Development of Novel Microsatellite DNA Markers from the Pacific Oyster Crassostrea gigas

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2–20; observed heterozygosity, 0.119–0.925; unbiased expected heterozygosity, 0.139–0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.     

Effects of extender composition, cooling rate and freezing on the fertilisation viability of spermatozoa of the Pacific oyster (Crassostrea gigas)

The aim of this research was to optimise protocols for freezing spermatozoa of the Pacific oyster. All the phases of the cryopreservation procedure (choice of cryoprotectant, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa to restore on thawing the morphological and physiological characteristics of fresh semen. The choice of type and concentration of cryoprotectant in which semen is incubated before freezing is fundamental for a successful cryopreservation: the cryoprotectants (dimethylsulfoxide--Me(2)SO, ethylene glycol--EG, propylene glycol-PG, and glycerol in concentrations between 5 and 15%) were tested for their toxicity on the semen exposed up to 30 min at +26 degrees C (room temperature) by evaluating its ability to fertilise and the embryo development to the regular D larval stage. The best cryoprotectants, Me(2)SO, EG, and PG 5, 10, and 15% respectively, were used for the pre-cooling (adaptation/cooling) tests. Two different adaptation/cooling procedures were tested: (A) from +26 degrees C to 0-2 degrees C (2.6 degrees C/min) and (B) at +26 degrees C for 15 min. Lastly, using the cryoprotectants and the adaptation procedure (B) that had given the best results in the preceding stages of the experiment, four cooling rates were tested: 6, 11, 16, and 21 degrees C/min. It was seen that the semen that was incubated with EG 10%, adapted at +26 degrees C for 15 min, and then cooled at a rate of 6 degrees C/min showed a percentage of regular D larvae on thawing comparable to that of fresh semen (p > 0.05).     

Individual relationship between aneuploidy of gill cells and growth rate in the cupped oysters Crassostrea angulata, C. gigas and their reciprocal hybrids

The Portuguese oyster, Crassostrea angulata, is taxonomically close to the Pacific oyster, C. gigas, but there are clear genetic and phenotypic differences between these taxa. Among those differences, the faster growth of C. gigas compared with C. angulata has often been observed in the field. Crosses between C. angulata and C. gigas were performed to investigate the relationship between growth variation and somatic aneuploidy at the individual level in the two taxa and their reciprocal hybrids. The different progenies were reared in Ria Formosa (Portugal) under standard farming conditions. Growth rate and survival were significantly higher in C. gigas than in C. angulata, and the hybrids showed intermediate performances. Significant differences were also observed in the proportion of aneuploid cells (PAC) and of missing chromosomes (PMC) between the two taxa, C. angulata showing the highest values. Intermediate values of PAC and PMC were observed in the hybrids, supporting additive genetic bases of these parameters. Our results also confirm the negative correlation between somatic aneuploidy and growth rate at the individual level, as previously reported in C. gigas.     

Sterol composition of dark-grown Isochrysis galbana and its implication in the seed production of Pacific oyster, Crassostrea gigas

Isochrysis galbana was cultured heterotrophicallywith glucose and mannose as a potential food for larval Pacific oyster,Crassostrea gigas. The food was evaluated in terms offeeding experiments and changes in sterol composition. The larvae showed delayedgrowth and higher mortality compared with ones fed light-grown material, with asignificant difference in mortality from day 8. Light-grown I.galbana contained three major sterols (24-oxocholesterol acetate,ergost-5-en-3-ol, and cholest-5-en-24-1,3-(acetyloxy)-,3-ol) and twominor sterols (24-methylcholesta-5,22-dien-3-ol and24-methylcholest-5-en-3-ol). The sterol content decreased markedly aftertransfer to dark culture, especially in two of the major sterols,24-oxocholesterol acetate and ergost-5-en-3-ol. The other major sterol,cholest-5-en-24-1,3-(acetyloxy)-,3-ol, fall to about 50% of theautotrophic control by day 12. These results indicate that heterotrophicallygrown I. galbana is not a favorable alternative to normallygrown material for larval C. gigas culture as far as sterolcomposition is concerned.     

Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia)

The aim of this study is to describe the early stages of spermatogenesis of the Pacific oyster Crassostrea gigas using both light and electron microscopy. The gonad is formed by gonadal tubules invaginated in a connective tissue constituting a storage tissue. Myoepithelial cells surround each gonadal tubule and are associated with an acellular matrix delimiting the outer part of the tubule, the inner part is composed by intragonadal somatic cells associated with germinal lineage. Two types of spermatogonia are identified, where type I spermatogonia (Spg I) are large, scarce and pale cells leaned against the base of the tubule (nuclear diameter: 5.5+/-0.5 microm). Type II spermatogonia (Spg II) are clustered and dark cells which appear smaller than type I (nuclear diameter: 4.3+/-0.3 microm). The aspect of nuage-like material in cytoplasm is described from pale spermatogonia to primary spermatocytes (nuclear diameter: pachytene 3.6+/-0.3 microm, diplotene 3.4+/-0.3 microm), while no structure related to a chromatoid body was observed in oyster spermatocytes and spermatids.     

Mitochondrial and nuclear DNA phylogeography of European grayling (Thymallus thymallus): evidence for secondary contact zones in central Europe

Mitochondrial and microsatellite DNA markers were applied to infer the phylogeography, intraspecific diversity and dynamics of the distributional history of European grayling (Thymallus thymallus) with focus on its central and northern European distribution range. Phylogenetic and nested clade analyses revealed at least four major mtDNA lineages, which evolved in geographical isolation during the Pleistocene. These lineages should be recognized as the basic evolutionary significant units (ESUs) for grayling in central and northern Europe. In addition, and in contrast to previous work on grayling, the results of Bayesian analysis of individual admixture coefficients, two-dimensional scaling analysis and spatial analysis of molecular variance provided evidence for a high level of admixture among major lineages in contact zones between drainages (e.g. the low mountain range of Germany), most likely resulting from glacial perturbations and ancient river connections between drainages during the Pleistocene glaciations. Even within river systems, a high level of differentiation among populations was revealed as indicated by the microsatellite data. Grayling sampled from 29 sites displayed high levels of differentiation (overall F(ST) = 0.367), a high number of private alleles and high bootstrap support for the genetic distance-based population clusters across 12 loci. We specifically discuss our results in context of phylogeograpic studies on other European freshwater fish species with habitat preferences similar to those of grayling. Our study shows that both large-scale phylogeographical and detailed genetic analyses on a fine scale are mandatory for developing appropriate conservation guidelines of endangered species.     

黄河口西南侧小岛河河口天然牡蛎礁的牡蛎种群结构

牡蛎礁是生态系统服务价值高、但退化最严重及受关注度最高的海洋生境之一,牡蛎礁修复亦成为国际海洋生态修复的热点。掌握牡蛎自然种群状况及动态变化是评估牡蛎礁修复效果的基础。目前,我国天然牡蛎礁的牡蛎种群状况相关的背景资料较为缺乏。在黄河口西南侧的小岛河河口新发现天然活体牡蛎礁,但该牡蛎礁曾被大规模的商业采捕,亟需推进针对性的保护和修复研究工作。基于2021年11月对该牡蛎礁开展的牡蛎种群生态调查,分析其种类组成、年龄结构及生长特征。结果显示:该牡蛎礁分布有近江牡蛎(Crassostrea ariakensis)和长牡蛎(Crassostrea gigas)。牡蛎礁上以活体牡蛎为主,死亡牡蛎壳体数仅占6.1%-6.7%。活体牡蛎的密度和生物量分别为(2811±778)个/m²和(21.97±30.43)kg/m²,近江牡蛎较多,其密度和生物量分别占比55.7%和76.4%。近江牡蛎和长牡蛎的年龄分别介于0+-4+ 龄和0+-2+ 龄,它们都以壳高介于30-40 mm及壳质量<5 g的0+ 龄个体数量居多(>80%)。近江牡蛎的壳体形态参数均值都高于同龄组长牡蛎的相应值。两种牡蛎壳体均呈负异速增长,不同龄级的壳体延展方向不同。拟合von Bertalanffy生长方程得到,近江牡蛎和长牡蛎的渐近壳高分别为286 mm和173 mm,估算的拐点年龄分别为5.47 龄和2.56 龄,两种牡蛎的生长曲线分布存在极显著差异(P<0.001)。以上结果表明,小岛河河口的天然牡蛎礁的牡蛎自然种群资源较丰富,具有高密度、低龄和低死亡率等特点,有较好的活力和扩张潜力,有利于被采捕后的礁体的恢复。两种牡蛎中,近江牡蛎因其具有较高的生物量和较长的生长年龄,对礁体形成和扩繁可能更为重要。建议对该天然牡蛎礁及牡蛎种群开展周期>3年的原位保护、修复和连续监测计划。     

