(Marine Biology) Edgar Raymond Banks - Sea Urchins - Habitat, Embryonic Development and Importance in The Environment-Nova Science Pub Inc (2014)

MARINE BIOLOGY
SEA URCHINS
HABITAT, EMBRYONIC
DEVELOPMENT AND IMPORTANCE
IN THE ENVIRONMENT
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MARINE BIOLOGY
SEA URCHINS
HABITAT, EMBRYONIC
DEVELOPMENT AND IMPORTANCE
IN THE ENVIRONMENT
EDGAR RAYMOND BANKS

EDITOR
New York
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Library of Congress Cataloging-in-Publication Data

Sea urchins : habitat, embryonic development and importance in the environment / editor,
Edgar Raymond Banks.
pages cm. -- (Marine biology)
Includes index.
ISBN: (eBook)
1. Sea urchins. I. Banks, Edgar Raymond.
QL384.E2S45 2014
593.9'5--dc23
2014026719
Published by Nova Science Publishers, Inc. † New York
CONTENTS
Preface vii
Chapter 1 Phenotypic Variation and Resilience in Sea
Urchin Morphogenesis 1
Dimitri Fabrèges
Chapter 2 Response of Sea Urchin to Environmental Stress 29
Oriana Migliaccio, Immacolata Castellano,
Giovanna Romano and Anna Palumbo
Chapter 3 Nonparametric Regression Applied to Sea
Urchin Growth 53
Isabel Martínez-Silva, Marta Sestelo,
Gorka Bidegain, Altea Lorenzo-Arribas
and Javier Roca-Pardiñas
Chapter 4 Sea Urchin Immune System: From Basic
Concepts to Environmental Biomonitoring 85
Paola Cristina Branco,
Débora Alvares Leite Figueiredo,
Andrews Krupinski Emerenciano,
Douglas Amaral dos Santos,
Marcelo González-Aravena and
José Roberto Machado Cunha da Silva
Index 135
PREFACE
Sea urchins play a key role in marine ecosystems, controlling through its
grazing activity the dynamic, structure and composition of seaweed and sea
grasses. Moreover, it is a crucial component of the food web, as prey for fishes
and other marine animals. Due to its sedentary habits and sensitivity to
pollutants, adult sea urchin has been used in several studies as a biological–
biochemical indicator of local pollution. This book provides several topics on
sea urchins. It discusses the phenotypic variation and resilience in sea urchin
morphogenesis; response of sea urchins to environmental stress;
nonparametric regression applied to sea urchin growth; and sea urchin immune
systems.
Chapter 1 - Sea urchins have long been used as model organisms for
investigations in embryology. Thanks to their availability, accessibility and
transparency, sea urchin eggs and embryos have helped scientists to decipher
processes underlying fertilization, cell division and other generic
morphogenetic events such as epithelium-to-mesenchyme transition or cell
apical constriction in gastrulation. It is also in sea urchin models that the
paradigm of gene regulatory network (GRN) architecture and dynamics
underlying early morphogenesis were developed and the most systematically
explored. In addition, sea urchin embryos display extensive regulative
capacities as shown by their response to experimental manipulations. Hans
Driesch showed that the separation of 2-cell stage blastomeres leads to perfect
twins. Theodore Boveri observed the development of triploid embryos and
Giovanni Giudice the reaggregation and further development of fully
dissociated early embryos. These seminal studies opened the way to
deciphering the processes underlying variation, robustness and resilience.
Current approaches in developmental biology in the context of complex
viii Edgar Raymond Banks
system science rely on the in vivo multiscale observation of biological

processes to achieve their multilevel reconstruction. In this context, sea urchin
model organisms will serve an emerging integrative biology and help to
achieve the ultimate synthesis of genetics, embryology and evolution.
Chapter 2 - Sea urchin is an ―opportunist‖ organism with great relevance
in different ecosystems, controlling locally the dynamic of seaweed and sea
grasses populations. Anyhow, in coastal zones this organism can be influenced
by human activities. Because of its sedentary habits and acknowledged
sensitivity to pollutants, sea urchin has long been recognized as a good model
system to detect environmental stress. Sea urchin embryos and larvae are very
sensitive and often used for embryo toxicity tests and in monitoring or risk
assessment programs. A critical survey of the available literature will be
presented with particular focus on the effects of different contaminants
(cadmium, manganese, mercury), ocean acidification and natural toxins, such
as palytoxin-like compounds produced by benthic dinoflagellates and diatom-
derived polyunsaturated aldehydes. These studies allowed the identification of
signaling pathways involved in the stress response activated by sea urchins.
Particular attention will be focused on the role played by the versatile
signaling molecule nitric oxide, which has emerging as an important mediator
of environmental stress.
Chapter 3 - An adequate nonparametric regression model is able to record
specific patterns in the data that cannot be detected by a parametric model. In
addition, quantile regression can provide a more complete description of
functional changes than an exclusive focus on the least square regression. This
chapter assesses the adequacy of a variety of nonparametric models to analyze
the growth patterns of sea urchins by means of the length-weight relationship.
For this purpose, data from fishery landings of green sea urchin
Strongylocentrotus droebachiensis are used to analyze this relationship for
lengths within the legal catch-size range (52.4 mm - 76.0 mm) at two depths
(i.e., shallow waters, 4.6 m, and deep waters, 7.6 m). Overall, this gives insight
into the study of the minimum capture size. The authors apply a Kernel
nonparametric regression model to determine both its suitability and
applicability as an alternative to the classic allometric model, for the
estimation of a minimum catch size directed to obtain the maximum yield in
weight from the fishery. The results demonstrate the suitability of the Kernel
nonparametric regression model as an alternative approach to the classic
allometric model to analyze the length-weight relationship in sea urchins and
to estimate a minimum capture size, particularly for deeper waters sea urchins.
Additionally, a boosting based quantile regression technique is successfully
Preface ix
applied which detects variability in sea urchin growth patterns throughout the
length distribution and between depths. Differences in food availability and
wave exposure between depths may explain these results.
Chapter 4 - Since the genome of sea urchin had been sequenced, the
phylogenetic proximity of echinoderms and chordates was reinforced, based
mainly on the report of a wide range of immune genes with high degree of
similarity to mammalian ones. Besides being a well-documented research
model, the sea urchin immune system became a source of investigation of cell
biology whose main objective is to understand the, at same time, simple and
highly complex and coordinated immune response; simple because innate
response in the only immune response that sea urchins possess and complex
due to the wide diversity of innate immune receptors reported, which indeed
outnumber the receptors reported for C. elegans, D. melanogaster and even H.
sapiens. All these facts contributed for the ―genome era‖ of sea urchin.
In sea urchins, the innate immune response is orchestrated by the immune
cells, also referred to as coelomocytes. Composed of four different cell types,
coelomocytes has been studied since the 1960s, and still today, many
physiological roles remain obscure. For some cell types, even their function is
still controversial. The best studied cell type is the phagocytic amoebocyte, the
most abundant cell type in the coelomic fluid and the only one that is capable
of performing phagocytosis. Many studies have been conducted to this cell
type and molecular tools revealed that phagocytic amoebocyte possess
different subpopulation with distinct diversity of cytoskeleton components
besides accessory proteins.
Not only coelomocytes play an important role in immunity of sea urchins,
humoral factors are also important pivots of their immune response. Recently,
two antimicrobial peptides have been reported, besides other molecule with
bioactive properties. These discoveries not only help to elucidate how this
complex system acts, but, widen the horizons of immune system of sea urchins
as a potent pharmacological source of research.
Lastly, the sea urchin immune system has been also used as a useful tool
for environmental biomonitoring. Different stressors and different responses,
including cellular components used today as biomarkers, were reported in
literature and altogether reached the same conclusion that sea urchins are
excellent environmental bioindicators.
The aim of this chapter is to discuss about the most relevant topics of
innate immune system of sea urchin, involving the genetic homologies that
prove the phylogenetic proximity to chordates, the cellular and humoral
x Edgar Raymond Banks
components and the use of immune system as an important tool for biomedical
research and environmental biomonitoring.
In: Sea Urchins ISBN: 978-1-63321-517-7
Editor: Edgar Raymond Banks © 2014 Nova Science Publishers, Inc.
Chapter 1
PHENOTYPIC VARIATION AND RESILIENCE

IN SEA URCHIN MORPHOGENESIS
Dimitri Fabrèges1,2,3
1
Team Multiscale Dynamics in Animal Morphogenesis,
Gif-sur-Yvette, France
2
BioEmergences, Gif-sur-Yvette, France
3
Institut des Systèmes Complexes Paris Île-de-France, Paris, France
Abstract
Sea urchins have long been used as model organisms for

investigations in embryology. Thanks to their availability, accessibility
and transparency, sea urchin eggs and embryos have helped scientists to
decipher processes underlying fertilization, cell division and other generic
morphogenetic events such as epithelium-to-mesenchyme transition or
cell apical constriction in gastrulation. It is also in sea urchin models that
the paradigm of gene regulatory network (GRN) architecture and
dynamics underlying early morphogenesis were developed and the most
systematically explored. In addition, sea urchin embryos display
extensive regulative capacities as shown by their response to
experimental manipulations. Hans Driesch showed that the separation of
2-cell stage blastomeres leads to perfect twins. Theodore Boveri observed
the development of triploid embryos and Giovanni Giudice the
reaggregation and further development of fully dissociated early
embryos. These seminal studies opened the way to deciphering the
processes underlying variation, robustness and resilience. Current
2 Dimitri Fabrèges
approaches in developmental biology in the context of complex system

science rely on the in vivo multiscale observation of biological processes
to achieve their multilevel reconstruction. In this context, sea urchin
model organisms will serve an emerging integrative biology and help to
achieve the ultimate synthesis of genetics, embryology and evolution.
I. Introduction
1. The Sea Urchin in the History of Science
Echinoderms are widely distributed in the world and many of them are
easily accessible. For more than 150 years, over 30 species of sea urchins have
served as models for embryological studies (Figure 1). In 1847, the
fertilization and embryonic development of sea urchin species explored by
Alphonse Derbès, Adolphe Dufossé and Karl Ernst von Baër led to the first
published works (for a review see Briggs and Wessel, 2006). Despite recent
progress in microscopy, fertilization processes remained very difficult to
investigate. The transparent sea urchin embryo provided a unique opportunity
to observe oocyte fertilization. Taking advantage of the same good optical
properties, Oscar Hertwig published in 1876 his seminal observation of sperm
and egg pronuclear fusion, settling the respective roles of sperm and oocytes in
fertilization. This major breakthrough helped to establish the sea urchin as a
valuable model for embryological studies.
In 1891, Hans Driesch proposed a method to isolate single blastomeres
from 2-cell or 4-cell stage embryos. Isolated blastomeres were able to develop
and form larvae altogether normal looking although of smaller size. Although
Hans Driesch supported the principle of entelechy (i.e., vitalism), his work,
demonstrating the regulative properties of the sea urchin embryo, considerably
influenced the field of biology. Ten years later, Theodor Boveri‘s work on
fertilization and early development (for a review see Baltzer, 1964) showed
that the embryo develops correctly if cells inherit a complete set of
chromosomes that segregate symmetrically, although haploid and triploid
embryos may form larvae in rare cases.
The accessibility, small size and transparency of sea urchin eggs, as well
as their fast development, are major reasons for choosing them for studies of
fertilization and embryogenesis.
Phenotypic Variation and Resilience in Sea Urchin Morphogenesis 3
Figure 1. Phylogenetic tree displaying sea urchin species studied in scientific literature.
The size of species name is proportional to the species‘ occurrence in studies.
Strongylocentrotus purpuratus (18.4%) and Paracentrotus lividus (12.2%) are the
most studied sea urchin species. Data from NCBI taxonomy database and Google
Scholar.
In the late 20th century, the sea urchin was the animal model behind two
fundamental discoveries. In 1983, the discovery by Tim Hunt and
collaborators (Evans et al., 1983) of a family of proteins called cyclins in sea
urchin eggs opened the way to the deciphering of cell cycle regulation. Tim
Hunt was awarded the Nobel Prize in 2001 for his discoveries of key
regulators of the cell cycle. At the same time, the question of gene regulation
in embryonic development was central to the field of developmental biology.
In 1996, Eric Davidson and collaborators (Yuh et al., 1996) demonstrated the
modular organization of the cis-regulatory sequences upstream of the DNA
4 Dimitri Fabrèges
sequences encoding the Endo16 protein, specifically expressed in the sea

urchin endoderm. Each module controls Endo16 expression during a specific
period of embryonic development. The concept of modular organization of
cis-regulatory sequences was the basis of a systemic approach to the gene
regulatory networks (GRN) underlying development. In 2002, 25 scientists
published the GRN of the endomesoderm specification in the sea urchin
(Davidson et al., 2002), providing the first systemic description of the
architecture of a gene regulatory network underlying early embryonic
morphogenesis. More than 150 genes have been integrated into the sea urchin
GRN, now thought to be close to completion (Peter & Davison, 2011).
The next challenges in developmental biology include the integration of
cellular, genetic and molecular dynamics in predictive and explanatory formal
models. The latter should be based on the multiscale and multimodal
microscopy observation of living specimens. Despite the relative transparency
of embryos from a number of species and their small size and fast
development, cilia-driven movements of the sea urchin blastula largely impair
the possibilities of long-term time lapse imaging. However, this challenging
issue should be solved, so that sea urchins will remain among the most
insightful models in developmental biology for understanding the basis of
morphogenesis, homeostasis, robustness, variation and evolution.
2. Defining Normal Development
Investigating the processes underlying development requires a definition

of the events under study and agreement about the ―normal‖. We assume here
that the development of an organism encompasses all its changes at all scales
throughout its life cycle from oocyte fertilization to death. Defining the normal
does not mean that development has a finality. What is needed here is to
describe what the normal developmental paths are in defined genetic and
environmental conditions. Predicting that the individual will die is trivial.
Predicting its path until death is never trivial. Under so-called physiological
conditions, the viability of sea urchins can be compromised at various stages
during embryogenesis, and non-fertile adults are considered to be outside the
normal space. In laboratory conditions, 95% of embryos will lead to fertile
adults in the best cases and the remaining 5% are rarely considered. However,
so-called failures are part of the system dynamics and should be informative in
terms of processes. We take the perspective of phenotypic diversity as
reflecting the exploration of a parameter space. This exploration is constrained
by viability (at the scale of a lifetime) and variation (at the scale of species
evolution). Robustness and resilience are part of the system dynamics and its
viability domain, even when challenged by external cues.
3. Introducing Developmental Variability
In living systems, variability is a major force which drives evolution and

allows adaptation (Darwin, 1859). It is generally accepted that variability is an
advantageous process without which species could not adapt to an ever-
changing environment, and would therefore become extinct. Variability at the
macroscopic scale is believed to reflect the stochasticity of events at the
microscopic scales. Whatever its description and explanation, variability is
intrinsic to biological processes and it should not be neglected in our approach
to developmental processes.
The development of individuals within a population is variable.
Differences may be observed at the macroscopic scale (embryo size, viability,
developmental path, etc.), the mesoscopic scale (cell lineage, cell
compartments, organs developments, etc.) and the microscopic scale
(metabolism, DNA polymorphism, etc.). The set of differences observed in a
population is the variation while the variability is the theoretical aspect of the
variation. Finally, the variance is the mathematical entity representing the
variation of one measured parameter.
Biological observables can be described with the probability distribution
of multiple parameters. The mean or the median value of probability
distributions are useful to describe the phenotype of a population of
individuals with defined genetic and environmental conditions.
Depending on the experimental context, it can, however, be misleading to
limit the description of a population to a set of mean values. Using the mean
and the variance gives a Gaussian approximation which is a more faithful
description of the probability distribution. Each observation can be described.
Indeed, biological observables can be described as a range of weighted values.
And the development path, rather than being described as a straight line,
should be considered as a viability tube gathering live specimens.
6 Dimitri Fabrèges
II. Early Development of Wild Type Sea Urchins

1. Overview
The typical development of a normal wild type sea urchin is depicted in

Figure 2.
Figure 2. Overview of the typical development of a sea urchin (Paracentrotus lividus)

from fertilization to pluteus larva. A-G: pre-hatching stages. The first two cleavages
form four identical cells (C). The third cleavage is asymmetrical, forming four animal
cells and four vegetal cells (D). The fourth cleavage generates three cell types in E:
eight mesomeres (blue), four macromeres (orange) and four micromeres (pink). The
next cleavage symmetrically divides mesomeres and macromeres (F). The micromeres
divide asymmetrically in four large micromeres (pink) and four small micromeres
(purple). Cells compact prior blastula hatching (G). The vitelline layer is digested
giving the swimming blastula (not shown). H-L: primary mesenchymal cells ingress
(PMCs, in red) into the blastocoel. PMCs form two clusters on both sides of the
blastocoel (H). macromeres‘ progeny (orange) invaginates forming the archenteron (I).
The tip of the gut fuses to the future ectoderm (J, blue) and completes its shaping in
three parts by the pluteus stage. Meanwhile, PMCs surround the gut, fuse to form a
syncytium and start the spiculogenesis which shapes the pluteus larva (J-L). Pluteus
larva shows three axes: left/right axis, oral/aboral axis and ventral/dorsal axis. In L,
light green curve corresponds to oral/ventral side, green curve corresponds to
aboral/ventral side and dark green curve corresponds to aboral/dorsal side. Pluteus
larva is represented in lateral and dorsal view showing the complex skeleton
organization. The secondary mesenchymal cells (SMCs) stay at the tip of the gut (I and
J in red) and migrate into the blastocoel or over the ectoderm during mouth formation
(K and L). Blue: ectoderm; Orange: future endoderm; Green: uncertainties of lineage
between ectoderm and endoderm; Red: future mesoderm; Pink: germ line.
2. Spawning and Reproduction
Sea urchins are iteroparous and most of them follow a seasonal

reproduction. The reproductive season depends on the species, winter for
Strongylocentrotus purpuratus, spring for Paracentrotus lividus, summer for
Diadema antillarum and fall for Lytechinus variegatus. The reproductive
season may be extended, shortened, preponed or postponed depending on the
environmental conditions. Some species (e.g., Sphaerechinus granularis) can
spawn at any time of the year. Spawning is synchronized by temperature
(Pearse, 1974; Fujisawa, 1989), lunar cycle (Pearse, 1975; Iliffe et al., 1982)
and chemical signaling (Kaupp et al., 2006) so that mature Paracentrotus
lividus may be found at any time of the year in different places (Hörstadius,
1973).
a. Female Gametes
Spawning of oocytes is highly variable from one species to another. The
abundance of eggs typically depends on the gonad size and can vary from a
million eggs (e.g., Paracentrotus lividus) to 20 million eggs (e.g., Echinus
esculentus, Hörstadius, 1973). The egg size varies from 80µm (e.g.,
Strongylocentrotus purpuratus) to 160µm (e.g., Stongylocentrotus
droebachiensis). The egg size within a batch of eggs may vary (e.g., ± 10% in
Paracentrotus lividus, D. Fabrèges, unpublished observation).
b. Male Gametes
Sperm is released in water and swims toward the oocytes, guided by
chemotaxis (Kaupp et al., 2006). Among some 80 sperm-activating peptides
(SAP) identified in the sea urchin Arbacia punctulata, a 14 amino-acid peptide
named resact has been characterized as a chemoattractant for the
spermatozoids (Suzuki et al., 1984; Kaupp et al., 2003). Whatever the
mechanisms favoring spermatozoa displacement toward the oocytes, their
efficiency is limited and the fertilization rate is very low (~5%) when gametes
are more than 2m away (Levitan et al., 1992) in laboratory conditions. The
fertilization rate drops even lower when strong water current is applied. Thus,
the fertilization rate is greatly improved by spawning synchrony and by
maintaining a high density of individuals. Studies in natura conclude that on
average, the fertilization rate is close to 1%.
8 Dimitri Fabrèges
c. Fertilization
In vitro fertilization typically shows a success rate of 80% to almost
100%, depending on the experimental conditions and gamete quality. The first
contact between spermatozoa and oocytes leads to the oocyte membrane
depolarization. It has been proposed that membrane depolarization is the first
shield against polyspermy, although recent analysis suggests the implication of
actomyosin contraction, as spermatozoa were found to enter the oocyte
through actin-enriched sites (Dale, 2014). Then a first striking consequence of
fertilization is the cortical reaction. Cortical granules near the spermatozoa
entry point fuse with the oocyte membrane and the fusion of cortical granules
propagates. The content of cortical granules is released into the perivitelline
space to form a layer between the hyalin and the zygote membrane. This
reaction is the slow blocking process preventing the entrance of other
spermatozoa.
Meiosis is completed in the ovary. Pronuclei meet, fuse and the first
mitosis is triggered. The first cleavage happens 90 minutes (Paracentrotus
lividus) to several hours (Strongylocentrotus droebachiensis) after fertilization.
Timing of cleavages depends on temperature and may vary up to 5% between
individuals in the same experimental conditions (Stephens, 1972).
The animal-vegetal axis is already established in unfertilized eggs through
an asymmetrical distribution of cellular components (Hörstadius, 1973;
Schroeder et al., 1980; Di Carlo et al., 1994; Romancino et al., 2001;
Romancino et al., 2004). Although the oral/aboral axis is also already
established in unfertilized eggs (Cameron et al., 1989; Vlahou et al., 1996), it
is labile until the third cleavage (Cameron et al., 1987). The first cleavage is
hollow and parallel to the animal-vegetal axis forming two cells, described as
being identical in terms of potentialities.
3. Cleavage Stages and Formation of the Blastula
a. Pre-hatching Development
The first asymmetrical cleavage in terms of cell size and cell fate is the
third one, perpendicular to the animal-vegetal axis, forming four animal cells
and four vegetal cells. Animal cells give eight mesomeres equally distributed
(Figure 2D-E), and the vegetal cells divide asymmetrically to form four
macromeres (about 1.7 times bigger than the mesomeres) and four micromeres
(about one third of the size of mesomeres). The 16-cell stage is therefore
composed of three cell types: mesomeres, macromeres and micromeres,
leading to the germ layers ectoderm, endoderm and mesoderm respectively.

The blastomeres‘ fate may, however, vary in particular at the layers‘ boundary
such as the ectoderm-endoderm boundary (Logan et al., 1997) or the
endoderm-mesoderm boundary, ill-defined at early stages.
At the next stage, the asymmetrical division of micromeres leads to the
formation of four large micromeres and four small micromeres. Small
micromeres are the precursors of the germ cells (Pehrson et al., 1985; Yajima
et al., 2011). They divide once and stay quiescent until the end of gastrulation.
Large micromeres divide three or four times, establishing a population of 32
cells (Paracentrotus lividus, Sphaerechinus granularis) or 64 cells (Lytechinus
variegatus), depending on the species.
At the 32-cell stage, the embryo consists in a single-layer epithelium
surrounding the blastocoel cavity. The major morphogenetic events observed
at blastula stages are the compaction of the embryo with outside surface
flattening, enhancement in cell-to-cell surface contact and adhesion strength,
and increase in the blastocoel size.
b. The Swimming Blastula

By the end of the blastulation period, the embryo hatches. The vitelline
envelope surrounding the blastula is digested by a specific protease (Ishida,
1936; Gache et al., 2004), synthesized by animal cells (Lepage et al., 1992).
By the end of the cleavage period, each blastomere carries one motile cilium
beating to generate a current. Freed from the vitelline envelope, the blastula
uses this current to move in the medium.
Later during development, the animal plate cells bear an apical ciliary tuft,
which is, however, not motile and functionally ill-defined (Horstadius, 1973;
Bisgrove, et al., 1986). Recent work suggests a mechano-sensory role (Jin et
al., 2013).
4. Gastrulation Stages
a. The Early Gastrula

Soon after hatching, the vegetal pole flattens (Figure 2H) to form the
vegetal plate encompassing about 100 cells (Ettensohn, 1984). This event
marks the onset of gastrulation and may happen a few minutes before hatching
in some species (Sphaerechinus granularis). The embryo is regionalized and
the map displays a ring-shaped organization. The small micromeres (SMic),
precursors of the germ cells (Yajima et al., 2011; Wessel et al., 2013), are
10 Dimitri Fabrèges
located at the most vegetal region of the embryo. SMic are surrounded by
large micromeres (Figure 2F, pink), forming the external ring of the vegetal
plate. Large micromeres undergo a first epithelium-to-mesenchyme transition
(EMT) giving rise to the primary mesenchymal cells (PMCs, for a review see
Wu et al., 2007). Within this population, some cells called secondary
mesenchymal cells (SMCs) undergo a second EMT during gastrulation.
The first EMT is a cell autonomous process and the ingression of PMCs is
observed according to its intrinsic timing in ectopic and heterochronous
transplantations (McClay et al., 1992; Peterson et al., 2003) or in vitro cultures
(Okazaki, 1975). Before PMC ingression, the sea urchin embryo is a single
layer epithelium. On its apical side, cells are attached to the hyaline layer
composed of hyalin fibrillar glycoprotein, laminin, echinonectin and collagens
(Spiegel et al., 1979; Spiegel et al., 1983; Alliegro et al., 1988; Matese et al.,
1997; Wessel et al., 1998), and cilia pass through the hyaline layer towards the
external environment. On the blastocoel side, cells lie on a basal lamina
composed among other proteins of laminin, fibronectin and collagens (Wessel
et al., 1984; McCarthy et al., 1987). Cell-cell lateral adhesion depends on
cadherin and specialized junctions (Miller et al., 1997). Prior to ingression, the
large micromeres lose their adhesion to the hyaline layer and cadherin cell
surface expression, and reinforce their adhesion to the basal lamina (Fink et
al., 1985). Meanwhile, PMCs become motile and move to the blastocoel
through the basal lamina. Whether PMCs squeeze through the loose basal
lamina or locally proteolyse it remains unclear.
b. The Mid Gastrula

