sandeep K. P.���������������������������������������������������������������������������������� 403
Kajal Chakraborty 315, 328, 335, 341, 375, 446, 448, 456 Sandhya Sukumaran������������������������������������� 174, 193, 198, 202
Kaladharan P.����������������������������������������������������������������������������������� 99 Sanil N. K.��������������������������������������������� 265, 267, 274, 280, 286
Krupesha Sharma S. R.������������ 180, 265, 267, 274, 278, 421 Srinivasa Raghavan V.�������������������������������������������� 129, 135, 220
Kumaraguru Vasagam K. P.��������������������������������������������� 403, 409 Suja C. P.������������������������������������������������������������������������������������������ 224

Training manual in Molecular Biology and Biotechnology for Fisheries Professionals

Lakshmi Pillai S.������������������������������������������������������������������������������� 67 Sumithra T. G.���������������������������������������������������������������������� 301, 305
Linga Prabu D.������������������������������������� 416, 424, 432, 443, 462 Syama Dayal J.�������������������������������������������������������������������� 403, 409
Madhu K.������������������������������������������������������������������������������������������� 29 Thomas P. C.����������������������������������������������������������������������������������� 116
Maheswarudu G.���������������������������������������������������������������������������� 67 Venkatesan V.����������������������������������������������������������������������������������� 76
Mohamed K. S.�������������������������������������������������������������������������������� 76 Vidya Jayasankar��������������������������������������������������������������������������� 206
Nagpure N. S.�������������������������������������������������������������������������������� 148 Vijayagopal P.������������������������������������������������������������ 353, 371, 421
Nandini Menon N.������������������������������������������������������������������������� 92 Vijayan K. K.������������������������������������������������������������������������ 239, 466
Pani Prasad K.�������������������������������������������������������������������������������� 298 Wilson Sebastian�������������������������������������������������������������������������� 202
Paulton M. P.��������������������������������������������������� 109, 113, 170, 190

515
 This training manual is a compilation of the lecture notes delivered in the areas

of marine biology and molecular biology related to fisheries research, fish

genetics and genomics, fish health management, microscopy, fish nutrition

and marine bioprospecting as part of the DBT sponsored 3 months National

Training on Molecular Biology and Biotechnology for Fisheries Professionals

at CMFRI, Kochi. Its uniqueness is in integrating marine biology with marine

biotechnology where the stress was on introducing the marine biodiversity to

the participants to begin their journey in marine biotechnology. This manual

also documents standard operating procedures and laboratory protocols in

marine biotechnology. The support from Department of Biotechnology, Govt.

of India for putting this together was a need of time.

DBT-HRD

Indian Council of Agricultural Research Ministry of Science and Technology

Central Marine Fisheries Research Institute Department of Biotechnology

Post Box No.1603, Ernakulam North P.O., Kochi-682 018, Kerala, India.
Phone: +91 484 2394357, 2394867 Fax: +91 484 2394909 E-mail: [email protected] www.cmfri.org.in