Spatio-temporal variations in biological performances and summer mortality of the Pacific oyster Crassostrea gigas in Normandy (France)

Mortality and biological performances of half-grown Crassostrea gigas were studied from spring 2000 to autumn 2001 at six instrumented stations located in two areas (Gefosse and Grandcamp) of the Bay of Veys (Normandy). Shell and meat growth, condition indexes and a macroscopic maturity index were determined on oysters deployed at the six stations in order to assess spatial variability in the influence of environmental conditions. In addition, histological and biochemical analyses were performed in order to determine the sex and establish the reproductive cycle (at all six sites) and the biochemical composition (at four stations). The data set including monthly mean temperatures and data provided by examination of 2,837 oysters were analysed by Principal Component Analysis and a Hierarchical Ascending Clustering which resulted in the formation of four clusters. The highest station on the shoreline belonged to a cluster characterized notably by low total weight due to a short immersion/feeding period, whereas all other stations belonged to another single cluster. Nevertheless, various biological differences were found between these stations, e.g. the reproductive cycle was generally synchronized throughout the bay but some differences relative to spawning occurrence were observed. In 2000, oyster mortality was higher at Gefosse than at Grandcamp, the latter being a more marine area. In 2001, oyster mortalities were significantly higher and all stations were strongly affected. In the Bay of Veys, oyster biological performances and mortality thus showed spatio-temporal variations which were worthy to be discussed.     

Mitochondrial DNA and nuclear microsatellites reveal high diversity and genetic structure in an avian top predator,the white‐tailed sea eagle,in central Europe

We analysed 123 white‐tailed sea eagles (Haliaeetus albicilla) from (primarily central) Europe with respect to variability and differentiation based on 499 bp of the mitochondrial control region and genotypes at seven unlinked nuclear microsatellites. Variability was high (overall expected heterozygosity, haplotype and nucleotide diversity being 0.70, 0.764 and 0.00698, respectively) and both marker systems showed a subdivision into two main genetic clusters (microsatellites) or haplogroups (mtDNA). In line with earlier analyses focusing on populations from northern and eastern Europe, as well as from Asia, we found a high level of admixture in Europe and no signs of a bottleneck – despite a severe decline of white‐tailed sea eagle populations during the 20^th century. Europe is thus a global stronghold for this species not only with respect to the number of breeding pairs but also regarding the proportion of species‐wide genetic diversity. Our dense sampling revealed a possibly clinal variation within central Europe from north‐west to south‐east that was reflected by the distribution of mtDNA haplotypes as well as the two microsatellite‐based clusters. This population differentiation in central Europe probably originated from a geographically structured postglacial colonization and was later enhanced by recent demographic fluctuations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 727–737.     

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min⁻¹ and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.     

Mitochondrial DNA diversity and phylogeography of endangered green turtle (Chelonia mydas) populations in Africa

We analysed the genetic structure of seven nesting sites of the endangered green turtle (Chelonia mydas) in Africa using mitochondrial DNA control region sequences. Tissue samples were collected from 188 nesting females at six sites in West Africa and one in the Indian Ocean. A 488 bp fragment of the control region revealed 14 different haplotypes, 10 of which are previously undescribed. The most common haplotype (CM8) was observed in 157 individuals. All other haplotypes were closely related, except two divergent lineages: CM38, removed by four substitutions, and the three Indian Ocean haplotypes, distinguished by 31 substitutions. Significant differences in haplotype and nucleotide diversity were observed between Atlantic rookeries and among ocean basins. Analysis of molecular variance revealed high levels of differentiation between the Atlantic and the Indian Ocean populations but a much shallower Atlantic substructuring. Green turtle population genetic structure is thought to have been shaped by a dynamic succession of extinction and recolonisation of rookeries, by natal homing and occasional breakdown in nest-site fidelity. Mismatch distributions of pairwise differences between haplotypes at each rookery were found to be consistent with recent population expansion. We argue that demographic histories can be explained by scenarios at several temporal scales, including geological events, sea level fluctuations and more recent patterns of exploitation. We discuss management and conservation implications of our results for these threatened populations, identifying two ESUs (one in the Atlantic and one in the Indian ocean) and three MUs within the Atlantic.     

