Research Proposal: Analysis of Larvicidal Activity Against Aedes Aegyptii Using Extracts of Medicinal Plant in Bangladesh

RESEARCH PROPOSAL
Analysis of Larvicidal Activity Against Aedes aegyptii Using Extracts of

Medicinal Plant in Bangladesh

Course Code: GEB 413
Course Title: Medical and Pharmaceutical Biotechnology
Submitted by
Jarin Taslem Mourosi
Registration: 2013 431 004
Department of Genetic Engineering & BiotechnologyShahjalal University of Science
& Technology Sylhet 3114, Bangladesh
Submitted to
Date of Submission: 14 September, 2017
Department of Genetic Engineering and Biotechnology
Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh

Title
Analysis Of Larvicidal Activity Against Aedes aegypti Using Extracts of Medicinal Plant In
Bangladesh
Abstract
The bio-efficacy of Andrographis paniculate and Citrullus colocynthis leaf extract and
larvicidal activity will be assessed against the first to fourth instars larvae of Aedes aegypti,
under the laboratory conditions. The plant material will be shade dried at room temperature and
powdered coarsely. Andrographis paniculate and Citrullus colocynthis will show varied degrees
of larvicidal activity against various instars larvae of A. aegypti. The LC50 of plant extract
against the first to fourth instars larvae and the LC90 need to be calculated. From the crude
extracts of medicinal plants several polar, non-polar solvents, such as water, hexane, methanol,
chloroform, ethanol, and acetone, are reported to exhibit larvicidal activity [1–4].
Introduction
Aedes aegypti is a vector which can transmit a lot of viral disease. Among them Dengue,
Chikungunya are highly notable. Recent in Bangladesh, we have faced a massive outbreak of
Chikungunya viral disease in Dhaka. It is an acute febrile illness that is associated with severe
debilitating arthralgias and rash. Mosquito can develop in both aquatic stage and terrestrial life.
When female lays egg in moist condition aquatic life starts. After undergoing instar larvae, pupa
they will become flying adult. The best time for controlling the population is 8-10 days aquatic
cycle. Bangladesh is a country considered to be rich in medicinal plants genetic resources. By
virtue of its favorable agro-climatic condition, it has a large genetic resources medicinal plants.
About 5000 species of phanerogams and pteridophytes grow in the country’s forests, wetlands,
farms and even roadsides as indigenous, naturally-occurring or cultivated plants. Of these, more
than a thousand have been claimed to possess medicinal or curative properties. Recently, 546
species have been identified as having medicinal properties. Secondary metabolites obtained
from plant act as important source of phytochemicals that can be directly used as drug or after
proper modification it can be used as drug.
Andrographis paniculate is a medicinal plant widely cultivated in tropical regions of Asia.

Traditionally it is used for several applications including as an antidote for snakebite in folk
medicine and poisonous stings of some insects and to treat dyspepsia, influenza, dysentery,
malaria, cold, fever, laryngitis, and respiratory infection in many Asian countries. The extract of
the plant is reported to possess immunological, antibacterial, anti-inflammatory, antithrombotic,
hepatoprotective, antihypertensive, and antidiabetic activities [6]. The extract of the plant is a
rich source of flavonoids and labdane diterpenoids [7]. Methanolic extracts of these plants were
tested in vitro on choloroquine sensitive and resistant strains of P. falciparum for their
antimalarial activity [8]
Citrullus colocynthis is an annual herb found in the wild in Bangladesh. It is locally known as
Makal in Bangla, bitter apple in English. In traditional medicine, this plant has been utilized to
treat constipation, diabetes, edema, fever, jaundice, bacterial infections as well as cancer. The
leaf petroleum ether extract of C. colocynthis showed larvicidal activity. The fatty acids, oleic
acid and linoleic acid were isolated and identified in C. colocynthis petroleum ether extract. As
mosquito larvicidal compounds oleic and linoleic acids were quite potent against fourth-instar
larvae.
Objective
o To identify the best plant which has the larvicidal nature against Aedes aegypti
o To identify the proper doses in which highest larvicidal will be done.
Rationales
These synthetic insecticidal has adverse effect on the environment and mosquitoes and these
issues can be overcome by environmental friendly mosquitocidals. Non- biological insecticidal
can kill other beneficial organism along with this it has capacity to enter food chain responsible
for an adverse effect on ecological system [5]. Moreover, the acceptance rate of synthetic
insecticide is very poor to human. In the context of above fact, bioactive compound from the
medicinal plant can be used an alternative of synthetic insecticidal.
Methodology
 Collection of eggs and Larva maintenance:

