First evidence of a potential antibacterial activity involving a laccase-type enzyme of the phenoloxidase system in Pacific oyster Crassostrea gigas haemocytes

Phenoloxidases (POs) are a group of copper proteins including tyrosinase, catecholase and laccase. In several insects and crustaceans, antibacterial substances are produced through the PO cascade, participating in the direct killing of invading microorganisms. However, although POs are widely recognised as an integral part of the invertebrate immune defence system, experimental evidence is lacking that these properties are conserved in molluscs, and more particularly in the Pacific oyster Crassostrea gigas. In the present study, Vibrio splendidus LGP32 and Vibrio aestuarianus 02/041 growths were affected, after being treated with C. gigas haemocyte lysate supernatant (HLS), and either a common substrate of POs, l-3,4-dihydroxyphenylalanine (L-DOPA), to detect catecholase-type PO activity, or a specific substrate of laccase, p-phenylenediamine (PPD), to detect laccase-type PO activity. Interestingly, a higher bacterial growth inhibition was observed in the presence of PPD than in the presence of L-DOPA. These effects were suppressed when the specific PO inhibitor, phenylthiourea (PTU), was added to the medium. Results of the present study suggest, for the first time in a mollusc species, that antibacterial activities of HLS from C. gigas potentially involve POs, and more particularly laccase catalysed reactions.     

Screening and selection of stress resistant Lactobacillus spp. isolated from the marine oyster (Crassostrea gigas)

We attempted to isolate Lactobacillus spp. from the marine oyster (Crassostrea gigas) and select stress resistant strains for development of a future marine aquaculture feed adjuvant. A total of 83 lactobacilli strains were isolated from oyster. They were all Gram-positive, rod-shaped and catalase-negative. By performing a stress resistance assay, we selected eighteen isolates. Based on 16S rRNA gene sequencing, Lactobacillus paracasei was the most prevalent species among the selected isolates. The in vitro antagonistic effect of the selected strains against fish pathogens was assayed by measurement of inhibition diameters. Except for MH44, MH51, MH53 and MH62, most of the isolates showed inhibition of Vibrio alginolyticus and Vibrio proteolyticus (diameters over 15 mm). Lactobacillus rhamnosus MH22 was selected as the most stress resistant strain showing the MICs of 1.8 M NaCl, 14% ethanol and 0.014% hydrogen peroxide. L. rhamnosus MH22 isolated from oyster has a potential to be applied as a microbial feed adjuvant for marine aquaculture.     

Comparison of quantitative gonad maturation scales in a temperate oyster (Crassostrea gigas) and a sub-tropical oyster (Crassostrea corteziensis)

Quantitative scales to evaluate maturity of female gonads were compared for a temperate oyster, Crassostrea gigas and a tropical oyster, Crassostrea corteziensis grown in the same locality. C. gigas had well-defined maturation and spawning periods and a resting phase in winter; in C. corteziensis mature individuals occurred most of the year and there were several spawning peaks. Of the quantitative scales used here, average oocyte diameter and gonad coverage area, much used for C. gigas, and ovary maturity index, less used, were inadequate to distinguish the reproductive pattern of C. corteziensis, since they both skewed the degree of maturation in vitellogenic stages in favor of C. gigas. Maximum oocyte diameter and maximum cytoplasm area were different among species at vitellogenic stages and also in previtellogenic stages. Nucleus:cytoplasm ratio was significantly different in previtellogenic and spawned stages between C. gigas and C. corteziensis. The Index of gonadal development was skewed in favor of C. gigas in postvitellogenic stage. The only scale that was comparable between species was reproductive potential, but it also was one of the most laborious. Other ordinal scales commonly used, such as a visual external evaluation of the gonad, only classified correctly a quarter of the stages.     

Cloning,characterization and chromosomal location of a satellite DNA from the Pacific oyster,Crassostrea gigas

We report the cloning and characterization of a high-copy-number, tandem-repeat satellite DNA sequence from the genome of the Pacific oyster, Crassostrea gigas (Cg). The monomeric unit was found to be 166 (±2) bp in length with 79–;94% homology between monomers of the array. The sequence is A+T-rich (60%) and lacks internal repetition and substructural features. The repeat was estimated to account for 1–;4% of the Cg genome. Fluorescence in situ hybridization (FISH) studies mapped the repeat to two distinct heterochromatic regions of two pairs of homologous chromosomes on Cg embryonic metaphases. Also, the number of metaphase chromosomes containing this repeat varied with the ploidy of the cell.     