Several hours after PMC ingression, the vegetal plate cells invaginate by
apical contraction (Ettensohn, 1984; Nakajima et al., 1996; Kimberly et al.,
1998) to form the archenteron. Extension of the presumptive gut in the
blastocoel toward the animal pole takes several hours. Endomesodermal
tissues organized along the vegetal-animal axis include the presumptive
hindgut, midgut and foregut, germ cells (SMic) and secondary mesenchymal
cells (SMCs).
The elongation of the gut rudiment takes place in two steps. First, gut cells
intercalate by means of convergent-extension movements. Then additional
cells integrate the archenteron, consequently contributing to the extension of
the forming gut. The gut elongation also serves to transport SMic to the animal
pole (Yajima et al., 2012). Although no direct evidence has been provided, this
translocation is thought to be passive.
By mid-gastrulation, SMCs extend filopodia towards the animal pole,

exploring the inner surface of the oral plate where the mouth will open (Dan et
al., 1956; Hardin et al., 1990; Miller et al., 1995).
Meanwhile, PMCs adhere to the basal lamina and move, driven by a
signal coming from the animal pole (Okazaki, 1965; Ettensohn et al., 1986).
PMCs stop slightly beyond the equator of the blastula to move back to the
vegetal pole and settle in a ring shape around the archenteron, close to the
ectoderm-endoderm boundary (Figure 2H, I). PMCs form two ventrolateral
clusters, probably driven by VEGF signaling secreted by ectoderm-endoderm
boundary cells adjacent to the cluster sites (Duloquin et al., 2007). PMCs then
fuse to form a syncytium (Hodor et al., 1998) which seems to exchange
positional information with ectodermal cells by direct contact through long
filopodia that are 200 to 400 nm thick and 5 to 30 µm long, up to 80 µm in
rare cases (Miller et al., 1995).
PMCs destined to form the larval skeleton are maintained in vitro
(Okazaki, 1965) or in ectopic and heterochronous transplantation experiments
(Ettensohn et al., 1986; Ransik et al., 1993). However, PMC organization is
not strictly autonomous, since they depend on external signaling to correctly
migrate in place and time (Armstrong et al., 1994).
The differentiation of the precise number of PMCs seems to be a very
robust process, as their ablation is compensated by the so-called trans-
differentiation of SMCs (Ettensohn et al., 1988; Ettensohn, 1992; Ruffins et
al., 1993; Kiyomoto et al., 2007). The endodermal and ectodermal lineages are
not fully distinct yet (Ruffins et al., 1996; Logan et al., 1997; Martins et al.,
1998; Piston et al., 1998; Ransick et al., 1998). It has been shown, in
archenteron ablation experiments, that both ectoderm and endoderm cells
contribute to restoring a partial or even complete gut (Logan et al., 1997).
c. Late Gastrula and Early Larval Stages

While PMCs are migrating to their final location, the forming
ventrolateral clusters start to synthesize the larval skeleton. The PMCs‘
syncytium shapes two cavities in which cells secrete calcium carbonate,
spicule matrix proteins and other compounds (Wilt, 1999; Mozingo, 2014)
forming several mineralized aggregates leading to two triradiate spicule
rudiments (Okazaki, 1960). Aggregates are essentially made of 95% CaCO3,
~5% MgCO3 and less than 0.1% matrix proteins (Wilt, 1999). For both
rudiments, branches point to ventral, dorsal and animal sides. Later, the two
rudiments elongate and branch by the addition of material to the tips, leading
to the complete larval skeleton (Decker et al., 1988).
Figure 3. Focusing on the prospective mouth region during mouth formation,

secondary epithelial to mesenchymal transition and gut differentiation. A: invagination
(black arrow) of the ectoderm (blue) coincides with the endoderm (orange) bending
driven by filopodia of secondary mesenchymal cells (SMCs, not shown) at the tip of
the archenteron. B: archenteron tip meets the ectodermal invagination. A first
constriction of the gut (dash line) separates the coelom (dark green, derived from
SMCs) from the rest of the gut. Meanwhile, SMCs not involved in coelom formation
(red) migrate into the blastocoel and differentiate in pigment cells or blastocoelar cells.
C1-C2: as ectoderm (blue) continues to invaginate and gut further elongates, the
coelom (dark green) is pulled apart into two coelomic pouches. A second constriction
of the gut (dash line) appears between the oesophagus (light green) and the rest of the
gut. A-C1: lateral view from the left. C2: animal view. Adapted from Gustafson et al.,
1963.
The skeleton that shapes the sea urchin larva is built in about two days by
a small number of cells (32 cells in most species). Spiculogenesis as well as
skeleton branching and elongation have been shown to be cell autonomous
(Okazaki, 1975). However, the precise shaping of the skeleton requires the
correct location of PMCs, which depends on the ectodermal expression of
growth factors (Duloquin et al., 2007; Adomako-Ankomah et al., 2013).
Concurrently with spiculogenesis, the archenteron extends through the
addition of endodermal cells. SMCs attached to the prospective mouth opening
through their filopodia contribute to archenteron elongation and bending by
pulling the apical plate (Dan et al., 1956; Gustafson et al., 1960; Hardin, 1988;
Latham et al., 1999). Meanwhile, the apical plate invaginates (Figure 3A)
through apical constriction, as observed earlier for the vegetal plate. Some
SMCs escape from the tip of the gut and migrate through the blastocoel to
their final destination. SMCs lead to at least four different cell types:
1) pigment cells; 2) blastocoelar cells; 3) coelomic pouches and
4) circumesophageal muscles. Pigment cells and blastocoelar cells come from

the archenteron during its expansion (Figure 3B in red). Pigment cells display
an intense protrusive activity and invade the ectoderm (Young, 1958; Gibson
et al., 1987). They show large vacuoles containing the red pigment
echinochrome in proportion to the sea water (Bay et al., 1983; Ageenko et al.,
2011). Blastocoelar cells extend thin filopodia across the blastocoel, creating a
multicellular mesh between gut, skeleton, larval arms and ectoderm.
Blastocoelar cells play different roles in immunity (Pancer et al., 1999) such as
aggregation at sites of injury to form cellular clots and exogenous compound
clearance (including bacteria and allografts). After making contact with the
apical plate, the archenteron continues to elongate, exerting increasing
pressure on the ectoderm (Gustafson et al., 1963). A first constriction of the
archenteron forms between the coelom (Figure 3B, oral side) and the gut. As
the archenteron continues to elongate, the coelom organizes on both side of the
oral plate to form the two coelomic pouches (Figure 3C). Meanwhile, a second
constriction forms between the presumptive oesophagus and stomach. The
mechanism underlying the fusion of the ectoderm and endoderm during the
opening of the mouth remains ill-known, although it has been proposed that
the tensions generated by archenteron growth, ectodermal invagination and the
beginning of peristaltic contractions of the oesophagus may be sufficient to
separate cells and trigger the mouth opening (Gustafson et al., 1963). The
progenitors of circumesophageal cells are found in coelomic pouches. They
extend filopodia around the oesophagus to meet the contralateral coelomic
pouch (Burke, et al., 1988). Circumesophageal cells migrate out of the
coelomic pouches and show autonomous contractions.
The last cell type to emerge during gastrulation are the primordial germ
cells originating from the eight small micromeres located at the vegetal pole
and born through the asymmetrical cleavage of micromeres (5th cleavage from
16 cells to 32 cells). During gastrulation, small micromeres stay at the tip of
the archenteron (Yajima et al., 2012). Whether their displacement is active or
passive is unclear, although the absence of detectable filopodia argues in favor
of passive movement (Yajima et al., 2012). Small micromeres segregate into
the coelomic pouches with a 5:3 ratio most of the time, although equal
distribution may be observed (Pehrson et al., 1985). Although proliferation
along the small micromere lineage is not extensively documented, it has been
shown that they divide at least once more in Strongylocentrotus purpuratus
(Pehrson et al., 1985).
5. Pluteus Larva
The larval skeleton continues to grow, regulated by the PMCs‘

developmental clock and ectodermal cues. Skeletal rods grow to form the
larval arms, meeting at the aboral extremity and shaping the larva from prism
stage to pluteus (Ettensohn et al., 1983; Figure 2L, dorsal view). Four arms are
formed, two of which are close to the oral ectoderm and the two others on the
aboral side. Oral and aboral ectoderms are separated by a band of
differentiated cells forming the ciliary band. The ciliary band serves to bring
food to the mouth and contributes to the larva movements. Pluteus larvae are
autonomous, swim and feed as part of the zooplankton and their
morphogenesis continues, in particular at the level of the cytoskeleton. During
their planktonic life, larvae will undergo metamorphosis through the activity
of one coelomic pouch, in principle the left one (Duboc et al., 2005), although
historical studies have shown that the right coelomic pouch can be active in
vitro and in natura (Ohshima, 1921).
III. Experimental Perturbation of Sea Urchin Early

Embryogenesis
Sea urchin development is often described as stereotypical, i.e., without
significant differences between individuals or species (Davidson et al., 1998;
McClay, 2011; Hinman et al., 2014). This stereotypical description mainly
concerns the first division axis, early asymmetries and early fate map. To my
knowledge there is no precise quantification of phenotypic variation in normal
sea urchin early development. However, a number of studies have pointed to
significant interindividual variations and extensive cell plasticity (Khaner et
al., 1990; Livingston et al., 1990; McClay et al., 1996; Logan et al., 1997). In
many cases, some of which will be described in this section, sea urchins are
somehow able to overcome disturbances and form larvae with no obvious
differences from normal ones.
1. Fractionation of Unfertilized Eggs
In the first part of the 20th century, researchers were looking for cell
components that are essential to good development. In 1932, Ethel Harvey
proposed a simple and reproducible way to produce halves and quarters of sea
urchin oocytes prior to fertilization. By means of strong centrifugation (10,000
x g for 30 minutes) in a sucrose gradient, sea urchin oocytes were separated
into two halves with different contents (Harvey, 1934). Clear partitioning
happened along the centrifugation axis with, starting from the centripetal end:
lipid droplets, pronucleus, clear zone, mitochondria, yolk and pigment. The
cytological content of oocytes halves or quarters and their development when
activated by sperm were further analyzed 40 years later (Anderson, 1970).
Although Anderson did not reproduce the high rate of successful development
observed by Harvey, both authors agreed that sperm-activated halves and
quarters, even those containing the male pronucleus only, were able to form
pluteus larvae.
The centripetal half inherits the maternal pronucleus, but largely lacks
food materials. Despite this lack of resources, mitoses occur in time compared
to control embryos. According to Ethel Harvey and Anderson, these halves
can form blastulae, gastrulae and normal plutei except for size and color.
The centrifugal half inherits food materials, but lacks the maternal
pronucleus. Development is strongly affected as only a few embryos divide.
Most of them replicate the male nucleus without cell division and die. In rare
cases, embryos reach the blastula stage and even the pluteus stage with four
well-developed arms. How this wide range of phenotypic variation is
generated has not been investigated. So we do not know where in the system‘s
parameter space, the embryo has a chance to properly develop. In any case,
this centrifugation protocol reveals striking aspects of developmental
plasticity.
2. Separated Fertilized Eggs: Twinning
Experimental twinning in sea urchin was introduced by Hans Driesch in

1891. He showed that blastomeres isolated from the 2-cell stage embryos were
able to develop and form adult larvae. Although anatomical aberrations may
be observed depending on the technique used (Marcus, 1979), blastomere
isolation can lead to fertile adults (Hinegardner, 1975; Cameron et al., 1996).
We present here a brief description of twin development from the 1-cell stage
to the pluteus stage, focusing on developmental variation.
a. Twins’ Development from the 1-Cell Stage to Hatching

To the best of my knowledge, the only description of early development
of sea urchin twins was made by Hans Driesch himself (Driesch, 1891). The
most striking difference compared to normal embryos is in terms of cell
number. At the 1-cell stage, twin embryos have the cytoplasmic clock of 2-cell
stage blastomeres. Therefore, at the 2-cell stage, blastomeres have the
cytoplasmic clock of normal 4-cell stage cells. In fine, asymmetrical divisions
happen at the same age post-fertilization. The usual three different cell types
are formed at the 8-cell stage rather than at the 16-cell stage (Figure 4B-D).
Hans Driesch reported that 75% of the twin embryos looked precisely like one
half of normal ones (Figure 4E1) until the blastula stage. Interestingly, he
observed that a few hours later, sea urchin twins formed perfectly shaped
blastulae (Figure 4F). He did not describe how embryos were able to catch up
by closing their half-sphere shape. 25% of the embryos followed a different
developmental path (Figure 4E2 and 4E3). Some of them had 8 cells of
identical size (Figure 4E3). It was not clear for Hans Driesch whether these
observations were experimental artefacts (e.g., 4-cell stage embryo
aggregation) or the result of particular biological mechanisms (e.g., delayed
asymmetrical division).
Figure 4. Early development of sea urchin twins. A: first cleavage in whole embryo; B:
mechanical isolation of 2-cell stage blastomeres; C: 2-cell stage of one blastomere
corresponding to the 4-cell stage of a normal embryo; D: 4-cell stage of one
blastomere corresponding to an 8-cell stage of a normal embryo; E1: 75% of the
observed phenotypes are described by Driesch as half-embryos. E2 and E3: 25% of the
observed phenotypes can be further categorized: E2: three cell types distinguished by
their size are formed, but cell position are shuffled, and E3: cell types cannot be
distinguished or two 4-cell stage twins aggregate. F: a percentage of embryos from E1,
E2 and/or E3 is able to form a small swimming blastula.
b. Twins’ Developmental Variation: An Ambiguous Fate Map

Hans Driesch concluded from these experiments that ―das Princip der
organbildenden Keimbezirke [ist] widerlegt‖ (the principle of organ-forming
territories is refuted) (Driesch, 1891, p. 178) rather than acknowledging the
regulative properties of the sea urchin embryo, as done later (for review see
Angerer et al., 1999). The contemporary perspective would actually be that of
resilience.
Resilience is the ability to recover from injuries while robustness is the
ability to compensate for variation without macroscopic phenotypic difference.
We analyze that sea urchin twins differ from normal embryos, but overcome
the perturbation of blastomeres‘ separation through three possible
developmental paths (Figure 4E1, E2 and E3). Twins vary in terms of cell type,
cell number, and cell organization (Figure 4E2 and E3). These morphological
types are likely to correlate with major changes in terms of embryonic fate
map. Despite these striking differences, a high percentage of twins are resilient
and form viable and fertile adults (Hinegardner, 1975; Cameron et al., 1996).
Resilience in sea urchin has been observed and described in a number of
experimental cases, including blastomere ablation and disaggregation (Harvey,
1932; Giudice, 1962; Anderson, 1970; Spiegel et al., 1975; Ettensohn et al.,
1986; Ettensohn et al., 1988; Ransik et al., 1993; McClay et al., 1996; Logan
et al., 1997; Davidson et al., 2002).
3. Blastomere Ablation and Transfating
Ablation experiments are classic experimental protocols to investigate the

role of cells or cell populations in development and the ability of the embryo
to regulate. At the early gastrula stage (Figure 2H), Charles Ettensohn and
David McClay (1988) removed the PMCs from the blastocoel and observed
further development. Interestingly, archenteron invaginated and SMCs
migrated into the blastocoel to form two ventrolateral cell clusters performing
spiculogenesis. The same phenomenon was observed after ablation of the
micromeres at the 16-cell stage (Sweet et al., 1999). Although mouth opening
and spiculogenesis happened with delay, this experiment revealed, once again,
the embryonic potential for resilience. SMC transfating is not an isolated case.
Transfating of endodermal cells to SMCs has been observed after ablation of
the archenteron tip (McClay et al., 1996).
Recent work (Ettensohn, 2007) has deciphered the mechanism by which
SMC-to-PMC transfating may occur. alx1 is a PMC-specific gene sufficient
and necessary for PMC differentiation. Future SMCs normally repress alx1
expression by means of a direct or an indirect signaling from PMCs. In the
absence of PMCs, this signal is abolished. Therefore, alx1 expression in SMCs
is no longer repressed and transfating occurs. Interestingly, removal of both
PMCs and archenteron tip lead to the same result, suggesting that transfating
may successively occur from endodermal cells to SMCs, and from SMCs to
PMCs (McClay et al., 1996).
4. Reaggregation of Blastomeres and Cell Sorting
The complete disaggregation followed by reaggregation of embryonic

cells was first explored by Giovanni Giudice in 1962. Giovanni Giudice
isolated cells from sea urchin blastula (Figure 2G) and gastrula (Figure 2I).
Putting the cells back together in regular sea water was sufficient to lead to
cell reaggregation and larva formation. It was however shown that dissociation
at gastrula stage did not lead to such a high percentage of successful
development. In addition, cells from different species never mixed, indicating
that cell reaggregation is species-specific. More recent experiments have
shown that disaggregation by the 16-cell stage (Figure 2E) followed by
reaggregation led to fertile larvae (Hinegardner, 1975).
Whether the underlying mechanism is active or passive is not clear.
However, there is strong evidence that a hybrid mechanism operates with
active cell-cell recognition and passive cell sorting (Spiegel et al., 1978;
Takeichi, 1991; Ghersi et al., 1993). Reaggregating cells have been shown to
expand filopodia, to adhere to other cells and migrate, actively sorting out
species-specific cells (Spiegel et al., 1975). Cell polarity is maintained through
active processes and, within a few hours, cells reorient in the aggregate so that
their apical surfaces face the outside (Nelson et al., 1988). Cell sorting has
been correlated to the cell type, showing that aggregation is homotypic (i.e.,
cells of the same type aggregate together; Spiegel et al., 1978). This
observation has been confirmed in mice (Takeichi, 1991) and later in sea
urchin (Ghersi et al., 1993) and shown to rely on a cadherin-dependent
mechanism.
IV. Discussion and Perspectives

The sea urchin remains a major model in developmental biology. Recent
work on the sea urchin gene regulatory network has dramatically changed
developmental descriptions. However, the GRN architecture concept fails to
either fully capture spatiotemporal features of developing embryos or to take
into account intra-individual and inter-individual variation of gene expression.
Both older and more recent studies have shown that experimental
perturbation of development reveals inter-individual variation and potential
developmental paths. There is no scientific reason to assume that normal
development does not follow multiple developmental paths, as perturbed
embryos do. Current challenges include deciphering the development of single
individuals at all possible levels: from DNA polymorphism and gene
expression to cellular and tissular morphogenesis and evolution.
Technical advances in microscopy and imaging provide tools to perform
high throughput imaging of the development of single individuals at the
cellular level. An ontological description of living embryos through
generations could highlight intra-individual and inter-individual variations. We
expect such an investigation to integrate the GRN dynamics underlying
morphogenetic processes. Unification of genetic, ontological and phylogenetic
approaches may be the key to understanding the resilience mechanisms
operating during the development and evolution of sea urchin and other
species. The major challenge of the 21st century in developmental biology is
certainly to understand how, despite highly variable environments, life finds a
way (Crichton, 1990. p. 160).
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Chapter 2
RESPONSE OF SEA URCHIN

TO ENVIRONMENTAL STRESS
Oriana Migliaccio, Immacolata Castellano,

Giovanna Romano and Anna Palumbo
Stazione Zoologica Anton Dohrn, Naples, Italy
Abstract
Sea urchin is an ―opportunist‖ organism with great relevance in

different ecosystems, controlling locally the dynamic of seaweed and sea
grasses populations. Anyhow, in coastal zones this organism can be
influenced by human activities. Because of its sedentary habits and
acknowledged sensitivity to pollutants, sea urchin has long been
recognized as a good model system to detect environmental stress. Sea
urchin embryos and larvae are very sensitive and often used for embryo
toxicity tests and in monitoring or risk assessment programs. A critical
survey of the available literature will be presented with particular focus
on the effects of different contaminants (cadmium, manganese, mercury),
ocean acidification and natural toxins, such as palytoxin-like compounds
produced by benthic dinoflagellates and diatom-derived polyunsaturated
aldehydes. These studies allowed the identification of signaling pathways
involved in the stress response activated by sea urchins. Particular
attention will be focused on the role played by the versatile signaling

Corresponding author: Laboratory of Cellular and Developmental Biology, Stazione Zoologica
Anton Dohrn, Villa Comunale, 80121 Naples, Italy. E-mail: [email protected].
30 O. Migliaccio, I. Castellano, G. Romano et al.
molecule nitric oxide, which has emerging as an important mediator of

environmental stress.
Introduction
Sea urchin plays a key role in marine ecosystems, controlling through its
grazing activity the dynamic, structure and composition of seaweed and sea
grasses (Tomas et al., 2004). Moreover, it is a crucial component of the food
web, as prey for fishes and other marine animals and finally for humans, that
consider gonads a culinary delicacy.
Due to its sedentary habits and sensitivity to pollutants, adult sea urchin
has been used in several studies as a biological–biochemical indicator of local
pollution (Pancucci et al., 1993; Soualili et al., 2008). Moreover, the embryo
sensitivity to pollution, its transparency useful to detect malformations, the
relative synchrony and rapidity of development prompted to consider sea
urchin development a useful tool to set up toxicity assays in monitoring or risk
assessment programs (Beiras et al., 2003).
A series of experiments performed in laboratory have revealed that sea
urchin responds to a variety of stress agents, including heavy metals (Roccheri
and Matranga, 2009; Pinsino et al., 2010), natural toxins (Romano et al., 2011;
Marrone et al., 2012; Privitera et al., 2012; Varrella et al., 2014) and
acidification (Dupont and Thorndyke, 2013). Activation of MAP kinases
(Pinsino et al., 2011; 2014), induction of metallothioneins (Ragusa et al.,
2013), caspase 3 (Agnello et al., 2007) and different heat shock proteins (hsp)
(Roccheri et al., 2004, Pinsino et al., 2010; Romano et al., 2011; Marrone et
al., 2012) have been reported to occur. All these characteristics make the sea
urchin one of the best model system to investigate the response to
environmental stress.
In this chapter we will focus the attention on the effects of metals, such as
cadmium, manganese and mercury, ocean acidification and natural toxins,
such as palytoxin-like compounds produced by benthic dinoflagellates and
diatom-derived polyunsaturated aldehydes on sea urchins. Moreover, we will
highlight the involvement of nitric oxide in the stress response of this marine
organism.
Response of Sea Urchin to Environmental Stress 31
1. Cadmium, Manganese and Mercury

For many years the effects of different toxicants have been studied on
marine invertebrates and in particular on sea urchin. Among the various
contaminants, some of them, such as cadmium and mercury, has been
investigated since many years, whereas manganese have only recently begun
to be explored as an emergent factor in the environmental contamination. A
complete review on the effects of cadmium on sea urchin was recently
published (see Roccheri and Matranga, 2009). In this paragraph we will
provide an update on cadmium and we will focus the attention on manganese
and mercury.
There is great interest in using adult sea urchins to assess the extent of
seawater pollution. However, despite their abundance and wide distribution,
only few studies have been performed on natural sea urchin populations. Table
1 shows the concentration of cadmium, mercury and manganese in the gonads
of different sea urchin species.
Exposure to metals can impair gamete functionality and reproductive
success. Treatment of Arbacia punctulata spermatozoa with different mercury
and manganese concentrations induced variation in the swimming speed and
respiration (Barron et al., 1948; Young and Nelson, 1974). Moreover,
exposure of Paracentrotus lividus fertilized sea urchin eggs to mercury caused
alteration in the intracellular pH and increase in Ca2+ influx (Allemand et al.,
1993).
Regarding the effect of these metal ions on sea urchin development, a
series of studies have revealed that cadmium affects P. lividus development,
exhibiting a large spectrum of actions. Developmental delay, skeletal
malformations, apoptosis, reactive oxygen species production and synthesis of
heat shock proteins have been reported to occur after cadmium treatment (for a
review see Roccheri and Matranga, 2009). Recently, cadmium has been
reported to induce also autophagy and the expression of specific
metallothioneins (Chiarelli et al., 2011; 2014; Ragusa et al., 2013).
Developmental delay was also observed in the case of mercury. This metal has
been reported to be more toxic on embryogenesis and early larval growth of P.
lividus, compared to copper, lead and cadmium (Fernandez and Beiras, 2001).
In Sphaerechinus granularis, mercury exposure arrested embryo development
at the level of the first cell cycle (Marc et al., 2002). In the sea urchin
Strongylocentrotus purpuratus, both inorganic and organic mercury impaired
mitosis (Bosnjak et al., 2009), interfering with the mitotic apparatus and
blocking embryos in prophase and metaphase.
Table 1. Comparison of metal concentrations (µg.g-1 of tissue) in sea

urchins gonads
Source Species Site Cd Hg Mn

Bohn, 1979 Strongylocentrotus spp. 0.9–1.5 - -
Buchanan et al., Echinocardium Clyde Sea
1980 cordatum (Scotland) 0.40 - -
Warnau et al.,
1995 Paracentrotus lividus Calvi 0.24 0.05 -
Paracentrotus lividus Naples (Italy) 0.45 0.25
Warnau et al.,
1998 Paracentrotus lividus Calvi 3.47 - -
Paracentrotus lividus Ischia 3.41 - -
Paracentrotus lividus Marseille 3.51 - -
Bargagli et al.,
1998 Sterechinus neumayen Terra Nova Bay - 0.02-0.21 -
Guillou et al., Strongylocentrotus
2000 granularis Brest Bay 5.00 0.52 -
Storelli et al.,
2001 Paracentrotus lividus Adriatic sea 5.19 ND -
Bayed et al., 2005 Atlantic Morocco
Paracentrotus lividus Rabat
F 25.15 - -
M 2.58 - -
Paracentrotus lividus Bouznika
F 2.21 - -
M 1.51 - -
Paracentrotus lividus Mohammedia
F 2.24 - -
M 2.32 - -
Soualili et al.,
2008 Paracentrotus lividus Algiers Beach
F 0.14 - -
M 0.08 - -
Paracentrotus lividus Tamenfoust
F 0.12 - -
M 0.05 - -
Paracentrotus lividus Sidi Fredj
F 0.14 - -
M 0.05 - -
Ahn et al., 2009 Strongylocentrotus spp. 0.52–1.6 - 1.1–3.8
F = female, M= male. Where not indicated, data are the mean values between male and females. -
= no data available, ND = undetectable.
In the same work, the ABCC/multidrug resistance associated protein

