Echinoderm immunity

Abstract

Echinoderms are exclusively marine animals that, after the chordates, represent the second largest group of deuterostomes. Their diverse species composition and singular ecological niches provide at the same time challenges and rewards when studying the broad range of responses that make up their immune mechanisms. Two types of responses comprise the immune system of echinoderms: a cellular response and a humoral one. Cell-based immunity is carried by the celomocytes, a morphologically heterogeneous population of free roaming cells that are capable of recognizing and neutralizing pathogens. Celomocytes present diverse morphologies and functions, which include phagocytosis, encapsulation, clotting, cytotoxicity, wound healing among others. Humoral immunity is mediated by a wide variety of secreted compounds that can be found in the celomic fluid and play important roles in defense against infection. Compounds such as lectins, agglutinins, perforins, complement and some cytokines make up some of the humoral responses of echinoderms. Recent advances in the field of molecular biology, genomics and transcriptomics have allowed for the discovery of new immune genes and their products. These discoveries have expanded our knowledge of echinoderm immunity and are setting up the stage for future experiments to better understand the evolution of the immune mechanisms of deuterostomes

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ISJ 7: 211-220, 2010 ISSN 1824-307X
REVIEW
Echinoderm immunity
F Ramírez-Gómez1, JE García-Arrarás2
1Department of Biology, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA
02747, USA
2Department of Biology, University of Puerto Rico, P.O. Box 23360, UPR Station, Río Piedras, San Juan, PR
00931-3360, USA
Accepted September 27, 2010
Abstract
Echinoderms are exclusively marine animals that, after the chordates, represent the second
largest group of deuterostomes. Their diverse species composition and singular ecological niches
provide at the same time challenges and rewards when studying the broad range of responses that
make up their immune mechanisms. Two types of responses comprise the immune system of
echinoderms: a cellular response and a humoral one. Cell-based immunity is carried by the
celomocytes, a morphologically heterogeneous population of free roaming cells that are capable of
recognizing and neutralizing pathogens. Celomocytes present diverse morphologies and functions,
which include phagocytosis, encapsulation, clotting, cytotoxicity, wound healing among others.
Humoral immunity is mediated by a wide variety of secreted compounds that can be found in the
celomic fluid and play important roles in defense against infection. Compounds such as lectins,
agglutinins, perforins, complement and some cytokines make up some of the humoral responses of
echinoderms. Recent advances in the field of molecular biology, genomics and transcriptomics have
allowed for the discovery of new immune genes and their products. These discoveries have expanded
our knowledge of echinoderm immunity and are setting up the stage for future experiments to better
understand the evolution of the immune mechanisms of deuterostomes.
Key Words: comparative immunology; echinoderm; immunity; celomocytes; genes
Introduction
The phylum Echinodermata is a very diverse
group of marine animals that have sparked the
interests of scientists for over a century. Significant
discoveries have been made using echinoderms in
the areas of cell biology, developmental biology and
immunology. Five classes comprise the phylum:
Asteroidea (sea stars or starfish), Crinoidea
(crinoids or feather stars), Ophiuroidea (brittle stars),
Echinoidea (sea urchins and sand dollars) and
Holothuroidea (sea cucumbers or holothurians).
Even though research has been done on all
echinoderm classes, one group excels as the
favorite of scientists: the echinoids. Thus, sea
urchins have become one of the classical animal
models and have been particularly exploited in
studies of fertilization and developmental biology.
Similarly, in the field of echinoderm immunology,
sea urchins comprise the group that has been most
_____________________________________ ______________________________________
Corresponding author:
Francisco J Ramirez-Gomez
University of Massachusetts Dartmouth,
285 Old Westport Road,
North Dartmouth, MA 02747-2300, USA
E-mail: framirez@umassd.edu
extensively studied (Smith et al., 2006).
Furthermore, the availability of the genome
sequence for the purple sea urchin
(Strongylocentrotus purpuratus) has allowed for in
depth studies into the genetic aspects of its immune
response (Hibino et al., 2006; Rast et al., 2006).
