Project description:Background The Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa) is an alternative and irregular protandrous hermaphrodite. Little is known about the genetic and phenotypic bases of sex determinism in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011) representing the oyster transcriptome. The application of this microarray to the study of mollusks gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction. Results Expression of the 31,918 ESTs was studied in gonads of oysters cultured along the French Atlantic coasts over a yearly reproductive cycle. Principal component analysis and hierarchical clustering first showed a significant divergence in gene expression patterns of males and females beginning when gonial mitosis started. Early expressed male-specific genes included bindin and female-specific genes included foxL2, a pancreatic lipase related protein, cd63 and vitellogenin. ANOVA analysis of the data further revealed 2482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of gonads to the transcriptomes of striped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals. Genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Additional lists of genes more specifically involved in either spermatogenesis (meiotic phase, in spermatozoids and mature sperm formation and in flagella structure and movement) or oogenesis (female sex differentiation, transcriptional regulation, cell cycle regulation and oocytes maturation) were also generated. Conclusions Our study provides novel insight into spermatogenesis and oogenesis in an alternative hermaphrodite bivalve, the Pacific oyster C. gigas. We identified genes opening new perspectives for functional studies of the signaling pathways implicated in gonad differentiation and development.

Project description:Background The Pacific oyster Crassostrea gigas (Mollusca, Lophotrochozoa) is an alternative and irregular protandrous hermaphrodite. Little is known about the genetic and phenotypic bases of sex determinism in oysters, and little more about the molecular pathways regulating reproduction. We have recently developed and validated a microarray containing 31,918 oligomers (Dheilly et al., 2011) representing the oyster transcriptome. The application of this microarray to the study of mollusks gametogenesis should provide a better understanding of the key factors involved in sex differentiation and the regulation of oyster reproduction. Results Expression of the 31,918 ESTs was studied in gonads of oysters cultured along the French Atlantic coasts over a yearly reproductive cycle. Principal component analysis and hierarchical clustering first showed a significant divergence in gene expression patterns of males and females beginning when gonial mitosis started. Early expressed male-specific genes included bindin and female-specific genes included foxL2, a pancreatic lipase related protein, cd63 and vitellogenin. ANOVA analysis of the data further revealed 2482 genes differentially expressed during the course of males and/or females gametogenesis. The expression of 434 genes could be localized in either germ cells or somatic cells of the gonad by comparing the transcriptome of gonads to the transcriptomes of striped oocytes and somatic tissues. Analysis of the annotated genes revealed conserved molecular mechanisms between mollusks and mammals. Genes involved in chromatin condensation, DNA replication and repair, mitosis and meiosis regulation, transcription, translation and apoptosis were expressed in both male and female gonads. Additional lists of genes more specifically involved in either spermatogenesis (meiotic phase, in spermatozoids and mature sperm formation and in flagella structure and movement) or oogenesis (female sex differentiation, transcriptional regulation, cell cycle regulation and oocytes maturation) were also generated. Conclusions Our study provides novel insight into spermatogenesis and oogenesis in an alternative hermaphrodite bivalve, the Pacific oyster C. gigas. We identified genes opening new perspectives for functional studies of the signaling pathways implicated in gonad differentiation and development. Individual gonad oysters were sampled in 3 sites: site 1 = Locmariaquer (Brittany, France), site 2 = Baie des Veys (Normandy, France), and site 3 = Argenton (Brittany, France). 8 undifferentiated stage 0 gonads from site 1 were processed. 4 individuals from site 1 were processed for each gonad developmental stage and sex: stage 1 male, stage 1 female, stage 2 male, stage 2 female, stage 3 male, and stage 3 female. Pools of stage 3 females were prepared with individuals from site 1 and site 2 for biological validation of the results. Striped gonads from stage 3 females were prepared for prediction of gene expression localization.

Project description:BackgroundThe Pacific oyster, Crassostrea gigas, has developed special mechanisms to regulate its osmotic balance to adapt to fluctuations of salinities in coastal zones. To understand the oyster's euryhaline adaptation, we analyzed salt stress effectors metabolism pathways under different salinities (salt 5, 10, 15, 20, 25, 30 and 40 for 7 days) using transcriptome data, physiology experiment and quantitative real-time PCR.ResultsTranscriptome data uncovered 189, 480, 207 and 80 marker genes for monitoring physiology status of oysters and the environment conditions. Three known salt stress effectors (involving ion channels, aquaporins and free amino acids) were examined. The analysis of ion channels and aquaporins indicated that 7 days long-term salt stress inhibited voltage-gated Na(+)/K(+) channel and aquaporin but increased calcium-activated K(+) channel and Ca(2+) channel. As the most important category of osmotic stress effector, we analyzed the oyster FAAs metabolism pathways (including taurine, glycine, alanine, beta-alanine, proline and arginine) and explained FAAs functional mechanism for oyster low salinity adaptation. FAAs metabolism key enzyme genes displayed expression differentiation in low salinity adapted individuals comparing with control which further indicated that FAAs played important roles for oyster salinity adaptation. A global metabolic pathway analysis (iPath) of oyster expanded genes displayed a co-expansion of FAAs metabolism in C. gigas compared with seven other species, suggesting oyster's powerful ability regarding FAAs metabolism, allowing it to adapt to fluctuating salinities, which may be one important mechanism underlying euryhaline adaption in oyster. Additionally, using transcriptome data analysis, we uncovered salt stress transduction networks in C. gigas.ConclusionsOur results represented oyster salt stress effectors functional mechanisms under salt stress conditions and explained the expansion of FAAs metabolism pathways as the most important effectors for oyster euryhaline adaptation. This study was the first to explain oyster euryhaline adaptation at a genome-wide scale in C. gigas.

Project description:As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation-reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity.

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Gonadal transcriptomes associated with sex phenotypes provide potential male and female candidate genes of sex determination or early differentiation in Crassostrea gigas, a sequential hermaphrodite mollusc.

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