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260
CHAPTER 14
ECHINODERM IMMUNITY
L. Courtney Smith,*,1 Julie Ghosh,1 Katherine M. Buckley,2
Lori A. Clow,3 Nolwenn M. Dheilly,4 Tor Haug,5 John H. Henson,6
Chun Li,5 Cheng Man Lun,1 Audrey J. Majeske,1 Valeria Matranga,7
Sham V. Nair,4 Jonathan P. Rast,2 David A. Raftos,4 Mattias Roth,4
Sandro Sacchi,1 Catherine S. Schrankel1 and Klara Stensvåg5
1Department of Biological Sciences, George Washington University, Washington DC, USA; 2Department
of Medical Biophysics and Department of Immunology, Sunnybrook Health Sciences Centre, University of
Toronto, Toronto, Ontario, Canada; 3US Patent and Trademark Ofce, Alexandria, Virginia, USA; 4Department
of Biological Sciences, Macquarie University, Sydney, Australia; 5Norwegian College of Fishery Science,
University of Tromso, Tromso, Norway; 6Department of Biology and the Biochemistry and Molecular Biology
Program, Dickinson College, Carlisle, Pennsylvania, USA; 7CNR, Institute of Biomedicine and Molecular
Immunology “Albert Monroy”, Palermo, Italy
*Corresponding Author: L. Courtney Smith—Email: csmith@gwu.edu
Abstract: A survey for immune genes in the genome for the purple sea urchin has shown that
the immune system is complex and sophisticated. By inference, immune responses
of all echinoderms may be similar. The immune system is mediated by several types
of coelomocytes that are also useful as sensors of environmental stresses. There
are a number of large gene families in the purple sea urchin genome that function
in immunity and of which at least one appears to employ novel approaches for
sequence diversication. Echinoderms have a simpler complement system, a large
set of lectin genes and a number of antimicrobial peptides. Proling the immune
genes expressed by coelomocytes and the proteins in the coelomic uid provide
detailed information about immune functions in the sea urchin. The importance
of echinoderms in maintaining marine ecosystem stability and the disastrous
effects of their removal due to disease will require future collaborations between
ecologists and immunologists working towards understanding and preserving
marine habitats.
Invertebrate Immunity, edited by Kenneth Söderhäll.
©2010 Landes Bioscience and Springer Science+Business Media.
261ECHINODERM IMMUNITY
INTRODUCTION
Echinoderms are a phylum of ubiquitous benthic marine invertebrates, found in a
continuous distribution from the intertidal zone to the deepest depths of the ocean explored
to date. They are the sister group to the chordates and are the basal deuterostomes.1
Approximately 7000 extant echinoderm species have been described, falling into ve
classes—echinoids (sea urchins and sand dollars), holothurians (sea cucumbers), asteroids
(sea stars), crinoids (sea lilies and feather stars) and ophiuroids (brittle stars), as well as
a number of extinct classes known from the fossil record. Many members of the phylum
are large and long lived.2
Echinoderms are central players in many benthic ecosystems, variously as herbivores,
carnivores and detritivores. They possess a simple gut which opens into an anus on
the aboral surface, or, for crinoids, on the oral surface of the animal.1 All echinoderms
have basic pentameric radial symmetry in their adult forms and most metamorphose
from bilateral larvae found in the plankton that function as the dispersal stage, feed
and produce the adult rudiment. Adults lack obvious sensory organs or any kind of
head and locomote largely by means of tube feet, which function by means of a water
vascular system unique to the phylum. Activities are coordinated by a simple neural
ring that communicates to ve nerve bundles into the ve ambulacral grooves that run
longitudinally along each radial segment of the body. All echinoderms have some form of
calcied skeleton composed of aragonite, which is more or less extensive in the various
classes. At one extreme, echinoids are completely enclosed in a calcied test, with only
a thin layer of muscular tissue lining the interior of the body cavity (or coelom) while
holothurians possess a highly muscularized body wall in which the exoskeleton is reduced
to microscopic ossicles dispersed throughout the dermis. The coelomic cavity contains the
gonads and gut of the animal, bathed in coelomic uid. This coelomic uid is key to the
immunological capabilities of echinoderms, being the medium in which the immunocytes
or coelomocytes reside, and which also contains antimicrobial molecules.
A larval sea star was rst used by Metchnikoff 3 to demonstrate that invertebrate
immune cells could recognize the presence of and respond to (encapsulate) a rose prickle.
Subsequent characterization of the echinoderm immune system in adults relied on allograft
rejection assays in sea cucumbers, sea stars,4,5 and sea urchins,6‑8 and demonstrated the
ability of echinoderms to recognize self from nonself. Later evaluation of the sea urchin
graft rejection kinetics illustrated the innate characteristics of echinoderm immunity.9
Although the immune system is entirely innate, it has recently been found to be highly
complex and sophisticated10,11 and is clearly effective given that echinoderms have
survived as a phylum for 450‑500 million years and that individual echinoderms can
survive the constant assault by pathogens in the marine environment (reviewed by ref. 12)
for upwards of 100 years.2
COELOMOCYTES, THE IMMUNE MEDIATORS IN ECHINODERMS
The body cavity of echinoderms is lled with coelomic uid,13 which bathes the
internal organs and forms the uid medium in which the coelomocytes are suspended.
The composition of coelomic uid (CF) is similar to sea water in terms of minor dissolved
salts and other minerals and contains proteins, the best‑characterized of which are those
involved in antipathogen responses. Indeed, whole CF (wCF) is a complex tissue that
262 INVERTEBRATE IMMUNITY
mediates responses to wounding and microbial infections by undergoing reactions such
as opsonization, coagulation, encapsulation and phagocytosis. Initial publications on
echinoderm coelomocytes appeared in the late 19th century (e.g., ref. 14) and since then
many reports have appeared describing the morphologies and functions of the various
coelomocytes9,15‑17 (reviewed by refs. 18,19). Observations of live cells in CF from sea
urchins suggest three basic categories of coelomocytes; phagocytes, spherule cells (also
called amoebocytes,20,21 or morula cells) and vibratile cells (Fig. 1); however further
analyses suggest additional categories (Table 1) (reviewed in ref. 22). Within the spherule
cell category, there are red and colorless spherules within the cells (Fig. 1B,C) and there
are three different categories of phagocytes depending on cytoskeletal morphology and
size (Fig. 2). Additional cell types have been noted in nonechinoid species including
crystal cells, fusiform cells and progenitor cells, about which very little is known. The
descriptions below refer to sea urchin coelomocytes, which are the best studied.
Phagocytes
Phagocytes have been variously referred to as leukocytes, or as bladder, petaloid or
loform phagocytes in the literature and constitute the most abundant type of coelomocyte
(Table 1) in the coelomic uid. In suspension the cells appear in a petaloid form (Fig. 1A;
Figure 1. Live coelomocytes from the sea urchin, Paracentrotus lividus. A) petaloid phagocyte.
B) red spherule cell. C) colorless spherule cell. D) vibratile cell. Scale bar = 5 microns. Images taken
by R. Bonaventura.
263ECHINODERM IMMUNIT Y
Figure 2. Coelomocytes from the sea urchin S. droebachiensis. Cells were withdrawn in anticoagulant
and settled onto a glass coverslip. All cell types are shown and labeled with numbers. Large phagocytes;
1 = discoidal phagocyte; 2 = polygonal phagocyte; 3 = red spherule cell; 4 = colorless spherule cell;
5 = vibratile cell (the lower cell has lost the prominent agellum seen in the upper cell). Inset; 6 = small
phagocyte. Bar = 10 microns.
Table 1. Coelomocytes in sea urchins
Cell Type % in Coelomic Fluid Function
Phagocyte Types Total phagocytes Encapsulation, Opsonization,
Type 1—Discoidal cells Sp* 40‑80% Graft rejection, Chemotaxis,
Type 2—Polygonal cells Sd 67% Phagocytosis, Antibacterial
Type 3—Small phagocytes Pl 80% activity, Cellular clotting
Red spherule cells Sp 7‑40% Oxygen transport. Antibacterial
Sd 8% activity from echinochrome A.
Pl 4.7%
Colorless spherule cells Sp 3.7‑25% Cytotoxicity, Clotting?
Sd 6.5%
Pl 7.8%
Vibratile cells Sp 11.9 ‑20% Movement or agitation of
Sd 18.5% coelomic uid? Associated
Pl 7.5% with clotting.
*Sp, Strongylocentrotus purpuratus. Sd, Strongylocentrotus droebachiensis. Pl, Paracentrotus lividus
Information collected from references 7, 16, 17, 19, 22, 24, 28, 33, 34, 38, 39, 50, 95, 222 and 223.
264 INVERTEBRATE IMMUNITY
Fig. 3A) in which numerous cytoplasmic lamellipodia give the impression of the petals
of a ower.23 More recent work indicates that the phagocyte population actually consists
of several different cell types that can be distinguished based on cytoskeletal morphology
and organization, actin‑based motility patterns,24‑27 and differential gene and protein
expression.19,28,29 Phagocytes are most easily distinguished as three morphologies in spread
cells; two types of large phagocytes24‑27 and small phagocytes.28,29 The discoidal‑shaped
phagocytes (Fig. 2) are large phagocytes that display rapid, actin‑based retrograde/
centripetal ow over their entire diameter, and contain a dense cortical actin meshwork
rich in the Arp2/3 complex (Fig. 4C), radial actin bundles that connect the cortex to the
perinuclear region, and a centralized distribution of myosin II (Fig. 4B).25,27 The retrograde/
centripetal ow activity in discoidal cells tends to restrict the distribution of microtubules
(Fig. 4A), organelles (endoplasmic reticulum, mitochondria, lysosomes and granules) and
Figure 3. Phagocytes undergo an inducible petaloid/lamellipodial to lopodial/liform shape change
that is important in the coelomic uid clotting process. This shape change occurs in cells in suspension;
panel (A), petaloid cell; panel (B), lopodial cell. Shape change also occurs when cells are attached
to glass substrate; panel (C), petaloid (lamellipodial) cell; panel (D), lopodial cell. Shape change was
induced by hypotonic shock. Bar = 10 microns; magnications of panels (A‑D) are equivalent.