This trend has helped advance the field and at
present, echinoderms are catching the attention of
comparative immunologists. However, due to the
inherent diversity of the echinoderm phylum,
general assumptions cannot be easily established
and what is true for one specific class may not apply
to others.
Interest in echinoderm immunobiology also
originates form the aquaculture field. Although little
known in the western hemisphere, holothurian and
echinoid cultures are an important economic activity
in Asia. With an increasing demand for sea urchin
roe and trepang (a generic name for sea
cucumbers), commercial culture venues have
increased in order to maintain the demands for
these organisms. With increase in aquacultures one
observes an increase in diseases, mainly infections,
and therefore an increase interest in understanding
how the organisms protect themselves from

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pathogenic threat. The present review will attempt to
summarize the latest published research work on
the echinoderm immune system with a special
emphasis on non-echinoid groups. The review
focuses on the different immune components and
mechanisms present in the phyla and highlights how
rich, diverse and complex this group of animals can
be.
General aspects of echinoderm immunity
In terms of their immune systems, echinoderms
display the same basic types of responses that most
multicellular (including vertebrates) animals do.
They can recognize self from non-self and, if a
foreign material (e.g., microorganism/pathogen)
enters the body cavity, they can readily neutralize it
and dispose of it (Yui and Bayne, 1983; Dybas and
Fankboner, 1986; Jans et al., 1996; Glinski and
Jarosz, 2000). Additionally, echinoderms possess
very good wound-healing capabilities, a key feature
that also plays an important role in one of the best
known characteristics of the group: regeneration of
lost body parts.
These defense mechanisms are mediated by
cellular and humoral responses, with several
homologous and analogous components found in
other invertebrates and vertebrates alike. In fact, it
is their key position in the evolutionary tree, being
invertebrate deuterostomes (thus sharing a common
evolutionary branch with vertebrates) that makes
the study of their immune system a very interesting
and exciting field. Therefore, this advantageous
phylogenetic position allows for comparisons
between immune mechanisms that have been well
studied in vertebrates with those of their echinoderm
counterparts. Thus, echinoderms can provide
important information on the evolution of the
immune response.
As in many other systems, echinoderm immune
responses can be divided between cellular and
humoral responses. Cellular responses are
mediated by the celomocytes, which are free
roaming cells that occupy the celomic cavity but can
also infiltrate tissues and organs. These cells
circulate in the celomic fluid and exert the vast
majority of immune functions. On the other hand,
humoral responses are defined by the broad variety
of molecules present in the celomic fluid. These
molecules are capable of recognizing and
neutralizing foreign material, promoting cell
migration and agglutination and also playing roles in
wound healing ( Ryoyama, 1973; Kanungo, 1982;
Canicatti et al., 1992; Smith and Davidson, 1992).
Cellular components
Celomocytes are a very abundant and diverse
cell types that are present in all echinoderms. These
cells are heterogeneous in morphology, size,
relative abundance and function, which make a
single standard classification for all echinoderms a
difficult task. Extensive research has been done
during the past century on the morphological
aspects of celomocytes. Comprehensive reports on
the celomocyte types of different echinoderms
classes have also been published (Kindred, 1924;
Boolootian and Giese, 1958, 1959; Boolootian,
1962; Endean, 1966). These studies clearly show
the wide variety of cell morphologies present in the
echinoderm celomic fluid. However, the absence of
a standard reference among groups and
particularly, differences in terminology and even
specimen preparation, contribute to the existing
heterogeneity. Nonetheless, some types of
celomocytes can be found in all classes, while
others have been considered to be specific to
certain classes. These cell types are summarized in
Table 1 along with the particular functions that have
been ascribed to certain cells.