The oyster vasa-like gene: a specific marker of the germline in Crassostrea gigas

The vasa gene is a key determinant for germline formation in eukaryotes. This gene, highly conserved through evolution, encodes a RNA helicase protein member of the DEAD-box family. To understand the germline formation in oyster, we report here the isolation and the characterization of a vasa orthologue in Crassostrea gigas (Oyvlg). OyVLG contained the eight consensus domains of the DEAD-box including those providing RNA unwinding activity. The expression pattern of Oyvlg was examined in adult oyster tissues at different reproductive stages. Its expression was restricted to germline cells both in males and females, including germinal stem cells and auxiliary cells. The expression of Oyvlg, strongest in early germ cells, decreased as the maturation proceeded. These data and the evolutionary conservation observed suggested the role of Oyvlg in germline development. Oyvlg is the first germ cell specific marker in oyster and will be very useful in studies of oyster germline formation.     

Molecular and phenotypic characterization of Vibrio aestuarianus subsp. francensis subsp. nov., a pathogen of the oyster Crassostrea gigas

Eleven Vibrio isolates invading the hemolymph of live and moribund oysters (Crassostrea gigas) collected in the field and from a hatchery in France, were characterized by a polyphasic approach. Phylogenetic analysis of 16S rRNA, gyrB and toxR genes indicated high homogeneity between these strains and the Vibrio aestuarianus type strain (ATCC35048(T)), and confirmed previous 16S rRNA analysis. In contrast, DNA:DNA hybridization was from 61% to 100%, while phenotypic characters and virulence tests showed a large diversity between the strains. Nevertheless, several common characters allowed the isolates to be distinguished from the reference strain. On the basis of several distinct phenotypic characteristics, it is proposed to establish two subspecies within the V. aestuarianus spp. group, V. aestuarianus subsp. aestuarianus [D. Tison, R. Seidler, Vibrio aestuarianus: a new species from estuarine waters and shellfish, Int. J. Syst. Bacteriol. (1983) 699-702] and V. aestuarianus subsp. francensis for these French isolates. The characters that differentiate the new strains from V. aestuarianus subsp. aestuarianus(T) are virulence (positive for 63% of the isolates) and 12:0 fatty acid content. The colonies were smaller and uncoloured, whereas no growth occurred at 35 degrees C or on TCBS, and the strains did not utilize several substrates, including L-serine, alpha-cyclodextrin, D-mannitol, alpha-glycyl-L-aspartic acid, L-threonine and glucose-1-phosphate.     

Exploiting EST databases for the development and characterization of EST-SSRs in the Pacific oyster (Crassostrea gigas)

A total of 147 microsatellite-containing expressed sequence tags (ESTs) (3.63%) were detected from 4053 ESTs of the Pacific oyster (Crassostrea gigas) in GenBank. The average density of simple sequence repeats (SSRs) was 1 per 8.25 kb of EST after redundancy elimination. Dinucleotide repeat motifs appeared to be the most abundant type. Sixteen new polymorphic EST-SSRs were developed. The number of alleles per locus varied from 3 to 12, with an average of 5.9 alleles per locus. Marker transferability was tested on 2 other Crassostrea species, and 14 loci gave successful amplifications in both species. Twenty EST-SSRs were tested on 3 families of C. gigas for examination of inheritance mode of EST-SSRs. Thirty-five tests of segregation ratios revealed 5 significant departures from expected Mendelian ratios, 4 of which confirmed Mendelian expectations when accounting for the presence of null alleles. Null alleles were detected at 3 loci (15.0%) of the 20 loci, and the frequency of null alleles at EST-SSRs was lower than that in genomic SSRs in C. gigas. The results obtained in this study suggest that C. gigas EST-SSRs will complement the currently available genomic SSR markers and may be useful for comparative mapping, marker-assisted selection, and evolutionary studies.     

