Genetic divergence accessed with microsatellite markers reflects the time of Crassostrea gigas genetic breeding in Brazil.

The Pacific Oyster was introduced on Santa Catarina Island in 1987, experiencing processes of selection and genetic breeding since then. Such procedures may have led to the establishment of specific strains, given the saltier and warmer conditions of the Atlantic Ocean. This study employed microsatellite markers to compare allelic patterns of oysters cultivated in Santa Catarina, the USA, and Asia. Specific allelic patterns were revealed in the Santa Catarina samples, reflecting the time of selection/breeding of the oyster in this region. This result supports the effectiveness of the selection/breeding procedures and the demand for protection of this commercially important genetic resource.

Transient Receptor Potential (TRP) Channels in the Pacific Oyster (Crassostrea gigas): Genome-Wide Identification and Expression Profiling after Heat Stress between C. gigas and C. angulata.

Transmembrane proteins are involved in an array of stress responses, particularly in thermo-sensation and thermo-regulation. In this study, we performed a genome-wide identification and characterization of the Transient Receptor Potential (TRP) genes in the Pacific oyster (Crassostrea gigas) and investigated their expression profiles after heat stress to identify critical TRPs potentially associated with thermal regulation. A total of 66 TRP genes were identified in the C. gigas, which showed significant gene expansion and tandem duplication. Meta-analysis of the available RNA-Seq data generated from samples after acute heat stress revealed a set of heat-inducible TRPs. Further examination of their expression profiles under chronic heat stress, and comparison between C. gigas and C. angulata, two oyster species with different tolerance levels to heat stress, led to the identification of TRPC3.6, TRPC3.7, and TRPV4.7 as important TRPs involved in thermal regulation in oysters. This work provided valuable information for future studies on the molecular mechanism of TRP mediated thermal tolerance, and identification of diagnostic biomarker for thermal stress in the oysters.

A novel tumor necrosis factor in the Pacific oyster Crassostrea gigas mediates the antibacterial response by triggering the synthesis of lysozyme and nitric oxide.

Tumor necrosis factors (TNFs) are a group of multifunctional inflammatory cytokines involved in various pathological and immune processes. Recently, a few primitive TNFs have been characterized from molluscs, which play important roles in modulating cell apoptosis, phagocytosis and production of immune-related enzymes. In the present study, a novel TNF (named as CgTNF-2) with the activity to mediate antibacterial response was identified from the Pacific oyster Crassostrea gigas. The open reading frame of CgTNF-2 was of 783 bp encoding a putative polypeptide of 261 amino acids with a typical TNF domain. The deduced amino acid sequence of CgTNF-2 shared high identity with that of TNFs previously identified from other molluscs, such as 96.1% identity with that in oyster C. hongkongensis, 33.7% identity with that in scallop Mizuhopecten yessoensis and 33.0% identity with CgTNF-1 in oyster C. gigas. There were two distinct TNF branches of vertebrate and invertebrate in the phylogenetic tree, and CgTNF-2 was firstly clustered with TNF-14 from C. hongkongensis, and then clustered with other molluscan TNFs. The mRNA transcripts of CgTNF-2 were widely expressed in various oyster tissues, with the highest expression level in hemocytes. The expression level of CgTNF-2 increased significantly at 6 h (2.45-fold and 6.20-fold, respectively, p < 0.05) after peptidoglycan and lipopolysaccharides treatments, and peaked at 12 h (31.86-fold and 7.90-fold, respectively, p < 0.05). The recombinant protein of CgTNF-2 (rCgTNF-2) inhibited the growth of human alveolar basal epithelial (A549) cells at a concentration of 800 ng/mL. After the oysters received an injection of rCgTNF-2, the serum from those oysters exhibited significantly higher antibacterial activity compared to that from control group, evidenced by inhibiting the growth of Vibrio splendidus. Moreover, the lysozyme activity as well as the contents of nitric oxide in the oyster serum also increased significantly. The above results collectively suggested that CgTNF-2 was a novel member of bivalve TNF-α family, which could prompt the antibacterial activity by inducing the lysozyme activity and the production of nitric oxide in the innate immune response of oyster.

Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

Cloning, characterization and comparative analysis of four death receptorTNFRs from the oyster Crassostrea hongkongensis.

Apoptosis plays an important role in homeostasis of the immune systems. The tumor necrosis factor receptors (TNFRs) play critical roles in the extrinsic apoptosis pathways and in determining cell fate. In this study, four death receptors (DR) named ChEDAR, ChTNFR27, ChTNFR5, and ChTNFR16 were identified from the oyster Crassostrea hongkongensis. These ChDRs proteins had 382, 396, 414 and 384 amino acids, respectively, with the typical domains of death receptors, such as the signal peptide (SP), transmembrane helix region (TM) and death domains. Phylogenetic analysis showed that the ChDR proteins clustered into three distinct groups, indicating that these subfamilies had common ancestors. mRNA expression of the ChDRs were detected in all 8 of the selected oyster tissues and at different stages of development. Furthermore, expression of all the genes was increased in the hemocytes of oysters challenged with pathogens or air stress. Fluorescence microscopy revealed that the full-length proteins of the ChDRs were located in the plasma membrane of HEK293T cells. Over-expression of the ChDRs activated the NF-κB-Luc reporter in HEK293T cells in a dose-dependent manner. These results indicate that the ChDRs may play important roles in the extrinsic apoptotic pathways in oysters.

[Effect of temperature and salinity on the reproductive cycle of males and females of Crassostrea virginica (Bivalvia: Ostreidae)]. / Efecto de la temperatura y la salinidad en el ciclo reproductor de hembras y machos de Crassostrea virginica (Bivalvia: Ostreidae).

The American oyster, Crassostrea virginica, is one of the most important economic coastal fishery activities in Mexico. As for other bivalve molluscs, its sustainability is based on healthy populations, and additional information on current reproductive cycles, will reinforce local management strategies. The objective of this study was to evaluate the effect of temperature and salinity on the gonadal development stages in both sexes and in two size groups (40-60 mm and ≥ 61 mm) of C. virginica from Tamiahua Lagoon, Gulf of Mexico. Monthly surveys of 90 organisms were undertaken, for a yearly total of 1 080 oysters sampled from the lagoon in 2011. Both field and laboratory work used standard procedures. Our results showed that reproduction was continuous in both sexes, but some peaks of the reproductive cycle were observed along the year. Peaks registered from January to July were for gametogenesis; those of June and September were for mature individuals; and in January, February, and from September to December, for spawning/spent oysters. Maturity of females of both range sizes was positively correlated with temperature, but not for males. For spawning, spent individuals, males of sizes ≥ 61 mm were positively correlated with salinity, whereas smaller males (40-60 mm) and females of both sizes, did not show any correlation. In this study, we observed that temperature and salinity had a different effect in the reproduction of the American oyster of different sizes and sexes of Tamiahua lagoon.

Pacific oyster polyamine oxidase: a protein missing link in invertebrate evolution.

Polyamine oxidases catalyse the oxidation of polyamines and acetylpolyamines and are responsible for the polyamine interconversion metabolism in animal cells. Polyamine oxidases from yeast can oxidize spermine, N(1)-acetylspermine, and N(1)-acetylspermidine, while in vertebrates two different enzymes, namely spermine oxidase and acetylpolyamine oxidase, specifically catalyse the oxidation of spermine, and N(1)-acetylspermine/N(1)-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylpolyamine oxidases have arisen from an ancestor invertebrate polyamine oxidase with lower specificity for polyamine substrates, as demonstrated by the enzymatic activity of the mollusc polyamine oxidase characterized here. This is the first report of an invertebrate polyamine oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to oxidase both N(1)-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.

Molecular cloning and expression analysis of a pearl oyster (Pinctada martensii) heat shock protein 90 (HSP90).