(MRP) transporters were shown to be involved in the detoxification processes
against mercury, by causing an increased intracellular accumulation of
inorganic mercury without any effect on the organic mercury. Differently from
cadmium and mercury, manganese exposure caused more specific
abnormalities. In P. lividus an increasing number of delayed embryos or
embryos with a poor symmetry and/or underdeveloped arms were observed
with increasing manganese concentration (Pinsino et al., 2010). No apoptotic
events were detected and no correlation between manganese exposure and
oxidative stress was found, whereas an increase in the protein level of hsp70
and hsp60 was detected (Pinsino et al., 2010). Interference with calcium and
perturbation of phosphorylation of the MAP kinases ERK and P38, resulting in
the formation of embryos without skeleton, was recorded after manganese
exposure (Pinsino et al., 2011; 2014).
The picture emerging from overall these studies is complex, especially for
the different sea urchin species and the experimental conditions used, (i.e.,
metal concentration, time of exposure, embryo density). Major efforts should
be focused on protocol standardization to better compare the results from
different studies. Moreover, future research should be directed to deeply
investigate the effects of metals on adults and on the offspring, as well as sea
urchin response to single versus mixed metal combination, mimicking the
conditions present in nature.
2. Ocean Acidification
Atmospheric carbon dioxide (CO2) levels are rising as a result of human
activities, leading to ocean acidification and consequent reduction of carbonate
ion concentrations and saturation state in seawater (Sabine et al., 2004; Feely
et al., 2004; Dupont and Pörtner, 2013). Recent global climate change models
predicted that ocean surface pH will be reduced of about 0.3 to 0.5 pH units by
year 2100 and 0.8 and 1.4 pH units by year 2300 (Caldeira and Wicket, 2003).
Ocean acidification adversely impacts marine fauna, directly affecting
calcification rates and disturbing acid–base physiology (Fabry, 2008; Doney et
al., 2009). Particular attention has been devoted to sea urchins, as typical
calcified organism.
A critical examination of the existing literature on the impact of ocean
acidification on sea urchin is difficult, considering that the majority of the
experiments have been performed in laboratory under different experimental
conditions and using various life-history stages, including gametes,

embryos/larvae, juveniles and adults. In addition, the impact of ocean
acidification is highly species-specific, even in closely related taxa. The effects
of acidification on sea urchins are mostly negative but sub-lethal. A critical
review on the impacts of near-future ocean acidification on sea urchins has
been recently published (Dupont and Thorndyke, 2013), updating a previous
one (Dupont et al., 2010).
In this chapter, some important and general aspects will be summarized.
In particular, under acidification an overall remarkable plasticity was
observed, with a slower somatic and gonadal growth and a concomitant shift
in energy budgets necessary to regulate the intracellular and extracellular pH
values (Stumpp et al., 2011a; 2012). Noteworthy, no direct impact on
calcification was observed. Larvae exposed to decreased seawater pH also
exhibited digestion impairment (Stumpp et al., 2013). Moreover, a series of
genes have been reported to be up or down-regulated by acidification
(Todgham and Hofmann, 2009; O‘Donnell et al., 2010; Stumpp et al., 2011b;
Hammond and Hofmann, 2012; Kurihara et al., 2012; Padilla-Gamino et al.,
2013). However, to better understand the processes occurring at molecular
level, it has been suggested to refer the data to developmental stage rather than
to developmental time (Portner et al., 2010). The growth delay caused by
acidification has important implications because the prolonged permanence of
larvae in the plankton resulted in a higher mortality with consequent reduction
of the number of juveniles and adults.
Compared to studies on other species, less is known on the Mediterranean
species P. lividus. This species appears resistant to low pH. In fact, when eggs
and larvae were reared for a short period in sea water at different pH values,
no effect on fertilization or larval survival was observed (Martin et al., 2011).
The development was normal but delayed at pH values below 7.25.
Interestingly, a substantial molecular plasticity was observed. Recently, the
effect of ocean acidification was also studied in an artificial community of P.
lividus juveniles and macroalgae, revealing a reduced ability of sea urchins‘
defence from predators (Asnaghi et al., 2013).
Although the efforts made to delineate at physiological and molecular
level the effects of acidification on sea urchins, the picture emerging from all
these studies is fragmentary, in some cases even contradictory and difficult to
interpret. To what extend the information derived from laboratory experiments
may be extrapolated to in situ studies is questionable. Therefore, it is important
to identify and characterize sites with naturally-elevated CO2 conditions.
Indeed, some recent studies have been carried out at natural CO2 vents in Italy,
Papua New Guinea and USA (Hall-Spencer et al., 2008; Arnold et al., 2012;
Johnson et al., 2012; Boatta et al., 2013; Evans et al., 2013). Differences in the
distribution and in the ion-regulatory abilities of the sea urchins Arbacia lixula
and P. lividus have been reported in the CO2 vent system in the volcanic island
of Vulcano in the Aeolian archipelago of south Italy (Calosi et al., 2013).
Another natural CO2 vent in Italy is off the Ischia island, where three different
pH zones, ambient, low and extreme low, have been identified, corresponding
the last two zones to near-future (i.e., 2100) and more future (i.e., 2300)
scenarios, respectively (Hall-Spencer et al., 2008; Martin et al., 2008; Cigliano
et al., 2010; Kerrison et al., 2011; Kroeker et al., 2011; Porzio et al., 2011;
Kroeker et al., 2013; Porzio et al., 2013). It has been reported that P. lividus is
absent in the extreme low zone, whereas there are no differences in the
abundance or size of sea urchins between the ambient and low pH zones,
although a reduced grazing rate was observed under low acidic conditions
(Kroeker et al., 2013).
The development of studies performed at natural CO2 vents might give us
in the next future a more detailed picture of the processes going on with ocean
acidification in the single organisms, as well as in whole ecosystems.
3. Natural Toxins
Sea urchin embryos have been shown to be sensitive not only to chemical
pollutants but also to toxins of natural origins, such as those produced by some
microalgal species forming massive blooms at sea. Several toxins isolated
from marine dinoflagellates are known to block cell divisions in sea urchin
embryos. These include goniodomin-A from Goniodoma pseudogoniaulax
(Murakami et al., 1988), amphidinolide-A from Amphidinium sp. (Kobayashi
et al., 1986), and okadaic acid and its derivatives (Fujiki et al., 1988). Among
natural toxins, our attention will be focused on palytoxin-like compounds
produced by benthic dinoflagellates and diatom-derived polyunsaturated
aldehydes.
3.1. Palytoxin-like Compounds
Natural toxins produced by dinoflagellates are receiving increasing

attention, due to worldwide occurrence of harmful algal blooms. These events
have attracted the scientific community interest for the deleterious effects on
marine organisms across multiple trophic levels and finally on human health
(Van Dolah, 2000). Among harmful algae, Ostreopsis spp., benthic
dinoflagellates generally living as epiphytes on macroalgae or on rocky
substrates, have been shown to produce palytoxin-like molecules, such as
ovatoxins (Ciminiello et al., 2010; Rossi et al., 2010). Ostreopsis blooms
occurred in temperate and tropical coastal waters, as well as in the
Mediterranean Sea, causing mortality of benthic organisms and human
intoxications by direct contact (dermatitis), inhalation of marine aerosol
(Zingone et al., 2006; Mangialajo et al., 2011) and consumption of
contaminated food. Bioaccumulation of palytoxin and ovatoxin-a have been
recently reported in mussels and sea urchins (Amzil et al., 2012). Moreover,
Ostreopsis blooms in the Mediterranean Sea (Zingone et al., 2006) and New
Zealand have been shown to cause lethal damages to sea urchin with folding
over or loss of spines (Shears and Ross, 2009; 2010) and consequent decrease
of animal abundance. However, it is still unclear how long toxins may remain
in tissues after blooms and if sea urchins can recover. The future identification
of molecular targets upon intoxication should help to clarify these aspects.
Regarding the effect of Ostreopsis bloom on sea urchin development, it
has been reported that P. lividus juveniles are more sensitive to intoxication
with O. cf ovata cells respect to larvae (Privitera et al., 2012). In fact, juveniles
are affected by O. ovata at much lower concentrations, in the range of those
commonly recorded during blooms (Mangialajo et al., 2008; 2011). In the
Mediterranean Sea, blooms of O. ovata have been observed during summer
(Mangialajo et al., 2008) or early autumn (Mangialajo et al., 2011), when P.
lividus juveniles are largely present, while larvae are missing in the water
column, thus markedly affecting sea urchin population. On the other hand,
blooms of O. ovata may strongly affect also other Mediterranean Sea urchin
species, such as Arbacia lixula that spawns during summer, thus altering the
relative abundance between these two sea urchin species interplaying together
in the structure and dynamics of Mediterranean rocky shores (Privitera et al.,
2012).
3.2. Diatom-derived Polyunsaturated Aldehydes
In recent years the effect of teratogenic compounds produced by

planktonic diatoms has been thoroughly studied on the sea urchin developing
embryos. These groups of microalgae are dominant photosynthetic organisms
in the world’s oceans, shown to activate the production of lipid derivative

compounds which cause abortion and larval abnormalities in the offspring of
their grazers (Miralto et al., 1999; Ianora et al., 2004; Pohnert, 2005). These
compounds are collectively termed oxylipins (Fontana et al., 2007a, b) and
include polyunsaturated aldehydes (PUAs) such as 2-trans,4-trans-decadienal
(decadienal), 2-trans,4-trans-octadienal (octadienal), 2-trans,4-trans,7-
octatrienal (octatrienal) and 2-trans,4-trans-heptadienal (heptadienal). The
PUA decadienal, used as a model aldehyde, was shown to affect
gametogenesis, gamete functionality, fertilization, embryonic mitosis, larval
fitness and competence in several marine organisms, including sea urchins
(Ianora and Miralto, 2010; Adolph et al., 2004). A comprehensive review of
these studies was recently published (Caldwell, 2009), thus only a brief
overview of past studies on sea urchins is reported here with the addition of
more recent findings. In a study conducted on the species Psammechinus
miliaris, decadienal was shown to inhibit sperm motility, impair fertilization
success, embryogenesis and hatching success in a dose dependent manner
(Caldwell et al., 2002; 2004). This aldehyde was also found to block cell
division and to induce apoptosis in Paracentrotus lividus early embryos
(Romano et al., 2003). At concentration of 5µg/ml, a clear induction of
apoptosis was demonstrated by means of caspase 3 activity and TUNEL
staining (TdT-mediated dUTP nick end labeling) which specifically marks
apoptotic cells. Caspase3 activity increased progressively with time reaching a
maximum value at 90 minutes. Number of TUNEL positive embryos followed
similar increase. Figure 1 shows the appearance of TUNEL positive nuclei (in
green) starting from 90 minutes after incubation with decadienal. Moreover,
actin staining (in red) highlighted the formation of blebbing vescicles after 120
minutes of treatment. Hansen and co-workers (2004) demonstrated that
exposure to decadienal led to the arrest of the cell cycle progression in
Spaerechinus granularis early embryos through the inhibition of tubulin
polymerization, DNA synthesis and cyclin B/Cdk1 kinase activity. Embryonic
and larval development of sea urchins are affected by PUAs even at lower
doses. Recently, the longer chain aldehydes, such as decadienal, have been
recognized as the most deleterious PUA (Romano et al., 2010). Embryos
exposed at lower doses of this aldehyde (0.2-0.6 µg/ml) showed the presence
of apoptotic tissues at the pluteus stage, as revealed by TUNEL staining.
Figure 1. Paracentrotus lividus embryos after 10 (a), 90 (b) and 120 minutes (c) from
fertilization. d-f embryos incubated with 5 μg/ml decadienal and observed at the same
time as controls. Red fluorescence corresponds to actin labelled with fluorescent
phalloidin; green fluorescence represents TUNEL positive nuclear regions. Actin
localization and TUNEL staining were performed according to the methods described
in Tosti et al., 2003 and Romano et al., 2003, respectively.
The response activated by decadienal in sea urchin embryos was further

investigated at the molecular level. In detail, the response of P. lividus embryos
to decadienal is mediated by nitric oxide through the up regulation of heat
shock protein 70 (hsp70) (Romano et al., 2011). Hsp70 has been previously
recognized as a valid marker of stress, following exposure to pollutants and
UV-B radiation in embryos, as well as in adult immune cells of sea urchin
(Roccheri and Matranga, 2009 and ref therein).
Moreover, Marrone et al., (2012) showed that P. lividus embryos activated
a dose-dependent response of target genes following decadienal treatment. In
detail, hsp60, hatching enzyme and blastula protease 10 were up regulated at
the blastula stage and hsp56 and other genes (14-3-3 epsilon, p38 MAPK,
DNA-methyltransferase 1 and glutamine synthetase) at the prisma stage. At
this latter stage, all genes involved in skeletogenesis (nectin, univin, spicule
matrix protein 30 and spicule matrix protein 50) were down-regulated,
accompanied by developmental abnormalities mainly related to skeleton
morphogenesis. Authors suggested that this orchestrated defense system
against decadienal represents part of the defensome of P. lividus affording

protection from environmental toxicants.
Varrella et al. (2014) expanded the analysis of the stress response to the
toxic PUAs during P. lividus development, examining the effects of
heptadienal and octadienal, the most abundant aldehydes in nature, in
comparison with the better-known PUA decadienal. The expression levels of
several genes in P. lividus embryos were modulated by all three PUAs,
although these three aldehydes affected different classes of genes at different
times of development. Interestingly, octadienal acted earlier than the other
aldehydes, affecting already at blastula stage the expression levels of some
skeletogenic genes. This finding is in accordance with post-recovery
experiments showing that embryos exposed to octadienal were less able to
recover with respect to those exposed to other PUAs. Regarding the expression
of other genes, heptadienal and octadienal behave as decadienal in up
regulating the expression of hsp70 and in down regulating the expression of
Wnt6 gene, member of the canonical Wnt pathway.
Further studies are required to ascertain the real impact of these natural
toxins in the benthic ecosystem including also possible influences of epiphytic
benthic diatoms which are a source of food for benthic organisms. These
microalgae, in fact, are known to produce volatile compounds similar to those
identified in planktonic diatoms (Juttner, 2005, Juttner et al., 2010), but their
impact on the reproductive fitness of sea urchin has never been investigated.
4. The Role of Nitric Oxide in Mediating

Stress Response in Sea Urchin
Nitric oxide is an important physiological messenger, produced from L-
arginine by the action of the enzyme nitric oxide synthase, present in all
metazoans (Andreakis et al., 2011). In non-vertebrates nitric oxide is implied
in many processes, including development, neural transmission, defence
system (Palumbo et al., 2000; Fiore et al., 2004; Palumbo, 2005 and references
therein; Comes et al., 2007; Palumbo and d‘Ischia, 2007; Mattiello et al.,
2010; 2012; 2013; Ercolesi et al., 2012). Increasing evidence also indicates
that nitric oxide is a cellular signal of environmental stress. In fact, heat stress
activates nitric oxide production in sponges (Giovine et al., 2001) as well as
salinity and light gives rise to nitric oxide bursts in culture media of marine
microalgae (Zhang et al. 2006). Moreover, treatment of the marine diatom
Phaeodactylum tricornutum with the diatom aldehyde decadienal induced the

production of nitric oxide (Vardi et al., 2006). Recently, it has been reported
that nitric oxide also mediated the stress response induced by decadienal in the
sea urchin P. lividus (Romano et al., 2011). At low decadienal concentrations,
nitric oxide protects developing embryos against the toxic effects of this
aldehyde, through the expression of hsp70. Decrease of endogenous nitric
oxide levels by inhibition of nitric oxide synthase resulted in an increase of
abnormal plutei and a decrease of hsp70 expression. As expected, hsp70
expression increased in the presence of a nitric oxide donor. This finding
allowed to demonstrate the involvement of this gas in the stress response to
diatom aldehydes as well as to identify the targets of nitric oxide action.
In the future, pharmacological approaches aimed at modulating
endogenous nitric oxide levels using nitric oxide synthase inhibitors or nitric
oxide scavengers or donors might be useful to deeply investigate the role of
nitric oxide as universal messenger of environmental stress in sea urchins
(Figure 2). Morphological observation and gene and protein expression
analyses will allow to identify the processes and the molecular pathways
affected by nitric oxide.
Figure 2. Experimental protocol to assess the involvement of nitric oxide in the

response of sea urchin to different stress agents.
Conclusion
The data reported in this chapter represent a critical summary of the
available literature on the response of sea urchins to very different stress
agents. These include agents deriving from human activities impacting natural
environment, such as metal ions and ocean acidification, as well as natural
agents, such as toxins, produced by some microalgae. Most of the studies have
been performed on developing embryos, which have been shown to be able to
defend themselves from these agents through an orchestrated array of gene
families and biochemical pathways.
In each paragraph recommendations for future research are indicated. In
particular, major attention should be focused on adults and offspring as well as
on the use of experimental conditions, mimicking as much as possible the
situation in the field. In this regard, the investigation on natural CO2 vents
could be important to increase our knowledge on ocean acidification at
different levels of complexity from the organism to the ecosystem.
Overall the results here reviewed, suggest that metal pollution and sea
water acidification, together with the presence of toxins produced by
microalgae, can strongly reduce the survival of sea urchin thus causing
cascading effects on the whole ecosystem. Therefore, effective policies should
be adopted to preserve this key species in the marine environment.
An important issue which requires further investigation is the involvement
of nitric oxide in the stress response of sea urchin. Understanding if nitric
oxide is the messenger of different stress agents would allow us to highlight
unifying mechanism underlying sea urchin response.
Acknowledgments
This work has been funded by SZN and by the Flagship RITMARE-
coordinated by the Italian National Research Council, funded by the Italian
Ministry of Education, Univ and Res, National Research Program.
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Editor: Edgar Raymond Banks c 2014 Nova Science Publishers, Inc.
Chapter 3
N ONPARAMETRIC R EGRESSION A PPLIED

TO S EA U RCHIN G ROWTH
Isabel Martínez-Silva1,∗, Marta Sestelo1,2†, Gorka Bidegain3,‡,

Altea Lorenzo-Arribas4,§ and Javier Roca-Pardiñas1,¶
1
Department of Statistics and O. R., University of Vigo, Spain
2 Department of Mathematics,
Autonomous University of Barcelona, Spain

3 Department of Coastal Sciences, Gulf Coast Research Laboratory,
University of Southern Mississippi, MS, US

4 Biomathematics and Statistics Scotland (BioSS), UK
Abstract
An adequate nonparametric regression model is able to record spe-
cific patterns in the data that cannot be detected by a parametric model.
In addition, quantile regression can provide a more complete description
of functional changes than an exclusive focus on the least square regres-
sion. This chapter assesses the adequacy of a variety of nonparametric
models to analyze the growth patterns of sea urchins by means of the
length-weight relationship. For this purpose, data from fishery landings
∗ E-mail address: [email protected]
† E-mail address: [email protected]
‡ E-mail address: [email protected]
§ E-mail address: [email protected]
¶ E-mail address: [email protected]
54 Isabel Martínez-Silva, Marta Sestelo, Gorka Bidegain et al.
of green sea urchin Strongylocentrotus droebachiensis are used to ana-

lyze this relationship for lengths within the legal catch-size range (52.4
mm - 76.0 mm) at two depths (i.e., shallow waters, 4.6 m, and deep wa-
ters, 7.6 m). Overall, this gives insight into the study of the minimum
capture size. We apply a Kernel nonparametric regression model to deter-
mine both its suitability and applicability as an alternative to the classic
allometric model, for the estimation of a minimum catch size directed to
obtain the maximum yield in weight from the fishery. The results demon-
strate the suitability of the Kernel nonparametric regression model as an
alternative approach to the classic allometric model to analyze the length-
weight relationship in sea urchins and to estimate a minimum capture
size, particularly for deeper waters sea urchins. Additionally, a boosting-
based quantile regression technique is successfully applied which detects
variability in sea urchin growth patterns throughout the length distribution
and between depths. Differences in food availability and wave exposure
between depths may explain these results.
PACS 05.45-a, 52.35.Mw, 96.50.Fm

Keywords: Sea urchin, nonparametric regression, bootstrap, quantile, testing
1. Introduction
The crash of many important fisheries worldwide has led to increased fishing
pressure on unexploited species and the development of new fisheries to save
displaced fishermen (Walters, 1998). At the same time, some of these recently
developed fisheries have been imprudent and initially based on a reactive man-
agement with lack of biological and stock data. Sea urchin fishery provides
a perfect example of emergent, increasing fishery overexploitation in certain
countries and regions. For instance, the fishery of the green sea urchin Strongy-
locentrotus droebachiensis in Maine (USA) began in the late 1980s, due to the
demand of urchin roe or uni in Japan, and quickly reached a peak as untouched
stocks were heavily fished. It has since experienced a steady declining trend
in yield (Taylor, 2004). Although it is believed that the sea urchin popula-
tions are quite volatile, the decline has been importantly attributed to decreasing
stock abundance over the 1990s (Chen and Hunter, 2003). Since then, a co-
management model including a variety of regulations has been established. A
cap on the number of new harvesters entering the fishery, a minimum and max-
imum size limits, and a restricted season ended the overfishing that had taken
Nonparametric Regression Applied to Sea Urchin Growth 55
place in the late 1980s and early 1990s, notwithstanding the population is not
recovering and the stock status remains relatively stable at low levels (DMR,
2010).
Despite the importance of the sea urchin fishery and its persistent plight far
below past abundances, as in the case of the green sea urchin, the knowledge on
its biology and ecology is sketchy and further research is still needed, especially
into those aspects which can supply information on the physiological condition
and/or the growth patterns. This could be aimed at obtaining a sustainable yield
of the fishery. The study of condition assumes that organisms with a higher
weight for a given length are in better physical condition than those with a lower
weight. Therefore, condition indices are used as indicators of the length-weight
relationship of a population or subgroup. The physiological state of an animal
is related to its evolutionary fitness, thus health status would be an indicator
of reproductive success and of the ability to cope with environmental factors
(Cone, 1989; Jones et al., 1999). One tried and tested approach to this subject is
to evaluate the growth by means of the relationship between weight and length.
Sestelo and Roca-Pardiñas (2011) demonstrated the potential benefit of using
this relationship to objectively estimate a suitable catch, in order to obtain, in
the long run, the maximum yield from the fishery.
The classic way to analyze this relationship in marine invertebrates, includ-
ing sea urchins, is by means of allometric models (Scheibling et al., 1999; Gros-
jean, 2001; Rahman et al., 2012, 2013), where the urchin weight (W ) is related
to its length, usually by measuring the test or skeleton diameter without spines
(D) (Huxley, 1924). Thus, the length-weight common allometric equation for
sea urchins is W = α Dβ , α and β being parameters. However, this relationship
has not been analyzed in detail for sea urchins. To the authors’ knowledge, the
only attempt to further analyze the length-weight relationship has been recently
conducted by Martínez-Silva et al. (2013) for the purple sea urchin.
The analysis of this relationship by means of allometric models leads to a
loss of valuable biological information due to the fact that, being a parametric
model, it specifies in advance the function that links the covariates to the re-
sponse (Rabaoui et al., 2007). Nonparametric regression analysis relaxes the
assumption of the specific parametric form of the allometric model, replacing
it with the more flexible assumption of a smooth population regression func-
tion. Consequences of relaxing this assumption are a greater computational
cost and, at times, a less straightforwardly interpretable result. The benefit is
that nonparametric analyses potentially provide more accurate estimates of the
regression function. It has been demonstrated for several commercial marine

invertebrates, such as gooseneck barnacles and clams, that an adequate non-
parametric model is able to record specific patterns in the data at the end of the
length-weight relationship regression curves that could not be detected by an
allometric model (Sestelo and Roca-Pardiñas, 2011; Bidegain et al., 2013).
Traditional shellfishery management attempts to maintain the maximum
sustainable yield of the fishery by placing limits on size, season, catch, and
gear. Albeit, these methods often do not consider variation in a population’s
growth, survivorship, or reproductive output and the probability of exceeding
or not reaching the maximum, sustainable yield is high. The estimation of ade-
quate catch sizes for commercial marine invertebrates integrates several biolog-
ical aspects such as individual size at sexual maturation, growth rate and length-
weight relationship (Donaldson and Donaldson, 1992; Coutures and Chauvet,
2001; Camacho-Mondragón et al., 2012). Sestelo and Roca-Pardiñas (2011)
studied the potential benefit of using the length-weight relationship to objec-
tively estimate a suitable catch size, in order to obtain, in the long run, the
maximum yield from the fishery. They analyzed the length-weight relationship
for the gooseneck barnacle Pollicipes pollicipes using a Kernel-based nonpara-
metric model, and proposed the minimum catch size as the one at which the first
derivative of the regression curve reached the maximum, ensuring that beyond
this point weight gain from one size to the next decreases, obtaining this way
the maximum sustainable yield. They adequately used a nonparametric model
for this purpose, considering the absence of a maximum in the first derivative
of the allometric model. Bidegain et al. (2013) recently applied this approach
to clams with identical aims. These results demonstrate the feasibility of using
nonparametric techniques to analyze in detail the length-weight relationship and
estimate the minimum capture size of commercial species that display differen-
tiated weight gain patterns throughout their development.
In addition, quantile regression has emerged as a useful supplement to or-
dinary mean regression. In a given regression curve, upper or lower quantiles
may depend on the covariates very differently from the center. Therefore, quan-
tile regression can yield a more complete description of functional changes than
focusing attention exclusively on the mean (Koenker, 2005). Quantile regres-
sion is used in cases where a study seeks to estimate the different percentiles
of a population of interest. One advantage of using quantile regression to esti-
mate the median rather than using ordinary least squares regression (to estimate
the mean), is that the former is less sensitive to the presence of atypical val-
ues (Martínez-Silva et al., 2013). Similarly to the mean-based regression, the

nonparametric quantile regression potentially provides a more accurate estimate
of the regression function than that obtained by the parametric analysis. This
nonparametric approach for quantile regressions has already been successfully
tested for the analysis of the length-weight relationship in the purple sea urchin
Paracentrotus lividus (Martínez-Silva et al., 2013) demonstrating its potential
applicability to other sea urchin species.
In this chapter, we assess the suitability of two types of nonparametric mod-
els to analyze the length-weight relationship of the green sea urchin Strongy-
locentrotus droebachiensis. For this purpose, data were collected on landings
into the port of Jonesport (Gulf of Maine) during the open fishery season from
December 2012 to March 2013. This allows us to analyze in detail the relation-
ship for individuals with lengths within the legal catch-size range, which gives
insight into the study of the minimum capture size. Firstly, we apply a Kernel
nonparametric mean-based regression model to analyze (i) the suitability of this
model as an alternative to the classic allometric model in order to investigate the
length-weight relationship (Section 2) and (ii) the applicability of this model to
estimate a minimum catch size directed to obtain the maximum yield in weight
from the fishery (Section 3). Secondly, we apply boosting-based quantile re-
gression to further investigate this length-weight relationship and estimate the
different percentiles (Section 4).
1.1. Data Collection and Measurements

