Sylvie Lapegue

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.

Ostreid herpes virus 1 infection in families of the Pacific oyster, Crassostrea gigas, during a summer mortality outbreak: differences in viral DNA detection and quantification using real-time PCR.

Ostreid herpes virus 1 (OsHV-1) infections, notably reported in Europe and the USA, are closely associated with significant mortalities of the Pacific oyster, Crassostrea gigas, especially during its early stages of life. In summer 2006, we monitored mortality by strict daily verification of three full-sib families of oysters reared under common conditions. We quantified OsHV-1 using real-time PCR in dead and living individuals during and after a mortality event. Mortality events were severe and brief, but significantly different between tested families (cumulative mortality ranging from 1.2 to 49%). Real-time PCR assays revealed different viral DNA loads in dead individuals from different families (P<0.001). Moreover, the mean level of infection among families was correlated with mortality (P<0.05). Living oysters showed a significantly lower amount of viral DNA compared with dead ones. This is the first experiment showing the daily changes of individual OsHV-1 DNA load during a mortality outbreak. Our results also support the previously reported high genetic basis underlying the variance of resistance of Pacific oyster to summer mortality, suggesting that there might be a possibility to improve resistance to OsHV-1 by selective breeding.

Phylogeography of the green turtle, Chelonia mydas , in the Southwest Indian Ocean

Patterns of mitochondrial DNA (mtDNA) variation were used to analyse the population genetic structure of southwestern Indian Ocean green turtle (Chelonia mydas) populations. Analysis of sequence variation over 396 bp of the mtDNA control region revealed seven haplotypes among 288 individuals from 10 nesting sites in the Southwest Indian Ocean. This is the first time that Atlantic Ocean haplotypes have been recorded among any Indo‐Pacific nesting populations. Previous studies indicated that the Cape of Good Hope was a major biogeographical barrier between the Atlantic and Indian Oceans because evidence for gene flow in the last 1.5 million years has yet to emerge. This study, by sampling localities adjacent to this barrier, demonstrates that recent gene flow has occurred from the Atlantic Ocean into the Indian Ocean via the Cape of Good Hope. We also found compelling genetic evidence that green turtles nesting at the rookeries of the South Mozambique Channel (SMC) and those nesting in the North Mozambique Channel (NMC) belong to separate genetic stocks. Furthermore, the SMC could be subdivided in two different genetic stocks, one in Europa and the other one in Juan de Nova. We suggest that this particular genetic pattern along the Mozambique Channel is attributable to a recent colonization from the Atlantic Ocean and is maintained by oceanic conditions in the northern and southern Mozambique Channel that influence early stages in the green turtle life cycle.

Mitochondrial and nuclear DNA sequence variation of presumed Crassostrea gigas and Crassostrea angulata specimens: a new oyster species in Hong Kong?

Abstract In several cases, oyster taxa have been misidentified owing to their high morphological plasticity, uncertain geographical range and undocumented introductions. Recently though, molecular techniques have been efficiently applied to discriminate between oysters and to quantify genetic divergence within and among species. In the present paper, we report mitochondrial (16S and COI) and nuclear (28S) DNA sequences of presumed Crassostrea gigas and Crassostrea angulata , two taxa of aquacultural importance. Mitochondrial DNA sequences are compared with previously published sequences and PCR-RFLP haplotypes. Within C. gigas , divergence was less than 0.5% for COI, and less than 0.2% for 16S. Within C. angulata , divergence was less than 1.1% for COI and 0.2% for 16S. Our results also confirm the close genetic relationship between C. gigas and C. angulata and further document their level of divergence: 2–3% for COI and 0.5–1% for 16S. However, the initially presumed C. gigas oysters farmed in Hong Kong (Pearl River delta), presented DNA sequences strongly divergent from both C. gigas and C. angulata : 13–14% for COI, 3–4% for 16S and 1.2–1.6% for 28S. The closest related species are C. gigas and Crassostrea nippona with a divergence of 12–13% for COI, 3–4% for 16S and 1.2–1.6% for 28S. Comparisons with existing DNA sequence data available in the nucleotide sequence databases shows that this is either a new species or that it corresponds to a species for which no DNA sequence is yet available. Further studies are required to document morphological characteristics and geographical range of this putative new species.