Heat shock protein 90 (HSP90) is an important molecular chaperone required for proper folding of cellular proteins, and thus, it plays an essential role in protecting cells from damage during stress. In this study, an HSP90 cDNA designated PmHSP90 was cloned from the mantle tissue of the pearl oyster Pinctada martensii using reverse transcription polymerase chain reaction (RT-PCR) coupled with the rapid amplification of cDNA ends (RACE) approach. PmHSP90 cDNA was 2584 bp in length, including an open reading frame of 2160 bp, which encodes a polypeptide of 719 amino acid residues, with predicted molecular mass and isoelectric point of 83.0 kDa and 4.87, respectively. Multiple-sequence alignment indicated that HSP90 is highly conserved among species, and PmHSP90 showed 89% sequence identity to Crassostrea gigas HSP90. Five conserved amino acid blocks defined as HSP90 protein family signatures were also observed in PmHSP90, indicating that PmHSP90 may be a cytosolic member of the HSP90 family. Expression levels of PmHSP90 were detected in various tissues of P. martensii and in hemocytes under three different stress conditions using quantitative real-time PCR (qPCR). The results demonstrate that PmHSP90 mRNA is constitutively expressed in all the tested tissues and may be involved in the immune response against thermal stress, lipopolysaccharide stimulation, and nucleus insertion operations. Studies on PmHSP90 are a valuable source to further explore the immune system in pearl oysters during the production of pearls, and may enhance our knowledge of molluscan innate immunity.

The nuclear receptor gene family in the Pacific oyster, Crassostrea gigas, contains a novel subfamily group.

BACKGROUND: Nuclear receptors are a superfamily of transcription factors important in key biological, developmental and reproductive processes. Several of these receptors are ligand- activated and through their ability to bind endogenous and exogenous ligands, are potentially vulnerable to xenobiotics. Molluscs are key ecological species in defining aquatic and terrestrial habitats and are sensitive to xenobiotic compounds in the environment. However, the understanding of nuclear receptor presence, function and xenobiotic disruption in the phylum Mollusca is limited. RESULTS: Here, forty-three nuclear receptor sequences were mined from the genome of the Pacific oyster, Crassostrea gigas. They include members of NR0-NR5 subfamilies, notably lacking any NR6 members. Phylogenetic analyses of the oyster nuclear receptors have been conducted showing the presence of a large novel subfamily group not previously reported, which is named NR1P. Homologues to all previous identified nuclear receptors in other mollusc species have also been determined including the putative heterodimer partner retinoid X receptor, estrogen receptor and estrogen related receptor. CONCLUSION: C. gigas contains a highly diverse set of nuclear receptors including a novel NR1 group, which provides important information on presence and evolution of this transcription factor superfamily in invertebrates. The Pacific oyster possesses two members of NR3, the sex steroid hormone receptor analogues, of which there are 9 in humans. This provides increasing evidence that steroid ligand specific expansion of this family is deuterostome specific. This new knowledge on divergence and emergence of nuclear receptors in C. gigas provides essential information for studying regulation of molluscan gene expression and the potential effects of xenobiotics.

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.

Characteristic and functional analysis of a ficolin-like protein from the oyster Crassostrea hongkongensis.

Ficolins are a group of soluble animal proteins with multiple roles in innate immunity. These proteins recognize and bind carbohydrates in pathogens and activate the complement system, leading to opsonization, leukocyte activation, and direct pathogen killing, which have been reported in many animal species but might not be present in the shellfish lineage. In the present study, we identified the first fibrinogen-related protein from the oyster, Crassostrea hongkongensis. This novel ficolin-like protein contains a typical signal peptide and a fibrinogen-related domain (designated ChFCN) at the N and C termini, respectively, but does not contain the additional collagen-like domain of ficolins. The full-length cDNA of ChFCN is 1105 bp, encoding a putative protein of 297 amino acids with the molecular weight of 35.5 kD. ChFCN is ubiquitously expressed in selected tissues, with the highest expression level observed in the gills. The temporal expression of ChFCN following microbe infection shows that the expression of ChFCN in hemocytes increases at 3 h post-challenge. The ChFCN protein expression was also examined, and fluorescence microscopy revealed that deChFCN (truncated signal peptide) is located in the cytoplasm of HeLa cells. Full-length ChFCN was detected in the medium supernatant by western blot analysis. Recombinant ChFCN proteins with the molecular weight about 50 kD bind Saccharomyces cerevisiae, Staphylococcus haemolyticus or Escherichia coli K-12, but not those from Vibrio alginolyticus. Furthermore, the rChFCN protein could agglutinate Gram-negative bacteria E. coli K-12 and enhance the phagocytosis of C. hongkongensis hemocytes in vitro. These results indicate that ChFCN might play an important role in the immunity response of oysters.