Specimens of the sea urchin Strongylocentrotus droebachiensis were collected
in Jonesport (Maine, USA) by the Maine Department of Marine Resources
(DMR) during the fishery open season from December 2012 to March 2013.
Sampling was performed once a week when weather conditions allowed har-
vesters’ activity. A random sample of 20 urchins was collected from each land-
ing of both divers and draggers when possible. Due to the origin of the data,
in general, expected length of individuals was within the legal catch-size range
(52.4 mm -76 mm). Individuals were measured (test diameter, mm), weighed
(fresh weight, g) and grouped by the factor “depth”. Two depth levels were
considered based on the results obtained during the interviews conducted by the
DMR, in which the fishermen were asked about depths fished. Data from divers
were labeled as SW (i.e., shallow waters) indicating that the average harvest
depth was 4.6 m, while individuals harvested by draggers were labeled as DW
(i.e., deep waters) indicating an average depth of 7.6 m. A random subsample

of 250 individuals per depth level was used for the analysis.
2. Classical Allometric Model vs Nonparametric Model

The relationship that defines the growth in a species’ weight with respect to
its length is one of the most common in fish biology and fisheries, and it is an
important element in population dynamics and stock assessment (Oniye et al.,
2006). Indeed, this length-weight relationship has been studied in various ma-
rine species, using different parametric models which are easy to apply and es-
timate, and have been extensively described in the literature (i.e., Froese, 2006;
Ismen et al., 2007; Neves et al., 2009; Pinheiro and Fiscarelli, 2009; Nieto-
Navarro et al., 2010; Ramón et al., 2010). One of the most widely used models
of this type is the allometric model proposed by Huxley (1924), which in the
case of sea urchins, relates the biometrical variables weight (W ) and skeleton
diameter (D) by the regression curve E[W |D] = α Dβ , with α and β being con-
stants.
Based on its extended use, in Subsection 2.1 we propose a procedure to
test if the data can be modelled by using this parametric model or, by contrast, it
would be more adequate to use a nonparametric one of the type E[W |D] = m(D),
with m being a smooth and unknown function.
The results of the testing procedure applied to the Strongylocentrotus droe-
bachiensis data are shown and discussed in Subsection 2.2.
2.1. Testing Procedure

The allometric model is a classical potential model, which is usually converted
into its logarithmic expression
logW = log α + β log D + ε,

or analogously,
W ∗ = α∗ + β∗ D∗ + ε, (1)
being ε the mean zero-error, W ∗ = log W , D∗ = log D, α∗ = log α and β∗ = β
. This conversion, which is quite simple, both conceptually and mathemati-
cally, facilitates the estimation of its parameters by linear regression. Once α̂∗
and β̂∗ have been obtained by fitting the model in (1), the original scale of the
parameters is returned to α̂ = exp(α̂∗ ) and β̂ = β̂∗ , and the estimated curve,
m̂(D) = α̂Dβ̂ , is thus obtained.
Despite the fact that such parametric models are appealing in many situa-
tions, there is a problem associated with their use. In certain circumstances, the
assumption of a given curve for the effects of the covariates is very restrictive
and is not supported by the data at hand and, consequently, if the parametric
model fails then the conclusions will be erroneous. In this setting, nonpara-
metric regression techniques are involved in modelling the dependence between
the response and the covariates, moreover without specifying in advance the
function which links the covariates to the response.
Hence, in order to facilitate the choice of a model appropriate to the data,
while at the same time trying to minimize the loss of information, we have
developed the following bootstrap-based procedure that tests the null hypothesis
of an allometric model versus a general nonparametric model. For this purpose,
the residuals of the fit of the parametric models are used.
Based on a general model of the type
W ∗ = m(D∗ ) + ε,
the aim here is to test the null hypothesis of an allometric model
H0 : m(d ∗ ) = α∗ + β∗ d ∗ , (2)
versus the general hypothesis H1 , with m being an unknown nonparametric
function; or analogously,
H1 : m(d ∗ ) = α∗ + β∗ d ∗ + g(d ∗ ), (3)

with g(d ∗ ) being an unknown function not equal to zero.
There now follows a detailed outline of the procedure to test H0 via the
bootstrap-based test. For this purpose, considering the L1 norm, we propose the
use of the following test statistic
n
Q = ∑ |ĝ(D∗i )|, (4)
i=1
with ĝ(D∗i ) being the nonparametric estimation of g(D∗i ) according to the ex-
pression in (3) with a sample {(D∗i ,Wi∗ )}ni=1 .
If the null hypothesis is verified, then Q should be positive and close to zero.
Thus, the test rule for checking H0 with a significance level α is that the null
hypothesis is rejected if Q is larger than its (1 − α)-percentile. To approximate
the distributions of the test statistic, resampling methods such as the bootstrap
introduced by Efron (1979) (see also Efron and Tibshirani, 1993; Härdle and
Mammen, 1993; Kauermann and Opsomer, 2003)) can be applied instead. Here
we use the wild bootstrap (Wu, 1986; Liu, 1988; Mammen, 1993) because this
method is valid for heteroscedastic models where the variance of the error is a
function of the covariate. The steps of the procedure are as follows:
Step 1. From the sample data {(D∗i ,Wi∗ )}ni=1 , obtain the estimates of
α∗ and β∗ according to the null model in (2), compute the residuals as
ri = Wi∗ − α̂∗ − β̂∗ D∗i and obtain the nonparametric estimates of g(D∗ ) accord-
ing to the model ri = g(D∗i ) + εi using the estimation algorithm exposed in
Subsection 3.1. Compute the Q value.
n
Step 2. For b = 1, . . ., B, generate bootstrap samples D∗i ,Wi∗•b

i=1
with
Wi∗•b = α̂∗ + β̂∗ D∗i + ε•b •b
i , and εi being
( √ √
(1− 5)
•b ε̂i · 2√ with probability p = 5+10 5
εi = √
ε̂i · (1+2 5) with probability p = 5−10 5
where ε̂i = Wi∗ − α̂∗ − β̂∗ D∗i are the residuals of the null model, and compute
Q•b the same way as in Step 1.
Since the bootstrap resamples are constructed under the null hypothesis, this
procedure approximates the distribution of Q under H0 . Consequently, the test
rule based on Q consists of rejecting the null hypothesis if Q > Q1−α, where
Q1−α is the empirical (1 − α)-percentile of the values Q•1 , . . ., Q•B previously
obtained.
2.2. Results
Figure 1 depicts the regression curves of the length-weight relationship esti-
mated by means of the two proposed models. Under the allometric model (left
panel), the regression curve shows the way in which individuals’ size increases
as their weight rises. The length-weight relationship seems to be a rising func-
tion across the entire range of values. On the other hand, under the nonparamet-
ric model (right panel), the regression curve is also rising and very similar to the
curve estimated with the allometric model. However, the final sections of these
curves seem to differ according to the model used. The nonparametric model
detects variations in the final part of the figure, which the allometric model is
not capable of discerning. However, a careful study of the so-called boundary
or edge effects (Hart and Wehrly, 1992; Müller, H. G., 1991) would be required
to specify the strength of this evidence.
When the study is repeated with the data being stratified by depth, it shows
the same behaviour as the overall study (Figure 2). Similarly, the allometric
model would seem to be incapable of detecting variations in the data which the
nonparametric model is able to record.
At this point, the above-mentioned test for the null hypothesis H0 : m(D) =
α Dβ is applied. In the case of the overall study, the result of this test is that,
for a 5% significance level, the null hypothesis is rejected (p-value < 0.01) thus
the use of the nonparametric model would seem to be a good alternative to the
classical model. However, if we split the data by depth, the null hypothesis is
only rejected (p-value < 0.01) with sea urchins harvested at shallow waters. The
relationship between W and D for sea urchins of deep waters seems to be a good
fit with an allometric model (p-value = 0.31).
Figure 1. Regression curves with bootstrap-based 95% confidence intervals

(dashed lines) for weight (W ) and test diameter (D). Left panel: allometric
model. Right panel: nonparametric regression model.
The different growth pattern between depths (Figure 2), in terms of weight
gain with respect to length, could be linked to a variety of factors such as wave
exposure, density of sea urchins, behavior and limitation of food. Green sea
urchins inhabiting wave exposed shallow waters allocate more energy to the
production of stronger body walls, maintenance and spine repair, and conse-
quently less remains available for growth (Ebert, 1968, 1982). Urchins from
exposed waters are usually difficult to detach while those in more calm wa-
ters are easily detached, indicating behavioral and morphological adaptations
of the urchins to increased water movement. Regarding feeding, this species is
omnivorous, however, laminarian kelps are usually the primary component of
the diet and can occur in the form of attached fronds, drifting fronds or detri-
tus (Scheibling and Hatcher, 2001). Individual growth rates vary considerably
due to food quality and availability and therefore, barren ground sea urchins
are usually smaller and less variable in size than those in kelp habitats (Vadas
et al., 1986). In very dense populations urchins overgraze the subtidal seaweed
community and food is limited, while in low density zones food availability is
higher, and consequently increasing urchin growth rates are found (Himmel-
man, 1986). Further research is needed to understand the relative importance of
these factors in explaining depth-associated differential growth patterns.
3. Minimal Size of Capture Based

on Nonparametric Regression
To establish the size of capture of any species subject to exploitation, a range
of biological and ecological aspects must be taken into account, such as indi-
vidual size at sexual maturation, growth rate and biological cycle. Additionally,
each specimen’s weight gain must be assessed. In this respect, the Food and
Agriculture Organisation (FAO) of the United Nations states that “The basic
purpose of fish stock assessment is to provide advice on the optimum exploita-
tion of aquatic living resources (...) and fish stock assessment may be described
as the search for the exploitation level which in the long run gives the maximum
yield in weight from the fishery” (Sparre and Venema, 1997). In line with this
guidance, we feel that the application of nonparametric regression to model the
data and the study of derivatives can be extremely useful. In particular, in cases
where the previous test (allometric vs. nonparametric) is rejected and where the
Figure 2. Regression curves with bootstrap-based 95% confidence intervals

(dashed lines) for weight (W ) and test diameter (D) using an allometric model
(left) and a nonparametric model (right). Upper panel: sea urchins from shallow
waters (SW = 4.6 m). Lower panel: sea urchins from deep waters (DW = 7.6
m).
nonparametric model is a suitable model, we propose that the minimum size cor-
responds to the point (or size) where the first derivative reaches the maximum.
From this point onwards, weight gain from one size to the next decreases, so that
the yield obtained ceases to be profitable (Sestelo and Roca-Pardiñas, 2011).
The details of the nonparametric estimation procedures carried out in order
to propose a possible size of capture for the green sea urchin Strongylocentrotus
droebachiensis are shown in Subsection 3.1. The result obtained jointly with
the derived discussion can be found in Subsection 3.2.
3.1. Nonparametric Estimation Procedures

In order to estimate the length-weight relationship for this species, the following
nonparametric regression model is considered
W = m(D) + ε, (5)
where m is an unknown smooth function and ε is the error that is assumed to
have mean zero and variance as function of the covariate D. It should be noted
that, in contrast to allometric models, in this type of model there is no need to
establish a parametric form of m.
The regression model in (5) is estimated using local polynomial kernel
smoothers (Wand and Jones, 1995; Fan and Gijbels, 1996). Given a sample
{(Di ,Wi )}ni=1 , be n independent and identically distributed (i.i.d.) observations,
the estimate of m at a point d is given by m̂(d) = γ̂0 (d), with γ̂0 (d) the first
position of the vector (γ̂0 (d) , γ̂1 (d) , . . ., γ̂R (d)) which is the minimizer of
n o2 D − d
n R r i
∑i=1 Wi − ∑r=0 γr (d) (Di − d) · K h , (6)
where K is a kernel function (normally, a symmetric density), h is the smoothing

parameter or bandwidth and R is the degree of the polynomial. Finally, the
estimated r-th (r ≤ R) derivative of m(d) is given by m̂r (x) = r!γ̂r (x).
As we have mentioned before, using this nonparametric model, it is possible

to register a maximum in the first derivative of m at a given size (d0 ), which
could be used for estimating a possible ideal size of capture. The size sought,
d0 , is given by the maximizer of the first derivative of m. Accordingly, we
defined this point, d0 , as
d0 = arg max m1 (d).

d
In practice, however, neither m nor m1 is known, so that the estimated d0

must be obtained on the basis of the estimates m̂ and m̂1 of the true m and m1
curves. A natural estimator of d0 can be obtained as the maximizer of
m̂1 (k1 ), . . ., m̂1 (kN ),

with k1 , . . ., kN being a grid of N equidistant points in a range of D values.
Needless to say, since d0 is only an estimate of the true d0 , the sampling un-
certainty of these estimates needs to be taken into account. Hence, a confidence
interval using the wild bootstrap is created for d at a specific level of confidence.
The steps for constructing this confidence interval for an R value obtained from
the model in (5) (for instance, R = d0 , R = m(d), or R = m1 (d) for a given d)
are as follows:
Step1. Obtain the estimated R̂ from the original sample. n
Step 2. For b = 1, . . ., B, generate bootstrap samples Di ,Wi•b i=1 with
Wi•b = m̂(Di ) + ε•b •b
i , and εi being
( √ √
ε̂i · (1−2√5) with probability p = 5+ 5
ε•b
i = 10
√
ε̂i · (1+2 5)
with probability p = 5− 5
10
where ε̂i = Wi − m̂(Di ) are the residuals of the general model in (5), and compute
R̂•b the same way as in Step 1.
Finally, the 100(1 − α)% limits for the confidence interval of R are given by

I = R̂α/2 , R̂1−α/2 ,
where R̂ p represents the percentile p of bootstrapped estimates R̂•1 , . . ., R̂•B.
Bandwidth selection. It is well known that the nonparametric estimates m̂r (D)
depend heavily on the bandwidth h used in the kernel-based algorithm. Vari-
ous methods for an optimal selection have been suggested, such as Generalised
Cross-Validation (GCV) (Golub et al., 1979) or plug-in methods (see e.g., Rup-
pert et al., 1995). For a good overview of this topic see Wand and Jones (1995).
However, optimal bandwidth selection is still a challenging problem.
As a practical solution, in the equation (6) of the estimation algorithm, the
bandwidth h is automatically selected by minimizing the following data-driven
cross-validation criterion
2
n
CV (h) = ∑i=1 Wi − m̂(−i) (Di ) ,
where m̂(−i) (D) indicates the fit at X, leaving out the i-th data point based on
the smoothing parameter h.
Computational aspects. Bootstrap resampling techniques are time-consuming

processes because it is necessary to estimate the model many times. Moreover,
the use of the cross-validation technique for the choice of the bandwidths used
in the nonparametric estimates implies a high computational cost, due to the ne-
cessity of repeating the estimation operations several times to select the optimal
bandwidths. Consequently, recourse to some computational acceleration tech-
nique is fundamental to ensure that the problem can be addressed adequately
in practical situations. In this chapter, we use binning techniques to speed up
the process. A detailed explanation of this technique can be found in Fan and
Marron (1994).
3.2. Results
Shown in Figure 3 are the estimated regression curves and their first derivatives
for the data split by depth. As we concluded in the previous section, a non-
parametric growth model offers a better fit for the data from the sea urchins in
shallow waters while an allometric growth model seems to be more appropriate
for fitting the individuals from deep waters. According to this, the regression
curves of both models are monotone increasing functions, and the value of W in-
creases with the values of D. In the nonparametric model, however, the increase
in weight per unit of D (given by the first derivative of m) registers a maximum
at a given size, denoted d0 , beyond which such weight gain declines (or at least
remains constant). This trend is not observed in the allometric model, in which
the first derivative always increases.
Based on this, a possible minimum size of capture is proposed only for
the sea urchins from shallow waters (Figure 3, upper panel). It is important to
underscore the fact that this curve is initially exponential, until it reaches a point
where the relationship between W and D continues with a more linear trend. The
first derivative of this curve increases as individuals grow in size, until it peaks
at a D of 64.3 mm (62.9, 66.0). Beyond this size the fishery yield in weight
may decrease. For deeper waters this maximum is not detected (Figure 3, lower
panel), denoting a monotone increase in weight per unit of size D. Therefore,
the yield obtained in weight in deep waters never ceases to be profitable.
The result obtained regarding the minimum size for shallow waters is more
conservative than the current catch-size for the green sea urchin in Maine, which
was increased in 2001-02 from 50.8 mm to 52.4 mm to reduce extraction of
small specimens. However, our result is quite similar to the median sea urchin
diameter in the Gulf of Maine, which since then has consistenly been about
60 mm (DMR, 2010). For deeper waters, we did not obtain a size where a
maximum yield in weight is achieved but a suitable minimum size may not be
larger than the current maximum capture size (76.2 mm).
Our results showing differences in growth patterns in terms of weight gain
per unit of size may also be interpreted in terms of a combination of two fac-
tors: food availabilty and wave exposure. The green sea urchin dive surveys
conducted recently by the DMR where the biomass of sea urchins and algal
cover were assessed in three different depth strata (0–5 m, 5–10 m and 10–15
m) showed, in general, higher biomass indices in the 0–5 m stratum. A higher
coverage of canopy, mostly kelp, and understory algal communities was found
in the shallower stratum than in the other strata. However, it seems that there is
not a food limitation in deeper waters (DMR, unpublished). In general, there are
higher urchin biomass indices in shallow waters than in deeper waters (DMR,
unpublished). Therefore, a more dense population in shallow waters, which in
some areas can overgraze the subtidal seaweed community, may lead to food
limitation for sea urchins. This potential food limitation, together with the en-
ergy allocated to maintenance in a higher wave exposure area, could explain the
maximum weight gain per unit of size in shallow waters. On the contrary, in
deeper waters, a sufficient food availability together with a lower wave energy
could result in the urchins allocating more energy to growth than to produc-
tion of stronger body wall, maintenance and spine repair. However, we need
to be cautious with the interpretation of these results since a full explanation
may need a parallel study of density, physical conditions and food availability.
Moreover, for deeper waters, a more detailed investigation including more study
zones may be essential to further analyze the first derivative of the nonparamet-
ric model and, probably, obtain the function’s maximum giving a suitable size
of capture.
4. New Approaches to Growth Study Through

Smoothed Quantile Regression
Quantile regression (QR) (Koenker and Bassett, 1978) is a form of regression
which models the relationship between certain variables in a more robust and
Figure 3. Regression curves and first derivatives with bootstrap-based 95% con-
fidence intervals (dashed lines) for weight (W ) and test diameter (D). Upper
panel: length-weight relationship for sea urchins from shallow waters (SW =
4.6 m) obtained by means of a nonparametric model. Lower panel: length-
weight relationship for sea urchins from deep waters (DW = 7.6 m) obtained by
means of an allometric model.
flexible manner than classical regression models. In the same way that the up-
per and lower quantiles give more information about our observations than the
mean, QR widens the focus of the classic regression (ordinary least square re-
gression - OLS). Mosteller and Tuckey (1977) state in this respect: “Just as
the mean gives an incomplete picture of a single distribution, so the regression
curve gives a correspondingly incomplete picture for a set of distributions”.
By estimating the conditional quantiles of the distribution of the response
variable, this methodology affords a robust analysis of the relationships between
this response and any other variables of interest. One of the main advantages of
QR (e.g., median estimation) versus OLS (i.e., mean estimation) is that median
regression is less sensitive to the presence of outliers. An additional advantage

is the possibility of estimating any other quantile required, therefore allowing
the evaluation of those extreme population values.
Although the initial applications of QR were in the field of Economics
(Fitzenberger et al., 2001), this technique soon appealed to other research ar-
eas. In Paediatrics, the use of growth curves is common practice to evaluate
unusual patterns in children’s growth. With this aim, several studies have been
developed by the World Health Organization (WHO), the most recent one being
the WHO Multicentre Growth Reference Study (MGRS) (de Onis et al., 2004)
which took place between 1997 and 2003. In the MGRS, QR models were used
to generate new growth curves for assessing the growth and development of tod-
dlers, children and adolescents between 0 and 19 years old worldwide in order
to adequately represent the current growth of the aforementioned population.
In Ecology and Biology, this technique has also become widely used. Cade
and Noon (2003) gather applications of QR to estimate rates of change for func-
tions along or near the upper boundary of the conditional distribution of re-
sponses. The authors highlight the fact that in populations with heterogeneous
variances - which are commonplace in ecological studies -, focusing exclusively
on changes in the means may underestimate, overestimate, or fail to distinguish
real nonzero changes in heterogeneous distributions. Scharf et al. (1998) argue
that in these populations the use of QR also “eliminates the need for an excess
of arbitrary decision-making by the researcher”, being only left to choose the
quantiles representing upper and lower bounds. In Scharf’s examples of pat-
terns in prey and predator size in piscivorous fishes, this choice is made based
on additional ecological background information as well as the data character-
istics and the nature of the research question.
Terrell et al. (1996) and Dunham et al. (2002) provide examples of QR as a
robust technique in modelling relationships between stream fish abundance and
habitat variables which OLS estimation fails to capture. Particular applications
to the exploitation of marine resources include, Planque and Buffaz (2008) and
Ourens et al. (2014), which focus on the relationships environment-recruitment
and recruits-adults densities, respectively.
Finally, Martínez-Silva et al. (2013) study the growth of purple sea urchin
populations in two Spanish locations. A similar approach will be taken in Sub-
section 4.3 which will aim to extend this analysis of the effect of the sea urchin
length on the weight average, developed in previous sections, to the study of the
effect of the length on the conditional distribution using smoothed QR models.
4.1. Smoothed Quantile Regression

Together with an increase in the range of areas of application of QR, the tech-
nique itself has also undergone substantial improvements and adaptations. Anal-
ogously to the developments in OLS (Hastie and Tibshirani, 1990), nonparamet-
ric QR techniques have been proposed that allow for the modelling of the rela-
tionship between variables in a flexible manner (Koenker, 2005; Fenske et al.,
2011; Yee and Wild, 1996; Rigby and Stasinopoulos, 2005). New techniques
such as smoothing the objective function (Kato and Galvao, 2010) tackle dif-
ficulties in the standard QR estimation. For instance, non-smoothness in the
objective function would complicate the asymptotic analysis of the estimator
particularly when working with panel data.
Using the same notation as in the nonparametric regression model (5) in
Subsection 3.1, the nonparametric quantile regression model can be defined as
follows
wi = g(di) + ετi , ετi ∼ Fτi

where the index i = 1, . . ., n denotes the individual, wi its weight value, and di
is the skeleton diameter. g denotes a smooth function of the skeleton diameter
di which is assumed to relate in a nonlinear way to the response quantile func-
tion. τ ∈ (0, 1) indicates a fixed and known quantile, and Fτi is the cumulative
distribution function with the only restriction that the distribution function at 0
is τ (Fτi (0) = τ).
This proposed model allows for the description of the quantile function of
the green sea urchin weight Wi conditional on a skeleton diameter Di at a given
quantile τ as
QWi (τ|di ) = FW−1

i
(τ|di) = g(di ),
where FWi is the cumulative distribution function of Wi.