The distribution of these cell types is highly
variable among species and also even at the
individual level. For example, in some sea star
species the vast majority (> 90 %) of celomocyte
types are amebocytes, while other cell types seem
to be exclusive of certain groups (e.g., holothurian
crystal cells). Table 2 summarizes the general
distribution of celomocytes in three echinoderm
classes (echinoids, holothuroids and asteroids) and
how they differ depending on the group and the
species. This cell distribution is also very dynamic,
changing in accordance to the physiological or
immune state of the animal. For example, in the sea
star Asterias rubens specific sub-populations of
amoebocytes increase in number after injection of
gram-positive bacteria while other sub-groups
remain unchanged (Coteur et al., 2002). Our studies
with the sea cucumber Holothuria glaberrima have
shown that the total number of celomocytes remains
unchanged after challenges with diverse pathogen
associated molecular patterns (PAMPs). However,
the distribution of particular sub-types changes after
immuno-stimulation, e.g., lymphocytes numbers
diminish, while phagocytes increase (Ramirez-
Gomez et al., 2010).
From all the celomocyte types, probably the
one that is present in all the echinoderm classes is
the phagocyte/amebocyte type. This cell ranges in
size from 3 to 20 μm and its main characteristic is its
ability to phagocytize other cells or foreign particles
(Endean, 1966). Other roles have been attributed to
phagocytes, most of them immune related,
demonstrating that this cell type is the main effector
of the echinoderm immune system. In fact, the
discovery of these cells in the sea star, back in the
late 1800’s by Russian zoologist Ilya Metchnikoff
gave rise to the field of cellular immunity, for which
he was awarded the Nobel prize in 1908
(Metchnikoff, 1891). Amebocyte roles include: graft
rejection, chemotaxis, reactive oxygen species
production, encapsulation, cytotoxicity, immune
gene expression, agglutination and clotting
reactions (Gross et al., 1999, 2000; Beck et al.,
2001; Coteur et al., 2001; Lin et al., 2001; Hillier and
Vacquier, 2003; Clow et al., 2004; Matranga et al.,
2005; Sun et al., 2008). Several authors sub-
categorize phagocytes according to their size and
morphology, but since these classifications are not
the same for all echinoderms, some sub-types may
overlap or on the other hand can be rendered as a
different cell type altogether. Lymphocytes are
another cell type that might be present in all
echinoderms (Endean, 1966), but it is most
frequently found in holothurians and some sea stars

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Table 1 Summary of celomocyte types reported for echinoderm classes. E: Echinoidea, H: Holothuroidea, A:
Asteroidea, C: Crinoidea, O: Ophiuroidea.
Cell type Present in class Role Reference
Discoidal cell E, H
Polygonal cell E
Small phagocyte E, H
Amebocytes
/Phagocytes E, H, A, C, O
Phagocytosis, clotting,
encapsulation, chemotaxis,
opsonisation, graft rejection
(Coteur et al., 2002; de Faria and
da Silva, 2008; Eliseikina and
Magarlamov, 2002; Endean,
1966; Matranga et al. 2005;
Ramirez-Gomez et al., 2010;
Smith et al., 2006)
Colored spherule E, H, C Antibacterial activity (de Faria and da Silva, 2008;
Endean, 1966; Smith et al.,
2006)
Colorless spherule E, H, A, C, O Antibacterial, inflammation,
Wound healing, ECM
remodeling
(Coteur et al., 2002; de Faria and
da Silva, 2008; Eliseikina and
Magarlamov, 2002; Endean,
1966; Garcia-Arraras et al.,
2006; Ramirez-Gomez et al.,
2010; Smith et al., 2006)
Lymphocyte E, H, A Progenitor cells
(Coteur et al., 2002; Eliseikina
and Magarlamov, 2002; Endean,
1966; Ramirez-Gomez et al.,
2010; Xing et al., 2008)
Vibratile E, H, A, O Celomic fluid movement,
clotting
(de Faria and da Silva, 2008;
Eliseikina and Magarlamov,
2002; Endean, 1966; Matranga
et al., 2005; Pinsino et al., 2008;
Ramirez-Gomez et al., 2010;
Smith et al., 2006; Xing et al.,
2008)
Crystal cells H Osmoregulation
(Eliseikina and Magarlamov,
2002; Endean, 1966; Ramirez-
Gomez et al., 2010; Xing et al.,
2008)
Hemocytes H, A, O Oxygen transport (Eliseikina and Magarlamov,
2002; Endean, 1966; Pinsino et
al., 2008)
(Smith, 1981). These are small cells (4-6 μm), with a
large nucleus and a thin layer of cytoplasm whose
only common characteristic with their vertebrate
namesakes is their morphology. Lymphocytes are
regarded as progenitor cells and may be the
precursors of other celomocyte types (Xing et al.,
2008; Ramirez-Gomez et al., 2010). They can show
phagocytic capabilities but this may reflect an
intermediate state of maturity before becoming
phagocytes (Ramirez-Gomez et al., 2010).