Alternative splicing and immune response of Crassostrea gigas tumor necrosis factor receptor-associated factor 3.

Diverse alternative splicing isoforms play an important role in immune diversity and specificity. Their role in molluscan host-defense is however poorly understood. We characterized two alternative isoforms of tumor necrosis factor receptor-associated factor 3 (TRAF3) in the Pacific oyster, Crassostrea gigas, which were named CgTRAF3-S and CgTRAF3-L. An intron was retained in CgTRAF3-L, introducing a premature termination codon. Comparison and phylogenetic analysis revealed that CgTRAF3 shared a higher identity with other species, suggesting the conservation of the two gene transcripts. Quantitative real-time PCR was performed and the expression levels of CgTRAF3 isoforms were found to be significantly changed after Vibrio anguillarum and ostreid herpesvirus 1 challenges. These two isoforms represented contrary trends, indicating that CgTRAF3-L might function as a negative regulator of CgTRAF3-S. We also investigated the expression level of the transcripts of the two CgTRAF3 isoforms, following the silence of C. gigas mitochondrial anti-viral signaling protein like gene (CgMAVS-like). We concluded that CgTRAF3 might be involved in a MAVS-mediated immune signaling pathway. This study suggests that CgTRAF3 may be a response to bacterial and viral stimulation and that the two isoforms may be involved in immune response pathways. It is also possible that the two alternative splicing isoforms could be inter-coordinated and may promote survival of these oysters under immune stress conditions.

Molecular cloning and differential expression in tissues of a tyrosinase gene in the Pacific oyster Crassostrea gigas.

Tyrosinases are a group of type-3 copper proteins that catalyze the first two reactions in the melanin biosynthesis in organisms ranging from bacteria, fungi, plants to animals. Tyrosinases are not only involved in pigmentation, but also play an important role in the innate immunity in invertebrates. Additionally, tyrosinases are also known to be involved in the biogenesis and pigmentation of shells. The recently published Crassostrea gigas genome sequences revealed that the Pacific oyster possesses at least 26 tyrosinase isoforms. However, their molecular features are largely understudied. In fact, the full-length mRNA sequence was determined for one of the tyrosinase genes (i.e., CgTry1; aka cgi-tyr1). Here we report the full-length transcript of a second C. gigas tyrosinase (CgTyr2) sequence and the determination of its sequence features characteristic to the tyrosinase family proteins. We also showed that CgTyr2 gene was differentially expressed with the highest level of expression in mantle edges, suggesting its potential role in the formation of periostracum/pigmentation. Our comprehensive phylogenetic reconstructions supported that hemocyanins possibly evolved from a tyrosinase by an ancient gene duplication followed by functional differentiation, and the current large number of tyrosinase isoforms in C. gigas and other mollusks were originated from multiple gene duplication events that took places before and after mollusk species were established.

Exploring the feasibility of multi-elements coupled with chemometrics for discriminating the geographical origins of oysters (Crassostrea ariakensis).

This study explored the efficacy of multi-elements combined with chemometrics to discriminate the geographical origins of oysters (Crassostrea ariakensi). We determined 52 elements in 166 samples from four regions along the southeast coast of China. Significant regional variations of 51 elements were revealed (P < 0.05), while the principal component analysis (PCA) provided no clear regional delineations. The training models (n = 117) established on linear discriminant analysis (LDA), partial least square discriminant analysis (PLS-DA), and random forest (RF) uniformly achieved 100% predictive accuracy. The cross-validation accuracies of the final models (n = 166) derived from LDA, PLS-DA, and RF were 100%, 100%, and 99.4%, respectively. Even with the models simplified to 8 elements (Zn, Al, K, Cd, Cu, Rb, B, and Ag), high predictive and cross-validation accuracies were maintained, underscoring the robustness and algorithm flexibility of elemental profiling for accurately identifying the geographical origins of oysters.

