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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 β-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of β-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 β-glucan receptors for phagocytosis. [ABSTRACT FROM AUTHOR]

Abstract: 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 54kDa, 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

For molluscs, including bivalves, circulating hemocytes serve an important role in the principal mechanisms of the host defense. The hemocyte adherence and mobility are essential factors in processes such as phagocytosis against foreign particles and migration toward inflammatory sites. It is described in crustaceans that these immune responses dependent on hemocyte adherence are mediated by the membrane receptor, such as integrins. In this article, the article authors demonstrated that the RGD peptide treatment resulted in the C. gigas hemocyte apoptosis that was induced irrespective of whether the hemocyte integrin is activated and bound to the plasma. This result indicates that the apoptosis induction by the RGD peptide in the C. gigas hemocytes was a different mechanism from that of the anchorage-dependent cells.

Highlights: [•] CgPGRP-S1S in the Pacific oyster specifically recognizes DAP-type PGN and Escherichia coli cells. [•] CgPGRP-S1S does not have bactericidal activity, but agglutinates E. coli cells. [•] CgPGRP-S1S functions as an immune mediator by inducing granulocyte degranulation. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 pH5.5, 7.0, and 8.5 tested in this study. RT-PCR showed that Cg-Chit1expression 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. [Copyright &y& Elsevier]