265ECHINODERM IMMU NIT Y
Figure 4. Large phagocytes of the discoidal (d) and polygonal (p) subpopulations can be differentiated
based on cytoskeletal organization. Actin (panels A‑D) in discoidal cells displays a broad cortical
meshwork containing Arp3 (panel C) that feeds into a radial array of centralized bundles. The
cortical actin and Arp3 meshwork is more limited in polygonal cells (panel C) and cells often contain
prominent actin bundles oriented either radially in discoidal cells (panel C) or parallel to the long axis
in polygonal cells (panels A,D). Both myosin II (panel B) and microtubules (panel A) are perinuclear
in discoidal cells and more widespread in polygonal cells. b integrin expression is restricted to the
polygonal cells (panel D). The significant difference between the structural organization of the
cytoskeletons of the two large phagocytes is best appreciated in TEM images of critical point dried
and rotary shadowed replicas of detergent extracted cells (panel E). Bar = 10 microns; magnifications
of panels A‑D are equivalent.
266 INVERTEBRATE IMMUNITY
associated kinesin motor proteins to the perinuclear region.26 The second type of large
phagocyte is polygonal‑shaped cells (Fig. 2, labeled 2), which display retrograde ow in
a thin rim along the cell margin corresponding to more limited distribution of the Arp2/3
complex‑containing actin meshwork (Fig. 4C). These large cells are integrin‑positive (Fig.
4D) and the interior cytoplasm contains elongate actin bundles associated with myosin
II (Fig. 4A‑D),25,27 as well as a broad array of microtubules (Fig. 4A) and associated
organelles and kinesin (Fig. 4E).26 Furthermore, both cell types undergo signicant shape
changes from a lamellipodial/petaloid to a lopodial form (Fig. 3B; see also ref. 20),
inducible by the calcium‑dependent clotting process, treatment with hypotonic shock23
or calcium ionophore.30
Large phagocytes display phagocytic activity associated with clearance of bacteria,
xenogeneic cells, foreign particles and proteins, both in vivo and in vitro (reviewed
in ref. 18). Phagocytes have also been implicated in encapsulation and graft rejection
responses, as well as cytolytic/cytotoxic reactions,8,31 (reviewed in refs. 18,19). Phagocytes
participate in the cellular clotting process, in which their lopodial form facilitates the
cell‑cell interactions leading to cellular clot formation and retraction.16,23 Based on their
size, preponderance in the CF and their phagocytic and encapsulation functions, the large
phagocytes appear to be the major mediators of cellular immunity in echinoderms.
The third and least common form of phagocyte is the small phagocyte (Fig. 2,
labeled 6)28,29 of which some express the Sp185/333 family of highly diverse immune
response‑related proteins (see below). These cells are characterized by their small size
relative to the large phagocytes, numerous small cytoplasmic granules and lopodial
morphology (Fig. 5). The morphology of small phagocyte lopodia appears different
from that of large phagocytes in that they are less numerous, thicker and often contain
periodic knobs and microtubule bundles (Fig. 5D).29
Spherule Cells
Red spherule cells (Fig. 1B; Fig. 2, labeled 3; Table 1) have been called amoebocytes,
spherulocytes, morula cells, pigment cells, granulocytes and eleocytes in the literature,
and are small cells lled with spherical red granules containing echinochrome A, a
naphthoquinone pigment with antibacterial properties. Red spherule cells have been reported
to move towards bacteria to initiate an encapsulation response and to degranulate in the
presence of bacteria.32‑35 They have been reported to migrate to and form a rim around
the edge of wounds, infections and tissue grafts.8,36,37 When settled onto a substrate, red
spherule cells exhibit dynamic actin‑based, amoeboid mobility, which may account for
their ability to surround wounds and infections. Colorless spherule cells (Fig. 1C; Fig. 2,
labeled 4; Table 1) contain granules and have therefore been referred to as morula cells
(from Latin for ‘mulberry’). Their properties and functions are not well known, however,
a recent study suggests that these cells have potent cytolytic activity that is augmented
by the presence of phagocytes.38
Vibratile Cells
Vibratile cells are round, highly motile, agellated cells that contain large
cytoplasmic granules (Fig. 1D; Fig. 2, labeled 5; Table 1). Exocytosis of these granules
may trigger the initiation of the clotting reaction32,39 (Sacchi and Smith, unpublished),
however this hypothesis will require additional testing.
267ECHINODERM IMMUNITY
Coelomocytes in Other Echinoderms
Asteroids clear their coelomic cavities efciently despite having only two major cell
types, phagocytes and colorless spherule cells.22,40 Rapid activation and differentiation of the
phagocytes into multiple functional cell types upon bacterial infection is likely responsible for
efcient microbial clearance.41 The phagocytes in the sea star, Asterias rubens, are present in
a continuum of small to large cells that change relative abundance upon immune stimulation.
Larger cells become more numerous and exhibit strong phagocytic reactions to bacteria
and the total number of phagocytes triple 24 hrs after challenge.41 Increased numbers of
total coelomocytes have also been noted in response to wounding and hypoxic conditions.42
Swift responses, immune challenges, and the possible differentiation of phagocytes into
cells with augmented phagocytic activity is an interesting approach to infection in sea stars
that have fewer morphotypes of coelomocytes compared to echinoids.
Figure 5. Small phagocytes in living preparations. Small phagocytes (SP) can be identied based on
their relative small size, unusual lopodial morphology and granular cytoplasm (panels A,B). These
cells are clearly labeled by antibodies against the Sp185/333 family of proteins (SP cell in panels C,D),
whereas the petaloid and lopodial discoidal large phagocytes do not stain for Sp185/333. The Sp185/333
labeling of the small phagocytes highlights the atypical nature of their lopodia which are often thick
and contain knobs, not common for lopodia of discoidal phagocytes (left‑hand cell in panel D). Note
that in some small phagocytes the lopodia become very elongate (panels A,C). Bar = 10 microns.
268 INVERTEBRATE IMMUNITY
Holothurians have lymphocytes, morula cells, amoebocytes, crystal cells, fusiform
cells and vibratile cells.22,40 In addition to phagocytosis, holothurian coelomocytes exhibit
brown body formation in response to multicellular parasites. These are pigmented
aggregates of phagocytes and spherule cells that encapsulate parasites that are too
large to be phagocytized.43 Brown bodies in the sea cucumbers, Holothuria polii and
H. tubulosa, are tissue nodules containing entrapped parasites that are rst surrounded
by a brous, electron‑dense, noncellular layer—probably melanin—and then covered by
layers of specialized cells44,45 including subsets of elongated phagocytes44 and spherule
cells.46 Spherule cells likely degranulate to chemically kill and/or degrade the invader,46
while other cells within the brown bodies possess active phenoloxidase resulting in
melanization.44 Brown bodies are progressively eliminated from the animal through
coelo‑rectal canaliculi.44,45,47 Essentially, coelomocytes appear to have two means for
clearing microbes and parasites; phagocytosis and encapsulation.
Crinoids have phagocytes, red spherule cells and colorless spherule cells (or morula
cells), whereas ophiuroids have phagocytes, colorless spherule cells and possibly crystal
and vibratile cells.22,40 Because little work has been done on these classes of echinoderms,
little is known about the functions of coelomocytes in these groups.
CLOT FORMATION
Clotting is mediated by a variety of agglutination factors. It is an important response
to injury in echinoderms functioning to block loss of CF resulting from wounds, and to
sequester pathogens and prevent their invasion throughout the body. A 220 kDa agglutinating
factor thought to be involved with coagulation is secreted from coelomocytes of the sea
cucumber Holothuria polii.48 Coelomocytes from the purple sea urchin express amassin‑1, a 75
kDa, multidomain protein with an olfactomedin domain that is stabilized by multiple disulde
Box 1. Larval immune cells.
While most investigations of echinoderm immunity have been carried out in the
adult, the larvae of indirectly‑developing species possess specialized mesodermal cells
derived from embryonic blastocoelar cells that display a variety of immune behaviors.
The immune activities of the larval immunocytes were recognized at the inception of
cellular immunity by Metchnikoff and his infamous experiment of encapsulation of a
rose prickle by blastodermal cells of a sea star larva.3 This was the rst demonstration of
phagocytosis and encapsulation and lead to a nobel prize. More recently, recognition and
phagocytic behavior of larval immune cells responding to bacteria or yeast injected into
the blastocoel has been demonstrated.218,219 The immune cells in the embryo and larvae are
the blastocoelar cells located in the blastocoel220 and the pigment cells in the ectoderm19
that produce echinochrome A.221 Embryonic and larval immune cells are also known to
express immune factors, including the complement homologue SpC392 and the immune
response genes Sp185/333.10,70,92 Besides immune effector functions, these cells carry
out a number of immune functions including surveillance‑like behavior, wound healing
and the expression of a complex suite of immune recognition, regulatory and effector
genes10 (Rast, unpublished). As more data are accumulated about this simple stage of
the sea urchin life cycle, postgastrula embryos and larvae are becoming an increasingly
valuable model system in which to investigate immunity.
