Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives.

For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker's yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1-5, 6-10, 11-15, and ≥16), and the phagocytic frequencies were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or ß-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of ß-laminarin-treated granulocytes decreased significantly for zymosan and zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of ß-glucan receptors for phagocytosis.

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galß1-4Glc). MytiLec revealed ß-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.

Characterization of GnRH-like peptides from the nerve ganglia of Yesso scallop, Patinopecten yessoensis.

There is yet no firm experimental evidence that the evolutionary ancient gonadotropin-releasing hormone GnRH (i.e., GnRH1) also acts in invertebrate gametogenesis. The objective of this paper is to characterize candidate invGnRH peptides of Yesso scallop Patinopecten yessoensis (i.e., peptide identification, immunohistochemical localization, and immunoquantification) in order to reveal their bioactive form in bivalves. Using mass spectrometry (MS), we identified two invGnRH (py-GnRH) peptides from the scallop nerve ganglia: a precursor form of py-GnRH peptide (a non-amidated dodecapeptide; py-GnRH12aa-OH) and a mature py-GnRH peptide (an amidated undecapeptide; py-GnRH11aa-NH2). Immunohistochemical staining allowed the localization of both py-GnRH peptides in the neuronal cell bodies and fibers of the cerebral and pedal ganglia (CPG) and the visceral ganglion (VG). We found that the peptides showed a dimorphic distribution pattern. Notably, the broad distribution of mature py-GnRH in neuronal fibers elongating to peripheral organs suggests that it is multi-functional. Time-resolved fluorescent immunoassays (TR-FIA) enabled the quantification of each py-GnRH form in the single CPG or VG tissue obtained from one individual. In addition, we observed greater abundance of mature py-GnRH in VG compared with its level in CPG, suggesting that VG is the main producing organ of mature py-GnRH peptide and that py-GnRH may play a central regulatory role in neurons of scallops. Our study provides evidence, for the first time, for the presence of precursor and mature forms of invGnRH peptides in the nerve ganglia of an invertebrate.

In Vivo Administration of Scallop GnRH-Like Peptide Influences on Gonad Development in the Yesso Scallop, Patinopecten yessoensis.

Existing research on the role of gonadotropin-releasing hormone (GnRH) in bivalve reproduction is inadequate, even though a few bivalve GnRH orthologs have been cloned. The objective of this paper was to elucidate the in vivo effect of GnRH administration in Yesso scallop reproduction. We performed in vivo administration of scallop GnRH (py-GnRH) synthetic peptide into the developing gonad, and analyzed its effect on gonad development for 6 weeks during the reproductive season. The resulting sex ratio in the GnRH administered (GnRH(+)) group might be male biased, whereas the control (GnRH(-)) group had an equal sex ratio throughout the experiment. The gonad index (GI) of males in the GnRH(+) group increased from week 2 to 24.8% at week 6. By contrast the GI of the GnRH(-) group peaked in week 4 at 16.6%. No significant difference was seen in female GI between the GnRH(+) and GnRH(-) groups at any sampling point. Oocyte diameter in the GnRH(+) group remained constant (about 42-45 µm) throughout the experiment, while in the GnRH(-) group it increased from 45 to 68 µm i.e. normal oocyte growth. The number of spermatogonia in the germinal acini of males in the GnRH(+) group increased from week 4 to 6. Hermaphrodites appeared in the GnRH(+) group in weeks 2 and 4. Their gonads contained many apoptotic cells including oocytes. In conclusion, this study suggests that py-GnRH administration could have a potential to accelerate spermatogenesis and cause an inhibitory effect on oocyte growth in scallops.

Roles of extracellular ions and pH in 5-HT-induced sperm motility in marine bivalve.