Morphological and Molecular Analysis Identified a Subspecies of Crassostrea ariakensis (Fujita, 1913) along the Coast of Asia.

Crassostrea ariakensis (Fujita, 1913) is one of the most important economic and ecological oysters that is naturally distributed along the coast of Asia, separated by the Yangtze River estuary. They are usually compared as different populations, while there is no consensus on whether C. ariakensis in northern and southern areas should be considered as two species or subspecies. Here, we analyzed morphological characteristics, COI, 16s rRNA, mitogenome sequences, and species delimitation analysis (ASAP and PTP) to resolve the intraspecific taxonomic status of the C. ariakensis. Phylogenetic and ASAP analysis highlight that C. ariakensis was divided into N-type and S-type. PTP was unable to differentiate between the two types of C. ariakensis. The divergence time of N-type and S-type C. ariakinsis is estimated to be 1.6 Mya, using the relaxed uncorrelated lognormal clock method. Additionally, significant morphological differences exist between the two groups in terms of the adductor muscle scar color. Despite these differences, the COI (0.6%) and 16S rRNA (0.6%) genetic distance differences between N-type and S-type C. ariakensis has not yet reached the interspecific level. These results suggest that N-type and S-type C. ariakensis should be treated as different subspecies and renamed as C. ariakensis ariakensis subsp. nov and C. ariakensis meridioyangtzensis subsp. nov.

Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality.

Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.

Molecular characterization of a novel tetraspanin from the oyster, Crassostrea ariakensis: variation, localization and relationship to oyster host defense.

We identified a tetraspanin family member gene, named Ca-TSP, in the oyster Crassostrea ariakensis and found that the transcription profiles of Ca-TSP were variable in the oyster hemocytes. Three distinct patterns of variation of Ca-TSP were observed. Using immunofluorescence and immunoelectron microscopy, we show that Ca-TSP was present in granules and in vesicular structures of the oyster hemocyte. Sequence analysis, structural features and immunogold electron microscopy showed that Ca-TSP is an integral membrane glycoprotein of granules of hemocyte and may be a novel CD63-like gene of the tetraspanin family of molluscs. The gene expression analysis of Ca-TSP using isolated oyster hemocytes, was done following challenge of the oysters with LPS and Poly I:C. The Ca-TSP mRNA levels increased in hemocytes in the first 12 h after LPS and Poly I:C stimulation, and decreased after the addition of H(2)O(2). Western blot analysis using anti-Ca-TSP antibody indicated that gene expression and protein levels were similar. The recombinant Ca-TSP was found to significantly inhibit hemocytes aggregation. Our results suggested that Ca-TSP participates in the innate immunity of the oyster.

Genetic evidence for the uncoupling of local aquaculture activities and a population of an invasive species--a case study of Pacific oysters (Crassostrea gigas).

Human-mediated introduction of nonnative species into coastal areas via aquaculture is one of the main pathways that can lead to biological invasions. To develop strategies to counteract invasions, it is critical to determine whether populations establishing in the wild are self-sustaining or based on repeated introductions. Invasions by the Pacific oyster (Crassostrea gigas) have been associated with the growing oyster aquaculture industry worldwide. In this study, temporal genetic variability of farmed and wild oysters from the largest enclosed bay in Ireland was assessed to reconstruct the recent biological history of the feral populations using 7 anonymous microsatellites and 7 microsatellites linked to expressed sequence tags (ESTs). There was no evidence of EST-linked markers showing footprints of selection. Allelic richness was higher in feral than in aquaculture samples (P = 0.003, paired t-test). Significant deviations from Hardy-Weinberg equilibrium due to heterozygote deficiencies were detected for almost all loci and samples, most likely explained by the presence of null alleles. Relatively high genetic differentiation was found between aquaculture and feral oysters (largest pairwise multilocus F(ST) 0.074, P < 0.01) and between year classes of oysters from aquaculture (largest pairwise multilocus F(ST) 0.073, P < 0.01), which was also confirmed by the strong separation of aquaculture and wild samples using Bayesian clustering approaches. A 10-fold higher effective population size (N(e)) and a high number of private alleles in wild oysters suggest an established self-sustaining feral population. The wild oyster population studied appears demographically independent from the current aquaculture activities in the estuary and alternative scenarios of introduction pathways are discussed.