The eggs of A. aegypti need to be collected. These eggs would be brought to the laboratory and
transferred to tray containing 500 mL of water for hatching. The mosquito larvae would feed
pedigree dog biscuits and yeast at 3:1 ratio. The feeding was continued until the larvae
transformed into the pupal stage.
 Maintenance of pupae and adult:
The pupae would be collected from the culture trays and transferred to plastic containers
containing 500-mL volume of water with the help of a dipper. The plastic jars need to keep in a
mosquito cage for adult emergence. Mosquito larvae would be maintained at 28 d C, 80%
relative humidity, under a photoperiod of 14:10 (light/dark). A 10% sugar solution must be
provided for a period of 3 days before blood feeding.
 Blood feeding of adult A. aegypti:
The adult female mosquitoes need to be allowed to feed on the blood of a mouse (a mouse per
day, exposed on the dorsal side) for 2 days, to ensure adequate blood feeding for 5 days. After
blood feeding, enamel trays with water from the culture trays would be placed on the cage as
oviposition substrates.
 Collection of plant and preparation of extract:
Plants need to collected. Then, tap water washing will be performed and dried at room
temperature. An electrical blender powdered the dried plant materials (leaves). From the powder,
300 g of the plant materials need to extracted with 1 L of organic solvents of petroleum ether for
8 h using a Soxhlet apparatus [11]. The extracts would be filtered through a Buchner funnel with
Whatman number 1 filter paper. The crude plant extracts need to be evaporated to dryness in a
rotary vacuum evaporator. One gram of the plant residue will be dissolved in 100 mL of acetone
(stock solution) and considered as 1% stock solution. From this stock solution, different
concentrations will be prepared.
 Larval toxicity test:
A laboratory reared colony of A. aegypti larvae will be used for the larvicidal activity. Twenty-
five individuals of first, second, third, and fourth instars larvae would be kept in a 500 mL glass
beaker containing 249 mL of dechlorinated water and 1-mL of desired concentration of leaf
extracts. Larval food will be given for the test larvae. At each tested concentration, two to five
trials need to be perform and each trial consists of five replicates. The control was setup by
mixing 1 mL of acetone with 249 mL of dechlorinated water. The larvae exposed to
dechlorinated water without acetone served as control. The control mortalities need to be
corrected by using Abbott’s formula [9]
(Observed mortality∈treatment−Observed Mortality ibnControl) X 100
Corrected Mortality=
100−Control Mortality
Number of dead larvae X 100

Percentage Mortality=
Number of larvae introduced
The LC50 and LC90 will be calculated from toxicity data by using pro bit analysis[10].
These tests will be done for methanol, ethanol, hexane extracts also.
Expected Outcome
After analysis of mortality rate of acetone, methanol, ethanol and hexane extracts we will get a
clear idea in which extracts and doses maximum mortality occur.
Estimated Budget (in BDT)
Investigator honorarium 15,000

Lab expenditure 8000
Field work expenditure 2000
Travel Expense 1000
Report printing and Binding 500
Miscellaneous 1000
Total cost 27,500
Discussion
Mosquitoes in the larval stage are attractive targets for pesticides because mosquitoes breed in
water, which makes it easy to deal with them in this habitat. The use of conventional pesticides
in the water sources, however, introduces many risks to people and the environment. Natural
pesticides, especially those derived from plants, are more promising in this aspect. Aromatic
plants and their essential oils are very important sources of many compounds that are used in
different respects.
Conclusion
This approach could not only improve the bio-efficacy of the extracts but also substantially
reduce the possibilities of physiological resistance development in mosquito population.
Therefore, the present strategy should be promoted in the other vector control program. The
mode of action and larvicidal efficiency of the plant extract under the field conditions should be
scrutinized and determined. Besides, further investigation regarding the effect on non-target
organism is extremely important and imperative in the near future.
References
1. Ghosh A, Chowdhury N, Chandra G. Plant extracts as potential mosquito larvicides.

Indian J Med Res (2012) 135:581–98.
2. Bansal SK, Singh KV, Sharma S, Sherwani MR. Laboratory observations on the larvicidal
efficacy of three plant species against mosquito vectors of malaria, dengue/dengue
hemorrhagic fever (DF/DHF) and lymphatic filariasis in the semi-arid desert. J Environ Biol
(2012) 33:617–21.
3. Jayanthi P, Lalitha P, Aarthi N. Larvicidal and pupicidal activity of extracts and
fractionates of Eichhornia crassipes (Mart.) Solms against the filarial vector Culex
quinquefasciatus say. Parasitol Res (2012) 111:2129–35. doi:10.1007/ s00436-012-3061-0
4. Panneerselvam C, Murugan K, Kovendan K, Mahesh KP. Mosquito larvicidal, pupicidal,
adulticidal, and repellent activity of Artemisia nilagirica (family: Compositae) against
Anopheles stephensi and Aedes aegypti. Parasitol Res (2012) 111:2241–51.
doi:10.1007/s00436-012-3073-9
5. Mrema EJ, Rubino FM, Brambilla G, Moretto A, Tsatsakis AM, Colosio C. Persistent
organochlorinated pesticides and mechanisms of their toxicity. Toxicol (2013) 307:74–88.
doi:10.1016/j.tox.2012.11.015
6. Mishra SK, Sangwan NS, Sangwan RS (2007) Andrographis paniculata (Kalmegh): a review.
Pharmacogn Rev 1:283–298
7. Mishra K, Dash AP, Swain BK, Dey N (2009) Anti-malarial activities of Andrographis
paniculate and Hedyotis corymbosa extracts and their combination with curcumin. Malar J 12:8–
26
8. Mishra K, Dash AP, Swain BK, Dey N (2009) Anti-malarial activities of Andrographis
paniculate and Hedyotis corymbosa extracts and their combination with curcumin. Malar J 12:8–
26
9. Abbott, W.S., 1925. A method of computing the effectiveness of insecticides. J. Ecol.
Entomol. 18, 265–267.
10. Finney, D.J., 1971. Pro bit Analysis. Cambridge University Press, London, pp. 68–78.
11. Vogel, A.I., 1978. Textbook of Practical Organic Chemistry. The English Language Book
Society and Longman, London, pp. 1368.

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RESEARCH PROPOSAL

Analysis of Larvicidal Activity Against Aedes aegyptii Using Extracts of

Date of Submission: 14 September, 2017

Department of Genetic Engineering and Biotechnology

Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh

Andrographis paniculate is a medicinal plant widely cultivated in tropical regions of Asia.

 Collection of eggs and Larva maintenance:

Number of dead larvae X 100

Estimated Budget (in BDT)

Investigator honorarium 15,000

1. Ghosh A, Chowdhury N, Chandra G. Plant extracts as potential mosquito larvicides.

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