Spherule cells (spherulocytes) are present
mostly in echinoids and holothuroids (Endean,
1966; Eliseikina and Magarlamov, 2002; Smith et
al., 2006; de Faria and da Silva, 2008; Xing et al.,
2008; Ramirez-Gomez et al., 2010) and in at least
one sea star species (Penn, 1979). They are
characterized by the presence of vesicles in their
cytoplasm, some containing pigment (red, yellow,
green, brown) other being colorless. Spherulocytes
range in sizes from 8 to 20 μm and their distribution
varies substantially between species. They have
been associated with antibacterial activity (Johnson,
1969; Service and Wardlaw, 1984; Haug et al.,
2002), inflammatory responses (Pagliara and
Canicatti, 1993), extracellular matrix remodeling
(Garcia-Arraras et al., 2006), and wound healing
(San Miguel-Ruiz and Garcia-Arraras, 2007).
Another cell type present in echinoids and
holothuroids are the vibratile cells. These are cells
whose size ranges from 6 to 20 μm and are highly
motile due to the presence of a flagellum. Their
distribution varies accordingly to the species and
their function is still not completely determined. They
have been associated with clotting reactions
(Bertheussen and Seijelid, 1978) and are also
thought to be involved in the movement of the
celomic fluid (Xing et al., 2008).
Crystal cells seem to be exclusive of
holothurians, these cells display a very regular
geometric morphology (rhomboidal or hexagonal)
and present a crystal inclusion within their
cytoplasm (Endean, 1966). Their role is still not well
defined, but it is likely that they play osmoregulatory
roles (Xing et al., 2008).

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Table 2 Summary of celomocyte distribution in the celomic fluid in three echinoderm classes and six different
species. L.var: Lytechinus variegatus; S. purp: Strongylocentrotus purpuratus; E. luc: Echinometra lucunter; H.
glab: Holothuria glaberrima; A. jap: Apostichopus japonicus; A. rub: Asterias rubens.
Cell type Echinoidea Holothuroidea Asteroidea
L. var
(Borges et
al., 2005)
S. purp
(Smith et
al., 2006)
E. luc
(de Faria and
da Silva, 2008)
H. glab
(Ramirez-
Gomez et al.,
2010)
A. jap
(Xing et
al., 2008)
A. rub
(Coteur et al.,
2002)
Lymphocytes n.f. .n.f n.f. 60 % 59 % n.f.
Phagocytes/
amebocytes > 60 % 40-80 % 77 % 30 % 17 % 80-95 %
Colored
spherules < 40 % 7-40 % 1 % N.F. N.F. N.F.
Colorless
spherules + 3-25 % 3 % 5 % 23 % N.F.
Vibratile cells + 11-20 % 19 % < 1 % N.A. N.F.
N.F., not found; N.A., not accounted.
n.f., not found.
+ spherules and vibratile cells were accounted together.