Diseases of oysters Crassostrea ariakensis and C. virginica reared in ambient waters from the Choptank River, Maryland and the Indian River Lagoon, Florida.

To assess potential benefits and liabilities from a proposed introduction of Asian Suminoe oysters, susceptibilities of exotic Crassostrea ariakensis and native C. virginica oysters were compared during exposures to pathogens endemic in temperate, mesohaline waters of Chesapeake Bay and sub-tropical, polyhaline Atlantic waters of southern Florida, USA. Cohorts of diploid, sibling oysters of both species were periodically tested for diseases while reared in mesocosms receiving ambient waters from the Choptank River, Maryland (>3 yr) or the Indian River Lagoon, Florida (10 to 11 mo). Haplosporidium sp. infections (e.g. MSX disease) were not detected in oysters from either site. Perkinsus sp. infections (dermo disease) occurred among members of both oyster species at both sites, but infections were generally of low or moderate intensities. A Bonamia sp. was detected by PCR of DNAs from tissues of both oyster species following exposure to Florida waters, with maximum PCR prevalences of 44 and 15% among C. ariakensis and C. virginica oysters respectively during June 2007. Among C. ariakensis oysters sampled during April to July 2007, a Bonamia sp. was detected in 31% of oysters by PCR (range 11 to 35%) and confirmed histologically in 10% (range 0 to 15%). Among simultaneously sampled C. virginica oysters, a Bonamia sp. was detected in 7% by PCR (range 0 to 15%), but histological lesions were absent. Although this is the first report of a Bonamia sp. from Florida waters, sequences of small subunit (SSU) rDNA and in situ hybridization (ISH) assays both identified the Florida pathogen as Bonamia exitiosa, which also infects oysters in the proximate waters of North Carolina, USA.

A Crassostrea gigas Toll-like receptor and comparative analysis of TLR pathway in invertebrates.

Toll-like receptor (TLR) signaling pathway was an important and evolutionarily conserved innate immune pathway. Evolutionary lineage of this pathway in the Lophotrochozoans is still less understood. In this study, we cloned a novel TLR, a key component of TLR pathway, from Crassostrea gigas, and named it CgToll-1. The 4343 base pairs full-length cDNA was assembled with the 3' and 5' RACE (rapid amplification of cDNA ends) PCR results, and containing a 3540 bp open reading frame, which encoding a putative TLR protein of 1179 amino acid residues. Real-time reverse transcription polymerase chain reaction analysis revealed that the highest CgToll-1 expression level was in hemolymph, and the expression pattern in hemolymph dramatically increased in the presence of bacteria Vibrio anguillarum. Furthermore, TLR pathway core genes of mollusks were searched and compared with model invertebrates. Phylogenetic trees of two downstream genes (IκB, Rel) showed that mollusks genes were closer to Drosophila melanogaster than Strongylocentrotus purpuratus, while three upstream genes (MyD88, IRAK, TRAF6) showed the opposite propensity. We have also detected that these two downstream genes were significantly more conservative than the three upstream genes based on amino acid sequence alignment. We found no significant difference between the codon usage biases of TLR pathway genes. This study suggests that CgToll-1 was a constitutive and inducible protein and thus could play an important role in the immune responses against bacterium infection. Besides, comparative analysis of TLR pathway showed that gene loss and divergence might exist during evolution in invertebrate.