Factors that inhibit and stimulate the initiation of sperm motility were determined for Manila clam (Ruditapes philippinarum), Pacific oyster (Crassostrea gigas), and Japanese scallop (Patinopecten yessoensis). Compared with artificial seawater (ASW), serotonin (5-hydroxytryptamine creatinine sulfate, 5-HT) could fully trigger sperm motility and increase sperm velocity and motility duration. Sperm motility was decreased in ASW at pH 6.5-7.0 and suppressed at pH 4.0. In Manila clam and Pacific oyster, 5-HT could overcome the inhibitory effects of acidic pH on sperm motility. In the presence of nigericin (a K(+)/H(+) exchanger), sperm motility was only triggered at pH 8.3. Testicular fluid K(+) concentrations were two- to fourfold higher than that in ASW. Sperm motility and velocity were decreased in ASW or 5-HT containing ≥40  mM K(+) or ≥2.5  mM 4-aminopyridine, suggesting K(+) efflux requirement to initiate motility. Sperm motility and velocity were reduced in ASW or 5-HT containing EGTA or W-7, suggesting that extracellular Ca(2)(+) is required for Ca(2)(+)/calmodulin-dependent flagellar beating. Ca(2)(+) influx occurs via Ca(2)(+) channels because sperm motility and velocity were decreased in both ASW and 5-HT containing T-type and L-type Ca(2)(+) channel blockers. 5-HT-dependent initiation of sperm motility was associated with intracellular Ca(2)(+) rise, which was comparable to that seen in ASW but was not observed in the presence of EGTA or a Ca(2)(+) channel blocker. Extracellular Na(+) is also essential for sperm motility initiation via regulation of Na(+)/Ca(2)(+) exchange. Overall, 5-HT-dependent initiation of sperm motility in marine bivalve mollusks is an osmolality-independent mechanism and regulated by extracellular pH, K(+), Ca(2)(+), and Na(+).

Involvement of Pacific oyster CgPGRP-S1S in bacterial recognition, agglutination and granulocyte degranulation.

Peptidoglycan recognition protein (PGRP) recognizes invading bacteria through their peptidoglycans (PGN), a component of the bacterial cell wall. Insect PGRPs contribute to effective immune systems as inducers of other host defense responses, while this function has not been reported from PGRP of bivalves. In this study, recombinant CgPGRP-S1S (rCgPGRP-S1S), produced in the mantle and the gill, was synthesized and used to elucidate the immunological function of CgPGRP-S1S. rCgPGRP-S1S bound specifically to DAP-type PGN and to Escherichia coli cells, but not to other DAP-type PGN-containing bacterial species, Vibrio anguillarum, or Bacillus subtilis. Antibacterial activity was not detected, but E. coli cells were agglutinated. Moreover, in addition to these direct interactions with bacterial cells, rCgPGRP-S1S induced secretion of granular contents by hemocyte degranulation. Taken together, these results suggest for the first time that a PGRP of bivalves is, just as in insects, involved in host defense, not only by direct interaction with bacteria, but also by triggering other defense pathways.

Molecular characterization and expression analysis of chitinase from the Pacific oyster Crassostrea gigas.

Chitinases are necessary enzymes supporting functions as a host defense factor against chitin-coated pathogens. They also function as a digestive enzyme for the hydrolysis of dietary chitin. We conducted characterization and assessed the tissue expression of the encoding gene of a chitinase (EC 3.2.1.14), Cg-Chit1, and the production of recombinant protein of Cg-Chit1, from the Pacific oyster, Crassostrea gigas. Chitinase activity in mantle extracts was detected to a marked degree in samples collected in July and August. Mantle chitinase worked well at pH 5.5, 7.0, and 8.5 tested in this study. RT-PCR showed that Cg-Chit1 expression is highly tissue-specific in the hemocytes and mantle. We then determined the distribution of Cg-Chit1 mRNA in C. gigas hemocytes and mantle histologically using in situ hybridization. Of the two subgroups of oyster hemocytes, granulocytes (main phagocytes) and hyalinocytes, only the former were found to express Cg-Chit1. In the mantle, chitinase-2 was expressed at the inner lobe of the mantle edge. Recombinant Cg-Chit1 clearly showed chitinase activity in a wide range of neutral/basic pH. These findings suggest that Cg-Chit1 functions as a host defense factor to hydrolyze chitin-coated organisms after phagocytosis by granulocytes and to exclude foreign substances from the mantle cavity.

Isolation and functional characterization for oocyte maturation and sperm motility of the oocyte maturation arresting factor from the Japanese scallop, Patinopecten yessoensis.

In bivalves, serotonin (5-hydroxytriptamine, 5-HT) acts as a major promotional factor in oocyte maturation, sperm motility, and sequential spawning. The previously reported novel neuronal protein, oocyte maturation arresting factor (OMAF) that was found in the central nervous system and hemolymph of the Japanese scallop, Patinopecten yessoensis, has an inhibitory activity in the 5-HT-induced oocyte maturation via a receptor-mediated mechanism, resulting in an arrest of spawning [30]. In this study, OMAF protein was isolated from the supernatant of hemolymph of the scallop using gel and anion-exchange chromatography, and SDS-PAGE. Three digested partial peptides with 4, 11, and 16 amino acid residues were determined through reversed-phase HPLC and amino acid sequencing. The anti-OMAF antibodies generated against the obtained peptides with 11 and 16 amino acid residues were applied to immunohistochemistry and 5-HT-induced spawning and oocyte maturation assays. Fusiform OMAF neurons were localized in the external area of the anterior lobe of the cerebral ganglion, supporting our presumption that OMAF was secreted from the cerebral and pedal ganglia (CPG). Pretreatment with anti-OMAF antibody on three kinds of bivalve species showed a strong in vivo amplification of 5-HT-induced release of egg and sperm, and an in vitro restoration of 5-HT-induced germinal vesicle breakdown (GVBD) from inhibition by the CPG extract, suggesting the release from suppressive activity of OMAF due to the absorption with antibody. These results confirm that the isolated peptides are from OMAF and OMAF acts as an inhibitor of 5-HT-induced oocyte maturation and sperm motility as previously reported.