269ECHINODERM IMMUNITY
bonds (Table 2; Table 3;49 Dheilly, Raftos and Nair, unpublished). Amasin‑1 functions in
cellular clot formation through homooligomerization and cross‑linking coelomocytes.50 A
search of the sea urchin genome identied a putative receptor for amassin‑1, colmedin,
which also has an olfactomedin domain and is expressed in coelomocytes.51
Other molecules involved in clotting mechanisms, including transglutaminase
homologues, were identied during an expressed sequence tag (EST) study from gut
tissue of the sea cucumber Holothuria glaberrima.52,53 Many of the genes referenced from
Table 2. ESTs from the purple sea urchin and a sea cucumber
Category Examples
Defense Sp185/333, DD104, DD186, Complement C3, Complement factor B,
Galectin, SpEchinoidin and other C‑type lectins, Thrombospondin,
Amassin, Integrin‑bC, Ferritin, Melanotransferrin, Annexin, Serum
amyloid A, Echinonectin, Fibrinogen‑like protein, Ficolin‑like protein,
Kazal‑type serine proteinase inhibitors
Cytoskeleton Actin, Gelsolin, Colin, Thymosin b, Protein tyrosine kinase‑9, Fascin,
Tubulin, Microtubule‑associated protein
Signal
Transduction
SpTie1/2, Steroid hormone receptor, LPS‑induced TNFa, Protein tyrosine
phosphatase receptor, Adenylyl cyclase, Receptor for activated protein
kinase C, Guanine nucleotide binding protein b, Rho‑ GTPase, Rho‑GDP
dissociation inhibitor, GTPase‑activating protein, FK Binding Protein‑12,
GF14/14‑3‑3
Nuclear
Activities,
Splicing
RNA/DNA binding protein, DNA methyl transferase‑associated protein,
Nonhistone nucleic acid‑binding protein, Splicing factor 30, Paraspeckle
protein, ET putative translation product, Heterogeneous nuclear ribonucleo‑
protein R, PolyA binding protein
Transcription
factors
Steroid hormone receptor, LPS‑induced tumor necrosis factor a, Immediate
early‑response protein, SpRunt, SpNFkB
Protein
Metabolism,
Synthesis,
Degradation
Translation elongation factor 1a,1g, 2, Translation initiation factor, Peptide
chain release factor, Signal sequence receptor, Protein disulde isomerase,
Binding Protein, Heat shock protein, g p96, Presenilin, Proteosome subunit,
Ubiquitin
Molecular
Tra ns por t
Dynein, Kinesin, Vesicle trafcking protein, Coated vesicle membrane pro‑
tein, ER transport protein, Vacuolar protein sorting protein, Rab7, Rab5‑
interacting protein, Sec22, vSNARE, Mannose‑6 phosphate receptor
Endosomal
System
Vac u ola r H +‑ATPase, Cathepsins, Lysozyme, Arylsulfatase
Proliferation,
Apoptosis
Polo‑like kinase, Bax inhibitor‑1, Allograft inammatory factor‑1
Metabolism ATP synthase, Cytochrome C oxidase subunit 1, 4, Cytochrome b, NADH
dehydrogenase subunit 2, 4, 5, Fatty acid desaturase, ATP/ADP translocase,
Vacuolar ATP synthase subunit, Na+/phosphate cotransporter, Na+K+‑trans‑
porter, Citrate synthase, Malate dehydrogenase
From references 49, 52, 53, 88 and 165.
270 INVERTEBRATE IMMUNITY
these EST studies were originally studied in the context of the intestinal regeneration
that occurs after sea cucumbers expel their internal organs as part of an antipredation
response, and then regenerate the entire digestive tract in 30 days. As such, evisceration
and regeneration exposes the coelomic cavity to microorganisms from the surrounding
seawater and the animal’s own enteric microora. Thus, genes related to immune function
and tissue regeneration are upregulated in intestinal tissues undergoing regeneration
and are likely essential for the animal to survive the process.52‑55
KEY MOLECULES IN ECHINODERM IMMUNITY
Proteins with Leucine Rich Repeats
Toll‑Like Receptors
The Toll‑like receptors (TLRs) are best characterized in insect and mammalian
systems, in which they form small gene families of ~10 genes.56,57 These receptors have
an N‑terminal solenoid‑like leucine‑rich repeat (LRR) ectodomain, a transmembrane
region and a cytoplasmic Toll/interleukin 1 receptor (TIR) in the C‑terminus.58 The
genes are often encoded in a single exon. There are more than 200 TLRs in the
Strongylocentrotus purpuratus genome, far more than has been identied in any other
species to date.10 These genes fall into three categories: a large family of more than
200 genes with vertebrate‑like structure; a set of three genes with structure similar
to Drosophila Toll (which differs from that of the vertebrate TLRs and Drosophila
Toll‑959) and a family of ve genes with a short truncated ectodomain structure (Fig. 6).
In vertebrates, the ectodomain consists of canonical leucine‑rich repeats anked by
specialized, cysteine‑rich domains (designated single cysteine cluster, sccTLR). In
Table 3. Proteomic analysis of whole coelomic uid from Strongylocentrotus
purpuratus
Classication Examples
Immune Response Complement components (SpC3, SpBf), Scavenger receptors
cysteine‑rich, Sp185/333
Pathogen Destruction Arylsulfatase and other lysosomal enzymes, a‑2‑macroglobulin
Clotting Amassin, Annexin V, Von Willebrand factor
Metal binding Major yolk protein, Transferrin, Ferritin, Ceruloplasmin
Cytoskeleton Actin, Prolin, Fascin, Colin, Gelsolin, Myosin, Microtu‑
bule‑associated protein, Arp 2/3 complex proteins, Coronin,
Tubulin, a‑actinin, Tetraspannin, Talin, Vinculin, Rab
Cell Adhesion Integrin, NCAM, Selectin, Cadherin, Fibronectin
Signalling Ras
Cytoplasmic Enzymes Oxidative enzymes
From Dheilly, Raftos and Nair, unpublished.
271ECHINODERM IMMUNITY
contrast, the Drosophila Toll has specialized domains that are located in the center of the
ectodomain (multiple cysteine cluster; mccTLR).58 The mccTLRs may be the ancestral
form,10 as mccTLR genes are present throughout eumetazoans, with the exception of
the vertebrate lineage, in which it has apparently been lost. The sea urchin short TLRs
have distant similarity within the TIR domain to the ancient Toll genes.60
The large family of sea urchin TLRs is unusual in both its multiplicity and the apparent
rapid diversication of some of its subfamilies. The ~210 genes in this family can be
divided into seven subfamilies based on phylogenetic analysis of the TIR domain. Some
of these subfamilies are composed of many members that differ primarily within their
ectodomains while the TIR domains show greater conservation. Divergence within the
leucine‑rich repeats takes a number of forms, including point mutations, insertion‑deletions
between LRRs and insertion‑deletions of whole LRR units.10 The extensive diversity of
this class of sea urchin TLRs, along with a relatively large proportion of pseudogenes,
differs from the more conserved picture seen in vertebrate TLR evolution61 and suggests
that this complex family of receptors function fundamentally differently.60
Many of the sea urchin TLR families are expressed most highly in coelomocytes
in addition to gut tissue.10 Expression of sea urchin TLRs is not detected in the embryo
but many families are expressed in the feeding larvae. Thus, expression patterns and
diversity of sea urchin TLRs are consistent with an immune rather than developmental
function. Consistent with this is the fact that extensive investigations of vertebrate TLRs
only show immune functions.
Figure 6. Pattern recognition receptor gene families are signicantly expanded in the purple sea urchin
with respect to other model organisms. The numbers of gene models encoding Toll‑like receptors (TLR),
Nod‑like receptors (NLR), scavenger‑receptor cysteine‑rich (SRCR), peptidoglycan recognition proteins
(PGRP), Gram negative binding proteins (GNBP) and RIG‑I‑like receptors (RLR) for human (H.s.),
mouse (M.m.), purple sea urchin S. purpuratus (S.p.), fruit y Drosophila melanogaster (D.m.) and
nematode Caenorhabditis elegans (C.e.)212 are shown. Given the complexity associated with identifying
SRCR gene models accurately, the number of SRCR domains is shown, with the number of gene models
that contain multiple SRCR domains indicated parenthetically. In the electronic version of this chapter,
colors are used to illustrate protein domains and to emphasize the sea urchin gene model numbers. A
color version of this image is available at www.landesbioscience.com/curie.
272 INVERTEBRATE IMMUNITY
NOD‑Like Receptors
The second family of expanded immune receptors in the sea urchin is the NOD‑like
receptors (NLRs).10 NLRs are cytoplasmic pattern recognition receptors (PRRs) that recognize
a variety of microbial signatures, including LPS, peptidoglycan, dsRNA and agellin.62
Structurally, NLRs are composed of C‑terminal LRRs, a central NACHT domain and
one of several N‑terminal domains that function in protein‑protein interactions. NLRs in
mammals function in the immune response by serving as scaffolding proteins to assemble
protein complexes that lead to the activation of the NFkB and MAPK signalling pathways.
NLRs have also been shown to activate inammatory caspases, including caspase‑1, which
is responsible for the processing of pro‑interleukin‑1b.63 There is a signicant expansion
of the NLR gene family in the S. purpuratus genome, which has over 200 NLR gene
models compared to vertebrates that typically have ~20 NLR genes (Fig. 6).10 NLRs
appear to be restricted to the deuterostome lineage and have not been identied in any
of the sequenced protostome genomes, including Drosophila or Caenorhabditis elegans.
As in vertebrates, the sea urchin NLR genes contain LRRs, a central NACHT domain
and an N‑terminal protein‑protein interaction domain. However, unlike in the vertebrate
system, this N‑terminal domain is most commonly a DEATH domain, another member of
the death‑domain superfamily. A small number of sea urchin NLRs also contain CARD
domains at the N‑terminus, while the PYD domain is absent outside of the vertebrate
lineage. Although many of the NLR gene models encode C‑terminal LRR domains, these
domains are lacking from some sea urchin gene models, which may be due to problems
with accurate computational prediction of gene structure. The sea urchin NLR family
appears to be the result of a sea urchin‑specic gene expansion, which is seemingly more
diverse than the similarly expanded sea urchin TLR family. The extent of this diversity is
likely underrepresented given the incompleteness of the gene models, particularly within
the LRR region. Although the function of the sea urchin NLRs is unknown, it is notable
that they are most highly expressed in the gut,10 and therefore may be involved in managing
gut microora. This mimics the role of the NLR NOD2 protein in mammalian systems,
which when mutated, results in inappropriate inammatory reactions in the gut tissue and
leads to Crohn’s disease.64 The LRR gene families in the sea urchin are greatly expanded
compared to families in vertebrates and insects and it is noteworthy that the LRR families
in amphioxus are also expanded,65 suggesting that these receptors play an important role
in the innate immune functions of these deuterostome invertebrates.
Sp185/333—A DIVERSE FAMILY OF GENES AND PROTEINS EXPRESSED
IN RESPONSE TO IMMUNE CHALLENGE
The Sp185/333 Gene Family
The initial discovery of the Sp185/333 family was the result of an EST analysis of
transcripts that are upregulated in response to LPS challenge (see Box 2).49 The diversity
observed among the Sp185/333 transcripts is intriguing for a putative immune response
repertoire and resulted, in part, from an extraordinarily diverse gene family. The Sp185/333
genes are small with two exons, of which the rst is short and encodes a hydrophobic
leader sequence, while the second encodes the remainder of the highly variable protein.