Interestingly, echinoderm cellular immunology
has escaped the classical characterization of their
components by phenotyping (identification of cell-
specific epitopes expressed on cell membranes) as
vertebrate lymphocytes do. The lack of definite
surface markers for celomocytes has helped
maintain the confusion in distinguishing between
specific cell types and sub-types, limiting it to just
morphological observations. However, this trend is
slowly changing, as demonstrated by studies with
sea urchin celomocytes, in which a sub-population
of phagocytes was defined by NK cell surface
markers and characterized by their cytotoxic
properties (Lin et al., 2001). Furthermore,
monoclonal antibodies were generated against
these cells showing a successful identification of a
specific cytotoxic phagocyte sub-type (Lin et al.,
2007). Our research group has also identified sub-
groups of sea cucumber celomocytes using
monoclonal antibodies, each sub-population
showing distinct characteristics and different
responses to immunostimulation (Ramirez-Gomez
et al., 2010). Similarly, a recent study in the sea
cucumber Apostichopus japonicus, has also led to
the development of a monoclonal antibody that
specifically recognizes spherulocytes. Initial
characterization of the antigen being recognized by
this antibody resulted in the identification of a 136
kDa protein according to Western blotting (Li et al.,
2010). However, what is still missing is the full
characterization of the antigens these antibodies are
recognizing (protein sequences and cloning). Future
experiments where cell markers are used to
describe celomocyte populations and compare
these populations among the different echinoderm
classes should be the basis for a clear and universal
classification of echinoderm celomocytes.
Celomocyte origin
The origin of celomocytes is still a matter of
debate. Two theories have been proposed to
address this issue: one involving specific organs or
tissues as the source of celomocytes while the other
points at the celomocytes themselves as self-
replicating cells (Bossche and Jangoux, 1976;
Matranga et al., 2005). Potential cytopoietic organs
include the axial organ, Tiedemann bodies, Polian
vesicles, connective tissue and the celomic
epithelium (Endean, 1966). The latter has received
particular attention in studies involving the sea star
A. rubens, showing the epithelial origin of sea star
celomocytes (Bossche and Jangoux, 1976; Holm et
al., 2008). Even though no direct evidence have
been proposed for a self-replicating population of
celomocytes, the idea of a circulating stem cell has
not been ruled out and if anything has become more
attractive in view of recent findings of stem cells in
other metazoans (Handberg-Thorsager et al., 2008;
Watanabe et al., 2009; Funayama, 2010). It must be
stated that the evidence to ascertain the origin of
celomocytes is far from definitive, and would not be
acceptable by modern scientific standards. Thus, it
is necessary for scientists to use modern
methodologies to verify the celomocyte origins
proposed by past investigators.
Humoral components
Echinoderms present a wide rich variety of
secreted immune molecules. They have been the
subjects of extensive research, even to the point of
potential medical applications (Kelly, 2005). As
mentioned before, the humoral components present
in celomic fluid of echinoderms are capable of

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recognizing foreign matter, neutralizing or
destroying pathogens, inducing or enhancing
cellular responses (opsonization) and helping during
wound healing.
A well-known group of recognition molecules
are the lectins, which recognize carbohydrate
moieties on the surface of host cells (self) and of
bacteria and fungi (non-self). Several lectins have
been identified from the celomic fluid of
echinoderms, where they play important roles in
opsonization, lytic cytotoxicity, clot formation and
wound repair (Gross et al., 1999). Different lectins
with specific recognition abilities have been found in
asteroids (Kamiya et al., 1992), and holothuroids
(Matsui et al., 1994; Gowda et al., 2008a, b).
Echinoidin, a C-type lectin (calcium-dependent)
identified in a sea urchin also possess an RGD
sequence, suggesting an additional role in cell-to-
cell adhesion (Giga et al., 1987; Ozeki et al., 1991).
Moreover, the C-type lectin CEL-III from the sea
cucumber Cucumaria miniata, which possess a
strong hemolytic activity have been transgenically
expressed in mosquitoes and shown to successfully
impair malaria parasite development (Yoshida et al.,
2007).
