Arnaud Huvet

Generation and analysis of a 29,745 unique Expressed Sequence Tags from the Pacific oyster (Crassostrea gigas) assembled into a publicly accessible database: the GigasDatabase

BackgroundAlthough bivalves are among the most-studied marine organisms because of their ecological role and economic importance, very little information is available on the genome sequences of oyster species. This report documents three large-scale cDNA sequencing projects for the Pacific oyster Crassostrea gigas initiated to provide a large number of expressed sequence tags that were subsequently compiled in a publicly accessible database. This resource allowed for the identification of a large number of transcripts and provides valuable information for ongoing investigations of tissue-specific and stimulus-dependant gene expression patterns. These data are crucial for constructing comprehensive DNA microarrays, identifying single nucleotide polymorphisms and microsatellites in coding regions, and for identifying genes when the entire genome sequence of C. gigas becomes available.DescriptionIn the present paper, we report the production of 40,845 high-quality ESTs that identify 29,745 unique transcribed sequences consisting of 7,940 contigs and 21,805 singletons. All of these new sequences, together with existing public sequence data, have been compiled into a publicly-available Website http://public-contigbrowser.sigenae.org:9090/Crassostrea_gigas/index.html. Approximately 43% of the unique ESTs had significant matches against the SwissProt database and 27% were annotated using Gene Ontology terms. In addition, we identified a total of 208 in silico microsatellites from the ESTs, with 173 having sufficient flanking sequence for primer design. We also identified a total of 7,530 putative in silico, single-nucleotide polymorphisms using existing and newly-generated EST resources for the Pacific oyster.ConclusionA publicly-available database has been populated with 29,745 unique sequences for the Pacific oyster Crassostrea gigas. The database provides many tools to search cleaned and assembled ESTs. The user may input and submit several filters, such as protein or nucleotide hits, to select and download relevant elements. This database constitutes one of the most developed genomic resources accessible among Lophotrochozoans, an orphan clade of bilateral animals. These data will accelerate the development of both genomics and genetics in a commercially-important species with the highest annual, commercial production of any aquatic organism.

In vivo RNA interference in oyster –vasa silencing inhibits germ cell development

This study investigated the potential of RNA interference, which is technically challenging in bivalve mollusc species, to assess gene function in the oyster Crassostreau2003gigas. We designed dsRNA targeting the oyster vasa‐like gene (Oyvlg), specifically expressed in oyster germ cells. Inu2003vivo injection of oyvl‐dsRNA into the gonad provokes a knockdown phenotype corresponding to germ cell underproliferation and prematurely arrested meiosis througout the organ. The most severe phenotype observed is sterile. This knockdown phenotype is associated with a decrease in Oyvlg mRNA level of between 39% and 87%, and a strong reduction in OYVLG protein, to an undetectable level. Therefore, Oyvlg appears to be essential for germ cell development in Crassostreau2003gigas, particularly for mitotic proliferation and early meiosis. Our results demonstrate for the first time that inu2003vivo RNA interference works efficiently in a bivalve species, opening major perspectives for functional genetic studies.

Microarray-Based Identification of Gonad Transcripts Differentially Expressed Between Lines of Pacific Oyster Selected to Be Resistant or Susceptible to Summer Mortality

Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment, and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event, using a cDNA microarray that we designed. ANOVA analysis revealed that 34 genes were differentially expressed between R and S lines on four dates preceding the mortality event. Annotation of some of these genes highlights reproduction and its allocation and antioxidant defenses as the main pathways that operate differentially between R and S lines. This transcriptional analysis provides new indications to define markers for quantitative trait loci searches and functional studies and evaluate the potential role of each gene in the resistance to summer mortality.

Structural and functional characterizations of an Activin type II receptor orthologue from the pacific oyster Crassostrea gigas

Members of the Transforming Growth factor beta (TGF-beta) superfamily of cell signalling polypeptides are known to play important roles in cell proliferation and differentiation during development and in various physiological processes of most animal clades. Recent findings in the mollusc Crassostrea gigas demonstrate the occurrence of a diversity of TGF-beta signalling components including various ligands, three type I receptors but only a single type II receptor. This report describes the characterization of Cg-ActRII, a new type II receptor displaying homology with vertebrate and Drosophila Activin type II receptors. The use of zebrafish embryo as a reporter organism revealed that, in a way similar to its zebrafish counterpart, overexpression of Cg-ActRII or its dominant negative acting truncated form resulted in a dose dependent range of dorsoventral defects coupled with anterior disorders. Expression pattern of Cg-ActRII transcripts examined by real time PCR and in situ PCR in C. gigas showed high levels of Cg-ActRII transcripts in early embryonic stages and in the developing larval central nervous system. Except for a high expression in the visceral ganglia, most oyster adult tissues displayed rather low levels of transcripts. Altogether, the data suggest a high degree of conservation at both the structural and functional levels during evolution for this class of receptors.