The Sp185/333 genes are atypical for S. purpuratus in three respects: (1) the second exon
273ECHINODERM IMMUNITY
ranges in size from 771‑1431 base pairs (bp), which is notably larger than the average
exon length (100‑115 bp) as characterized from all gene models in the genome; (2) the
intron is smaller than average (~400 bp, compared to an average intron size of ~750 bp);
and (3) the average S. purpuratus gene has 8.3 exons.11,66 The most interesting aspect of
the Sp185/333 genes is the structure of the second exon. It is composed of contiguous
blocks of sequence called elements that are dened from sequence alignments that
require the insertions of large gaps (Fig. 7).49,66,67 Elements are variably present or absent
in different genes (and transcripts, see Box 2) in recognizable mosaic combinations that
have been called element patterns. Each element is actually a set of sequences that are
similar but not necessarily identical—elements in the genes and transcripts differ by single
nucleotide polymorphisms (SNPs) and small insertions or deletions (indels). Much of
the sequence diversity among the gene results from the element patterns in the second
exon. Although there are only six Sp185/333 genes assembled in the v. 2.1 build of the
S. purpuratus genome, three independent lines of evidence suggest that the gene family
is composed of 40 to 60 paralogous loci: quantitative PCR (qPCR) of genomic DNA,68
statistical estimates based on the frequency with which unique genes were cloned,69 and
an estimate based on results from screening two BAC libraries for Sp185/333 genes
combined with assumptions about gene linkage.70
The Sp185/333 genes have six types of repeats (Types 1‑6)69 in addition to elements
(Fig. 7). Individual genes contain between two and four Type 1 repeats, which are located
tandemly at the 5′ end of the second exon. In contrast, repeat types 2‑6 are arranged as
mixed, interspersed groups in the 3′ half of the second exon. The complexity of these
repeats facilitates multiple sequence alignments, two of which have been analyzed in
detail.66 The “cDNA‑based alignment” (Fig. 7A) was generated using the location of
gaps in the Sp185/333 transcript sequences as a guide.49 Alternatively, because of the
repeats within the sequences, genes (and transcripts) can also be aligned according to
the boundaries of the repeats, which results in the “repeat‑based alignment” (Fig. 7B).
Regardless of the criteria used to align the gene sequences, they are characterized by a
similar diversity in element pattern variation as is observed among the transcripts (see
Box 2). To date, 171 genes have been isolated from four animals, of which 121 have
unique coding sequences and have 33 different element patterns.66 Furthermore, identical
sequences have not been isolated from more than one individual, suggesting a level of
diversity that exceeds the estimated 4‑5% sequence divergence between S. purpuratus
individuals.71 Large, diverse gene families are a common theme in purple sea urchin
immunity (see above) and understanding the mechanisms by which this diversity is
generated and maintained is an important aspect of investigations of sea urchin immunity.
Gene‑Level Diversication through Recombination
Despite the striking number of unique genes that have been isolated to date, the
Sp185/333 sequences are surprisingly similar. Overall, the genes share >88% pairwise
identity. The number of different versions of each element is relatively low.72 The
complexity of the Sp185/333 gene family, therefore, is the result of a mosaic pattern of
these few element sequences distributed among the genes. Analysis of the evolutionary
histories of ve of the elements that were present in all of the genes revealed that each
element evolved independently. This level of incongruence suggests that the extant
Sp185/333 gene family is the result of rapid and recent diversication events.69 It is
peculiar, given this high rate of diversication that pseudogenes have yet to be identied.
274 INVERTEBRATE IMMUNITY
With one exception, all but one of the cloned genes are predicted to encode intact open
reading frames.66 This may reect a bias, however, in the primers that were designed in the
untranslated regions of expressed genes,49 so results do not conrm with certainty to a true
lack of pseudogenes within the genome. It should be noted that, extensive computational
analysis of elements and regions of the intron suggest that element boundaries do not
necessarily serve as “recombination hotspots”, but rather, that recombination appears to
occur throughout the length of the genes.
Figure 7. Two optimal alignments of the Sp185/333 genes. The sequence complexity and repeats within
the Sp185/333 genes facilitate multiple alignments, two of which have been analyzed in detail.66 The
alignments differ in the pattern of elements in the second exon; the rst exon and intron sequence do not
have elements. Phylogenetic analysis of the intron sequences dene ve major types66 (designated a‑e).
The type of intron associated with each element pattern is indicated. A) The “cDNA‑based alignment”
results from the gaps used to optimize alignments of the Sp185/333 ESTs and cDNAs.49,68 This alignment
divides the second exon into 25 elements. The terminal element is differentiated (designated a‑c) based
on the position of the rst of three possible stop codons. The locations of each of the repeat types is
indicated by the colored boxes under the alignments. B) An alternative alignment, the “repeat‑based
alignment”, results from inserting gaps so that the repeats and elements correspond as much as possible.66
This alignment divides the second exon into 27 elements based on the locations of gaps, as well as the
locations of the repeats. The elements in this alignment that correspond to the repeats are color coded
accordingly. The electronic version of this chapter shows the elements and repeats in color. A color
version of this image is available at www.landesbioscience.com/curie.
275ECHINODER M IMM UNITY
The mechanisms that promote this frequent recombination are unknown. However,
there are a few characteristics of the Sp185/333 gene family that may contribute to its
rapid diversication. Amplication of the regions between genes and preliminary analysis
of a sequenced BAC insert with six Sp185/333 genes show that many genes are closely
linked (~3 kb). In addition to the six types of repeats found within the coding regions,
the genes are anked on either side by stretches of di‑ and trinucleotide microsatellites66
(Miller, Buckley and Smith, unpublished). These repeats are closely associated with the
boundaries of two types of large segmental duplications that include the Sp185/333 genes
(Miller, Buckley, Easley and Smith, unpublished). Microsatellites have been associated
with genomic instability and increased recombination frequency73,74 and have been
implicated in mediating recombination of the variable surface glycoprotein (VSG) genes
from Trypanosma brucei75 and plant R genes.76 The genomic organization, high sequence
similarity and repeats within and surrounding the genes likely promote the diversication
of the Sp185/333 gene family through frequent recombination and thereby contributing
to the complex protein repertoire.
Sp185/333 Transcript Editing
Given the diversity of the Sp185/333 gene family, comparison of the gene and
message sequences from individual animals yielded the surprising result that the two
sets of sequences are very different.72 Specically, 148 messages and 53 genes were
isolated from a single animal, of which only ve of the sequences matched identically
to another. Similarly, there was little or no overlap in gene and message sequence in two
other animals. Furthermore, the gene and message repertoires were generally characterized
by different element patterns, such that the predominantly expressed element pattern
following immune challenge was E2, whereas the most common gene element pattern
was D1. Although about half of the messages isolated from immunoquiescent animals
had a truncated E2 element pattern, called E2.1, the SNP that introduced an early stop
codon was never found among the Sp185/333 genes. In fact, no genes were identied
with premature stop codons or indels resulting in frame shifts, which were both common
features of Sp185/333 transcripts.67 When genes and message from individual animals
were compared, the large majority of messages expressed both before and after immune
challenge were the likely product of a few genes.72 Conversely, most of the genes that
comprise the large Sp185/333 gene family were not transcribed. Notably, the pattern of
nucleotide substitutions between the messages and the genes from which they were most
likely transcribed indicated a bias towards transitions, specically a uridine in the message
at a position in which the gene contained a cytidine. This pattern of nucleotide substitution
is consistent with cytidine deaminase activity. A number of cytidine deaminase‑like
molecules have been annotated within the sea urchin genome, but phylogenetic analysis
of these sequences fails to identify homologues of activation‑induced cytidine deaminase
(AID).10 AID, which has only been identied in vertebrates, is involved in class switch
recombination and somatic hypermutation of immunoglobulins in B cells.77 Alternatively,
it is possible that the observed differences between Sp185/333 gene and message sequences
result from low‑delity polymerase activity and the sea urchin genome does contain a
homologue of terminal deoxytransferase and polymerase m (Tdt/Polm).10,78 In higher
vertebrates, this enzyme is also involved in immunoglobulin diversication, as well as
low‑delity DNA replication.79,80
276 INVERTEBRATE IMMUNITY
Two Levels of Diversity
The Sp185/333 gene family is an intriguingly diverse facet of the sea urchin immune
response.66 In response to immune challenge, this gene family is highly expressed and
produces a diverse message repertoire.49,67,68 The genes are believed to diversify through
frequent recombination that does not appear to be limited to element boundaries and may
be mediated by repeats within and anking the coding sequences.69 Given the diversity
within the gene family, it is surprising that a second tier of diversication appears to affect
the message sequences. That the majority of the messages appear to be derived from a
few genes suggests that many of the Sp185/333 genes may be nonexpressed pseudogenes
and serve as a source of sequence diversication to the expressed genes. Alternatively,
it may be that these genes are simply not expressed under the limited immunological
challenges with which the animals have been presented.67,68 Thus, the complex Sp185/333
gene family represents a novel form of invertebrate immunological diversication both
at the genomic and, also, possibly, at the posttranscriptional level.
In addition to diversity that appears to be generated by gene recombination,
duplication, deletion, conversion,69 and mRNA editing,72 the array of Sp185/333 proteins
show unexpectedly greater structural complexity than predicted from the genes and
messages,81 Not only are the arrays of Sp185/333 proteins different among different
individual sea urchins, but the majority of sizes are at least twice as large as predicted
and up to > 200 kDa (Fig. 8).29 The pI values range from 3‑10 although the majority of
isoforms have a pI more acidic than predicted. There are up to 260 discrete isoforms in
Figure 8. Over 260 spots for Sp185/333 proteins are present in CF from a single sea urchin. Total
proteins from CF were separated by two dimensional gel electrophoresis and analyzed by Western blot
with anti‑185 antisera. The image is a composite of different regions of the blot that received different
exposure times to optimize the spot intensities. pI units are shown at the top and molecular masses
(kDa) are shown to the left. Reproduced from reference 81 with permission, ©2009, The American
Association of Immunologists, Inc.