Other humoral factors include hemolysins, that
interact with plasma membranes and form holes in
the membrane of cells causing lysis of target cells
(Canicatti, 1990, 1991). Hemolysins have been
identified in sea stars (Leonard et al., 1990), sea
urchins (Ryoyama, 1973; Stabili et al., 1992) and
sea cucumbers (Canicatti and Parrinello, 1985).
Agglutinins are another type of humoral factor that
play roles in cell aggregation, encapsulation and
clotting and have also been studied in wound repair.
They have been found in echinoids (Ryoyama,
1973; Canicatti et al., 1992), the sea star Asteria
pectinifera (Kamiya et al., 1992) and in the sea
cucumber Holothuria polii (Canicatti and Parrinello,
1985).
In vertebrates a well-known group of effector
molecules are the cytokines, which play a wide
variety of roles in the immune response. In
echinoderms, homologues of cytokines have also
been identified. The first glance at an echinoderm
cytokine came from the sea star A. forbesi, in which
a humoral factor named the sea star factor was
isolated and found to possess cytokine-like
properties (Prendergast and Suzuki, 1970;
Prendergast and Liu, 1976; Kerlin et al., 1994).
Furthermore, interleukin-like molecules were
identified in the sea star, e.g., a protein with IL-1
activity and an IL-6 like molecule ( Beck et al., 1989,
1993; Beck and Habicht, 1991a, 1991b, 1996).
However, none of these findings resulted in a
definite identification of the cytokine factor or the
cloning of the corresponding gene(s). The issue
appears to be complicated since no IL-1
homologues were found in the sea urchin genome.
However, other members of the cytokine network
(mostly pro-inflammatory) have indeed been found,
e.g., TNF and IL-17 (Hibino et al., 2006).
Another important humoral factor present in
echinoderms is the complement protein family. Most
of the components of the alternative and lectin
pathways have been identified in the sea urchins,
being the purple sea urchin the first invertebrate in
which a complement system was identified (Smith et
al., 1996; Smith, 2001). Initial evidence gathered
from sea urchins, sea cucumbers and sea stars
hinted at the presence of a complement system in
echinoderms (Kaplan and Bertheussen, 1977;
Parrinello et al., 1979; Bertheussen 1981a, 1981b,
1982, 1983; Bertheussen and Seljelid, 1982), but
they were mostly from complement-derived or -
dependent activity and no definite identification of a
complement protein was achieved. Smith and
colleagues (1996, 1998) successfully identified the
first echinoderm (and invertebrate) homologue of
the C3 component and later another complement
protein was found (Factor B) (Smith et al., 1998). A
recent publication reported the finding of a C3
complement homologue in the sea star A. rubens,
whose expression is also induced by LPS
stimulation (Mogilenko et al., 2010). Additional
components of the system have been identified from
the sea urchin genome, suggesting that
echinoderms possess a complement pathway
mostly directed towards opsonization, since the
components of canonical terminal pathway could
not be found (Hibino et al., 2006).
Molecular studies and the genomic era
The vast majority of molecular studies have
been done in echinoids, particularly the purple sea
urchin S. purpuratus. A broad number of immune
genes have been identified from the sea urchin
since the early 1990’s and pinnacled with the
publication of the S. purpuratus genome (Sodergren
et al., 2006). An in depth analysis of the immune
repertoire contained within the sea urchin genome
can be found in the publications of Hibino et al.
(2006) and Rast et al. (2006).
However, other species of echinoderms have
also been the subject of molecular studies in order
to better understand their immune responses.
These studies altogether benefit greatly the
advancement of the field, providing further insights
into the genetic and molecular aspects of
echinoderm immunity.