Mollusc gonadotropin-releasing hormone directly regulates gonadal functions: a primitive endocrine system controlling reproduction.

Gonadotropin-releasing hormone (GnRH) is central to the control of vertebrate reproductive cycles and since GnRH orthologs are also present in invertebrates, it is likely that the common ancestor of bilateral animals possessed a GnRH-like peptide. In order to understand the evolutionary and comparative biology of GnRH peptides we cloned the cDNA transcripts of prepro GnRH-like peptides from two species of bivalve molluscs, the Yesso scallop Patinopecten yessoensis and the Pacific oyster Crassostrea gigas. We compared their deduced uncleaved and mature amino acid sequences with those from other invertebrates and vertebrates, and determined their sites of expression and biological activity. The two molluscan GnRH sequences increased the number of known protostome GnRHs to six different forms, indicating the current classification of protostome GnRHs requires further revision. In both molluscs, RT-PCR analysis showed that the genes were highly expressed in nervous tissue with lower levels present in peripheral tissues including the gonads, while immunocytochemistry, using anti-octopus GnRH-like peptide, demonstrated the presence of GnRH-like peptide in neural tissue. Putative scallop GnRH-like peptide stimulated spermatogonial cell division in cultured scallop testis, but the scallop GnRH-like peptide did not stimulate LH release from cultured quail pituitary cells. This is the first report of the cloning of bivalve GnRH-like peptide genes and of molluscan GnRH-like peptides that are biologically active in molluscs, but not in a vertebrate.

Presence and characterization of multiple mantle lysozymes in the Pacific oyster, Crassostrea gigas.

Mantle tissue extracts from the Pacific oyster, Crassostrea gigas, exhibited anti-Gram-positive bacterial and lysozyme activities over a wide pH range, suggesting that multiple defensive mantle lysozymes were present. Degenerated reverse-transcription PCR detected the expression of two mantle lysozymes, CGL-1 and a novel lysozyme CGL-3, confirming the presence of multiple lysozymes in the mantle. Since CGL-3 is a cognate protein of the digestive lysozyme CGL-2, it is assumed that CGL-3 has evolved specifically a defensive function. Functional assays using recombinant CGL-1 and CGL-3 suggested that CGL-1 and CGL-3 play a major defensive role in the mantle tissue, and that they are responsible for lysozyme activity under different pH, ionic strength and temperature conditions. Based on these observations, we conclude that multiple mantle lysozymes in the Pacific oyster are better for host-defense under broader conditions than a single lysozyme.

The role in spawning of a putative serotonin receptor isolated from the germ and ciliary cells of the gonoduct in the gonad of the Japanese scallop, Patinopecten yessoensis.

Serotonin (5-hydroxytriptamin, 5-HT) triggers germinal vesicle breakdown (GVBD) of oocytes and the transporting of the mature oocyte through the gonoduct via cilia motility in bivalves. The 5-HT receptor in the oocyte membrane of the Japanese scallop, Patinopecten yessoensis, has been pharmacologically characterized as a mixed profile of 5-HT(1)/5-HT(2) and is induced by estradiol-17beta (E(2)). Here we report the isolation, cloning, and tissue expression of the 5-HT receptor from the gonad of the Japanese scallop. A full-length cDNA (1818 bp) encoding a putative 5-HT receptor (5-HT(py)) of 454 amino acid residues was isolated from the ovary and shared 53.3% and 40.2% homology with the Aplysia 5-HT(1ap) and mouse 5-HT(1A), respectively. The 5-HT(py) sequence possessed typical characteristics of 5-HT(1), including seven transmembrane domains, a long third inner loop, and a short fourth inner terminal. Phylogenetic analysis suggested that 5-HT(py) was classified into the 5-HT(1) subtype as well as other invertebrate 5-HT(1) receptors. Semi-quantitative RT-PCR showed the expression of the 5-HT(py) gene in both the nervous system and peripheral tissues and the induction of expression by E(2) in the ovarian tissue. In situ hybridization revealed a strong 5-HT(py) signal in the oocytes, spermatids, and ciliary epithelium of the gonoducts in the ovary and testis. These results suggest that the effects of 5-HT on the induction of oocyte maturation, sperm motility, and transport of mature oocytes and sperm through the ciliated epithelium of the gonoducts are mediated by 5-HT(py).