277ECHINODERM IMMUNITY
individual sea urchin coelomocytes and many of the expressed proteins are truncated,
likely a result of mRNA editing.67,81 Finally, the arrays of Sp185/333 proteins change in
response to different pathogen‑associated molecular patterns (PAMPs), which may result
from a combination of variations in gene expression, mRNA editing and posttranslational
processing of the proteins. How these several levels of diversication are coordinated
and perhaps directed towards different types or species of pathogens will be the focus
of future research.
COMPLEMENT IN ECHINODERMS
The complement system is a system of central importance in immunity for vertebrates
and comprises over 30 known humoral and cell surface proteins.82 Complement activation
occurs by three major pathways: the classical pathway that is activated by antigen‑antibody
interaction, the lectin pathway that is activated by mannose binding lectin (MBL) or colins
and the alternative pathway that is initiated through C3 autohydrolysis (reviewed in ref. 83).
C3, a thioester containing protein, is the central component of the cascade and is activated
by all three initiation pathways. It acts to coat the surfaces of pathogens, functioning as the
initiator of the terminal pathway, an adjuvant for activating the adaptive immune response84
and as an opsonin for direct pathogen recognition, phagocytosis and killing.
Investigations of opsonization and phagocytosis by coelomocytes from the green
sea urchin Strongylocentrotus droebachiensis show that opsonizing target cells with
mammalian C3 enhances the coelomocyte response,39,85‑87 suggesting that coelomocytes
have receptors for mammalian C3 and consequently, sea urchins themselves may express
C3‑like proteins. The identication of two expressed sequence tags (ESTs) from the
purple sea urchin, S. purpuratus with sequence homology to C3 and to factor B (Bf)
called SpC3 and SpBf
88‑90 was the rst evidence that invertebrates have a complement
system. Gene models annotated from the sea urchin genome reports several gene models
encoding thioester proteins, including a second C3 homologue, Sp‑C3‑2, several factor B
genes, MBL and colins (Table 4) (Rast, unpublished).10
SpC3
SpC3 has several conserved functional regions that are also present in other members
of the thioester protein family, including a conserved thioester site, a histidine that regulates
binding specicity, a putative C3‑convertase site, a two‑chain structure putatively disulde
bonded via cysteines in conserved positions, putative factor I cleavage sites and other
conserved regions.90 Immunoquiescent sea urchins responding to LPS generally show
dramatic increases in the amounts of SpC3 in the CF91 with localization to small transport
vesicles in subpopulations of discoidal phagocytes and polygonal phagocytes.28 Expression
of Sp064, the gene which encodes SpC3, responds to immune challenge in adult animals
and also in embryos cultured in the presence of heat‑killed bacteria.92 The function of
SpC3 could be predicted from the deduced amino acid sequence and through comparison
to mammalian C3 functions. The characteristics of the thioester, which forms covalent
bonds with target molecules that lack protection against complement attack (i.e., microbial
surfaces), suggests that SpC3 functions as an opsonin. Classic assays demonstrate that
SpC3 binds methylamine, a small nucleophile that interacts covalently with thioester.93
Furthermore, not only does SpC3 undergo autolysis, a reaction that, under appropriate
278 INVERTEBRATE IMMUNITY
conditions, results in the cleavage of the peptide bond between glutamic acid and glutamine
within the thioester (reviewed in ref. 94), but methylamine binding blocks SpC3 autolysis.93
When CF containing SpC3 is incubated with yeast, SpC3 can be detected on the yeast
surface and augmented phagocytosis of the yeast by coelomocytes is inhibited by addition
of anti‑SpC3 antibody.95
SpBf
The deduced amino acid sequence and domain structure of SpBf show signicant
similarity to the vertebrate Bf/C2 family of proteins.89 SpBf is a mosaic protein with
short consensus repeat (SCR) domains, a Von Willebrand Factor (vWF) domain and
a serine protease domain. SpBf has a conserved cleavage site for a putative factor D
that is conserved compared to cleavage sites in other Bf/C2 proteins. Members of the
Bf/C2 family are mosaic proteins and most have three SCRs, although some have more
than three96 including SpBf, which has ve.89 Sequence analysis of these small domains
shows that the rst two may be the result of a duplication event and that SCR4 may be the
result of a recombination between SCR3 and SCR5.89 Furthermore, alternative splicing
produces some mRNAs with three or four SCRs.97 The results suggest redundancies of
SCRs in the SpBf protein and that the predicted function may be conserved in SpBf,
even though additional SCRs are present, as the relative order of the SCRs in SpBf is
maintained.89 The gene encoding SpBf, Sp152, is expressed in the phagocyte fraction of
coelomocytes with low levels detected in ovary, testes, gut and esophagus,97 although it
is not clear whether expression in tissues is actually due to coelomocytes present in the
tissues rather than the tissue cells themselves. Unlike Sp064 expression, Sp152 expression
is not induced by LPS and appears to be constitutive.
Table 4. Complement proteins in the sea urchin, Strongylocentrotus purpuratus
Predicted
Gene Model or cDNA Encoded Protein Pathway
Sp‑064 SpC3 Alternative
Sp‑C3‑2 SpC3‑2 Alternative?
Sp‑TCP1, Sp‑TCP2 Thioester containing proteins ?
Sp‑thioester containing Thioester containing proteins ?
protein‑1, ‑2, ‑3/4
Sp‑factor B SpBf Alter native
Sp‑factor B‑2, ‑3 SpBf‑2, SpBf‑3 Alter native?
SpSM30‑F Mannose‑binding protein Lectin
Sp ‑ C1q‑lik e (4*) SpC1q Lectin
Sp‑MACPF (21*) Perforin‑like proteins ?
Sp‑CD59, Sca2‑like1, 2 CD59 Regulatory
Sp5 SpCRL Regulatory?
Sp5013 SpCRS Regulatory?
*Numbers of gene models.
From references 10 and 124.
279ECHINODERM IMMUNITY
Complement Phylogeny and Evolution
The identication of a C3 homologue in the purple sea urchin88 inspired searches
for complement components in a wide range of invertebrates. Phylogenetic analysis of
thioester proteins including homologues of C3 with a‑2‑macroglobulin homologues as
the outgroup shows that vertebrate C3, C4 and C5 cluster into three well supported clades
(Fig. 9). The invertebrate C3 homologues cluster in three paraphyletic clades at the base
of the vertebrate complement clades. SpC3 clusters with other invertebrate C3 sequences,
while the tunicate sequences form a chordate cluster with the vertebrate sequences (see
also ref. 98). The structure of the tree suggests that the thioester complement proteins
diverged after the separation of the major phyla. Phylogenetic analyses of members
of the Bf/C2 family show that the sea urchin homologue, SpBf, is positioned near the
base of the tree89 with cnidarian Bf/C2 being more ancient.98 A number of analyses of
the complement family of proteins have suggested that they evolved from a restricted
set of primordial genes99,100 (reviewed in refs. 101, 102). Conserved sequence motifs,
such as the thioester site (GCGEQ) and similar organization of domains suggest that the
ancestral complement system may have included a thioester protein, a Bf/C2 protein and
a mannose binding lectin. These are all present in a number of invertebrates including
the purple sea urchin (Table 4).
LECTINS
Lectins are a large and heterogeneous group of proteins and glycoproteins present in
plants, microorganisms and animals, that function to bind mono‑ and disaccharides.103,104
Lectins can be soluble proteins or integral membrane proteins that often exist as oligomers,
contain at least two carbohydrate recognition domains (CRDs) and are capable of
agglutinating cells and/or precipitating glycoconjugates.105 In immunity, lectins are key
molecules that function in cell‑cell interactions, self/nonself discrimination and interactions
between cells and the extracellular matrix (ECM), among other functions.106 Lectins in
invertebrates fall into four major groups based on similarity of structure to vertebrate
lectins. C‑type lectins are the most common type in various invertebrates107‑110 and consist
of both soluble and integral membrane proteins that require divalent cations (Ca2+) to
maintain the CRD structure to bind carbohydrates. A second major group of invertebrate
lectins are S‑type lectins that specically bind b‑galactosyl residues,111,112 S‑type lectins
are predominantly intracellular and employ free thiols for binding carbohydrates. A third
major group of invertebrate lectins resemble vertebrate pentraxins and share properties
with C‑reactive protein and serum amyloid protein.113‑115 Finally, a fourth group of
invertebrate lectins includes all those that cannot be otherwise categorized based on lack
of information on their primary structure.
Innate immune functions performed by lectins include recognition and specic
binding of microbial surface carbohydrates through the CRDs,116 which exhibit seven
different structural folding patterns for binding to different carbohydrate motifs.117 Both
mannose‑binding lectin (MBL) and colins have been identied in the sea urchin genome10
and a homologue of MBL has been characterized in a sea cucumber, Apostichopus
japonicus.118 It is speculated that these homologues may initiate the lectin pathway of
complement in echinoderms (Table 4). MBLs and colins selectively bind mannose,
280 INVERTEBRATE IMMUNITY
Figure 9. Please see the gure legend on the following page.