Amongst the different methodologies in QR, we will base our choice on
the results from the comparison study described in Martínez-Silva et al. (2013).
These can be further classified in two different perspectives. The first group
formed by Koenker’s approach and the new application of boosting in QR prob-
lems, proposes an independent estimation for each quantile curve. By doing so,
they model the data taking into consideration that they are working with the per-
centage of population indicated by the quantile under study. The second group
includes Cole’s Lambda Mean Standard deviation (LMS) technique and Gener-
alized Additive Models for Location, Scale and Shape (GAMLSS). The latter
methodology models the data and calculates the population distribution so that
specified quantiles can be inferred from it.
The Koenker and Basset technique applies linear programming approaches
that have evolved from the initial idea (Koenker and Bassett, 1978) to the intro-
duction of smoothing (Koenker et al., 1994). Requirements for the application
of this technique worth noting are as follows: i. the fitting is done individually
for each of the QR curves as previously stated; and ii. it is necessary to do a
preliminary study to find the appropriate smoothing of the curve and to prevent
the process from ending as either a linear fitting or a data interpolation.
The boosting algorithm for smoothed QR is based on machine learning pro-
cesses, specifically on the Adaboost algorithm (Freund and Schapire, 1997).
Improvements to this adaptive boosting algorithm have allowed for estimation
in smoothed regression (Hothorn et al., 2013) and subsequently estimation in
the smoothed QR framework (Fenske et al., 2011). Additive quantile regression
estimation is embedded in the widely studied class of boosting algorithms for
empirical risk minimization. These are an extension of the boosting algorithms
for additive models described in Kneib et al. (2009). The flexibility in estimat-
ing the nonlinear effects is considerably increased by using boosting algorithms
compared to the previously mentioned technique, since the specification of dif-
ferentiability of the nonlinear effects remains part of the model specification and
is not determined by the estimation method itself. They also allow more com-
plex models with a larger number of nonlinear effects. The variable and model
selection process is implicitly supported when using boosting for model esti-
mation. In particular, parameter estimation and variable selection are combined
into one single model estimation procedure (Fenske et al., 2011).
The initial versions of the two methodologies in this first group are faced
with the possibility of having two or more crossing QR curves, which makes
them less attractive than the ones in the second group. However, this limitation
has been solved (He, 1997; Howard et al., 2010), and current versions are as
equally appealing to researchers as the techniques which will be described next.
The idea behind the Lambda Mean Standard deviation (LMS) method (Cole,
1988) is to transform the response variable by means of a Box-Cox power trans-
formation (Box and Cox, 1964) in such a way that the resulting variable follows
a standard normal distribution. In order to fit the initial distribution of the re-
sponse variable, the following parameters are estimated: λ (parameter of the
Box-Cox transformation), the mean and the standard deviation. Cole and Green
(1992) introduced smoothing using an iterative smoothing spline to fit the LMS
method and years later, Yee and Wild (1996) proposed implementing the fitting
via vector generalized additive models (VGAM), which simultaneously esti-
mates the three parameters using a vector smoothing spline.
Finally, the generalized additive models for location, scale and shape
(GAMLSS) (Rigby and Stasinopoulos, 2005) arose as a response to the lim-
itations associated to both generalized linear models (GLM) and generalized
additive models (GAM). In this case, the fitting of the response variable distri-
bution is implemented from the estimation of the mean, standard deviation and
the parameters related to the kurtosis and the asymmetry of distributions.
4.2. R Implementation
The aforementioned methodologies can be applied to databases in order to nu-
merically and graphically implement the QR curves modelling with the free
software R (R Core Team, 2014). R packages corresponding to each of these
methodologies are quantreg (Koenker, 2013) which implements Koenker and
Basset technique; mboost (Hothorn et al., 2013; Buehlmann and Hothorn, 2007)
implements boosting algorithms for QR; VGAM package (Yee, 2013) performs
the LMS technique; finally, gamlss package (Rigby and Stasinopoulos, 2005)
applies GAMLSS methodology.
Given conclusions from previous work by the authors in Martínez-Silva
et al. (2013) on the choice of the most appropriate methodology to be applied
to sea urchins growth data, those commands relevant to boosting-based QR are
described next.
After the methodological assessment in Martínez-Silva et al. (2013), the
boosting-based technique has been selected to be applied in this chapter largely
because it avoids the previous study of the smoothing selection given that it is
automatically integrated within the boosting process itself.
Within the package mboost the gamboost function has been used in order to
implement the smooth fitting with bbs specifying the smoth effects. Additional
specification of the QuantReg family has been required to indicate that the QR
models have been applied.
4.3. Results
As observed in Martínez-Silva et al. (2013), all the methodologies but boosting
require the choice of the smoothing parameter by the researcher. This is one
of the most important steps in the analysis because it could lead to very vari-
able and not sufficiently accurate results. Consequently, we have chosen the
boosting-based methodology as the most appropriate for the fitting under study
in this chapter. It is often considered a drawback of this technique the fact that
its complex iterative nature causes higher computational times than those of the
other techniques. However, for data sets of the size of the ones considered in
this chapter, this only results in times of half a second, although we acknowl-
edge that this would increase with the size and shape of the point cloud.
With a view to achieve a complete study of the data distribution, the median
and several extreme quantiles have been selected; the former because it is the
central quantile and the latter so as to give additional information about the
distribution, as shown in Figure 5. The selected quantiles have also been found
consistently reported in the literature (Planque and Buffaz, 2008; Muggeo et al.,
2013).
Sea urchins growth has been modelled in two phases. In the first phase, a
model has been fitted to the entire sample. In the second phase, each of the two
depth locations has been analysed separately.
In Figure 4, QR curves have been plotted for the quantiles 0.10 , 0.50 (the
median curve) and 0.90 comprising data from both depths. It is notable that all
the curves show monotone increasing functions patterns. However, some dif-
ferences can also be detected: whereas the increase in W with respect to D is
similar for both the 0.90 quantile and the median, this is nor mirrored for the
0.10 quantile. This quantile shows a less sharp growth for sizes beyond approx-
imately 65 mm. This fact indicates a higher variability in the increase in weight
for diameters greater than 65 mm, having found that while the 50% of the pop-
ulation grows homogeneously, the remaining 50% presents great heterogeneity
caused by low values of W .
A more thorough analysis is shown in Figure 5, where separate quantiles
are plotted for SW and DW . The same pattern is observed where a more ho-
mogenous growth appears for higher quantiles above the median than that of
the quantiles below the median. The variability for lower quantiles is more ob-
vious in DW than in SW .
In Figure 6, clear differences between the two populations under study are

Figure 4. Quantile regression curves for weight (W ) and test diameter (D) us-
ing the boosting-based technique. Different quantiles τ ∼ 0.1, 0.5 (the median
curve) and 0.9 are plotted for the two depths in a whole.
revealed. W growth shows greater variability in DW than in SW and it is ap-

parent that the quantiles above the median corresponding to DW exceed those
of SW . However, the values for the lower quantiles (below the median) of SW
surpass those of DW .
The results in Table 1 show that for low quantiles (0.025, 0.10) the estima-
tions for W are always greater in SW than in DW , i.e., the QR curves of SW lie
above the QR curves of DW . On the other hand, for the median curve and higher
quantiles (0.90, 0.975), the behavior is not always the same. For instance, given
a certain quantile curve, e.g., the median curve, estimations for W in 50, 65, 70,
75 and 80 mm are higher in SW than in DW , whereas for values of D in 55 and
60 mm, estimations of W are higher in DW than in SW . This behavior of the
median curve clearly suggests the need for smoothing.
0 1 2 3 4 5 6 7 8
9 : ;
9 : <
9 : =
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&
- .
&
! ! " " #
( ) * * +
D
O P Q R S T U V W
X Y Z
X Y [
X Y \
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G H I I J
Figure 5. Quantile regression curves for weight (W ) and test diameter (D) using
the boosting-based technique. Upper panel: sea urchins from shallow waters
(SW = 4.6 m). Lower panel: sea urchins from deep waters (DW = 7.6 m).
c
n o p q r s t u v
w x y
w x z
w x {
b
m
k l
] ^ _ ^ ` ^ a ^
f g h h i
Figure 6. Quantile regression curves for weight (W ) and test diameter (D) using
the boosting-based technique. The black lines show sea urchins from shallow
waters (SW = 4.6 m) and the grey lines show sea urchins from deep waters
(DW = 7.6 m).
Table 1. W boosting-based estimations for different quantile curves (0.025,

0.10, 0.50, 0.90 and 0.0975) in DW and SW
D (mm)
quantile Depth 50 55 60 65 70 75 80
0.025 SW 55.02 62.67 74.87 90.54 99.75 102.76 104.55
DW 45.82 62.21 72.85 87.14 99.64 102.60 103.27
0.10 SW 59.83 67.47 79.78 98.10 111.57 119.17 125.67
DW 51.81 66.72 77.53 94.69 109.12 113.31 108.70
0.50 SW 67.60 74.97 91.28 116.49 142.44 158.46 165.10
DW 62.14 78.20 91.52 112.49 140.04 167.12 178.90
0.90 SW 67.65 86.09 106.31 133.46 159.50 175.32 185.99
DW 75.32 88.40 105.19 127.66 155.53 183.18 200.88
0.0975 SW 88.34 98.35 112.42 139.56 166.56 179.39 186.09
DW 89.16 98.03 112.37 135.07 164.13 191.83 211.16
5. Conclusion
The results demonstrated the suitability of the nonparametric Kernel-based and
quantile regression models to record specific behaviors in the growth patterns of
sea urchins, estimate a suitable catch-size and provide a more complete descrip-
tion of functional changes. Kernel nonparametric regression models detected
variations in length-weight curves that the allometric model was not capable of
discerning, although they seemed to be a better fit for sea urchin data from shal-
lower waters. By means of the first derivative of the nonparametric regression
model we were able to estimate a suitable minimum capture size (64.3 mm, CI
= [62.9, 66.0]) in shallow waters, which was more conservative than the cur-
rent one (52.4 mm) in Gulf of Maine. Due to the nature of the analysis, this
result may lead to the maximum yield in weight from the fishery. In addition,
the boosting-based quantile regression technique was successfully applied and
detected variability in sea urchin growth patterns throughout the length distribu-
tion and between depths. Differences between the two populations under study
were detected where growth in weight showed greater variability in deeper wa-
ters than in shallower waters. The quantiles above the median corresponding
to deep waters exceeded those of shallow waters while the values for the lower
quantiles of shallow waters surpassed those of deep waters. Differences in food
availability and wave exposure between depths may explain these results. We
need to be cautious with the interpretation of these results, particularly regard-
ing the minimum catch-size. A full interpretation of these results may need a
parallel study of density, physical conditions and food availability, including a
higher sampling coverage in both spatial and depth terms. Specific analysis of
the potential presence of edge effects in the modelling should also be considered
prior to reaching further conclusions.
Acknowledgments
We wish to thank the Maine Department of Marine Resources (Maine, USA)
for providing the data, and particularly to Margaret Hunter for sharing with us
her knowledge about the green sea urchin fishery in the Gulf of Maine, and
for her helpful comments. We would also like to thank Mark J. Brewer for
his valuable contributions. Martínez-Silva’s research was supported by grant
MTM2011-28285-C02-01 (FEDER support included) from the Spanish Min-
istry of Science and Innovation and by grant CN2012/273 from the Galician
Regional Authority (Xunta de Galicia). Sestelo’s research was supported by

grant MTM2011-23204 (FEDER support included) from the Spanish Ministry
of Science and Innovation and by grant CN2012/180 from the Galician Regional
Authority (Xunta de Galicia). Lorenzo-Arribas’s research was supported by the
Scottish Government’s Rural and Environment Science and Analytical Services
Division.
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Chapter 4
SEA URCHIN IMMUNE SYSTEM:

FROM BASIC CONCEPTS
TO ENVIRONMENTAL BIOMONITORING
Paola Cristina Branco1*,

Débora Alvares Leite Figueiredo¹,
Andrews Krupinski Emerenciano¹,
Douglas Amaral dos Santos1,
Marcelo González-Aravena2
and José Roberto Machado Cunha da Silva1
1
Department of Cell and Developmental Biology,
Institute of Biomedical Sciences, University of Sao Paulo, Brazil
2
Antarctic Bio-resources Laboratory, Chilean Antarctic Institute,
Punta Arenas, Chile
Abstract
Since the genome of sea urchin had been sequenced, the

phylogenetic proximity of echinoderms and chordates was reinforced,
based mainly on the report of a wide range of immune genes with high
degree of similarity to mammalian ones. Besides being a well-
*
Corresponding author. Address: Av Prof Lineu Prestes 1524, Cidade Universitária, São Paulo,
Brazil. Phone: 55-11-30917223. E-mail: [email protected].
86 P. C. Branco, D. A. Leite Figueiredo, A. K. Emerenciano et al.
documented research model, the sea urchin immune system became a

source of investigation of cell biology whose main objective is to
understand the, at same time, simple and highly complex and coordinated
immune response; simple because innate response in the only immune
response that sea urchins possess and complex due to the wide diversity
of innate immune receptors reported, which indeed outnumber the
receptors reported for C. elegans, D. melanogaster and even H. sapiens.
All these facts contributed for the ―genome era‖ of sea urchin.
In sea urchins, the innate immune response is orchestrated by the
immune cells, also referred to as coelomocytes. Composed of four
different cell types, coelomocytes has been studied since the 1960s, and
still today, many physiological roles remain obscure. For some cell types,
even their function is still controversial. The best studied cell type is the
phagocytic amoebocyte, the most abundant cell type in the coelomic fluid
and the only one that is capable of performing phagocytosis. Many
studies have been conducted to this cell type and molecular tools revealed
that phagocytic amoebocyte possess different subpopulation with distinct
diversity of cytoskeleton components besides accessory proteins.
Not only coelomocytes play an important role in immunity of sea
urchins, humoral factors are also important pivots of their immune
response. Recently, two antimicrobial peptides have been reported,
besides other molecule with bioactive properties. These discoveries not
only help to elucidate how this complex system acts, but, widen the
horizons of immune system of sea urchins as a potent pharmacological
source of research.
Lastly, the sea urchin immune system has been also used as a useful
tool for environmental biomonitoring. Different stressors and different
responses, including cellular components used today as biomarkers, were
reported in literature and altogether reached the same conclusion that sea
urchins are excellent environmental bioindicators.
The aim of this chapter is to discuss about the most relevant topics of
innate immune system of sea urchin, involving the genetic homologies
that prove the phylogenetic proximity to chordates, the cellular and
humoral components and the use of immune system as an important tool
for biomedical research and environmental biomonitoring.
Introduction
―The glorious sea urchin‖ was the sentence with which Jusny and Purnell
(2006), in a brilliant introduction, began the 2006 special issue of Science
magazine. Indeed, it is fabulous! The easily artificial spawning and
fertilization, associated with embryo transparency that allows morphogenetic
movements observation, make sea urchin an indubitable model for
Sea Urchin Immune System 87
developmental research (Hardin, 1995). Posterior studies involving molecular

embryology, fertilization biology and gene regulatory molecular biology
endorsed their application as a scientific model. Moreover, another area of
knowledge that uses sea urchin as a model is cell biology, in which their usage
was justified in studies comprising analysis of cytoskeleton components,
biomineralization, centrioles biology and function of extracellular matrix
(Morris et al, 2006).
After sea urchin genome had been annotated, their role as model for
immunological studies became even more evidenced. It is impressive how
immune genes outnumber those same genes for others species, including
vertebrate. Moreover, it became clearer how phylogenetically close to
vertebrates sea urchin immune system is (Sea Urchin Genome Consortium,
2006).
Sea urchins have also an ecological and environmental appeal. Pearse
(2006) demonstrated their ecological role, pointing that their abundance and
distribution can be affected by different factors, including the abundance of
algal food, predators, storm intensities and incidence of disease. Another
important issue lies in the fact that sea urchins‘ limited locomotion allows
them to reflect local environmental disturbances, which is why they are
considered bioindicators for environmental stress and are widespread used for
biomonitoring (Couteur et al, 2003; Soualili et al., 2008).
Innate Immune System: An Overview

The concept of immunity was first used referring to the exemption from
the laws to designate the Pope (Silva, 2013). It is also referred to exemption
from military service or paying taxes. Later on, the concept has been attributed
to the ability of an organism to defend itself from diseases (Cooper, 2001).
Despite being an embryologist, Elie Metchnikoff (1854 – 1916) was the
first to understand the importance of phagocytosis for the immune system. His
study was initially based on the observations of cells capable to migrate,
encapsulate and phagocytose foreign material; he was the first to propose an
immunologic theory. The work of Metchnikoff was the base for the
development of the field of cellular immunity, and in 1908 he won the Nobel
Prize (Tauber and Chernyake, 1988; Smith et al., 2006).
All multicellular organisms need to defend themselves against infection
and pathogens, and for that they have the immune system. The immune system
can be divided into innate immune system and adaptive immune system. The
last one is found only in vertebrates, displays a higher level of specificity and
can generate immunological memory, while the first one is common to all
organisms (Alberts, 2007), being considered the most important type of
immunity, once it is the first and most rapidly to act (Beutler, 2004; Turvey
and Broide, 2010).
Although the term immunology is, in most cases, associated with
mammalians, it is important to remember that mammals represent less than 1%
of all animals described, while invertebrates comprise over 95% of that
(Smith, 1991), and survive with only a non-adaptive immune system (Locker
et al., 2004). Thus, the invertebrate immune system has been extensively
studied and has provided information and models that have been used in other
areas of study in biology (Smith, 1991).
According to Mydlarz et al. (2006), the immune components / responses
are divided into three categories / steps: (1) recognition of self from non-self
particles, (2) building of a defensive response, to kill or neutralize the invader,
and (3) recognition and elimination of own damaged cells. For this, the innate
immune system acts through cellular and humoral components (a variety of
molecules with a wide spectrum of activities) (Beutler, 2004).
In sea urchins, as in all invertebrates, the main cellular response is done
via phagocytosis, a complex process which culminates in the degradation /
elimination of pathogens and acts through several steps. The humoral
components are molecules capable of sensing and destroying microbes even
before they are captured by cells (Smith, 1991; Beutler, 2004). These
molecules can be involved in different immune responses like encapsulation
(e.g., agglutinins), recognition of foreign molecules and agglutination (e.g.,
lectins) and specific binding to molecules such as LPS (e.g., hemolysins)
(Gross, 1999). These are just few examples of the importance and function of
these factors in the immune system.
Recent studies have shown that these molecules (like other immune
processes such as phagocytosis) may be useful in many other areas of science,
as in the environmental biomonitoring and in the development of drugs that
can be used in human medicine.
In this chapter, we will describe and discuss the sea urchin immune
system, including its multiplicity of uses in different areas of science.
Sea Urchin Identity

The concept of self and non-self is fundamental for the immune response.
The establishment of the identity of the organism is based on its ability to
recognize what belongs to the individual (self) and what is strange to it (non
self) (Tauber, 1994).
Self/non-self-recognition is achieved by the expression of specific cell
markers, which are recognized as self. Cells that do not express these markers
are recognized as non-self and destroyed by the immune system (Pradeu,
2012).
For sea urchin, the establishment of self happens during the mid-gastrula
stage of development, when the sea urchin embryo is capable of
phagocytosing microorganisms (Silva, 2000).
Homology to Vertebrates and

Phylogenetic Position
Phylogenetic proximity of echinoderms and chordates can be evidenced
once both of them are deuterostomate, which means that they share many
developmental features, including fate of the blastopore, the first embryonic
opening, which in deuterostomes originates the anus; initial cleavage plans and
origin of mesoderm (Field et al., 1988; Blair and Hadges, 2005).
Molecular phylogeny also confirmed their proximity. Studies involving
18S rRNA in the ‗90s confirmed the phylogenetic proximity of chordates and
echinoderms (Wada and Satoh, 1994). However, this study did not provide
strong evidence for interrelationship among deuterostome subgroups. Posterior
studies used the large subunit ribosomal RNA (LSU rRNA) and small subunit
(SSU) rRNA to better comprehend deuterostome phylogeny.
More recent analyses evaluated microRNAs, a diverse family of small,
non-coding regulatory genes that are considered reliable phylogenetic markers
and are recognized as key regulators of gene expression. They revealed that
some miRNAs are present both in sea urchins and in chordates which are the
deuterostome-specific miR-103/107/2013, the ambulacrarian-specific miRNAs
miR-2008, -2011 and -2012. Moreover seven echinoderm-specific miRNAs:
miR-2002, miR-2004, miR-2005, miR-2006, miR-2007, miR-2009 and miR-
2010 were reported (Pisani et al., 2012). The miRNAs have been identified in
Strongylocentrotus purpuratus (sea urchin), Patiria miniata (sea star) and
Apostichopus japonicus (sea cucumber) (Kadri et al., 2011; Chen et al., 2013).
Recently, the expression of miRNAs in immune cells of the Apostichopus
japonicus reveled differential expression of two conserved miRNAs (mir-31
and mir-2008) that should be involved in the disease named as skin ulceration
syndrome (Li et al., 2012).
It is not surprising that, due to their phylogenetic proximity, sea urchins
and vertebrates share many immune similarities. To begin with, there is a high
level of homology of their immune receptors and their ability to detect a
pathogenic invasion. Also, there is a huge repertoire of cytoskeletal
components that is similar to those found in vertebrates.
Immune Genes and Receptors

The main characteristic of innate immune system is to recognize not each
specific antigen, but few and highly conserved patterns present in
microorganisms so far studied. Innate immunity recognizes that these patterns
are non-self and initiate responses to control and completely remove
microorganism and thus control the homeostasis of the organism.
These conserved patterns are known as pathogen-associated molecular
patterns (PAMPs), and receptors responsible for their recognition are called
pattern-recognition receptors – PRRs (Medzhitov and Janeway,1997).
The main types of PRRs are Toll-like receptors (TLRs), NOD-like
receptors (NLRs) and Scavenger Receptor Cystein-Rich (SRCR). TLRs are
composed of an extracellular ligand domain and an intracellular domain, in
which accessory proteins may couple and initiate a signaling cascade (Takeda
and Akira, 2004); NLRs are responsible for recognizing intracellular PAMPs
(Rosenstiel et al., 2008) and SRCR that are highly conserved and are present
either soluble in the cytosol or anchored to the plasma membrane (Sarrias et
al., 2004).
Analyzing sea urchin genome, it is predicted that more than 1000 genes
show relevance to immunity (Hibino et al., 2006), which means that 4 to 5%
of the genes identified in the sea urchin genome are directly involved in
immune functions (Rast et al., 2006). Out of more than a thousand genes,
almost 650 are composed of genes related to TLR, NLR and SRCR (Hibino et
al., 2006).
Moreover, it is worth mentioning that gene expansion was observed after
sea urchin genome sequencing, and it surprisingly outnumbers the same genes
observed for other species, including humans (Rast et al., 2006). In a study
conducted by Hibino and co-workers (2006), the sea urchin genes involved in
immune receptors were compared to other 4 species: D. melanogaster, C.
elegans, C. intestinalis and H. sapiens. The complexity of innate immune
receptors of sea urchins is astonishing. For TLR, 222 genes were reported for
the sea urchin S. purpuratus in comparison to 10 for H. sapiens and 9 for D.
melanogaster. Considering NLR, S. purpuratus presents 203 genes in contrast
to approximately 20 found in H. sapiens and 0 for others species evaluated.
Sea urchin possesses 218 genes for SRCR compared to 16 for H. sapiens, 8 for
C. intestinalis and 7 for D. melanogaster. C. elegans presents 1, 0 and 1 gene
for TLR, NLR and SRCR, respectively.
Rast and co-workers suggest that such diversification of receptors is
analogous to that observed for the acquired immune system of vertebrates.
Authors still emphasize that this may be a more general characteristic of
animal immunity than has been previously supposed.
The study of Messier-Solek and co-workers (2010) analyzed the genome
of the sea urchin Strongylocentrotus purpuratus, and found the existence of a
wide range of immune receptors homologous to those found in humans and
rodents. They have also shown that these receptors are found in greater
number and greater complexity in sea urchin. The families of genes encoding
the TLR receptor types and NLR contain from 10 to 20 times more members
than at present in vertebrates and Drosophila. Moreover, proteins produced
from the transcription of these genes are also diverse and distinct from those
found in vertebrates.
The same research group also showed that the SRCR type receptors are
also present in greater quantity and variety in sea urchins when compared to
other invertebrate and vertebrate models. The genome of the sea urchin has
approximately 220 genes involved in transcription of this type of receptor
compared to only 16 genes in humans. In the sea urchin these receptors are
expressed in phagocytes and can be found both in transmembrane and
secretory forms (Pancer et al., 1999).
a. TLR
Toll-like receptors are transmembrane receptors that consist of a

transmembrane LRR (leucine rich repeat) domain and a cytoplasmic TIR
(Toll/IL1-receptor) domain. Toll receptors are characterized by having an
extracellular domain marked by repetitions of leucine (LRR: Leucine-Rich
Repeat), whose function is the recognition of PAMPs, and an intracellular
domain counterpart to the recipient of interleukin 1 (IRR) of mammals which

translates the signal that activates the transcription factors Rel / NF-kB,
(Pasare and Medzhitov, 2005). For instance, the LRR domain is responsible
for the recognition of the ligand, which can be protein (flagelin and porin),
sugar (zymozan), lipid (LPS, LTA) or nucleic acid (viral RNA). TIR domain
presents homology to cytoplasmic region of IL-1 receptor. This domain
interacts with accessory proteins, such as MyD88 (myeloid differentiation
response gene 88) and TRAM (TRIF-related adaptor molecule), promoting
activation of the MAPK (mitogen-activated protein kinase) signaling
pathways and transcription factors NF-kB (nuclear factor kB) and IRF
(IFN regulatory factors) that ultimately promotes inflammatory cytokine
production (Kumar et al., 2009).
In the fruit fly Drosophila melanogaster, Toll receptors induce the
activation of genes that trigger the synthesis of antimicrobials peptides and
antifungals (Hoffmann and Reichhart, 2002). The transcript of nuclear factor
kappa B (SpNF-kB) was the first protein-type described for sea urchins and
similarities of function were also observed in Drosophila and in vertebrates
(Pancer et al., 1999). In non-stimulated coelomocytes, the level of expression
of SpNF-kB is not detectable; however this transcription factor shows a high
expression after 6 and 12 hours of stimulation with bacteria or injury (Pancer
et al., 1999).
As previously mentioned, 222 genes for TLR have been reported for the
sea urchin S. purpuratus. Out of these, the majority was related to TLR 1 (109
genes) (Hibino et al., 2006). According to the same authors, this multiplicity of
genes can be explained through a great diversity in the LRR domain, which is
responsible for immune recognition, indicating that the specificity of immune
recognition is multiple.
Diverse TLRs are expressed by coelomocytes in sea urchin. According to
Rast and co-workers (2006), it is suggestive that such variety can be due to
isotype- and/or allele-restricted expression, cellular selection, and expansion.
This same research still points that these characteristics can be correlated to
what usually occurs in vertebrate receptors associated to adaptive immunity.
Not only TLR genes are expanded in the sea urchin genome, but also there
is an expansion of genes involved in adaptor proteins responsible for signaling
cascade after TLR activation (Hibino et al., 2006).
b. NLR
Nod-like receptors are intracellular receptors of immune cells. They are

characterized by an N-terminal domain, named CARD (caspase recruitment
domain), a pyrin domain (PYD) or a BIR domain (Baculovirus inhibitor
domain), a central NOD (nucleotide binding oligomerization domain) domain,
responsible for the auto oligomerization of the receptors after their activation
and a C-terminal LRR domain that recognizes PAMPs of intracellular
microorganisms. This recognition leads to the activation of the transcription
factor NF-kB and consequently the activation of caspase-1 and inflammatory
cytokine production, such as IL1B (Chen et al., 2009).
NLRs are responsible for detecting cytoplasmic pathogens (Inohara et al.,
2005). Interestingly, for sea urchins, the major site of expression of NLRs is
the gut (Hibino et al., 2006). For sea urchins, more than 200 genes have been
reported, in contrast to approximately 20 found in vertebrates (Rast et al.,
2006).
c. Scavenger Receptors
Coelomocytes also express a large and complex family of transcripts of