Relationship between Marteilioides chungmuensis infection and reproduction in the Pacific oyster, Crassostrea gigas

Marteilioides chungmuensis, a protozoan paramyxean parasite, infects the oocytes of the Pacific oyster, Crassostrea gigas. The effects of infection on the reproductive cycle of C. gigas were investigated over two consecutive years at Okayama Prefecture, Japan. In male oysters, gonadal development began during February/March, maturity was achieved in June and spawning activity extended from July to September. In November and December, male oysters were not seen, probably because their gonads regressed to connective tissue and they transformed into undifferentiated oysters. By contrast, female oysters, in which parasite spore formation occurred, were still carrying oocytes until the following March and the spawning process of female oysters took 5 months longer than that of males in epizootic areas. The prevalence of M. chungmuensis infection increased from July to September, when most female oysters had their spawning period, and declined from October to the following April when oysters were at the spent stage. The prevalence of infection increased again in May of the following year and high prevalence was observed in the following July. When prevalence was compared between oysters of different age classes, higher prevalence was detected in older than in younger oysters. Histological examination showed that infected oysters produced oocytes continuously and spawned repeatedly from October to March, during which period healthy oysters were reproductively inactive. Parasites can infect the oocytes of infected oysters throughout the longer spawning period. These observations suggest that M. chungmuensis extends the reproductive period of infected oysters for its own reproductive benefit.     

Genome-wide and single-base resolution DNA methylomes of the Pacific oyster Crassostrea gigas provide insight into the evolution of invertebrate CpG methylation

Background

Studies of DNA methylomes in a wide range of eukaryotes have revealed both conserved and divergent characteristics of DNA methylation among phylogenetic groups. However, data on invertebrates particularly molluscs are limited, which hinders our understanding of the evolution of DNA methylation in metazoa. The sequencing of the Pacific oyster Crassostrea gigas genome provides an opportunity for genome-wide profiling of DNA methylation in this model mollusc.

Results

Homologous searches against the C. gigas genome identified functional orthologs for key genes involved in DNA methylation: DNMT1, DNMT2, DNMT3, MBD2/3 and UHRF1. Whole-genome bisulfite sequencing (BS-seq) of the oyster’s mantle tissues revealed that more than 99% methylation modification was restricted to cytosines in CpG context and methylated CpGs accumulated in the bodies of genes that were moderately expressed. Young repeat elements were another major targets of CpG methylation in oysters. Comparison with other invertebrate methylomes suggested that the 5’-end bias of gene body methylation and the negative correlation between gene body methylation and gene length were the derived features probably limited to the insect lineage. Interestingly, phylostratigraphic analysis showed that CpG methylation preferentially targeted genes originating in the common ancestor of eukaryotes rather than the oldest genes originating in the common ancestor of cellular organisms.

Conclusions

Comparative analysis of the oyster DNA methylomes and that of other animal species revealed that the characteristics of DNA methylation were generally conserved during invertebrate evolution, while some unique features were derived in the insect lineage. The preference of methylation modification on genes originating in the eukaryotic ancestor rather than the oldest genes is unexpected, probably implying that the emergence of methylation regulation in these ''relatively young’ genes was critical for the origin and radiation of eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1119) contains supplementary material, which is available to authorized users.     

Improved attachment and spreading in primary cell cultures of the eastern oyster, Crassostrea virginica

At present, establishment of a cell line from bivalve molluscs has been unsuccessful, and in vitro work is limited to primary cell cultures. We sought to improve attachment and spreading of cells of the eastern oyster, Crassostrea virginica, to aid primary cultures and to assist development of a bivalve cell line. Our objectives were to examine the effects of substrate on ventricle cell viability, attachment, and spreading by testing of collagen I, collagen IV, fibronectin, laminin, poly-D-lysine, and two types of uncoated tissue culture plates (Falcon and Corning). Experiments were conducted by incubating cells with the various substrates for 24 h and 5 d. An assay with a tetrazolium compound (MTS) was used to estimate cell numbers based on metabolic activity. Although differences in MTS assay values for substrate effect on cell viability were detected at 24 h and at 5 d (P > 0.0001), these were attributed to variations in metabolic activity due to different levels of attachment and spreading among treatments. Differences among treatments were detected in attachment and spreading at 24 h and 5 d (for all, P > 0.0001). At 24 h, poly-D-lysine induced the highest levels of attachment and spreading; no other factor performed better than the uncoated Falcon substrate, and collagen I performed most poorly. At 5 d, poly-D-lysine and the uncoated Corning substrate induced significantly higher levels of attachment and spreading than did the uncoated Falcons substrate, and collagen I performed most poorly. From these results, poly-D-lysine best promoted cell attachment and spreading. Fibronectin (at 24 h) and laminin (at 5 d) warrant further study. Along with improvements in medium composition, future work should involve screening of other attachment factors and combinations of factors, including those of bivalve origin.     