281ECHINODER M IMM UNI TY
Figure 9, viewed on previous page. The phylogenetic relationships among members of the thioester
protein family. Amino acid sequences for the thioester family were obtained from GenBank. The
alignment was done in T‑coffee213 and manual editing was done with Mesquite.214 A consensus tree
was constructed in PAUP*215 using maximum parsimony. Bootstrap support was generated with 10,000
iterations. Similar cladogram results were obtained by the maximum parsimony method in PAUP*,
Neighbour‑joining distance method in PAUP* and the Bayesian method in Mr. Bayes.216 The Bayesian
method was used with default priors and the GTR+G+I as the nucleotide substitution model suggested
by jModelTest.217 The a2macroglobulin sequence from horseshoe crab (Limulus polyphemus) was
chosen as the outgroup. Sp‑C3, Strongylocentrotus purpuratus, NP_999686; Hs‑C3, Homo sapiens,
AAR89906; Rn‑C3, Rattus norvegicus, NP_058690.2; Me‑C3, Macropus eugenii, AAW69835; Gg‑C3,
Gallus gallus, NP_990736; Xl‑C3, Xenopus laevis, AAB60608; Lj‑C3, Lethenteron japonicum,
AAR13241; Ej‑C3, Entosphenus japonicus, Q00685; Bb‑C3, Branchiostoma belcheri, BAB47146;
Cr‑C3, Carcinoscorpius rotundicauda, AAQ08323; Se‑C3, Swiftia exserta, AAN86548; Hr‑C3,
Halocynthia roretzi, BAA75069; Ci‑C3, Ciona intestinalis, Q8WPD8; Eb‑C3, Eptatretus burgeri,
CAA77677; Om‑C3, Oncorhynchus mykiss, I51339; Cc‑C3, Cyprinus carpio, BAA36618; Es‑C3,
Euprymna scolopes, ACF04700; Vd‑C3, Venerupis decussates, ACN37845; Gg‑C4, Gallus gallus,
T28153; Xl‑C4, Xenopus laevis, BAA11188, Hs‑C4, Homo sapiens, AAB67980; Cc‑C4, Cyprinus
carpio, BAB03284; Mm‑C4, Mus musculus, CAA28936; Ts‑C4, Triakis scyllium, BAC82347; Hs‑C5,
Homo sapiens, AAI13739; Mm‑C5, Mus musculus, P06684 ; Gg‑C5, Gallus gallus, XP_415405;
Om‑C5, Oncorhynchus mykiss, AAK82852 ; Lp‑A2M, Limulus polyphemus, BAA19844; Lj‑A2M,
Lethenteron japonicum, BAA02762; Pt‑A2M, Pan troglodytes, XP_001139559; Xl‑A2M, Xenopus
laevis, AAY98517; Cc‑A2M, Cyprinus carpio, BAA85038; Cf‑a2M, Chlamys farreri, AAR39412;
Ci‑A2M, Ciona intestinalis, NP_001027688; Hs‑a2M, Homo sapiens, P01023.
fucose and other specic carbohydrates present exclusively on the surface of pathogens
resulting in the direct activation of complement.119‑122
Lectins were rst demonstrated in echinoderms in the early 1980s.123 Lectins have
been identied in asteroids, echinoids and holothurians and characterized essentially
from a functional and biochemical point of view. With improved molecular methods
greater numbers of lectins and proteins with putative lectin function have been identied.
Preliminary analysis of the sea urchin genome shows gene models encoding more than
100 small C‑type lectins, over 400 mosaic proteins with lectin domains, 34 galectins,
in addition to a few pentraxins and fucolectins124 (Cohen and Smith, unpublished). This
indicates that lectins in general are likely to have a variety of important functions in
echinoderms, including recognition of foreign cells. Many, but not all of the lectins that
have been identied in echinoderms have been C‑type lectins (Table 5). Many show
opsonin and agglutinin functions with the capability of binding carbohydrates on the
surface of pathogens. It is generally accepted that lectins in echinoderms play an important
role in the immune system, functioning as key molecules in immune responsiveness and
to augment coelomocyte functions in host defense.
SCAVENGER RECEPTORS
Another expanded gene family in the sea urchin genome encodes a large repertoire of
scavenger receptors containing multiple scavenger‑receptor cysteine‑rich (SRCR) domains
that are both membrane‑bound and secreted proteins.125,126 Receptors of this structure are
found throughout the animal kingdom but the sea urchin genome encodes more than 1000
SRCR domains in ~180 gene models, which greatly exceeds the multiplicity of these genes
in other characterized species (Fig. 6).10 These proteins are known to act as phagocytic
receptors and some family members in mammals have been shown to bind bacteria.127
These receptors are highly polymorphic in the population of purple sea urchins and show
282 INVERTEBRATE IMMUNITY
Table 5. Echinoderm lectins
Class Species Name Structure Specicity Agglutination Reference
Asteroidea Asterina pectinifera (‑) C‑type lectin a‑N‑acetyl‑
galactosamine
yes 224
Oreaster reticulatus (‑) C‑type lectin galactosyl (‑) 225
Echinoidea Strongylocentrotus purpuratus SpEchinoidin C‑type lectin galactose and derivates (‑) 88,124
Anthocidaris crassispina Echinoidin C‑type lectin N‑acetyl‑galactosamine yes 108
SUEL unique D‑galactoside (‑) 123
Lytechinus variegatus Echinonectin (‑) galactoside (‑) 226
Paracentrotus lividus (‑) C‑type lectin (‑) yes 146
PlSL (‑) D‑glucose, L‑rhamnose yes 227
D‑arabinose, L‑fucose
N‑acetyl‑D‑glucosamine
Toxopneustes pileolus SUL‑I (‑) D‑galactose D‑fucose (‑) 228
SUL‑II (‑) D‑galactose (‑) 228
Tripneustes gratilla TGL‑I C‑type lectin (‑) (‑) 228
continued on next page
283ECHINODERM I MMU NITY
Table 5. Continued
Class Species Name Structure Specicity Agglutination Reference
Holothuroidea Apostichopus japonicus SJL‑I (‑) N‑acetyl‑D‑galac‑
tosamine
yes 229
SJL‑II (‑) simple carbohydrates yes 229
SPL‑I C‑type lectin D‑glucuronic acid,
D‑galacturonic acid
(‑) 230
SPL‑II C‑ty pe lectin D‑galactosamine,
D‑galactose
(‑) 230
MLB‑AJ C‑t ype lectin a‑D‑mannans yes 118
Cucumaria echinata CEL‑I C‑type lectin N‑acetyl‑galactosamine yes 231
CEL‑II C‑t ype lectin (‑) no 232
CEL‑III C‑type lectin (‑) yes 231
CEL‑IV C‑type lectin N‑acetyl‑galactosamine,
a‑galactose
yes 232
Cucumaria japonica (‑) (‑) branched a‑D‑mannans yes 233
Holoturia scabra HSL (‑) galctose derivates,
T‑a n t ige n
yes 234
(‑), not known, not done, not named.
284 INVERTEBRATE IMMUNITY
pronounced variability in coelomocyte expression among individual animals suggesting a
complex expression control system.125 It is notable that representatives of all three of the
most expanded families of sea urchin receptors (TLR, NLR and SRCR receptors) form a
coregulated immune circuit that, in mammals, functions in gut immunity.128
ANTIMICROBIAL PEPTIDES
Antimicrobial peptides (AMPs) have been identied in a wide variety of species
including bacteria, fungi, plants, insects, tunicates, amphibians, birds, sh and
mammals.129‑132 Since the discovery of cecropins in insects133 and defensins in mammals,134
more than 1,200 different eukaryotic AMPs have been characterized.135 In eukaryotes they
form the rst line of host defense against pathogenic infections and are a key component of
the innate immune system. AMPs have an enormous variety of sequences and structures,
but certain features are common. Most have a net positive charge and are 12‑50 amino
acids long, of which approximately half are hydrophobic.136,137 However, a few peptides
of up to 100 residues are also recognized as AMPs.138
Early work to document antimicrobial activities of crude extracts from echinoderms
showed a wide range of activities against bacterial and fungal isolates45,139 of which
some functioned as antifoulants to deter the settlement of barnacle and bryozoan
larvae in addition to bacterial colonization.140 More recently, a variety of molecules
with antimicrobial properties have been isolated from echinoderms, including steroidal
glycosides,141‑143 polyhydroxylated sterols,141 naphthoquinone pigments such as
echinochrome A,33,144 and complement homologues.89,90 Lysozymes with antibacterial
activity have also been detected,145‑147 and concentrations up to 15 mg/ml have been
found in red spherule cells of the sea urchin Paracentrotus lividus.34 Lysozyme and other
antibacterial factors may act synergistically to provide effective defense against bacterial
infections. In the Strongylocentrotids, the antimicrobial pigment echinochrome A is
present in vesicles of red spherule cells and is bound to uncharacterized coelomocyte
proteins.148 A semi‑puried coelomocyte fraction from Paracentrotus lividus, included
fragments of beta‑thymosin that were proposed to have antibacterial activity.149 In
extracts from coelomocytes of the sea star Asterias rubens, a number of partial peptide
sequences were obtained and identied as fragments of actin, histone H2A and lamin
A.150,151 Antibacterial activity was detected in extracts of several tissues from the green
sea urchin Strongylocentrotus droebachiensis, the common sea star Asterias rubens,
and the sea cucumber Cucumaria frondosa,152 with most activity in coelomocytes and
body wall.
Strongylocins
Scans of the gene models in the purple sea urchin genome did not detect any
sequences encoding recognizable AMPs.10 This may be due to short exons that are
difcult to recognize computationally, but may also be due to the divergent nature of
these small proteins. However, two cysteine‑rich AMPs, called strongylocins, have
recently been isolated and characterized from the green sea urchin, S. droebachiensis.153
Homologues are also present in the sister species S. purpuratus, called SpStrongylocins.154
The strongylocin peptides are members of the cysteine‑rich AMP family, which have
six cysteines with three disulde bonds involved in peptide conformation, stabilization
285ECHINODERM I MMU NITY
and resistance to proteases,155 and which are crucial for the antimicrobial activity.156,157
The strongylocins have a novel cysteine pattern (Table 6) suggesting a different
conformation than the other members of the group, perhaps to resist proteolysis within
the coelomocytes and in the CF. Both the native and recombinant peptides show
antibacterial activity against both Gram‑positive and Gram‑negative bacteria (Table 7).
In addition, other peptides have been isolated and characterized from S. droebachiensis
that appear as heterodimers and have strong activity against Gram‑positive and
Gram‑negative bacteria (Stensvåg, unpublished). The heavy chain from one of the
heterodimeric peptides also has strong activity against fungi and yeast.