Scavenger Receptor Cysteine-rich Receptors (SRCR), which would be
associated with immune recognition (Hibino et al., 2006). These proteins
possess 90 to 110 amino acids and are characterized by their elevated cysteine
content. This family of receptors is also known to recognize PAMPs (Martínez
et al., 2011).
Scavenger receptors comprise a structurally heterogeneous group of
proteins often expressed on macrophages that functions to recognize
endogenous or microbial modified lipoproteins with polyanionic character
(Mukhopadhyay and Gordon, 2004). Dynamic expression of the sea urchin
SRCR genes by coelomocytes has already been reported (Pancer, 2000; Pancer
et al., 1999)
For sea urchins, more than 1000 domains and 218 genes have been
related. Great amount of them is expressed in coelomocytes after immune
challenge (Rast et al., 2006). In the non-stimulated sea urchin, the individual
level in the portfolio of transcripts for SRCR is highly variable and deeply
modified after stimulation with fungi and bacteria (Pancer, 2000). In non-
stimulated animals, changes in the expression of the SRCR were observed of
20 to 30 times over a period of three months. The animals challenged with
bacteria also showed variations in the expression of the transcript of the same
magnitude; however it is not possible to observe a specific pattern of
expression among different animals (Smith et al., 2006).
d. Sp185/333
Sp185/333 is a gene family, found in sea urchins, that was demonstrated

to be upregulated after bacterial challenge and after injection of LPS (Smith,
2012). According to Rast and co-workers (2006), their transcripts constitute up
to 6.5% in activated coelomocytes, reinforcing their importance in the immune
response.
Moreover, it was demonstrated that their expression varies depending on
the cell type. Sp185/333 is more expressed in the subpopulation of phagocytic
amoebocytes, small phagocytes and polygonal phagocytes (Brockton et al.,
2008). However, there is a high variability rate of these subpopulations that
express the Sp185/333. According to the same authors, their expression varies
from 3 to 14% for small phagocytes and from 2 to 18% for polygonal
phagocytes.
Frequently Sp185/333 is associated to perinuclear vesicles and in cell
surface, probably associated to integrins and RGD motifs (Whittaker et al.,
2006). In the cell surface, they are often localized to filopodia (Ghosh et al.,
2010).
The hypothesis has been that this group of transcripts codes for a family of
proteins that participates in the immune response and could represent a major
component of the immune system in S. purpuratus (Nair et al., 2005);
however, its precise function is still unknown. Apparently sea urchins are
capable of differentiating between different types of PAMPs by inducing
transcription of different groups of 185/333 genes (Terwilliger et al., 2007).
Regarding the protein sequence deduction for 185/333, no similarities to that
of other proteins have been found. These regions have three features: a
glycine-rich region, another region rich in histidine and a terminal region
(Smith et al., 2006).
Although their exact function remains unknown, there is evidence that
they can be associated to encapsulation response (Ghosh et al., 2010).
e. Other Genes
According to Rast and co-workers (2006), a large gene family that is

involved with immune response of sea urchins has been identified in their
genome. It includes C-type lectin and galectin genes. Lectins are involved in a
variety of functions including opsonization. In the sea urchin genome, Hibino
and co-workers (2006) reported more than 100 genes responsible for encoding
C-type lectins.
Additionally, Hibino and co-workers (2006) pointed out the presence of
RIG-I-like genes which are associated to antiviral response; peroxidase and
Nitric Oxide Synthase (NOS) genes and peptidoglycan recognition proteins
(PGRPs)
Two putative sequences were identified as Gram-negative binding
proteins (GNBPs) which are related to pathogens recognition (Rast et al.,
2006). However, the genetic annotation for these sequences was misassigned,
because these sequences shown more identity to LPS-binding proteins (LBP)
or Bactericidal/permeability-increasing protein (BPI). BPI is another LBP, a
55-kDa cationic protein specifically active against Gram-negative bacteria. It
increases the permeability of bacterial membranes. LBP and BPI are
structurally related, with 45% sequence identity. They have a coordinated
function in the response to invading bacteria. The antibacterial BPI displays
LPS neutralizing properties and suppresses LPS inflammatory activity,
whereas LBP is an acute-phase reactant that displays a concentration-
dependent modulation of LPS activity (Bachere et al., 2004). Interestingly, a
gene homologous to the LPS-binding protein, named bactericidal/
permeability-increasing protein (BPI) gene present in vertebrates and
invertebrates was found in the Antarctic sea urchin (S. neumayeri) using a
PCR approach. A partial cDNA sequence has been obtained (M. Gonzalez,
manuscript in preparation; accession number JQ 736681). After LPS and
bacterial challenge, the expression of this gene increased in coelomocytes and
several tissues (González-Aravena, personal communication). Further studies
should be done to determinate the functional characterization of this novel
protein in sea urchin and the determination of whether it acts as an LBP or as a
BPI will bring interesting insights into the anti-microbial defense in sea urchin.
Immune Challenge
Sea urchins immunity is activated by multiple stimuli, as it was already
reported in literature immune response to India ink (Mangiaterra and Silva,
2002; Tucunduva and Silva, 2008), which demonstrated an increase in
phagocytic response after challenge. However the most studied activation is
regarding LPS activation.
When sea urchins face an LPS infection, different responses occur, among
them the expression of SpEchinoidin is detected (Terwilliger et al., 2004), an
increased expression of SpC3 is also evidenced in S. purpuratus (Smith et al.,
1996). By contrast, it was detected a downregulation of the transcription factor
SpGATA after immune challenge (Pancer et al., 1999)
Besides, it was reported that there was an upregulation of a Kazal-type
protease inhibitor that may function to inactivate bacterial proteases. Other
ESTs involved in LPS-activated coelomocytes include cell surface proteins
and receptors, proteins involved in signaling cascades, lysosomal and secreted
proteins and cytoskeletal and cytoskeleton-modifying proteins (Smith et al.,
1996). A posterior study that also evaluated ESTs involved in LPS-activated
coelomocytes reported that genes involved in RNA splicing, protein
processing and targeting, secretion, endosomal activities, cell signaling, and
alterations to the cytoskeletal architecture including interactions with the
extracellular matrix were upregulated (Nair et al., 2005).
Another molecule that is upregulated after immune challenge is profilin, a
protein involved in cytoskeleton remodeling and signal transduction (Smith et
al., 1995).
Also, Nair and co-workers (2005) demonstrated an upregulation of
Sp185/333 after LPS stimuli, which represented 60% of the ESTs analyzed in
this study, reinforcing their role in the immune response of sea urchins.
Sea Urchin Innate Immune System:

A Link to Acquired Immune System
Adaptive immune system is characterized by high immune specificity,
specific recognition of proteins, carbohydrates, lipids, nucleic acids, and
pathogens, using the same activated, but not antigen-specific, effector cells
generated by innate immune recognition. Although invertebrates are deprived
of adaptive immunity, they possess alternative mechanisms for enabling
specificity, including high genetic diversity of receptors or effectors and

synergistic interactions among immune components (Schulenburg et al.,
2007). According to Rast and co-workers (2006), such mechanisms can be
correlated to what usually occurs in vertebrate receptors associated to adaptive
immunity.
The origin of adaptive immunity in vertebrates refers to the appearance of
the RAG genes (Smith et al., 2002). The proteins encoded by them are
responsible for the activation of immunoglobulin V-D-J recombination, which
is the specialized DNA rearrangement used by cells of the immune system to
assemble highly specific and unique immunoglobulin and T-cell receptors
(Gellert, 2002).
Unexpectedly, a homologous Rag1/2-like gene was identified in the sea
urchin genome (Fuggman et al., 2006; Rast et al., 2006). However, their
function in sea urchins remains unknown, but it consolidates the phylogenetic
proximity, reinforcing that sea urchin and vertebrates share a common
ancestor.
Cytoskeleton Homology
Cytoskeleton, a network composed of three protein filaments and
accessory proteins, plays an important role in many cellular processes such as
vesicle/organelle transport, cell-cell interaction, cell-extracellular matrix
interactions through association with surface receptors, cell cycle, cell
motility, and phagocytosis (Ridley, 2001).
After sea urchin genome sequencing, a large repertoire of cytoskeleton
components displaying homology to vertebrate cytoskeleton has been
reported, including genes encoding actin, specific actin-binding proteins,
myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins,
and intermediate filament proteins. Taking into consideration the cytoskeleton
components involved in immune response, it is possible to infer that proteins
involved in phagocytosis in vertebrates may play an important role in immune
response in echinoids. That is the case of actin and its accessory proteins (e.g.,
Arp complex, cofilin, paxilin, among others) and genes related to cell
signaling that are involved in phagocytic response including Cdc42, RhoA,
ROCK 1, PAK, among others. The same was observed for tubulin and its
accessory proteins (Morris et al., 2006).
Sea Urchin Immunity

Metchnikoff initially used echinoderms as a model of study, and the first
observations were made on the starfish larvae. Echinoderms are used as a
model in several areas including immunology, development and cell biology.
In the immunological field, sea urchins are among the most studied organisms
(Ramirez-Gómez and García-Arrarás, 2010).
Sea urchins belong to the phylum Echinodermata, class echinoidea; they
are coelomate deuterostomes and this phylogenic position makes them
important animals to understand and investigate the phylogeny of immunity
and to explain the evolution of the immune system (Yui and Bayne, 1983). On
the other hand, the immunology of these animals has become of interest in
aquaculture, mainly in regions where their culture are important for economic
activities, as in Asia, where they are largely used in cooking (Ramirez-Gómez
and García-Arrarás, 2010; Lawrence, 2006).
When it comes to immunity, we immediately think of adult subjects. We
must remember, however, that the embryos and planktonic larval stages of
these organisms are subject to infections in the same way as the adults. In
embryos, for example, Silva (2000) observed phagocytosis of yeast occurring
through mesenchymal cells in Lytechinus variegatus, demonstrating that at this
stage of development the embryos can already distinguish self from non-self-
particles.
In adult sea urchin, as in most organisms, immune response includes
cellular and humoral components, with a large amount of molecules
homologous and analogous to other invertebrate and vertebrate species.
(Beutler, 2004; Ramirez-Gómez and García-Arrarás, 2010). They have also
mechanisms of wound-healing and clotting.
Cellular Components
The body wall is an important mechanic barrier against parasite infection
(Ratcliffe et al., 1985), but when pathogens can penetrate this barrier, they find
large quantities of molecules and cells to limit infection (Matranga, 2005). In
sea urchins, coelomocytes are the major cellular components responsible for
immune response. These cells are found in the coelomic fluid, which fills the
spaces between organs and the coelomic cavity (Gross et al., 1999; Smith et
al., 2006, Ramirez-Gómez and García-Arrarás, 2010).
For sea urchins, literature describes four basic types of coelomocytes:

phagocytic amoebocyte (phagocytes), sphere cells (red and colorless) and
vibratile cells. (Johnson, 1969; Gross et al., 1999; Borges et al., 2005;
Matranga, 2005; Smith et al., 2006, Ramirez-Gómez and García-Arrarás,
2010) each one with a related function (Figure 1).
Figure 1. Coelomocytes from sea urchin tropical L. variegatus. A. Phagocytic

Amoebocyte spread onto glass slide and others cell types (Scale Bar 50 µm). B.
Vibratile Cell (Scale Bar 10 µm). C. Red spherule cell (Scale Bar 10 µm). D. Colorless
spherule cell (Scale Bar 10 µm). E. Petaloid phagocytic amoebocyte (Scale Bar 10
µm). F. Filopodial phagocytic amoebocyte (Scale Bar 10 µm). Photos were obtained in
a phase contrast microscopy. Photo: Emerenciano, A. K.
I. Phagocytic Amoebocyte
Phagocytic amoebocyte (PA) is the most abundant cell type found in the
coelomic fluid of sea urchins (Johnson 1969; Chia and Xing, 1996; Matranga
2005; Smith et al., 2006; Ramirez-Gómez and García-Arrarás, 2010). The cell
size varies between 10-40 µm (Chia and Xing, 1996), and the main functions
performed by these cells are phagocytosis, encapsulation, graft rejection,
chemotaxis, immune gene expression, production of reactive oxygen species,
agglutination, cytotoxicity (Ito et al., 1992; Smith et al., 2006; Ramirez-
Gómez and García-Arrarás, 2010).
Observations under light microscopy demonstrated a very characteristic
morphology, which is very similar to a macrophage. Relatively big, this cell
type presents cytoplasmic projections that can assume different
rearrangements (Matranga, 2005). Transmisson Electron Microscopy revealed
a cell with a large and central nucleus with loose chromatin and a well-
developed endoplasmic reticulum, Golgi apparatus and abundant vesicles,
which is characteristic of a phagocytic cell (Branco et al., 2014; Chia and
Xing, 1996).
Another feature that enables their recognition under microscopic
observations is the presence, in their nucleus, of an iron inclusion named
intranuclear crystalloid (Endean, 1966; Hobaus, 1978) whose function remains
unclear, however it seems to be related to iron transportation (Karasaki, 1965),
heavy metals clearance from the organism and urea synthesis (Bachmann and
Goldschimid, 1978).
Literature describes subpopulations of these cells, categorized according
to their morphology and size. Chia and Xing (1996) and Matranga (2005)
classified these cells in two different types: the petaloid and the filopodial
form. The petaloid form cells display projections into petal shapes, while the
filopodial form presents elongated branched pseudopodia. The transition from
petaloid to filopodial amoebocyte has already been detected (Edds, 1992,
Matranga, 2005). However, the opposite transition has not been documented
(Edds, 1992; Chia e Xing, 1996). Such differences may be attributed to the
protein fascin, responsible for organizing actin cytoskeleton and might shed
some light on these distinct morphologies (Otto et al., 1979).
On the other hand, Smith and co-workers (2006) classified the phagocytic
amoebocyte into three different sub-types: (1) discoidal cell, (2) polygonal cell
and (3) small phagocyte. According to this classification, discoidal cells
presents a disc-shaped cytoplasm with actin striations radiating from the
center, while the polygonal cell has a clear nucleus with actin bundles running
parallel to straight edges of the cell. The last type, the small phagocyte‘s size is
smaller than the previous two, with little cytoplasm, but its function is still
unknown. Additionally, these sub-populations of phagocytes present different
expression patterns of SpC3 and Sp185/333 (Gross et al., 2000; Brockton et
al., 2008).
Sub-populations of phagocytic amoebocytes were also reported by Borges
and co-workers (2002). This study demonstrated different cell populations in
oral and aboral regions which present different phagocytic capacities.
II. Spherule Cells
Also called sphere cells, morula cells, or spherulocytes, these cells size
ranges from 8 to 20 µm, they have large quantities of vesicles (granules) in
their cytoplasm and move via amoeboid movements (Johnson, 1969; Chia and
Xing, 1996; Matranga, 2005). The spherule cells can be divided into two
different types: the red spherule cells (RSC) and the colorless spherule cell
(CSC).
The RSCs contain in their cytoplasm Echinochrome A, a red pigment
responsible for the color of these cells. This molecule is related with
bactericidal activity (as we shall see), besides the antimicrobial properties;
these cells are accumulated in places of injury and infection or in infiltrates
with amoebocytes, also being related to clotting and wound healing (Gross et
al., 1999; Smith et al., 2006).
The CSC is very similar to the RSC, but without the presence of
Echinochrome A in their granules. The function of these cells remains unclear.
As well as the subtypes of phagocytic amoebocytes, Matranga and co-workers
(2000) proposed that the differences between the two types of sphere cells is
due to different states of maturation.
II.I. Echinochrome
Polyhydroxylated compounds of different coloration can be found in sea
urchins, like the echinochrome A (2,3,5,7,8 - pentahydroxy - 6 -ethyl - 1,4
naphthoquinone), whose activity was related with the elimination of peroxide
radicals in liposomes, trapping of superoxide anion radicals, and binding of
ferrous ions to inactive complexes in the aqueous phase, besides bactericidal
activity (Service and Wardlaw, 1984; Lebedev et al., 2001). As consequence
of their properties, a pharmacological interest has aroused, although their roles
are not fully understood. The echinochrome can be found on the perivisceral
coelomic fluid – specifically into red sphere cells, responsible for their
biosynthesis, and dispersed in the coelomic fluid - as well as in shells, spines
and other organs (Anderson et al., 1969; Kuwahara et al., 2010).
The echinochrome was initially described by McMunn (1885), and
regarded an oxygen transporter. In 1912, McClendon questioned about the
respiratory function of the pigment because their solutions did not contain
significant amounts of iron and, when subjected to vacuum, it was not capable
of absorbing any appreciable amount of atmospheric oxygen. In addition, the
oxygen affinity of the reduced pigment is so elevated that it was necessary to
be careful, otherwise solutions could be re-oxidized in the presence of air.
Pure samples of echinochrome were only isolated in 1934 (Ball 1934), and
its molecular structure was identified in 1940 by Kuhn and Wallenfells.
Among all the known pigments of echinoderms, the echinochrome is the one
that possesses the highest antioxidant activity (Gerasimenko et al., 2006). In
1939, Hartmann and co-workers reported that the echinochrome stimulates the
activation and agglutination of sperm in Arbacia pustulosa, but Tyler (1939)
studied the effects of purified echinochrome in the species S. purpuratus, and
did not find the same result.
In the species Echinus esculentus, the echinochrome is present inside
coelomocytes in a concentration between 3 and 60μg/ml, and it is active
against a number of gram-positive and gram-negative bacteria in a
concentration of 50μg/ml (Service and Wardlaw, 1984). Probably, the
bactericidal effects of the echinochrome are related to iron chelation.
Moreover, the pigment can be involved in the immunity associated to
reproduction, since it can be found in the eggs and larvae of sea urchins after
spawning and fertilization (Smith et al., 2010).
Sea urchins are not the only echinoderms that contain the echinochrome.
A study conducted by Smith and Smith (1985) demonstrated that the stress
response in sand dollars Mellita quinquiesperforata induces the release of
echinochrome by red sphere cells, similar to the release of allergy mediators
by basophils and mast cells from mammals. Such similarities may suggest a
possible correlation with the allergic response of mammals. Although a similar
study was not conducted with echinoids, and for them echinochrome function
remains unknown, this speculation could not be discarded.
III. Vibratile Cells
Vibratile cells (VC) are spherical cells with size ranging around 5-50 µm
once they have a single long flagellum, so they are highly motile. Its function
is not well understood but they are thought to be involved in the circulation of
the coelomic fluid and with clotting rejection (Chia and Xing, 1996; Matranga,
2005; Smith, 2006).
Coelomocytes Proportion in Different Species

The cell types described above are present in all sea urchins, but their
concentration can vary between species and even between individuals of the
same species. This variability can be related for example to nutritional state,
immunological state or individual size (Matranga, 2005; Smith et al., 2006;
Ramirez-Gómez and García-Arrarás, 2010).
PA is the most abundant cell type present in the coelomic fluid, followed
by VC; RSC and CSC represent the smaller portion of cells (Bertheussen and
Seljelid apud Silva, 2013; Mangiaterra and Silva 2001) as summarized in
Table 1.
Table 1. Coelomocytes proportion in sea urchins
Cell Type Proportion

Phagocytic amoebocyte 50 – 70%
Vibratille cell 15 - 20%
Red spherule cell 5 – 10%
Colorless spherule cell 5 – 10%
Coelomocytes Origin
The origin of coelomocytes is not fully understood. In the past, it was
proposed that coelomocytes were originated from the epithelium of the
peritoneum (Liebman, 1950)
Nowadays, they are believed to come from the axial organ, a complex
circulatory system which has been associated with an ancestral primary
lymphoid gland (Matranga 2005, Mydlarz 2006). In the sea star
(Asterias rubens), the main proliferative tissues are the coelomic epithelium
and axial organ (Holm et al., 2008).
Moreover, it is also unclear whether all types of coelomocytes are
originated from the same precursor or each cell type arises from different stem
cells (Chia and Xing, 1996).
Humoral Factors
IL-likes
Genes involved in immune signaling such as interleukin-like and

cytokines were almost not identified in the sea urchin genome. It was
previously reported that 17 genes associated to the interleukin family, besides
the receptors IL-1R and IL-17R had been identified in the sea urchin genome
(Rast et al., 2006). Moreover, three genes seem to be related to the receptor for
IL-1, IL1R (Hibino et al., 2006). Nowadays, two genes have been identified as
interferon (IFN)-γ-inducible Ca2+-binding cytokine in echinoderms. These
molecules are identified as Allograft inflammatory factor-1 (AIF-1) in sea
cucumber Apostichopus japonicas and the Antarctic sea urchin (Sterechinus
neumayeri). After bacterial challenge and physical injury, AIF-1 transcripts
were significantly upregulated in coelomocytes (Ovando et al., 2012; Ji et al.,
2014).
Interleukin is a family of cytokines which plays a central role in the
regulation of innate immune response besides inflammatory response. IL-1,
the most studied among interleukins, is involved in the induction of a wide
range of proinflammatory cytokines (Dinarello, 2011).
TNF
Tumor necrosis factor (TNF) is a family of cytokines involved in

physiological processes, systemic inflammation, tumor lysis, apoptosis and
initiation of the acute phase reaction. The mechanisms by which TNF
promotes those processes seems to be related to the activation of NF-kB and
mitogen-activated protein kinase (MAPK) pathways, which in turn modulates
the expression of proinflammatory cytokines (Chu, 2013). TNF is also present
in the sea urchin genome (Rast et al., 2006).
Antimicrobial Peptides
Antimicrobial peptides (AMP) are the effectors of the innate immune

system more widely distributed in the animal kingdom. These peptides are
present from insect to mammals. AMPs are the first barrier of a multisystem
defense of the organism against pathogens and they are key molecules related
to the immune system activation. AMPs show a big diversity of sequences and
structures, but certain features are common. Most antimicrobial peptides are
cationic peptides and are classified according to their protein structure. They
are classified into three major groups: (1) peptides with an a-helical
conformation lsuch as insect cecropins or magainins, (2) cyclic and open-
ended cyclic peptides with pairs of cysteine residues such as defensins and (3)
peptides with an over-representation of some amino acids such as proline rich
or histidine rich. The cysteine-rich peptides are some of the best characterized
groups of AMPs, including α- and β-defensins from mammals, insects
defensins, crustaceans defensins and tachystatin A (Bulet et al., 2004).
The innate immunity is the first line of defense against pathogens and
allows their recognition and the production of a series of signaling molecules
that trigger the synthesis of effector molecules, but also the activation of
immune cells. Coelomocytes of the sea urchin, which are circulating within
the coelomic cavity, are considered to be key cells for most of the defense
reactions and could produce a large repertoire of molecules including AMPs.
For instance, in the green sea urchin (Strongylocentrotus droebachiensis),
it has been characterized two novel genes that encoded antimicrobial peptides
named strogylocins and centrocins (Li et al.,, 2008, 2010). These molecules
are the first AMPs purified from echinoderms and show potent activities
against Gram-positive and Gram-negative bacteria. The strongylocins and
centrocins were obtained primarily from coelomocytes isolated from the
coelomic cavity in non-stimulated sea urchin. Both AMPs, strongylocins and
centrocines, show two types of peptides (1 and 2) having a mass of 5.6 and 5.8
kDa and 4.5 and 4.4 kDa, respectively. For the purified and identified peptides
from green sea urchin, biochemical approaches were used such as solid phase
extraction and HPLC. Finally, active fractions obtained from HPLC were
analyzed to determine the molecular mass using electrospray ionization mass
spectrometry (ESI-MS) and additionally native peptides were sequenced by
Edman degradation. Then, based on the partial peptide sequences, PCR was
carried out using degenerated primers (Li et al.,, 2008). From the strongylocins
sequences obtained from BLAST analysis revealed that two putative proteins
of the purple sea urchin, S. purpuratus, are similar to the strongylocins of S.
droebachiensis. The alignment of strongylocin 1 and 2 from S. droebachiensis,

with other AMPs from insect, mollusk, horseshoe crab, mammals and plant
show a special characteristic by their unique cysteine array. On the other hand,
centrocins have an intramolecular heterodimeric structure, containing a heavy
chain and a light chain. The genome of the purple sea urchin (S. purpuratus),
was shown to contain two putative proteins with high similarity to the
centrocins (Li et al.,, 2010). AMPs of S. droebachiensis are expressed mainly
in phagocytes. Beside, transcripts of strongylocin 1 were also detected in
vibratile cells and/or colorless spherule cells, while transcripts of strongylocin
2 were found in red spherule cells. The strongylocins and centrocins were also
expressed in the larvae; these findings confirm the fundamental role of AMPs
(Li et al., 2014).
Perforin
The way perforin acts is crucial for pathogen destruction. It forms pores in
the membrane of the pathogen which in turn make their elimination easier,
contributing to the host defense (Rosado et al., 2007). Biochemically, perforin
genes are characterized by the presence of a MACPG domain which was also
identified in the sea urchin genome (Hibino et al., 2006).
Complement System
In vertebrates, complement system is responsible for particle opsonization,

augmenting phagocytosis, acting as chemoattractants for other immune cells
and helping killing pathogens (Abbas and Litchmann, 2011).
In summary, complement cascade is composed by three pathways:
classical, alternative and lectin. All of them converge to a central component
of the complement system, C3, which acts as opsonin, binding covalently to
the pathogen and thereby leading to their destruction by phagocytes (Janeway
et al., 2005).
Since the early 1980‘s, Bertheussen and Seijelid (1982) demonstrated that
phagocytosis is enhanced after particle opsonization with mammalian C3,
suggesting that coelomocytes had cell receptors for C3 proteins (Clow et al.,
2004). Posterior studies demonstrated that sea urchin possess complement
components homolog to those found in vertebrates: a C3 homologue (SpC3;
Al-Sharif et al., 1998) and a B factor (Bf) homologue (SpBf; Smith et al.,
1998).
The most studied component corresponds to the SpC3 (Al-Sharif et al.,
1998), which has been identified as an important component in the
opsonization process, expressed exclusively in coelomocytes. Also, SpC3 acts
as a humoral inducible opsonin that augments the phagocytosis of target cells
(Clow et al. 2004). Moreover, Gross and co-workers (2000) demonstrated that
SpC3 displays a heterogeneous expression in the subpopulations of phagocytic
amoebocytes. Polygonal cells presented higher expression compared to the
other sub-types.
SpBf is a mosaic protein, composed of five short consensus repeats, a von
Willebrand Factor domain, and a serine protease domain. It is specifically
expressed in coelomocytes and, along with C3, is part of a simple complement
system that is homologous to the alternative pathway in higher vertebrates
(Smith et al., 1998).
Other Humoral Factors
Agglutinins (Ryoyama, 1974), hemolysins (Canicatti, 1991) and lectins

