ISJ 7: 211-220, 2010     211 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 mailto:framirez@umassd.edu     212 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     213 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).     214 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     215 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     216 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     217 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. 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