The strongylocins are composed of three regions: a signal peptide, a prosequence
and the mature peptide, and the strongylocin 1 peptides show high amino acid sequence
similarity throughout (Fig. 10). However, SpStrongylocin 2 shares an identical
signal peptide with strongylocin 1, instead of strongylocin 2. The prosequences are
negatively charged, which may act to neutralize and stabilize the positive charge of
the mature peptide153,154 and presumably function as an intracellular steric chaperone
during folding.158‑160 The peptides become active after the prosequences are cleaved
during maturation. The rst amino acid in the mature peptide of strongylocin 2 from S.
droebachiensis is a tryptophan which is likely brominated. Although the recombinant
SpStrongylocin 2 is not brominated, it shows equivalent antimicrobial activity to native
peptides. Therefore, the posttranslational modication of tryptophan may affect the
properties of the peptides by enhancing stability rather than mediating antimicrobial
activity. The site of strongylocin activity is likely to be intracellular based on membrane
integrity assays.154
Nonechinoids also synthesize a range of AMPs, of which many have been
characterized. For example, the antibacterial activity in the CF of the orange‑footed sea
cucumber, Cucumaria frondosa, has been traced to small peptides (<6 kDa) that appear
to be active at low pH (5.0‑6.5) and which may be similar to the clavanins found in
solitary tunicates.161 Other immune‑active chemical compounds with roles in maintaining
antiseptic environments in nonechinoids include saponins and saponin‑like compounds
in sea stars and brittle stars, which are active against some Gram‑positive bacteria.141 Sea
stars and brittle stars in particular express steroidal glycosides that exhibit antifungal
activity and toxicity against brine shrimp.162‑164 It is likely that multitudes of molecules
Table 6. Cysteine patterns in AMPs containing six cysteines
Peptide Family Cysteine Patterna Group of Organism
Strongylocins C – C – C – CC – C Echinoderms
Beta‑defensins C – C – C – C – CC Mammals, birds
Alpha‑defensins C – C – C – C – CC Mammals
Tachystatins C – C – CC – C – C Horseshoe crab
Knottin‑type AMPs C – C – CC – C – C Plants
Thionins Type III and IV AMPs CC – C – C – C – C Plants
Insect defensins C – C – C – C – C – C Insects
Mytilus defensin C – C – C – C – C – C Molluscs
aAdjacent double cysteine residues are highlighted. Information regarding cysteine ar rangements in
the different peptides was obtained from the Antimicrobial Peptide Database.135
286 INVERTEBRATE IMMUNITY
Table 7. Susceptibility of bacteria to strongylocins, recombinant SpStrongylocins and dimeric peptides from Strongylocentrotus droebachiensis
and S. purpuratus
Peptide
Minimal Inhibitory Concentration (mM)
Listonella
anguillarum Escherichia coli
Corynebacterium
glutamicum
Staphylococcus
aureus
Penicillium
rogveforti
Candida
albicans
Strongylocin 1a2.5 5.0 2.5 2.5 NtcNt
Strongylocin 2a1. 3 5.0 2.5 2.5 Nt Nt
Recombinant
SpStrongylocin 1b15.0 7. 5 7. 5 15.0 Nt Nt
Recombinant
SpStrongylocin 2b15.0 7.5 3.8 15.0 Nt Nt
Dimeric peptide 1a2.5 1.3 1. 3 2.5 6.25d6.25d
Dimeric peptide 2a2.5 2.5 1.3 5.0 Nt Nt
aMinimal inhibitory concentration was determined as the lowest concent ration of peptide causing an optical density less than 50% of the growth control without
any peptide present.
bMinimal inhibitory concentration was determined as the lowest concent ration of peptide causing 100% growth inhibition of the test organism compared to
the growth cont rol.
cNt = Not tested.
dThe peptide is the heavy chain of dimeric peptide 1. Growth inhibition was determined microscopically af ter 48 hrs of incubation.
287ECHINODERM I MMU NIT Y
Figure 10. Alignment of strongylocins from S. droebachiensis and SpStrongylocins from S. purpuratus. The predicted cleavage site between the signal peptide
and the proregion is indicated with a ▼. The rst amino acid in active strongylocin 2 and SpStrongylocin 2 are likely a modied tryptophan, as indicated with
a ♦. Identical amino acids are shown in boxes, similar amino acids are shown in gray and cysteines are highlighted in black. A color version of this image is
available at www.landesbioscience.com/curie.
288 INVERTEBRATE IMMUNITY
with activity against all types of pathogens function efciently in echinoderms and
have central importance for immune functions in this group.
GENE EXPRESSION IN SEA URCHIN COELOMOCYTES
Before it was possible to scan for gene models encoding proteins with putative immune
function in the sea urchin genome, the only feasible approach for understanding the echinoderm
molecular immunology was through EST proling of messages in coelomocytes under
challenged vs nonchallenged conditions.49,88,165 ESTs encoding proteins with putative immune
function have also been reported for the sea cucumber Holothuria glaberrima, undergoing
gut regeneration after evisceration.52,53 A variety of categories of proteins are expressed in
coelomocytes that illustrate the activities of these cells in an assortment of functions (Table
2). The main focus of these studies was to identify immune response genes (see Box 2),
which included protein matches suggesting several mechanisms for opsonization, clotting,
iron sequestration plus the activity of a variety of signaling pathways. Matches were found
to proteins with functions in the endosomal system (lysosomes) that may be involved in
killing phagocytosed pathogens. Coelomocytes express a number of genes encoding proteins
involved in splicing transcripts and translating proteins. They process, package, transport and
secrete proteins as suggested from the transcripts encoding proteins involved in trafcking
transport vesicles. Phagocytes have extensive cytoskeletons (as illustrated in Figs. 1‑5),
which is borne out by the number of ESTs matching proteins that function as cytoskeletal
elements and others that act to modulate the cytoskeleton. One of the benets of generating
Box 2. EST studies identify immune‑related transcripts; discovery of the Sp185/333
sequences.
Differential characterization of ESTs of coelomocytes from Strongylocentrotus
purpuratus before and after immune challenge provide a picture of global changes in
transcriptional activity. Genes encoding complement homologues,88‑90 transcription fac‑
tors,126 and a lectin,67,88 among many others are induced by immune challenge (Table 6).
Signicantly upregulated transcripts include a large and diverse set of novel transcripts
designated Sp185/333 that were rst discovered with the use of differential display165
and subtracted probes followed by EST analysis.49 Probes representing transcripts from
LPS‑activated coelomocytes were used to screen a high‑density arrayed, conventional
cDNA library made from bacterially‑activated coelomocytes. About 4.5% of the clones
in the library were positive and about 60% of clones selected for EST analysis showed
signicant sequence similarity to two previously uncharacterized cDNAs: DD185 and
EST333.70,88,126 Given the considerable number of related Sp185/333 sequences, the
coding regions of the ESTs could be aligned. However, optimization of the alignment
required the insertion of multiple, large articial gaps49 that dened 25 to 27 blocks of
shared sequence called elements, which are variably present or absent in element pat‑
terns (Fig. 7).66,67 The extraordinary diversity of the Sp185/333 transcripts is based on
the mosaic element patterns in addition to extensive single nucleotide polymorphisms
(SNPs). The induction of Sp185/333 gene expression in response to LPS and other
pathogen‑associated molecular patterns (PAMPs)67,165 plus the diversity of the sequences
suggest immune‑related functions for the encoded proteins in S.purpuratus.
289ECHINODERM I MMU NIT Y
ESTs is that the level of expression rather than the level of match to known sequences can
sometimes provide clues to putative immune function for unknown sequences. This has
been the case for the Sp185/333 sequences that are not known outside of the echinoids, but
for which there is strong evidence of immune function.70
PROTEIN EXPRESSION PROFILE OF SEA URCHIN COELOMOCYTES
In addition to genome scans and EST analyses, high throughput methods in
proteomics are being applied to analyzing proteins in echinoderms. In response to
LPS challenge, 319 proteins were identied in the wCF of the purple sea urchin,
Strongylocentrotus purpuratus, of which 284 were encoded by gene models in the
genome and 48 were encoded by hypothetical open reading frames (Table 3) (Dheilly,
Raftos and Nair, unpublished). The majority of the proteins were involved in modulating
the cytoskeleton and linkage between the cytoskeleton and cell adhesion molecules, all
of which are essential for intracellular transport and behaviors such as locomotion and
phagocytosis. Cell adhesion molecules and intracellular signaling proteins were also
identied, suggesting that coelomocytes respond to LPS with large‑scale alterations to
the cytoskeleton. Furthermore, the response to immune challenge implies the secretion
of cellular products, as well as the endocytosis of extracellular uids and phagocytosis
of pathogens. Proteins involved in clotting and coagulation suggest another mechanism
to sequester pathogens and to clear them from the CF, in addition to the prevention
of CF loss after trauma. A number of proteins involved in opsonization are present
in wCF including complement homologues. Metal‑binding proteins are present,
including ferritin, which has been identied in several echinoderm species under
various conditions and using different analytical approaches.49,52,88,166 Consequently,
sequestration of iron must be an important mechanism for controlling the proliferation
of invading microbes.
Proteomic analyses of coelomocytes from a second sea urchin species, Heliocidaris
erythrogramma, demonstrated different expression proles over time in response
to sterile injections compared to injection of microbes (Dheilly, Raftos and Nair,
unpublished). Results from shotgun proteomics showed two different proles of
proteomic changes. The proteins identied in the response to bacterial challenge
were different from those identied in the controls. The proteomic prole changed in
the coelomocytes from bacterially challenged animals within the rst 6 hrs, largely
due to the increased abundance of some proteins involved in cytoskeletal dynamics,
while others decreased such as F‑actin capping protein, advillin and a‑actinin. Other
cytoskeletal proteins that were absent in the controls, such as actins and annexin A7,
were present in the challenged coelomocytes. Other proteins that appeared in the
challenged coelomocytes included the complement homologue, C3 and SRCRs. The
protein expression prole in sea urchin coelomocytes indicates that these cells show
dynamic responses to wounding and immune challenge. Temporal analysis of proteomic
changes in coelomocytes indicates that cellular responses to wounding and infection are
biphasic. The initial phase, occurring within the rst 24 hrs after treatment, appears to
be a generalized response common to both types of insult. This phase involves reactions
including CF coagulation and coelomocyte cytoskeletal remodelling, which resolves
by 48 hrs to control levels. The second phase, which peaks at 48 hrs after injection,
appears to be specic to microbial infections and the proteins expressed in this phase
290 INVERTEBRATE IMMUNITY
are involved in pathogen recognition and opsonization as well as the destruction of the
invading microbes. A number of attendant sub‑cellular pathways involved in signal
transduction, endocytosis and exocytosis are also enhanced during this phase. These
two phases thereby function in an integrated manner to repair wounds and to neutralize
microbial infections.