(Giga et al., 1987) were also described for sea urchins. Hemolysins present the
capacity to specifically bind to non-self-particles such as erythrocytes,
zymosan particles, lipopolysaccharide, contributing to their damage and
destruction (Gross et al., 1999). Agglutinins, as their name suggest, promote
clotting, besides mediating wound repair and encapsulation through their cell-
cell interaction in sea urchin (Canicatti et al., 1992).
Another humoral member that is associated to cell adhesion is the lectin.
In sea urchin, it was described a C-type lectin named echinoidin. It is also
related to agglutination (Smith et al., 1996).
Other molecules that are important in the immune defense of sea urchin
are lectins. These molecules are characterized by having a large spectrum of
recognition; possess opsonization properties and agglutination. In sea urchin, it
has been identified 104 genes encoding models for a variety of small C-type
lectins, consisting of one or two domains with a variety of binding
oligosaccharides sites (Hibino et al., 2006). The SpEchinoidin is an example of
type C lectin that is expressed exclusively in phagocytic cells activated by LPS
(Multerer and Smith, 2004, Terwilliger et al., 2004).
Figure 2. Phagocytosis in sea urchin cells. Images demonstrate phagocytic amoebocyte

from the tropical sea urchin L. variegatus phagocytosing yeast cells. Photos were
obtained in a phase contrast microscopy. Scale Bar 20 µm. Photo: Emerenciano, A. K.
The phenoloxidase system (PO) is also important in invertebrate

immunity. In sea urchins this system was identified both in coelomocytes and
in coelomic fluid (Chia and Xing, 1996).
Phagocytosis
Metchnikkoff described phagocytosis as the capacity of cells to actively
engulf foreign material. Nowadays, with the development of molecular and
cellular techniques, the role of phagocytosis has also been related to other
extremely important functions in the body, such as tissue remodeling and
homeostasis (Flannagan et al., 2012).
Phagocytosis is an important cellular immune feature, crucial for the
survival of all vertebrates and invertebrates; through this process the organism
can neutralize and eliminate microbial and other non-self-particles. In
invertebrates, it is extremely relevant once they do not have the large
quantities of molecules and cells working together, as mammals do (Smith
1991). This process is defined as the engulfment of particles ≥ 0.5µm and is
responsible for the first line of defense in the organism (Botelho and Grinstein,
2011; Flannagan et al., 2012). In sea urchins, as in other echinoderm groups,

the only cell capable to phagocyte is the phagocytic amoebocyte (Figure 2)
(Johnson, 1969; Chia and Xing, 1996; Mangiaterra and Silva, 2001).
Didactically, phagocytosis can be divided into 5 general steps: (1)
chemotaxis to the foreign particle, (2) adhesion, (3) opsonization, (4)ingestion
(when PA extend and fuse pseudopodia around the opsonized material
followed by the formation of phagosome) and (5) digestion (phagocyted
material is digested through phagosome formation and association with
lysosome enzymes and acidic pH) (Matranga et al., 2005) (Figure 3).
1. Chemotaxis
Chemotaxis can be defined as directed/oriented cell migration towards a

specific (chemical) stimulus (Borges et al., 2005); this process and the
adhesion to the foreign particle are essential for the cellular immune response
and act prior to the cell activation (Ratcliffe, 1985). It acts through
chemotactic elements like LPS, complements and damaged cells.
Figure 3. Schematic drawing of phagocytic process. The scheme shows the steps of
phagocytosis. First there is a directional migration after chemotatic stimulus, then the
recognition and adhesion to the particle and its engulfing, forming a structure named
phagosome in the phagocyte cytoplasm. At this moment, the digestion of the particle is
initiated. After digestion, debris are released via exocytosis. Scheme: Dzik, L. M.
1a. Cell Migration

Cell migration is a very well-coordinated biological process composed of
multiple steps, which are: 1- lamellipodia extension, 2- adhesions sites
formation, 3- cell body retraction and tail detachment (Ridley et al., 2003).
This process is not only important for the immune response, but also for
embryonic development and wound repair (Ridley et al., 2003). Deregulated
cell migration can lead to a wide range of pathologies (Gardel, 2010) including
metastasis, vascular diseases and also severe inflammatory conditions
(Yamaguchi et al., 2005).
Cell migration steps are dependent of actin and tubulin cytoskeleton
(Horwitz and Webb, 2003). These events are coordinated by several signaling
molecules, including Rho GTPases (Ridley, 2011).
In addition to being involved in cell migration, Rho GTPases participate in
various other activities which are dependent on actin cytoskeleton, including
phagocytosis (Cox et al., 1997; Caron and Hall, 1998).
Rho GTPases are part of the Ras superfamily - related small GTPases
(Jaffe and Hall, 2005) which are subdivided into 5 smaller families: Ras, Rho,
Rab, Arf and Ran (Etienne - Manneville and Hall, 2002). There is evidence of
the phylogenetic proximity of these four families (Ras, Rho, Rab and Arf)
between sea urchins and chordates. More than 90% of these GTPases are
expressed during sea urchin embryogenesis (Sea Urchin Genome Consortium,
2006).
In general, GTPases act in signal transduction pathways cycling from an
inactive state - bound to GDP - to an active state - bound to GTP. When bound
to GTP, they interact with downstream effectors developing diverse cellular
responses (Ridley and Hall, 1992). This cycle of activation and inactivation of
GTPases is regulated by three molecules: GEF - guanine nucleotide exchange
factor, which promotes the exchange of GDP for GTP to activate the target
molecules; GAP - GTPase activating protein to inactivate the molecule
responsible for promoting the hydrolysis of GTP; and GDI - guanine
nucleotide dissociation inhibitors, which block the GTPase cycle by
sequestering and solubilizing the GDP form and thereby inhibiting the
exchange of GDP to GTP (Moon and Zheng, 2003; Schmidt and Hall, 2002).
GDIs are responsible for the sequestration of the protein in its inactive state,
and modulate the cycling of GTPases from the membrane to the cytoplasm,
thereby contributing to the maintenance of GTPases in the cytoplasm of cells
(inactive form) (DerMardirossian and Bokoch, 2005)
There are three main members of this family which actively participate in
cellular migration: Rho, Rac and Cdc42. They exert their function through
interaction with their effectors, which include kinases, phospholipases, adapter

proteins and actin nucleating proteins (Heasman et al., 2008; Ridley, 2012).
The GTPase activity of Rac and Cdc42 largely regulates the assembly of
protrusive organelles; it also promotes the formation of small adhesions near
the cell periphery (Nobes and Hall, 1995, Nobes et al., 1995). Likewise, Rho
activity promotes the assembly of contractile actomyosin structures (Nobes
and Hall, 1995, Ridley and Hall, 1992).
In the sea urchin Strongylocentrotus purpuratus, RhoA is expressed in
oocytes (Covian-Nares et al., 2004), eggs (Cuellar-Mata et al., 2000) and in
early stages of development of the embryo (Nishimura et al. 1998; Beane et
al., 2006). However, many answers remain unknown. It is not clear how Rac
and Cdc42 GTPases participate in the embryonic development. It is also
unclear the role of these GTPases in coelomocytes mediating, for instance, cell
migration and phagocytosis.
2. Adhesion and Opsonization
Cell adhesion is extremely important in immune response, for by this

process immune cells can adhere to parasites and form complexes for
encapsulation and opsonization (Johansson, 1999)
Opsonization is a process in which cells are coated with molecules being
prepared for the recognition by phagocytes; it enhances adhesion and
consequently phagocytosis.
The phagocytic amoebocyte surface, as all phagocytic cells, has
specialized receptors that recognize molecules present in the cell surface
of microbes that are not found in other eukaryotes (like lipopolysaccharide -
LPS). These molecules are called Pathogen-Associated Molecular Patterns
(PAMPs), and once inside the host body they can be detected by cells through
cell membrane receptors, like the Toll-like-receptors (TLRs), and by specific
molecules called opsonins (Flannagan et al., 2012, Botelho and Grinstein,
2011). Opsonins are deposited in the microbial surface and then they are
bound to the membrane receptor of phagocytes (Flannagan et al., 2012).
In the immune system of sea urchin, about 222 genes are related to TLRs,
besides that they present integrins, cathenins, hemolysins, agglutinins and
lectins - also related to opsonization (Sea Urchin Genome Consortium, 2006;
Gross et al., 1999). In addition, they display scavenger receptor proteins -
related to immune recognition (Hibino et al., 2006).
Another important feature of the sea urchin immune system is the

complement system which is also related to opsonization. This system, in
vertebrates, acts mainly through a C3 molecule, and in 1996 Smith and
colleagues identified, in the purple sea urchin, molecules homologue to the
vertebrate complement system e.g., SpC3 and SpBf. (Smith et al., 1996).
3. Ingestion
After adhesion and opsonization, cells need to engulf the particle. For this,
cell membrane undergoes a series of changes to internalize the particle, and in
this process lipids are essential once they are needed to remodel cell
membrane to bend around the target particle, forming the phagocytic cup,
besides being responsible for the signaling events of ingestion, including the
structure of the actin cytoskeleton. (Flannagan et al., 2012; Botelho and
Grinstein, 2011).
The actin cytoskeleton is important both in remodeling cell membrane and
in pseudopod extension. In sea urchins, there are five types of actin proteins
described, CyI, CyIIa, CyIIb, CyIIIb and muscle actin; In addition, these
animals have actin-binding proteins, responsible for regulating actin dynamics,
such as the Arp2/3 complex and WASP, that promote actin polymerization and
stimulates the actin-nucleating activity of the Arp2/3 complex, respectively
(Morris et al. 2006).
4. Digestion
According to Botelho and Grinstein (2011) the phagocytosis process

completes when pseudopods completely surround the particle and internalize
in the cell. However, little is known about the biochemical processes of
membrane fusion and phagosome closure (Ratcliffe, 1985) even in vertebrates,
but it is believed that proteins such as myosins are involved (Botelho and
Grinstein, 2011).
Within the cell, the phagosome goes through maturation processes via
intracellular signal determining change in its composition, and later the
phagosome will be merged to a lysosome, becoming a phagolysosome
(Desjardins et al., 1994). During the process of maturation, intravesicular
environment becomes more acidic and also oxidative due to the production of
hydrogen peroxide; degradative enzymes are also present (Flannagan et al.,
2012, Botelho and Grinstein, 2011). The production of hydrogen peroxide in

sea urchins was confirmed by Toshimitsu et al. (1992).
Rho GTPases are also involved in these processes, regulating the NADPH
oxidase system (Abo et al., 1991; Knaus et al., 1991), endocytosis (Lamaze et
al., 1996; Leung et al., 1999; Jou et al., 2000) and macrophage phagocytosis
(Caron and Hall 1998).
Immunity Versus Environmental Biomonitoring

Environmental biomonitoring can be defined as a set of biological
responses used to assess alterations in the environment, especially those
caused by anthropogenic activities. For that, both the use of organisms‘
responses, whether cellular, molecular or physiological alterations
(biomarkers); and the use of sentinels organisms (bioindicators) may help
predict how the environment is affected by human activities.
Sea Urchin Biomarkers
The sea urchin immune system, as described above, has a wide variety of
molecules, cells and processes used in the defense of the organisms. These
molecules and processes can also be used as a tool to monitor and indicate
changes in the environment, being many of these molecules and cells
considered environmental biomarkers.
The term ―biomarker‖ is used in the literature as any substance, cell or
biochemical alteration in body fluids that can be measured after exposition to a
substance or condition whose determination in body evaluate the intensity of
exposure as well as potential health risks (Decaprio, 1997; Amorin, 2003;
Bartell, 2006).
Biomarkers can be used for biological monitoring (to detect responses to
environmental challenge and stress) as well as for evaluating dose-effect
relationship in clinical diagnostics (Amorin, 2003), relating the exposure to
chemical substances or physical agents to the risk or susceptibility of disease
(Decaprio, 1997).
According to the World Health Organization (WHO, 1993), biomarkers
can be classified in three different groups: biomarkers of exposition, effect and
susceptibility. Biomarkers of exposition are used to evaluate the effect of a
substance correlating external exposition to internal quantification of the
substance. Biomarkers of susceptibility are related to the level of organism

response due to exposition, and biomarkers of effect are related to the onset of
adverse effects due to exposition. The last one is very appropriate for
evaluating specific physiological parameters once they promote responses to
the cellular level (Bartell, 2006). It is considered a biological parameter that
can be measured in the organism after the exposition to a substance or physical
agent (Amorin, 2003). The use of techniques to rapidly assess stress response
to marine pollution for example can be of great use to detect alterations and
monitor environmental hazards (Matranga and Bonaventura, 2002).
Sea urchins have several responses and/or physiological alterations that
are considered as biomarkers. Among the immune system-related alterations,
Branco et al. (2014) classified them into four groups related to immune
response: studies related to alterations in the proportion of coelomocytes, heat
shock response, immunological responses and molecules related to
inflammatory processes.
Regarding alterations in the proportion of coelomocytes, studies refer
mainly to the red spherule cell, in which the proportion of these cells proved to
be increased across waters polluted by industrial waste (Matranga et al. 2000),
soluble fractions of oil (Borges et al. 2010), and during heat stress (Branco et
al. 2012; 2013).
Increase of this cell type is indicative of cellular stress (Matranga et al.,
2000). It is believed that this cell type is increased in the coelomic fluid
reflecting the red spherule cell pigment function, the echinochrome. Due to its
bactericidal activity, their increase might be associated to an immune response.
Added to the fact that red spherule cell increase is positively correlated to
phagocytic response (Branco et al., 2012), it reinforces the role of spherule
cells in innate immune response.
Considering the heat shock response, these studies analyze mainly the
expression of HSPs (e.g., Hsp70), which are upregulated in elevated
temperatures (Matranga et al. 2000) and in response to exposition to acidic pH,
UV radiation and heavy metals (Matranga et al. 2002, 2006). The expression
of Hsp70 was also reported in sea urchin embryos (Sconzo et al.,, 1997;
Giudice et al., 1999).
Heat Shock Proteins are a phylogenetic conserved family of proteins that
are frequently associated to the correct folding and unfolding of proteins. They
are involved in organelles biogenesis and stress response (Ellis and Van Der
Vies, 1991).
Studies involving immunological responses as environmental biomarkers
mainly focus on the immunological function of phagocytic amoebocyte
(including the phagocytic capacity, as well as their adhesion and spreading

capability). Branco et al. (2012, 2013) showed impairment of these parameters
in sea urchins exposed to high temperatures. These findings might reflect
possible cytoskeleton impairment. Moreover, Falugi and co-workers (2012)
demonstrated the endocytosis of toxic nanoparticles and found that this
cellular process is also a tool to evaluate environmental disorders. These data
reinforce the use of phagocyte as a target to evaluate environmental stress in
marine communities.
Some studies point to the use of molecules related to inflammatory
processes as environmental biomarkers. One example is the cholinesterase, as
described by Falugi et al. (2012) and Angelini et al. (2003). Morphological
changes were also reported including changes in lysosomes and endoplasmic
reticulum of phagocytic amoebocytes (Falugi et al., 2012) and in the
intranuclear iron crystalloid that is used as biomarker for oil contamination.
These structures were found increased after exposition to oil soluble fraction
in the Antarctic sea urchin S. neumayeri (Borges et al., 2010). As previously
mentioned, the role of the intranuclear iron crystalloid is not yet fully
understood; in literature, authors associate their function with urea synthesis
(Bachmann and Goldschimid, 1978), iron transportation (Karasaki, 1965) and
other physiological functions, however the reasons why they are increased and
their probable role in the cellular stress caused by oil pollution is still unclear.
Sea Urchins as Environmental Bioindicators
Bioindicators are used as a tool to evaluate exposure and / or effects that

are measured to the level of organisms, community and populations (high
levels of organization) (Adams et al., 2001, apud Bartell et al., 2006).
Besides the wide range of molecules and cells described above used as
biomarkers, sea urchins (adults, larval and embryonic stages) are considered
good bioindicators of environmental alterations since they are low mobility
animals, and it is known that they are sensitive to pollutants, so they can
reflect the disturbances occurred in their incidence area. (Soualili et al., 2008;
Coteur et al., 2003). Furthermore, these animals inhabit coastal regions, which
are the regions with the highest concentration of pollutants arising from
anthropogenic activities (Coteur et al., 2001).
Sea urchins as environmental bioindicators have been used to evaluate

contaminations by metals and PCB (polychlorinated biphenyls) (Coteur et al.,
2003; Soualili et al., 2008), and for environmental pollution (Matranga and
Bonaventura, 2002).
Sea Urchin Immunity versus

Biomedical Application
Sea urchins themselves are an excellent scientific model option for
biomedical studies. Their application for embryology, efflux transport,
autophagy and apoptosis studies have been reviewed (Branco et al., 2014).
Moreover, it should be mentioned that echinoids are also extensively used for
ecotoxicological purposes.
Regarding sea urchin, not as a tool, but as a source of biomedical
compounds, it is worth mentioning the isolation and characterization of a
molecule extracted from their eggs, which presents antitumoral activity (Liu et
al., 2007). However, sea urchin immunity as a potential for biomedical
application remains obscure. Here we point some of the main findings
regarding this issue.
An early study demonstrated that coelomic fluid of the sea urchin E.
esculentus presented bactericidal activity against Pseudomonas sp. (Wardlaw
and Unkles, 1978).
Due to the growing interest in marine organisms, studies have led to a
significant increase in the amount of known natural products (Stonik, 2009).
Marine organisms are important producers of bioactive metabolites that may
be structurally unique, including some with unusual mechanisms of action, in
addition to various biosynthetic pathways that may include steroids,
terpenoids, alkaloids, polyketides, phenolic metabolites, different
carbohydrates, lipids and peptides (Stonik, 2009; Lotufo Costa et al., 2009).
Marine natural products are a potential source of development of new drugs
with different and often unique structures with different biological properties
(Fusetani, 2000).
The echinochrome constitutes a structurally diverse class of phenolic
compounds with a broad range of pharmacological properties and is widely
used in pharmacy and medicine (Sharma et al., 2003; Martinez and Benito,
2005). Thus, in 1988 it was reported that echinochrome presents antioxidant
and antimicrobial properties; it was also demonstrated function on stabilization
of erythrocytes membranes and reducing the level of serum cholesterol

(Lebedev et al., 2001). In the case of quinones, although the precise action
mechanisms are not fully understood, it is suggested that their main target is
the DNA (El - Najjar et al., 2011)
In 1999, it was developed in Russia a water soluble solution named
Histochrome (registered trademark), composed of echinochrome A as the
active ingredient. The solution was tested in different animals, and results
showed that histochrome was able to reduce the degree of reperfusive damage
in acute tests on dog with myocardial infarction models, influencing
contractility of rat hearts in the calcium paradox test, as well as producing
positive effects in tests with models of burns and ocular hemorrhage, being
indicated for the treatment of eye and heart diseases such as coronary artery
disease and heart attack (Mishchenko et al., 2003). Unlike endogenous
antioxidants (such as vitamin E and ubiquinone naphthoquinones), the
echinochrome is capable of neutralizing the catalysts of the oxidation of lipid
membranes, such as iron cations that accumulate in the region of ischemic
tissue damage (Lebedev et al., 2008).
Another study demonstrated that echinochrome ameliorated intraocular
inflammation, reducing oxidative stress and decreasing inflammation. Authors
still consider the pigment as a promising candidate for the safe treatment of
intraocular inflammation (Lennikov et al., 2014).
Although there are studies involving echinochrome, and its use as drugs
brings good results and new perspectives for the treatment of diseases, its role
in sea urchins remains unclear; studies are needed to unveil new functions.
Besides the necessity to discover new functions, there is still the question of
the mechanism by which this pigment would be acting, which is something
that remains unknown. Thus, the echinochrome presents itself as a good
subject for future studies.
The activity of antimicrobial peptides has also been reported for sea
urchins. Schialli and co-workers (2010) demonstrated an antistaphylococcal
biofilm activity in the sea urchin P. lividus, which may be attributed to three
peptides of a beta-thymosin. Beta-thymosin peptides present a wide range of
biological functions, such as chemotaxis, anti-inflammatory activity and
induction of metalloproteinases due to their direct or indirect effects on actin
cytoskeleton (Schialli and Arizza, 2013).
Sea Urchin Immune System as

an Inexhaustible Source of Research
Sea urchin immune system has been widely studied since the 18th century,
when Metchnikoff‘s discoveries changed the metaphysical concept of
phagocytosis, defining it as an active process instead of a passive one (Tauber
and Chernyak,1991).
Posteriorly, studies focused on cell populations‘ description, as well as
their proportion in the coelomic fluid (Bertheussen and Seijelid, 1978 Isaeva
and Korembaum, 1980); then, subpopulations of phagocytic amoebocyte were
described (Borges et al., 2005; Brockton et al., 2008).
After 2006, a genomic era of sea urchin immunity has begun. Studies
confirmed how proximal sea urchins and vertebrates are, evidenced by their
homolog immune genes, besides cytoskeleton and their accessory proteins
(Sea Urchin Genome Consortium, 2006; Morris et al., 2006).
Additionally, studies regarding the use of sea urchin immune system for
environmental biomonitoring have evolved (Matranga et al., 2000; Branco et
al., 2014).
Despite almost two centuries of intensive research, many questions remain
unanswered, from basic concepts to more complex ones. It is still not clear
how sea urchin coelomocytes are originated; as well as what determines the
phagocytic amoebocyte subpopulations. Is it possible that sea urchins display
different subtypes of cells similar to human adaptive immunity cells?
Regarding the use of immune system as biomonitoring tool, it is still a mystery
why red sphere cell is increased in such cases, the causes that determine its
increase, red sphere cell destination after the stressful condition is restored.
Indeed, sea urchin immune system is fabulous and certainly an endless source
of research.
Conclusion and Future Direction

As previously mentioned, innate immune system is the first to be activated
after antigen exposure, which can not only be microorganism activation, but
also particles. In summary, innate immune response can be defined as the
ability to recognize molecules that are non-self and mount a response in order
to eliminate such antigen.
Despite being relatively simple compared to acquired immune response,

innate immune response involves many steps: recognition of non-self-
particles, migration to the site of injury, phagocytosis of particles, production
of humoral components.
The variability in recognition receptors may guide the immune response
probably favoring a more specific recognition of pathogens. It is indeed
fascinating the high rate of variability that is larger than in any animal
previously described.
The forces that drive coelomocytes migration towards a gradient produced
by strange particles is also an interesting subject. Moreover, it is not clear and
many times controversial how coelomocytes are produced, how they reach
coelomic fluid, and how they infiltrate tissues. Mechanisms of their general
physiology and molecular characteristics are a big issue here.
Phagocytosis is per se an interesting topic. Many steps control and are
controlled by this response. Besides, it is possible that despite being a basic
and conserved phenomenon, there are differences among species, suggesting
that cellular and molecular mechanisms that regulate phagocytosis may suffer
variation at different conditions.
Humoral factors of sea urchins are much more obscure and a potential
source to better comprehend immune response in echinoids
Each of these subjects can be an inexhaustible source of research. Much
has been done but much more remains unclear. Here, we could make an
immeasurable list of questions whose answers remain unknown. But instead,
we present some general points that make us think of how fascinating innate
immune system of sea urchin is and how valuable it can be to use sea urchins
as a model for immunity research.
Acknowledgments
To grant 2011/06044, São Paulo Research Foundation (FAPESP), to Adam A
Martens for the English editing and to Luciana M Dzik for gently conceding
figure 3.
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INDEX
apoptosis, 31, 37, 44, 49, 104, 116