Mitochondrial activity,hemocyte parameters and lipid composition modulation by dietary conditioning in the Pacific oyster Crassostrea gigas

Several parameters can affect membrane lipid composition in bivalves, including diet. Although two fatty acids (FA) 22:6n-3 and 20:5n-3 are essential membrane components, they are sparingly synthesized by bivalves and must be obtained from their diet. Here, effects of dietary modifications of membrane lipid composition were studied at both cellular and subcellular levels in the oyster Crassostrea gigas. To this end, we compared oysters fed two monoalgal diets that differed markedly in their FA composition and a mix of both. As expected, algae impacted phospholipids, in particular 22:6n-3 and 20:5n-3, reflecting differences of dietary microalgae FA composition. Meantime, total saturated FA, total monounsaturated FA, total polyunsaturated FA and total non-methylene-interrupted FA varied little and phospholipid class composition was only slightly affected by diets. Measures made in hemocytes indicated that only mitochondrial membrane potential was affected by diets. Total ROS production as well as mitochondrial superoxide production did not differ with diet. There was no difference in phosphorylating (state 3) and non-phosphorylating (state 4) rates of oxygen consumption rates or in cytochrome c oxidase activity of mitochondria isolated from gills between the three diets. Similarly, neither cytochromes a, b, c or c ₁ content nor citrate synthase activities were changed, suggesting that number and morphology of mitochondria were not affected by dietary treatment. These results suggest that oysters could possess high homeostatic capabilities, at both cellular and subcellular levels, to minimize the effect of dietary FA and related membrane lipid FA modifications on mitochondrial functions. These capabilities could be a means to face variations in diet composition in their natural environment and to preserve important oyster physiological functions such as growth and reproduction.     

Identification and expression of a factor of the DM family in the oyster Crassostrea gigas

The Pacific oyster Crassostrea gigas is a successive not systematic protandric hermaphrodite. Searching for an ortholog to Dmrt1, a conserved sex determinism factor, we have identified the first complete cDNA of a DM factor in Lophotrochozoa which we have called Cg-DMl (Crassostrea gigas DMRT-like). It is 359aa long, with the DM domain common to all the family factors, and one DMA domain specific to members such as Dmrt4 and Dmrt5. Its gene presents one intron of 598 bp. Real time PCR and in situ hybridization have shown that Cg-DMl was expressed in both sexes, with a significantly higher expression in male than in female gonads at the end of the adult gametogenetic cycle and that a significant peak of expression was observed in spat between 1 and 2 months of age. These results suggest that Cg-DMl may be involved in the development of the gonad and may constitute preliminary clues for future work in order to better understand DM protein evolution.     

Characterization of a defensin from the oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial activities, and gene expression

In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized alpha-beta motif (CS-alphabeta) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.     

Development, characterization, and inheritance of 113 novel EST-SSR markers in the Pacific oyster (Crassostrea gigas)

A total of 113 novel EST-derived simple sequence repeat (EST-SSR) markers were developed in the Pacific oyster (Crassotrea gigas). Polymorphisms of these markers were evaluated in a wild population of 30 individuals. The number of alleles per locus ranged from 2 to 27 with an average of 6.3, and the observed and expected heterozygosities varied from 0 to 0.9667 and from 0.0333 to 0.9701, respectively. Mendelian segregations were tested for 24 of the markers that were polymorphic in one family produced by single-pair mating. Null alleles were discovered at four loci. Nine tests of segregation ratios revealed significant departures from expected Mendelian ratios. As a useful addition to the collection of the microsatellites that are now available for C. gigas, these EST-SSR markers will help the advance in investigation of QTL mapping and genetic diversity in this species.     