REGENERATION
Many classes of echinoderms are remarkably plastic in their abilities for repair and
regeneration resulting from both proliferation and transdifferentiation of circulating cells
of mesenchymal origin (see Microscopy Research and Technique vol 55 no. 6, 2001). New
evidence suggests that cells required for regeneration originate from coelomocytes.167‑169
Unfortunately, sea urchins have limited regenerative capabilities170 compared to sea stars
and crinoids, which have received more attention.168 Sea stars are generally known for
arm regeneration and levels of heat shock protein 70 (Hsp70) increase in the common
sea star Asterias rubens, following arm tip amputation.169 Furthermore, manganese
treatment induces proliferation of coelomic epithelial cells that is coupled to traumatic
stress responses including increased expression of Hsp70.171 Red spherule cells (or
amoebocytes) and polygonal phagocytes appear to be involved in regeneration.169 These
ndings suggest a role for increased coelomocyte numbers and the expression of classic
stress molecules in the early repair phase of tissue damage and regeneration.
Toposome, which is also referred to as the major yolk protein (MYP), is the most
abundant protein in the CF (Table 3).172 A monoclonal antibody to embryonic toposome
also recognizes sea star coelomocytes and the coelomic epithelium.169,173,174 Although
previous efforts in cloning the MYP gene in Strongylocentrotus purpuratus suggested
its relationship to vitellogenin,175 protein fragments were too short for unambiguous
identication. The cDNA sequences encoding MYP from the sea urchins, Pseudocentrotus
depressus, Paracentrotus lividus and Tripneustus gratilla, show that they are members
of the transferrin family, lack iron‑binding sites, and are not homologous to vertebrate
vitellogenins.173,176 The toposome precursor, which has been postulated to serve multiple
functions, is synthesized exclusively in the gut of the adult animal as a 180‑190 kDa
glycoprotein and the mature protein is found in the CF. The amount of toposome protein
increases in response to traumatic stresses in agreement with increased gene expression
as deduced from ESTs matching toposome in sea cucumbers regenerating gut tissue.177
Toposome is expressed with other genes encoding proteins that function in wound
healing, cell proliferation, differentiation, morphological plasticity, cell survival, stress
response, immune challenge and neoplastic transformation.55 It is emerging that circulating
coelomocytes from some echinoderms originate from coelomic epithelia and are able to
differentiate into a few tissue types, including nerve and muscle cells.178 Accordingly,
research on stem cells in marine organisms is becoming important for both comparative
studies and for future applications.
291ECHINODER M IMM UNI TY
ECOTOXICOLOGY AND THE ECHINODERM IMMUNE SYSTEM
Because coelomocytes are sensitive, stress‑activated effectors of the echinoderm
immune response,19,179 they are good candidate biosensors for monitoring environmental
stress in an environmental management context. Several examples of using
coelomocytes as indicators employ both analyses of specic proteins and of cell
function. Unfractionated coelomocytes from the sea urchin Paracentrotus lividus
cultured at stress‑inducing temperatures, (4°C or 35°C), or exposed to acidic pH, or high
levels of cadmium, express high levels of Hsp70,180,181 a well‑recognized stress marker
induced in response to a wide range of biological and physicochemical stresses.106,182
In addition to serving as molecular chaperones, secreted and membrane‑bound heat
shock proteins (particularly Hsp60, Hsp70, Hsp90 and gp96) are potent activators of
the innate immune system capable of inducing the production of proinammatory
cytokines by the monocyte‑macrophage system.183 Sp‑gp96, which is expressed in
sea urchin coelomocytes88 is present on the surface of coelomocytes.184 High levels of
Hsp70 are also observed in coelomocytes obtained from sea urchins collected from
waters heavily polluted with urban run‑off and industrial waste181 and, more recently,
with the explosive 2,4,6‑trinitrotoluene (TNT) from conventional weapons dumped at
sea at the end of World War II.185 Increased levels of Hsp70 levels in coelomocytes
from specimens of the sea star Asterias rubens, collected along a transect from inland
waters to the open sea along the Norwegian ords correlate with a natural concentration
gradient of heavy metals.186 Increased production of reactive oxygen species (ROS)
by coelomocytes from A. rubens is also observed after cadmium exposure in a
dose‑dependent manner.187‑189 Cadmium, on the other hand, leads to reduced phagocytic
activity by coelomocytes.190 Other heavy metals, including cadmium, as well as UV light
can also result in single strand DNA breaks in coelomocytes.20,191,192 Exposure to lead
increases the phagocytic activity of sea star coelomocytes, though another pollutant,
polychlorinated biphenyls (PCBs), do not have this effect.190 Responses to temperature
stress levels can also be detected in coelomocytes as increases in acetylcholine esterase
(AChE) activity.193 Overall, there are several approaches for monitoring protein levels
and enzyme activities that can be employed as signs of environmental stress both in
eld and in laboratory studies.
Another approach for employing echinoderms as biosensors has been to evaluate
the numbers of coelomocytes in the CF, which can be altered by environmental stressors.
For example, the percentage of red spherule cells increase from 5% to 40% of total
coelomocytes in animals collected from polluted seawaters or that are subjected to accidental
injury.181,185 Similarly, the numbers of total coelomocytes increase in immunoquiescent
sea urchins after simple immune challenge in the lab.29 Therefore, levels of red spherule
cells and perhaps total coelomocytes may be used as a practical marker of environmental
stress in animals collected in costal surveys for marine management.
CONCLUSION
Echinoderms are important members of marine ecosystems and are required for
the stable maintenance of habitats. This has been illustrated by the aftermath of the
disappearance of the long‑spined black sea urchin, Diadema antillarum, the top herbivore
292 INVERTEBRATE IMMUNITY
on Caribbean coral reefs. The population crash in 1983‑1984, with repeat crashes in
1985 and 1991‑1992, reduced the population in the Caribbean and Western Pacic by
95‑99%.194,195,196 General destabilization of the coral reef ecology resulting from herbivore
release that was exacerbated by hurricane damage, lead to a change in the trophic cascade
resulting in a swift and sustained switch to an alternative ecological state; reef cover of
mostly coral switched to mostly soft algae.197‑200 The effect of the disappearance of D.
antillarum was predicted by Sammarco201 and modeling these population changes show
the same outcome.202
The population crash of D. antillarum progressed from west to east in the
Caribbean and was speculated to have been due to a pathogen198 that may also have
been a commensal.203 Disease outbreaks and mass mortalities have also been noted
in the green sea urchin, S. droebachiensis, along the Atlantic coast of Nova Scotia.204
Little is known of the bacterial pathogens of sea urchins205‑209 and much less is known
of the fungal and viral pathogens of any class of echinoderm. This general lack of
knowledge regarding pathogenesis in echinoderms is an important problem that will
require coordinated efforts of both ecologists and immunologists. Understanding
the population dynamics in a complex ecosystem such as coral reefs should include
investigations of the host‑pathogen interactions that are involved in the stability of the
ecosystem or lack thereof.
Until recently, workers interested in the immune system of echinoderms were
hobbled by identifying and analyzing one gene or protein at a time. Cross‑phylum searches
for genes encoding proteins involved in microbial recognition and immune effector
functions were complicated by the rapid pace of immune gene evolution. However, the
availability of the purple sea urchin genome11 has greatly increased the sensitivity with which
immune gene homologues can be identied, in addition to the efcient characterization
of complex multigene families. Immune transcription regulators are generally very well
conserved and BLAST type sequence identity searches are typically adequate to identify
these factors. In the case of many immune receptors and effectors, primary sequence is
poorly conserved but domain structure can be used as a unique identier and combinatorial
domain prole searches can be useful. A nal class of immune mediators is encoded
by genes that have novel structure relative to other phyla. These are typically identied
in experimental surveys, but in some cases multiplicity of domains that are common to
proteins with immune functions can be identied in purely bioinformatic surveys for
candidates of novel immune mediators.
Analysis of the purple sea urchin genome has revealed a complex repertoire of immune
receptors, regulators and effectors unlike those known in other phyla.10,60,78,210 In addition
to the LRR‑containing proteins and the Sp185/333 gene family described above, the
genome encodes a virtually complete set of homologues of (i) vertebrate hematopoietic and
immune transcription factors, (ii) candidate effector proteins and (iii) genes with distant
homology to key adaptive immune mediators of the jawed vertebrates.10 Transcription
regulators of haematopoiesis include a nearly complete set with respect to the vertebrate
homologues and include representatives of some subfamilies such as the PU.1/SpiB/SpiC
Ets factors that are important regulators of vertebrate myeloid and lymphoid immunocyte
development not found outside of deuterostomes.211 Transcription factors involved in
the regulation immune response genes include GATA1/2/3, SCL and NFkB have also
been identied.
Future work on echinoderm immunity will focus on the purple sea urchin, but additional
echinoderm genomes are needed. Preliminary 1X sequence coverage of the genomes for
293ECHINODERM IMMUNITY
S. franciscanus and Allocentrotus fragilis, sister species of S. purpuratus are available
(http://www.hgsc.bcm.tmc.edu/project‑species‑o‑Strongylocentrotus%20purpuratus.
hgsc?pageLocation = Strongylocentrotus purpuratus) and preliminary analyses suggest
a similar repertoire of immune genes. Additional genomes from species from other
classes are needed and will not only promote phylogenetic and comparative evolutionary
genomics, but will enable the characterization of the immune gene repertoire for a different
echinoderm species. This will be of particular interest given that the immune genes and
the mechanisms of immune gene diversication are dictated by the life history, pathogens
and habitat of individual species.
ACKNOWLEDGEMENTS
The authors are grateful to Ms. Margaret Throckmorton for collecting and
formatting the references. This work was supported in part by funding from the US
National Science Foundation (MCB‑0744999) to LCS, the US National Institutes of
Health (GM60925) to JHH, the Australian Research Council (DP0556486) to SVN.
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