Setting tools for the early assessment of the quality of thawed Pacific oyster (Crassostrea gigas) D-larvae

Parameters used to assess the survival of larvae after cryopreservation generally misestimate the damages that prevent larval development. The objectives of the present study were to 1) define the reliability of the survival rate, assessed at 2 and 7 days post fertilization, to estimate Pacific oyster larval quality after thawing, and 2) select complementary tools allowing an early and reliable estimation of their quality. Oyster larvae were reared for 25 h after fertilization at 19 °C and cryopreserved at early D-stage. Then, thawed larvae were incubated in 2-L beakers. At 2 days post fertilization, the survival rate of thawed Pacific oyster larvae was lower than that of fresh larvae for only one experiment (Experiment 3) among the four identical experiments carried out in this work (Experiments 1-4). By contrast, the survival of thawed larvae, as assessed 7 days after fertilization, was lower than that of fresh larvae for the four experiments. These results confirm that the quality of thawed larvae is lower than that of fresh larvae and that the survival rate, estimated 2 days post fertilization, is not adapted to a reliable estimation of the subsequent development ability of thawed larvae. Then, complementary parameters were tested at 2 days: the movement characteristics (Experiments 1 and 2) and the morphologic features (Experiments 3 and 4) of thawed larvae. Compared to values observed on fresh larvae, the percentage of thawed motile larvae was different for only one experiment (Experiment 2) of the two. Compared to control, a reduced Average Path Velocity (VAP) of larvae (determined at the D-larval stage using a CASA-Computer Assisted Sperm Analysis-system) was observed after thawing for both experiments (Experiments 1 and 2), suggesting the ability of larval movement velocity to assess the decrease of the quality of thawed oyster larvae. Using an ASMA (Automated Sperm Morphology Analysis) device, a lower area of thawed larvae was observed, compared to control and for the two experiments (Experiments 3 and 4). By contrast, the Crofton perimeter of thawed larvae was lower than that of control larvae for only one experiment (Experiment 3) and no significant difference of circularity between fresh and thawed larvae was recorded for Experiments 3 and 4. In conclusion, changes in the movement velocity (assessed by CASA) and in the area (measured by ASMA) of D-larvae allow an early and reliable estimation of the quality of thawed Pacific oyster larvae.     

Pathogenicity of the protozoan parasite Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas

Marteilioides chungmuensis is an ovarian parasite that causes nodule-like structures to appear on the gonads of female Pacific oysters, Crassostrea gigas. It is known that the prevalence of infection increases in summer and decreases from autumn to spring. To investigate the decrease in prevalence of infection and pathogenicity of the parasite, a biopsy method was developed to detect infected oysters, which were then monitored to calculate the mortality rate. Mortality of infected oysters was recorded monthly and changes in reproductive development observed histologically. Compared with control groups, a significant difference in mortality was observed in infected oysters in September and October. Histological observations showed that infected oysters produced oocytes continuously, even in autumn when healthy oysters were reproductively inactive. This prolonged spawning activity of infected oysters resulted in nutritional wasting and mortality. From December onwards, however, almost all infected oysters survived, though the infection persisted. Infection intensity decreased gradually from December. Histological observations revealed that, in winter, infected oysters released infected and uninfected oocytes through the genital canal. The gonad subsequently degenerated and was replaced with connective tissue, as in normal, healthy spent oysters. The results revealed that prevalence of infection decreased from September to May. It is hypothesised that the decline in prevalence within the epizootic area in autumn occurred because infected oysters died and that the winter decrease was due to recovery from infection.     

Cytometric investigations on hemocytes of the American oyster, Crassostrea virginica

Pericardial hemolymph was obtained from American Oysters (Crassostrea virginica) and the hemocytes characterized by flow cytometry. The cells were found to have a broad unimodal size distribution with a median diameter of 7 micrometers. Total protein measured by flow cytometric fluorescence of dansylated cells also revealed a broad unimodal distribution similar to that obtained for size. The proportion of hemocytes in each stage of the cell cycle was measured using DNA-specific DAPI fluorescence. Histograms showed a single peak representing the G(0)/G(1) population. There was no evidence of S or G(2)+M phases of the cell cycle, nor was polyploidy seen. The forward and orthogonal light scatter of fixed hemocytes showed no evidence of sub-populations on the basis of cytoplasmic granularity. Thus, in terms of these parameters, oyster hemocytes appear to represent a single population exhibiting graded cellular differences.     

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.