Demonstration of a true phenoloxidase activity and activation of a ProPO cascade in Pacific oyster, Crassostrea gigas (Thunberg) in vitro

The prophenoloxidase (ProPO) system is the origin of melanin production and is considered to be an innate defence mechanism in invertebrates. In different bivalve species, phenoloxidase (PO) is present in the haemolymph as an inactive form of ProPO. The present study focuses on the Pacific adult oyster, Crassostrea gigas, an economically important bivalve species along French coasts. The results indicate that many factors may inhibit the PO-like activity. These include: phenylthiourea (PTU), sodium diethylthiocarbamate (DETC), beta-mercaptoethanol and tropolone, which repressed the spontaneous PO activity. The activation of PO-like activity in C. gigas acellular fraction by lipopolysaccharide (LPS) involved participation of other factors, including at least one serine protease. PO was present as proPO in the acellular fraction of haemolymph and haemocytes of C. gigas and could be activated by an exogenous protease (trypsin-N-tosyl-l-phenylalanine chloromethyl ketone) when used at 1 gL(-1). Treatment of acellular fractions with other modulators/activators namely LPS (1 gL(-1)), zymosan (0.6 gL(-1)) or laminarin (0.6 gL(-1)) also increased PO-like activity but to a less important way. The study demonstrated the evidence of a true phenoloxidase activity in Pacific oyster, C. gigas (Thunberg). The activation of a proPO system by non-self molecules suggests the role played by PO in vivo in the internal defence mechanisms. Understanding the activation of the ProPO system could enable the evaluation of the health of oyster stocks.     

Identification and characterization of 18 novel polymorphic microsatellite makers derived from expressed sequence tags in the Pacific oyster Crassostrea gigas

We report the development of 18 new polymorphic microsatellite DNA markers derived from Crassostrea gigas expressed sequences tags. Genotyping of 48 wild adult oysters sampled from Marennes-Oléron bay (France) revealed 12 to 48 alleles per locus. Observed and expected heterozygosity levels ranged from 0.64 to 1 and from 0.77 to 0.97, respectively. The development of these new markers creates a useful complementary tool for population genetics studies, parentage analysis and mapping in Pacific oyster, a species of major aquacultural and ecological importance.     

Molecular characterization of the glutamine synthetase gene in the Pacific oyster Crassostrea gigas: expression study in response to xenobiotic exposure and developmental stage

In this study, we characterized the full-length cDNA and genomic sequence of the gene encoding cytosolic glutamine synthetase (CgGSII) in the Pacific oyster, Crassostrea gigas. A phylogenetic analysis of GS sequences showed that CgGS clustered with the invertebrate group as expected. We analyzed the expression of mRNA CgGSII using RT-PCR to follow the expression of this gene in gills and digestive gland of oysters exposed, under experimental conditions, to hypoxia and to several contaminants (hydrocarbons and two pesticide treatments, glyphosate and a mixture of atrazine, diuron and isoproturon). We also investigated the expression of CgGSII in different developmental stages of C. gigas. Our results show that CgGSII expression was highly regulated in xenobiotic-exposed oysters compared to the control for all the treatments. Likewise, CgGSII expression was highly regulated according to the developmental stage of C. gigas. Finally, use of CgGSII as a possible marker to monitor xenobiotic exposure in disturbed ecosystems is discussed.     

Identification of three singular glycosyl hydrolase family 18 members from the oyster Crassostrea gigas: Structural characterization,phylogenetic analysis and gene expression

Glycoside hydrolase family 18 (GH18) includes chitinases and non-enzymatic chitinase-like proteins (CLPs) with representatives among eukaryotes (animals and plants), prokaryotes and viruses. In Lophotrochozoa, one of the three clades of bilaterian animals, only three members (Cg-Clp1, Cg-Clp2 and Cg-Chit) have been reported from the bivalve mollusc Crassostrea gigas. Here, we describe the cloning and the characterization of two additional chitinases (Cg-Chit2 and Cg-Chit3) and a new CLP (Cg-Clp3) from this species. Cg-Chit2 presents an atypical C-terminal hydrophobic region acting probably as a GPI-anchor signal for plasma membrane attachment. On the contrary, Cg-Chit3 displays a C-terminal truncated structure leading to a possible sequestration in lysosomes. Phylogenetic analyses suggest that CLPs have appeared independently in the three main branches of bilaterian animals, as a result of convergent evolution. Gene expression profiles analyzed by quantitative RT-PCR support the involvement of Cg-Clp3 in embryonic development, adult oyster growth and tissue remodelling during metamorphosis and gonadal restructuring.