Identification and characterization of multiple beta-glucan binding proteins in the Pacific oyster, Crassostrea gigas.

The present study reports on the characterization of two cDNAs coding beta-glucan binding proteins (betaGBPs), designated as Cg-betaGBP-1 and Cg-betaGBP-2, from the Pacific oyster, Crassostrea gigas. Cg-betaGBP-1 consists of 555 amino acid residues and possesses two possible integrin recognition sites. The other protein, Cg-betaGBP-2, is composed of 447 amino acid residues without integrin recognition sites. Domain structures of both Cg-betaGBPs are similar to other invertebrate betaGBPs, but phylogenetic positions and major expression tissues for these proteins are different. Cg-betaGBP-1 is expressed in circulatory hemocytes and Cg-betaGBP-2 in digestive glands. Functional assays using recombinant proteins revealed that Cg-betaGBP-2 enhanced the phenoloxidase (PO) activity of hemocyte suspensions under the presence of laminarin, but Cg-betaGBP-1 did not show this enhancement. It is suggested that Cg-betaGBPs in the Pacific oyster have evolved to obtain different immunological functions. Cg-betaGBP-1 possibly evolved for hemocyte-related functions through integrin, and Cg-betaGBP-2 for the PO activation system.

A novel peptidoglycan recognition protein containing a goose-type lysozyme domain from the Pacific oyster, Crassostrea gigas.

Peptidoglycan recognition protein (PGRP) is considered an essential molecule for effective immunity in invertebrates by its detection and clarification of invading bacteria. Bivalve mollusks also possess PGRP systems for self-defense, however, their functions in bivalves remain to be understood. In the present study, cDNA of a novel PGRP was identified from the Pacific oyster, Crassostrea gigas, using EST-based RACE PCR. This novel PGRP is homologous to short PGRPs and the presence of a signal peptide was predicted. The PGRP is classified into the short PGRP group, although its molecular weight was estimated as 54 kDa, close to that of long PGRP groups. A conserved domain search detected amidase_2/PGRP and goose-type (g-type) lysozyme domains in this PGRP structure, and thus this novel PGRP was designated as CgPGRP-L. Catalytic residues for PGRP and g-type lysozyme are well conserved, suggesting that CgPGRP-L may have both binding and lytic functions against bacteria. Reverser transcription PCR (RT-PCR) detected CgPGRP-L mRNA expression in circulatory hemocytes, and quantitative real-time RT-PCR revealed that its expression increased after Marinococcus halophilus and Vibrio tubiashii exposure. These results indicate that CgPGRP-L is expressed in hemocytes by bacterial invasion, and then may play roles of a short PGRP and bacterio-lytic lysozyme.

Distribution of multiple peptidoglycan recognition proteins in the tissues of Pacific oyster, Crassostrea gigas.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors that specifically bind to peptidoglycans, a major component of bacterial cell wall. Generally, PGRPs are responsible for recognition of bacterial invasion in invertebrates. Full length cDNAs of PGRP, designated as CgPGRP-S1S, -S1L, -S2 and -S3, were identified from the Pacific oyster, Crassostrea gigas. Homology and domain searches classified these CgPGRPs as short-type PGRPs for extracellular PGN recognition. Amidase activity was predicted in all CgPGRPs, and defensin-like domains were found in CgPGRP-S1S and -S1L, suggesting that they may also function as antimicrobial proteins. Although phylogenetic analysis indicated that CgPGRPs are closely related to each other, they showed different tissue expression patterns; CgPGRP-S1S in the mantle and the gill, -S1L in the mantle, -S2 in the hemocytes and -S3 in the digestive diverticula. The CgPGRPs seem to survey bacterial invasion in their corresponding expression tissues. This is the first report of the possibility that bivalve mollusks have PGN recognition systems as suggested by the identification of multiple PGRPs distributed in various tissues.

Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum.

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.

Mechanisms and immunological roles of apoptosis in molluscs.