Echinoderm molecular immunogenetics has
evolved in parallel with the technologies available
for its study. Starting with gene-by-gene
approaches, in which single genes were analyzed at
a time and their immune roles determined. An
example of this is the case of the Profilin gene, an
actin binding and cytoskeletal modification protein,
expressed in celomocytes and up-regulated after
injury and LPS injections (Smith et al., 1992, 994,
1995). Then, when sequencing technologies
became accessible, high-throughput sequencing
projects were launched, mostly screening cDNA
libraries. In the late 1990’s a survey of a cDNA
library from LPS-activated celomocytes provided the
first glimpses of the immune repertoire of an
echinoderm (Smith et al., 1996). Several interesting
findings were made in this study on sea urchins,
beginning with the discovery of an echinoderm
complement and a collection of putative immune
effector genes that set the basis for future
comparative studies between echinoderm species.
Our research group has been dwelling into the
molecular immune aspects of holothurians since the

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year 2000, when a homologue of the acute phase
response protein serum amyloid A (SAA) was
identified for the first time in an invertebrate
(Santiago et al., 2000). Its expression was found
mostly in intestinal tissues during the regeneration
process but also after immune stimulation with LPS.
SAA mRNA was found to be overexpressed
following an immune challenge not only in the
intestine (Santiago-Cardona et al., 2003) but also in
celomocytes (Ramirez-Gomez et al., 2008).
Additionally, a series of immune-related genes were
identified in the holothurian from intestinal cDNA
libraries. This identification was mostly done by
sequence comparison with other immune genes
present in other organisms and whose immune role
was clearly defined. The expression of these
holothurian immune genes was corroborated in
celomocytes to determine if they were part of the
gene repertoire of these cells. In addition their
expression was analized after an LPS challenge
(Ramirez-Gomez et al., 2008). Among these genes
we found a C-type lectin, ferritin, cathepsin,
toposome and an alpha 2 macroglobulin domain
(A2M)-containing protein. One sequence of
particular interest was a homologue of the DD104
protein from the sea urchin, which is up-regulated in
celomocytes after injury and infection (Rast et al.,
2000) with the holothurian DD104 following a similar
pattern but with higher expression levels in
celomocytes after LPS injection. Recently, analysis
of the expression of immune-related genes has also
been done in embryos and larvae of the sea
cucumber, A. japonicus. Nine genes were studied:
six of them (heat shock proteins -70, -90 and -gp96;
thymosin-beta, ferritin and DD104) showed no
changes upon LPS challenge while the remaining
three (mannan-binding C-type lectin, lysozyme and
serine proteinase inhibitor) were found to be up-
regulated upon challenge (Yang et al., 2010).
The advent of array technologies that allow for
studies on the expression of multiple genes at the
same time, have opened the door for the
identification of potential novel genes. Many of
these genes were missed in previous approaches
probably due to their lack of homology to known
genes. This approach was first carried out with the
sea urchin, comparing immune-stimulated and
immunoquiescent animals. An unexpected diversity
of genes was found to be differentially expressed
and, more interestingly, a set of novel genes, the
185/333 family of proteins, were then identified (Nair
et al., 2005). This family of genes represents a
highly variable set of proteins that are involved in
the immune response of the sea urchin (reviewed in
Ghosh et al., 2010).
We have also used immune activation and
microarray technologies to compare LPS-injected
sea cucumbers with seawater-injected controls and
thus, identify immune-responsive genes in the
holothurians. We have found 50 unique sequences
differentially expressed after LPS stimulation. The
vast majority of these sequences showed no known
homologies in the databases (Ramirez-Gomez et
al., 2009). Ongoing efforts are being done to further
characterize these unknown genes. By complete
sequencing of the mRNAs we expect to find
similarities and/or conserved domains that will
provide either proper identification, or the
characterization of novel holothurian LPS-
responsive genes.
An interesting case derived from our microarray
study, is the echinoderm mayor yolk protein (MYP)
and its closely related protein, toposome. In our
microarray, the holothurian MYP gene was one of
the top genes that showed differentially expression
following LPS injection. Echinoderm MYP was
initially identified as an unconventional iron-binding
vitellogenic protein making up to 50 % of the protein
content of the sea urchin celomic fluid (Brooks and
Wessel, 2002). It is synthesized in the digestive tract
and also binds zinc ions (Unuma et al., 2007, 2009).