# aquaculture, 98, 127
archenteron, 6, 10, 11, 12, 13, 17, 18, 22, 24
20th century, 3, 14
Asia, 98
21st century, 19
aspartate, 48
assessment, 43, 48, 58, 62, 72, 79, 84, 123
A asymmetry, 21, 72
Austria, 82
acid, 7, 33, 35, 43 autooxidation, 126
acidic, 35, 109, 112, 114
adaptations, 5, 62, 70
B
adaptive immunity, 97, 118
adhesion, 9, 10, 20, 25, 27, 107, 109, 111,
background information, 69
112, 115, 122, 125
bacteria, 13, 92, 93, 95, 102, 105, 125, 130
adhesion strength, 9
bandwidth, 64, 65, 83
adhesions, 110, 111, 129
basal lamina, 10, 11, 24, 27
adults, 4, 15, 17, 33, 34, 41, 69, 98, 115
base, 33, 43, 70, 87
adverse effects, 114
basement membrane, 26
agglutination, 88, 100, 102, 107
basophils, 102
aggregation, 13, 16, 18, 84
benthic diatoms, 39
aldehydes, viii, 29, 30, 35, 37, 39, 40, 46,
benthic dinoflagellates, viii, 29, 30, 35, 36,
47, 49, 51
50
algae, 36, 43, 48, 49, 131
bioaccumulation, 43
alkaloids, 116
biochemical processes, 112
allele, 92
bioindicators, ix, 86, 87, 113, 115, 116, 122
alters, 48
bio-indicators, 127
amino acids, 93, 105, 124
biological activities, 123
amoeboid, 101
biological processes, viii, 2, 5
animal pole, 10, 11
biological responses, 113
antigen, 90, 96, 118
biomarkers, 113, 114, 115, 120, 121
antioxidant, 102, 116, 126
136 Index
biomass, 67 Chile, 85
biomonitoring, ix, x, 86, 87, 88, 113, 118 cholinesterase, 115
biosensors, 121 chordates, ix, 85, 86, 89, 110
biosynthesis, 45, 102 chromatophore, 47
biosynthetic pathways, 116 cilia, 4, 10
blastomeres, vii, 1, 2, 9, 15, 16, 17, 20, 24 cilium, 9
blastopore, 89 classification, 100
blastula, 4, 6, 9, 11, 15, 16, 18, 38, 39 cleavage, 6, 8, 9, 13, 16, 20, 22, 89
blood, 128 closure, 112
body fluid, 113 clusters, 6, 11, 17
bounds, 69 coastal region, 115
Brazil, 85 Cocconeis, 46
coding, 89
coelom, 12, 13
C coelomocytes, ix, 25, 86, 92, 93, 94, 95, 96,
98, 99, 102, 103, 104, 105, 106, 107,
C. elegans, ix, 86, 91
108, 111, 114, 118, 119, 120, 121, 123,
Ca2+, 31, 104
124, 125, 126, 127, 128, 129
cadmium, viii, 29, 30, 31, 33, 43, 44, 45, 49
collagen, 26
calcification, 33, 34, 50
colonization, 49
calcium, 11, 33, 45, 48, 51, 117
commercial, 56
calcium carbonate, 11
community(ies), 34, 35, 42, 46, 49, 62, 67,
carbohydrates, 24, 96, 116
115
carbon, 33, 43, 45
complement, 106, 107, 112, 120, 122, 124,
carbon dioxide (CO2), 33, 34, 35, 41, 43,
128, 130, 131
44, 45, 46, 47, 48, 49, 50
complexity, 41, 91, 131
cascades, 20, 50, 96
composition, vii, 30, 112
case studies, 82
compounds, viii, 11, 29, 30, 35, 36, 39, 46,
cDNA, 95
101, 116
cell apical constriction, vii, 1
construction, 24
cell biology, ix, 86, 87, 98, 123, 124
consumption, 36
cell body, 110
contaminated food, 36
cell cycle, 3, 31, 37, 97
contamination, 45, 115, 121
cell division, vii, 1, 15, 26, 35, 37
controversial, ix, 86, 119
cell fate, 8, 21, 22, 24, 26
cooking, 98
cell fusion, 23
copper, 31, 45
cell line, 5
coronary artery disease, 117
cell organization, 17
correlation, 33, 102
cell signaling, 96, 97
cost, 55, 66
cell size, 8, 100
crystal structure, 124
cell surface, 9, 10, 94, 96, 111
crystalline, 120
cellular immunity, 87
cues, 5, 14
changing environment, 5
culture media, 39, 51
chaperones, 128
cumulative distribution function, 70
chemical, 7, 35, 48, 109, 113, 123
cycling, 110
chemotaxis, 7, 23, 100, 109, 117
Index 137
cyclins, 3
cysteine, 93, 105, 106, 129, 130
E
cytochrome, 120
echinoderms, ix, 23, 27, 43, 44, 85, 89, 98,
cytokines, 104
102, 104, 105, 125
cytoplasm, 100, 101, 109, 110
ecology, 55, 82, 83, 131
cytoskeleton, ix, 14, 86, 87, 96, 97, 100,
economics, 80
110, 112, 115, 117, 118
ecosystem, 39, 41, 45, 46, 51
cytotoxicity, 100
ecotoxicological, 116
ectoderm, 6, 9, 11, 12, 13, 14, 21, 24
D egg, 2, 7, 123, 127
elongation, 10, 12, 123
D. melanogaster, ix, 86, 91 embryogenesis, 2, 4, 21, 27, 31, 37, 45, 110
data distribution, 73 embryology, vii, 1, 87, 116
data set, 73 embryos, vii, viii, 1, 2, 4, 15, 16, 17, 19, 20,
database, 3 21, 22, 24, 25, 26, 29, 31, 33, 34, 35, 36,
defence, 34, 39, 45, 122 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
degradation, 88, 105 49, 98, 114, 126, 130
depolarization, 8 encapsulation, 88, 94, 100, 107, 111
depth, 57, 58, 61, 62, 66, 67, 73, 77 encoding, 4, 27, 91, 95, 97, 107, 128, 130
derivatives, 35, 62, 66, 68 endoderm, 4, 6, 9, 11, 12, 13, 24, 25
dermatitis, 36 energy, 34, 62, 67
destruction, 106, 107 England, 19, 20, 21, 22, 23, 24, 25, 27, 28
detectable, 13, 92 environment, 41, 44, 50, 69, 82, 112, 113
detection, 123 environmental conditions, 4, 5, 7
detoxification, 33 environmental contamination, 31
developmental process, 5 environmental factors, 55
diatoms, 36, 39, 46, 47, 51 environmental stress, vii, viii, 29, 30, 39,
diet, 42, 62 40, 48, 87, 115, 121
digestion, 34, 109 environments, 19
diseases, 87, 117 enzyme(s), 23, 24, 38, 39, 109, 112
displacement, 7, 13 epithelium, vii, 1, 9, 10, 103, 104
dissociation, 18, 110 erythrocytes, 107, 117
distribution, ix, 8, 13, 21, 24, 31, 35, 54, 60, ESI, 105
68, 69, 70, 71, 73, 87 European Union (EU), 48
distribution function, 70 evolution, viii, 2, 4, 5, 19, 23, 98, 122, 124
diversity, ix, 4, 86, 92, 105, 121, 131, 132 excretion, 42
DNA, 3, 5, 19, 37, 38, 45, 97, 117, 127 exocytosis, 24, 109
DOI, 46 experimental condition, 8, 33, 34, 41
Drosophila, 91, 92 exploitation, 62, 69
drugs, 88, 116, 117 exposure, ix, 31, 33, 37, 38, 49, 54, 62, 67,
77, 113, 115, 118
external environment, 10
extracellular matrix, 24, 26, 87, 96, 97
extraction, 66, 105
extracts, 43
138 Index
genes, ix, 4, 25, 34, 38, 39, 49, 85, 87, 89,
F 90, 91, 92, 93, 94, 95, 96, 97, 104, 105,
106, 107, 111, 118, 124, 129, 131, 132
families, 41, 91, 110
genetic diversity, 97
fauna, 33
genetics, viii, 2, 23
ferrous ion, 101
genome, ix, 85, 87, 90, 91, 92, 95, 97, 104,
fertilization, vii, 1, 2, 4, 6, 7, 8, 15, 16, 20,
106, 123, 125, 128, 130
23, 24, 34, 37, 38, 43, 51, 86, 102, 123
genus, 50
fibers, 129
germ cells, 9, 10, 13
filament, 97
germ layer, 9
fish, 58, 62, 69, 79, 81, 82, 84, 131
germ line, 6, 27
fisheries, 54, 58, 79, 84
gland, 45, 48, 103
fishing, 54
global climate change, 33
fitness, 37, 39, 55
global warming, 121
flagellum, 103
glutamate, 47
fluid, ix, 86, 98, 100, 102, 103, 108, 114,
glutamine, 38
116, 118, 119, 121, 122, 124, 130, 132
glycine, 94
fluorescence, 38
gonads, 30, 31, 32
food, vii, ix, 14, 15, 30, 39, 42, 54, 62, 67,
granules, 8, 101, 123
77, 79, 87
grasses, vii, viii, 29, 30
food web, vii, 30, 42
grazers, 37, 45, 46
force, 5, 23
grazing, vii, 30, 35, 83
formation, 6, 9, 12, 18, 21, 24, 25, 33, 37,
growth, vii, viii, 12, 13, 31, 34, 42, 45, 50,
51, 71, 109, 110, 111, 123
53, 54, 55, 56, 58, 62, 66, 67, 69, 72, 73,
France, 1
74, 77, 79, 81, 82, 83, 124, 129
functional changes, viii, 53, 56, 77
growth factor, 12, 129
fungi, 93
growth rate, 56, 62
fusion, 2, 8, 13, 112
GTPases, 110, 111, 113, 122, 123, 129, 130
guanine, 110
G guidance, 19, 62
Guinea, 35
gamete, 8, 31, 37 Gulf Coast, 53
gametogenesis, 37
gastrula, 17, 18, 22, 89
H
gastrulation, vii, 1, 9, 11, 13, 19, 21, 22, 24,
25, 121
H. sapiens, ix, 86, 91
GDP, 110
habitat(s), 50, 62, 69, 79, 82, 84
GEF, 110
haploid, 2
gene expression, 19, 46, 48, 50, 89, 100,
healing, 98
125, 128
health condition, 42
gene regulation, 3, 45
health effects, 51
gene regulatory network (GRN), vii, 1, 4,
health risks, 113
19, 22, 23, 25
health status, 55
heart attack, 117
heart disease, 117
Index 139
heat shock protein, 30, 31, 38, 124 inhibition, 37, 40, 43
heavy metals, 30, 50, 100, 114, 131 inhibitor, 93, 96
helical conformation, 105 injury(ies), 13, 17, 92, 101, 104, 119, 125
heterogeneity, 73 innate immunity, 105, 125, 126, 128
histamine, 131 insects, 105
histidine, 94, 105 integrins, 94, 111
homeostasis, 4, 90, 108 interferon (IFN), 92, 104
host, 106, 111, 122, 125 intoxication, 36
human, viii, 29, 33, 36, 41, 88, 113, 118, intraocular, 117
130 invaginate, 10, 12
human health, 36 invertebrates, 23, 31, 43, 48, 49, 55, 56, 88,
humoral factors, ix, 86 95, 96, 108, 120, 122, 130, 131, 132
Hunter, 54, 77, 78 ionization, 49, 105
hyaline, 10, 26, 27 Ireland, 48
hybrid, 18 iron, 43, 100, 102, 115, 117, 125
hydrogen peroxide, 112, 125 iron transport, 100, 115
hydrolysis, 110 isolation, 15, 16, 116, 127
Italy, 29, 32, 35, 43, 46, 50
I
J
IL-17, 104
immune defense, 107 Japan, 54
immune function, 90 juveniles, 34, 36, 49
immune response, ix, 86, 88, 89, 94, 95, 96,
97, 98, 104, 109, 110, 111, 114, 118,
119, 130, 131, 132 K
immune system, vii, ix, 25, 86, 87, 88, 89,
kill, 88
90, 91, 94, 96, 97, 98, 105, 111, 112,
kinase activity, 37
113, 114, 118, 119, 121, 124, 127, 129,
130, 131
immunity, ix, 13, 25, 86, 87, 88, 90, 91, 92, L
96, 98, 102, 108, 116, 118, 119, 124,
125, 128, 129, 132 landings, viii, 53, 57
immunoglobulin, 97 L-arginine, 39
impact assessment, 123 larva, 6, 12, 14, 18, 28
improvements, 70 larvae, viii, 2, 14, 15, 18, 21, 22, 25, 29, 34,
in vitro, 10, 11, 14, 121 36, 49, 50, 98, 102, 106
in vivo, viii, 2, 48 larval development, 37, 44, 50
India, 96 larval stages, 98
indirect effect, 117 laser ablation, 22
induction, 30, 37, 104, 117 Leahy, 20
infection, 87, 96, 98, 101 leucine, 91
inflammation, 104, 117 life cycle, 4
inflammatory disease, 123, 125 ligand, 90, 92, 131
ingestion, 109, 112 light, 6, 12, 39, 100, 106
140 Index
lineage conversion, 21 metabolites, 49, 116

linear model, 72 metal ion, 31, 41, 51
linear programming, 71 metalloproteinase, 48
lipids, 96, 112, 116 metals, 30, 31, 33, 43, 50, 51, 116
lipoproteins, 93 metamorphosis, 14, 44
liposomes, 101 metaphase, 31
liquid chromatography, 44, 49, 126 metaphor, 132
localization, 38, 129 metastasis, 110
locus, 124 methodology, 68, 71, 72, 73, 79
LTA, 92 mice, 18
lymphoid, 103 micromere, 13, 27
lysis, 104 microorganism, 90, 118
lysosome, 109, 112 microorganisms, 89, 90, 93
microRNA, 126
microscopy, 2, 4, 19, 99, 100, 108
M migration, 21, 109, 110, 111, 119, 129, 133
Ministry of Education, 41
machine learning, 71
mitochondria, 15
macroalgae, 34, 36, 46
mitogen, 92, 104
macrophages, 93
mitosis, 8, 31, 37
magnitude, 94
model specification, 71
malnutrition, 79
model system, viii, 29, 30, 43
mammals, 88, 92, 102, 105, 106, 108
modelling, 59, 69, 70, 72, 77, 79
management, 54, 56, 78, 84
models, vii, viii, 1, 2, 4, 33, 53, 55, 58, 59,
manganese, viii, 29, 30, 31, 33, 48
60, 64, 66, 67, 69, 71, 72, 80, 83, 84, 88,
marine animals, vii, 30
91, 107, 117
marine diatom, 39, 43, 45, 49, 51
molecular biology, 87
marine ecosystems, vii, 30
molecular dynamics, 4
marine environment, 41
molecular mass, 105
mass, 23, 44, 49, 105
molecular structure, 102
mass spectrometry, 44, 49, 105
molecules, 23, 36, 49, 88, 98, 104, 105, 107,
mast cells, 102
108, 110, 111, 112, 113, 114, 115, 118,
maturation process, 112
123, 125
median, 5, 56, 66, 68, 73, 74, 77
Montana, 21, 26
medicine, 88, 116
Moon, 110, 128
Mediterranean, 34, 36, 42, 47, 49, 50, 51,
Morocco, 32
81, 83
morphogenesis, vii, 1, 4, 14, 19, 21, 22, 38
melanin, 48
morphogenetic events, vii, 1, 9
membranes, 95, 117
morphology, 48, 100
mercury, viii, 29, 30, 31, 33, 42, 45
mortality, 34, 36
Mercury, 31, 43
morula, 101
mesenchyme, vii, 1, 10, 20, 21, 22, 24, 26,
mosaic, 107, 128
27, 124
mRNA, 22, 27
mesoderm, 6, 9, 27, 89
multicellular organisms, 87
messengers, 21
muscles, 13, 20
metabolism, 5
Index 141
mussels, 36 oxidation, 117

myocardial infarction, 117 oxidative stress, 33, 44, 117
oxide nanoparticles, 123
oxygen, 102
N oyster, 120
Na+, 42
nanoparticles, 115 P
National Academy of Sciences, 21
National Research Council, 41 parameter estimation, 71
natural killer cell, 131 parasites, 98, 111
natural selection, 21 pathogenesis, 123
necrosis, 104, 122 pathogens, 87, 88, 93, 95, 96, 98, 105, 106,
Netherlands, 126 119, 130
neurons, 20 pathways, 40, 41, 45, 92, 104, 106, 110
neurotransmission, 47 PCBs, 122, 123
New Zealand, 36, 50 PCR, 95, 105
Nigeria, 82 peptide(s), ix, 7, 27, 86, 92, 105, 116, 117,
nitric oxide, viii, 30, 38, 39, 40, 41, 44, 45, 122, 126, 127, 130
47, 48, 51 peritoneum, 103
nitric oxide synthase, 39, 40, 45 permeability, 95
Nobel Prize, 87 peroxide, 101, 113
normal development, 4, 19, 27 peroxide radical, 101
normal distribution, 71 personal communication, 95
nuclei, 23, 37, 125 pH, 31, 33, 34, 35, 42, 43, 44, 46, 50, 109,
nucleic acid, 92, 96 114
nucleus, 15, 100 phagocyte, 100, 109, 115
null hypothesis, 59, 60, 61 phagocytic cells, 107, 111
phagocytosis, ix, 86, 87, 88, 97, 98, 100,
106, 107, 108, 109, 110, 111, 112, 113,
O 118, 119, 122, 123, 124, 131
phenolic compounds, 116
ocean acidification, viii, 29, 30, 33, 34, 35,
phenotypes, 5, 16
41, 42, 43, 44, 45, 46, 48, 49, 50
phosphorylation, 33, 48
oceans, 37, 45
phylum, 98
oil, 114, 115, 121
physiology, 33, 48, 119
oligomerization, 93
phytoplankton, 46
oocyte, 2, 4, 8, 123
plankton, 34, 48
oogenesis, 21
plants, 42
operations, 66
plasma membrane, 27, 90
optical properties, 2
plasticity, 14, 15, 22, 34, 47
organ, 13, 17, 103
polar, 26
organelles, 97, 111, 114
polarity, 18, 25
organism, viii, 4, 29, 30, 33, 41, 87, 89, 90,
pollutants, vii, viii, 29, 30, 35, 38, 115
100, 105, 108, 114
pollution, vii, 30, 31, 41, 43, 45, 50, 114,
organs, 5, 47, 98, 102
115, 116, 127, 131
142 Index
polychlorinated biphenyl, 116 recombination, 97, 124

polyether, 47 reconstruction, viii, 2
polymerization, 37, 112 recovery, 39
polymorphism, 5, 19 regression, vii, viii, 53, 54, 55, 56, 57, 58,
population, 5, 9, 10, 36, 55, 56, 58, 67, 69, 59, 60, 61, 62, 64, 66, 67, 68, 69, 70, 71,
70, 71, 82 74, 75, 76, 77, 78, 79, 80, 81, 82, 83
population density, 82 regression analysis, 55
Portugal, 82 regression model, viii, 53, 54, 57, 61, 64,
predators, 34, 87 68, 70, 77, 81, 82
probability, 5, 56, 60, 65, 79 regulations, 54
probability distribution, 5 regulatory systems, 50
proliferation, 13, 46, 47, 125 rejection, 100, 103
proline, 105 relevance, viii, 29, 90
prophase, 31 repair, 62, 67, 107, 110, 124
protection, 39 replication, 46
protein sequence, 94 reproduction, 7, 42, 43, 47, 82, 102
protein structure, 105 residuals, 59, 60, 65
protein synthesis, 127 residues, 105
proteins, ix, 3, 10, 11, 22, 49, 86, 90, 91, 92, resilience, vii, 1, 5, 17, 19
93, 94, 95, 96, 97, 105, 106, 111, 112, resistance, 33
114, 118, 121, 124, 125, 128 resources, 15, 62, 69, 85
pseudopodia, 100, 109 respiration, 31, 42, 132
purification, 126 response, vii, ix, 1, 23, 30, 33, 38, 40, 41,
42, 45, 48, 50, 51, 59, 68, 70, 71, 72, 86,
88, 92, 94, 95, 96, 97, 102, 104, 114,
Q 118, 119, 122, 125, 128, 129, 132
reticulum, 100, 115
quantification, 14, 46, 113
ribosomal RNA, 89
quinones, 117, 123, 127
risk, viii, 29, 30, 71, 113, 120, 132
risk assessment, viii, 29, 30, 120, 132
R RNA(s), 26, 92, 96, 126
RNA splicing, 96
Rab, 110 rodents, 91
radiation, 38 rods, 14
radicals, 101 Russia, 117
reactant, 95
reactions, 105
S
reactive oxygen, 31, 100, 122
reagents, 42
salinity, 39
reception, 23
SAP, 7
receptors, ix, 27, 86, 90, 91, 92, 93, 96, 97,
saturation, 33
104, 106, 111, 119, 126, 128, 129, 130
scatter, 83
recognition, 18, 22, 88, 89, 90, 91, 92, 93,
scavengers, 40, 126
95, 96, 100, 105, 107, 109, 111, 119,
SEA, 53
124, 125, 128
sea grasses, vii, viii, 29, 30
Index 143
sea urchin eggs, vii, 1, 2, 3, 22, 23, 26, 27, statistics, 79

31, 42, 128 stem cells, 104
seafood, 42 steroids, 116
seaweed, vii, viii, 29, 30, 62, 67 stimulation, 48, 92, 93
secrete, 11 stimulus, 109
secretion, 96 stock, 54, 55, 58, 62, 78, 84
sediment(s), 43, 50, 123 stomach, 13
sensing, 88 stress, viii, 29, 30, 38, 39, 40, 41, 43, 45, 48,
sensitivity, vii, viii, 29, 30 49, 51, 102, 113, 114, 115, 120, 127,
sequencing, 90, 97, 126, 127 128, 129
serine, 107 stress response, viii, 29, 30, 39, 40, 41, 48,
serum, 117 49, 102, 114
shape, 11, 16, 72, 73, 83 stressors, ix, 50, 86
shellfish, 78 Strongylocentrotus droebachiensis, viii, 8,
shock, 114 27, 54, 57, 105, 124, 127
shores, 36 structure, vii, 30, 36, 106, 109, 112, 120
showing, 6, 18, 39, 67 subgroups, 89
signal transduction, 48, 96, 110 substrate(s), 20, 27, 36
signaling pathway, viii, 29, 44, 123, 132 sucrose, 15
signalling, 20, 48 survival, 34, 41, 108
signals, 129 Switzerland, 124
significance level, 60, 61 syncytium, 6, 11
skeleton, 6, 11, 12, 13, 14, 21, 27, 33, 38, syndrome, 90, 127
48, 55, 58, 70 synthesis, viii, 2, 31, 37, 48, 92, 100, 105,
skewness, 78 115
skin, 45, 90, 127
smoothing, 64, 65, 70, 71, 72, 73, 74, 81
smoothness, 70 T
solid phase, 105
target, 38, 107, 110, 112, 115, 117
solution, 65, 117
taxa, 34
Spain, 53, 84
taxes, 87
species, 2, 3, 4, 5, 7, 9, 12, 14, 18, 19, 21,
taxonomy, 3
25, 31, 33, 34, 35, 36, 37, 41, 43, 50, 54,
T-cell receptor, 97
56, 57, 58, 62, 64, 78, 87, 90, 98, 100,
techniques, 56, 59, 66, 70, 71, 73, 108, 114
102, 103, 119, 122
temperature, 7, 8, 121
speculation, 102
tensions, 13
sperm, 2, 7, 15, 23, 37, 42, 43, 102, 132
test statistic, 59, 60
spicule, 11, 25, 38
testing, 54, 58, 80
spine, 62, 67, 79
TIR, 91
stability, 24
tissue, 23, 32, 108, 117
stabilization, 116
TLR, 90, 91, 92, 132
standard deviation, 72
TNF, 104
standardization, 33
toxic effect, 40
state, 33, 55, 68, 82, 103, 110
toxicity, viii, 29, 30, 43, 45, 47, 48
states, 62, 101
toxin, 43, 45
144 Index
transcription, 25, 91, 92, 93, 94, 96, 127 vertebrates, 39, 87, 88, 90, 91, 92, 93, 95,
transcription factors, 25, 92 97, 106, 107, 108, 112, 118, 122, 132
transcripts, 93, 94, 104, 106, 132 vesicle, 97
transformation(s), 72, 78, 123 vitalism, 2
translocation, 10, 123 vitamin E, 117
transmission, 39
transparency, vii, 1, 2, 4, 30, 86
transplantation, 11 W
transport, 10, 97, 116
waste, 114
treatment, 31, 37, 38, 39, 117, 123
water, 7, 13, 18, 34, 36, 41, 43, 62, 117
triploid, vii, 1, 2
weight gain, 56, 62, 63, 66, 67
tumor, 45, 104, 133
World Health Organization (WHO), 69,
twinning, 15, 24
113, 132
twins, vii, 1, 16, 17
wound healing, 101
U
Y
United Nations, 62
yeast, 98, 108
United States (USA), 21, 35, 54, 57, 77,
yield, viii, 54, 55, 56, 57, 62, 63, 66, 67, 77
124, 129, 131
yolk, 15
urea, 100, 115
UV radiation, 114
uveitis, 126 Z
zinc, 43
V
zooplankton, 14, 45
zygote, 8, 24
vascular diseases, 110
vector, 64, 72

(Marine Biology) Edgar Raymond Banks - Sea Urchins - Habitat, Embryonic Development and Importance in The Environment-Nova Science Pub Inc (2014)

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(Marine Biology) Edgar Raymond Banks - Sea Urchins - Habitat, Embryonic Development and Importance in The Environment-Nova Science Pub Inc (2014)

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MARINE BIOLOGY

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EDGAR RAYMOND BANKS

NOTICE TO THE READER

Independent verification should be sought for any data, advice or recommendations

Library of Congress Cataloging-in-Publication Data

system science rely on the in vivo multiscale observation of biological

PHENOTYPIC VARIATION AND RESILIENCE

Sea urchins have long been used as model organisms for

approaches in developmental biology in the context of complex system

sequences encoding the Endo16 protein, specifically expressed in the sea

2. Defining Normal Development

Investigating the processes underlying development requires a definition

3. Introducing Developmental Variability

In living systems, variability is a major force which drives evolution and

II. Early Development of Wild Type Sea Urchins

The typical development of a normal wild type sea urchin is depicted in

Figure 2. Overview of the typical development of a sea urchin (Paracentrotus lividus)

2. Spawning and Reproduction

Sea urchins are iteroparous and most of them follow a seasonal

3. Cleavage Stages and Formation of the Blastula

leading to the germ layers ectoderm, endoderm and mesoderm respectively.

b. The Swimming Blastula

a. The Early Gastrula

b. The Mid Gastrula

By mid-gastrulation, SMCs extend filopodia towards the animal pole,

c. Late Gastrula and Early Larval Stages

Figure 3. Focusing on the prospective mouth region during mouth formation,

4) circumesophageal muscles. Pigment cells and blastocoelar cells come from

The larval skeleton continues to grow, regulated by the PMCs‘

III. Experimental Perturbation of Sea Urchin Early

1. Fractionation of Unfertilized Eggs

2. Separated Fertilized Eggs: Twinning

Experimental twinning in sea urchin was introduced by Hans Driesch in

a. Twins’ Development from the 1-Cell Stage to Hatching

b. Twins’ Developmental Variation: An Ambiguous Fate Map

3. Blastomere Ablation and Transfating

Ablation experiments are classic experimental protocols to investigate the

4. Reaggregation of Blastomeres and Cell Sorting

The complete disaggregation followed by reaggregation of embryonic

IV. Discussion and Perspectives

Harvey, E. (1932). The development of half and quarter eggs of Arbacia

McClay, D. R. (2011). Evolutionary crossroads in developmental biology: sea

Peterson, R. E., & McClay, D. R. (2003). Primary mesenchyme cell patterning

Stephens, R. E. (1972). Studies on the development of the sea urchin

Young, R. (1958). Development of pigment in the larva of the sea urchin,

RESPONSE OF SEA URCHIN

Oriana Migliaccio, Immacolata Castellano,

Sea urchin is an ―opportunist‖ organism with great relevance in

molecule nitric oxide, which has emerging as an important mediator of

1. Cadmium, Manganese and Mercury

Table 1. Comparison of metal concentrations (µg.g-1 of tissue) in sea

Source Species Site Cd Hg Mn

In the same work, the ABCC/multidrug resistance associated protein

conditions and using various life-history stages, including gametes,

3.1. Palytoxin-like Compounds

Natural toxins produced by dinoflagellates are receiving increasing

3.2. Diatom-derived Polyunsaturated Aldehydes