Molluscan defense mechanisms are regulated to innate immunity, which is largely dependent on cellular components such as hemocytes possessing phagocytic and bactericidal activities. Among immune responses, apoptosis is an indispensable process because it enables the adequate clearance of damaged, senescent and infected cells without inflammation. Available information related to the molecular mechanisms of apoptosis has been accumulated for many molluscan species during the last decade. Almost all molluscan species live in an environment that changes incessantly according to microorganisms, industrial pollutants, temperature, and salinity. Such environmental factors might directly or indirectly induce apoptosis in molluscan cells. One type of apoptotic agent, reactive oxygen intermediates (ROIs), which are produced by a stress signal or phagocytosis, triggers apoptotic cell death in molluscan hemocytes. Dysfunction of ROI-mediated hemocytic apoptosis putatively causes disease morbidity and/or mortality when molluscan organisms are infected by pathogens. Furthermore, integrins have attracted attention for their unique functions because integrins regulate the phagocytic ability of molluscan hemocytes and induce hemocytic apoptosis. That process might be the result of ROI-generation. In this review, we summarize the roles and molecular mechanisms of apoptosis related to immunological functions of molluscan hemocytes.

Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas.

As an initial step in the functional analysis of lectins in the Pacific oyster, Crassostrea gigas, we attempted to obtain the full coding sequences of C. gigas lectins and conduct tissue expression analyses. To obtain lectin genes quickly, we identified C. gigas expressed sequence tags that coded for lectins in GenBank, and selected three encoding partial sequences of C-type lectin 1 (CgCLec-1), galectin (CgGal) and fucolectin. We obtained full open reading frames of CgCLec-1 and CgGal cDNAs by RACE-PCR. CgCLec-1 is a typical C-type lectin with a signal peptide and C-type lectin domain. CgCLec-1 mRNA was expressed only in specialized basophilic cells involved with digestive enzyme secretion in the digestive gland, suggesting that CgCLec-1 is secreted into the lumen of the digestive diverticula. CgGal is a prototype galectin with a single galactose-binding domain that was expressed in all of the tissues examined. As suggested for vertebrate galectin-1 (prototype galectin), CgGal may function in general cell activities such as cell adhesion. Fucolectin in C. gigas was expressed specifically in the gonads, indicating a possible function in gonadal development. CgCLec-1 and CgGal expression in hemocytes was not upregulated after injecting Vibrio tubiashii into adductor muscle, suggesting that bacterial infection does not induce synthesis of these lectins. Of the three lectins examined, CgCLec-1 is an interesting target for future investigations of innate immunity in the digestive system of C. gigas.

cDNA cloning and in situ hybridization of a novel lysozyme in the Pacific oyster, Crassostrea gigas.

A novel lysozyme cDNA from the Pacific oyster, Crassostrea gigas, was identified. This second lysozyme from the Pacific oyster was designated as CGL-2. The complete CGL-2 cDNA sequence comprises of 536 bp, and 429 bp of the open reading frame encodes 147 bp of amino acid residues. Estimated CGL-2 molecular characteristics (isoelectric point and numbers of peptide recognition sites) resembled those of cv-lysozyme 2, a digestive lysozyme of the eastern oyster, Crassostrea virginica. Moreover, CGL-2 is phylogenetically homologous to the cv-lysozyme 2, indicating that CGL-2 and cv-lysozyme 2 evolved from the same ancestor protein for adaptation to the digestive environment. In situ hybridization revealed that the CGL-2 gene is expressed in digestive cells. It is noteworthy that the other Pacific oyster lysozyme, CGL-1, was also transcribed in the same cells. Presence and expression of multiple lysozymes in the digestive diverticula suggest that CGL-1 and CGL-2 might play complementary roles in digestive organs.

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis.

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.

Differences in integrin-dependent phagocytosis among three hemocyte subpopulations of the Pacific oyster "Crassostrea gigas".

Integrins play a key role in immunoresponses such as attachment, spreading, and phagocytosis in invertebrate hemocytes. This study was designed to identify integrin expression patterns at the hemocyte subpopulation level, and correlate the expression levels with phagocytic ability. First, we cloned a beta integrin from Crassostreagigas hemocytes and used real-time RT-PCR to analyze the quantitative expression level of its encoding mRNA. The expression level in hyalinocytes was significantly higher than that in granulocytes and agranulocytes. Subsequently, we investigated the phagocytic ability of each subpopulation using anti-alpha(5)beta(1) integrin antibody, and found that phagocytosis of hyalinocytes was inhibited by neutralization with the antibody but enhanced against the antibody-conjugated microspheres. In contrast, phagocytic abilities of granulocytes and agranulocytes showed high and zero levels, respectively, regardless of the antibody. These results suggest that phagocytosis of hyalinocytes is regulated by an integrin-dependent mechanism and that of granulocytes is elicited by other functional receptors.