A possible immune role for this protein had been
suggested due to its affinity for iron, making it an
excellent bacteriostatic agent. Our results from the
holothurian microarray, have shown that MYP
mRNA is up-regulated after LPS stimulation in the
digestive tract but its expression remains
unchanged in celomocytes (Ramirez-Gomez et al.,
2009). Nonetheless, anti-MYP labeling is found in
phagocytic lymphocytes (Ramirez-Gomez et al.,
2010). The toposome protein (which is closely
related to MYP), functions as an adhesion protein in
the sea urchin embryo (Cervello and Matranga,
1989; Scaturro et al., 1998; Noll et al., 2007), but is
also related to stress and injury respones (Cervello
et al., 1994; Matranga et al., 2005; Pinsino et al.,
2007). We have found toposome mRNA to be
expressed in H. glaberrima celomocytes at relatively
high levels that remain unchanged after LPS
stimulation (Ramirez-Gomez et al., 2008) as well as
in intestinal tissues, in which its levels remained
unchanged also (Ramirez-Gomez et al., 2009).
These results show that both MYP and toposome
are indeed associated with the immune response
but also suggest additional roles that might not be
part of the traditional functions associated with
celomocytes but might be associated with the
immune functions of the digestive tract.
Now that we have entered the genomic era,
further advances are expected as genome
sequencing technologies become faster and more
economically accessible. The S. purpuratus genome
represents a cornerstone in echinoderm research
that can be used to compare findings from other
echinoderm species. However, as presented here,
the great diversity of the animals within the
echinoderm phylum suggest that having the
genome of only one member of only one
echinoderm class will not be enough to understand
the echinoderm immune system. Take for example
one of the most diverse set of genes found in the
sea urchin genome: the NLR gene family
(nucleotide-binding domain, leucine-rich repeat
containing proteins). These genes encode
cytoplasmic pattern recognition proteins, which in
humans are represented by about 20 genes
(Inohara and Nunez, 2003). However, in the sea
urchin 203 NLR predicted genes can be found.
Similar to the vertebrate counterparts, the major site
of expression of the sea urchin NLRs is the gut
(Hibino et al., 2006). Nonetheless, we were not able
to identify sequences for this gene family in any of
our holothurian intestinal cDNA libraries nor in our
intestinal microarray studies. This may reflect key

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differences in gene repertoires between these two
species related to their phylogenetic divergence.
This variety of gene repertoires may also be
attributed to differences in habitat and
developmental history, and to differences in the
microbe flora that challenges the organisms. These
differences will eventually shape the type of immune
responses that organisms react to.
Therefore, we still need more information on
immune-related genes present in other species from
as many different groups as possible in order to
have a better understanding of the molecular events
that are involved with the echinoderm immune
response.
Concluding remarks
Echinoderm immunity is a challenging yet
promising field to study. The large diversity of
echinoderm species, with different internal organs
(most of them with little physiological information as
to their functions) and different lifestyles make it
difficult to identify those tissues or cells that might
be playing an immune role. Moreover, different
species might be responding to different immune
challenges not usually associated with other animal
groups (Think about the fact that echinoderms
occupy large number of niches in the benthic zone).
An additional complication is the difficulty in
establishing the immune status of the animals used
in experimentation. For example, in studies by Smith
and colleagues, sea urchins were kept in aquaria in
what appeared to be an immunoquiescent status. In
this scenario it is difficult to compare the LPS
response of these animals to that of animals that
have been directly collected from the wild.
Nonetheless, overcoming these difficulties can
provide exciting and rewarding goals. Among these,
are the identification of novel immune associated
genes and proteins and the characterization of new
immune signaling pathways. Moreover, the key
phylogenetic position of echinoderms in the tree of
life assures that whatever we learn about
echinoderm immunity will help us understand the
evolution of metazoan immune systems.
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