Trans-atlantic distribution of a mangrove oyster species revealed by 16S mtDNA and karyological analyses.

Three species of mangrove oysters, Crassostrea rhizophorae, C. brasiliana, and C. gasar, have been described along the Atlantic shores of South America and Africa. Because the distribution of these molluscs is of great biological and commercial interest, their taxonomy and distribution deserve further clarification. Therefore, 15 populations were sampled from both continents. Their 16S mitochondrial polymorphism was studied by sequencing and PCR-RFLP analysis. Two haplotypes were identified. Haplotype a was the only one observed in Africa, but it was also observed in South America together with haplotype b. Because C. gasar is the only mangrove oyster identified on the west coast of Africa, haplotype a was attributed to this species, which has thus been shown to occur in South America. Haplotype b is attributed to C. rhizophorae. The karyotypes of specimens of C. gasar, from Africa and from South America, were very similar, and both species were observed at the same location in Brazil. The occurrence of C. gasar in South America adds a third species—in addition to C. rhizophorae and C. brasiliana—to the list of species present along these coasts. The predominant surface circulation patterns in this part of the Atlantic Ocean favor the hypothesis that C. gasar was transported from Africa to America. Finally, a phylogenetic tree built with seven 16S sequences from Crassostrea and Saccostrea species showed that C. gasar is intermediate between the American Crassostrea species (C. virginica and C. rhizophorae) and the Asian species (C. gigas and C. ariakensis).

Mitochondrial and nuclear DNA phylogeography of Crassostrea angulata, the Portuguese oyster endangered in Europe

The respective status of the Portuguese oyster, Crassostrea angulata, and the Pacific oyster, Crassostrea gigas, has long been a matter of controversy. Morphological and physiological similarities, homogeneity of allozyme allelic frequencies between populations of the two taxa and the demonstration of hybridization lead most authors to suggest that they should be regrouped within the same species. The risk of introgression and the present expansion of C. gigas aquaculture in Europe raises the question of the need for preservation of C. angulata in Europe, as only a few populations remain. We studied European and Asian populations of C. gigas and C. angulata using microsatellite and mitochondrial DNA markers to estimate their genetic diversity and differentiation. The analysis of genetic distances and the distribution of allelic and haplotype frequencies revealed significant genetic differences between taxa, showing two clusters: (1) C. gigas French and Japanese populations and (2) C. angulata Portuguese and Taiwanese populations. The Asian origin of the Crassostrea angulata taxa is therefore confirmed. Unlike previous studies based on allozymes, significant nuclear genome differences were noted between C. angulata and C. gigas. Despite the presumed history of the introduction of C. angulata into Southern Europe, these populations did not show any significant reduction of variability compared to Taiwanese populations. Any conservation plans for European C. angulata populations should take its non-native origin into account. They represent a valuable genetic resources for European breeding program.

QTL for resistance to summer mortality and OsHV-1 load in the Pacific oyster (Crassostrea gigas).

Summer mortality is a phenomenon severely affecting the aquaculture production of the Pacific oyster (Crassostrea gigas). Although its causal factors are complex, resistance to mortality has been described as a highly heritable trait, and several pathogens including the virus Ostreid Herpes virus type 1 (OsHV-1) have been associated with this phenomenon. A QTL analysis for survival of summer mortality and OsHV-1 load, estimated using real-time PCR, was performed using five F(2) full-sib families resulting from a divergent selection experiment for resistance to summer mortality. A consensus linkage map was built using 29 SNPs and 51 microsatellite markers. Five significant QTL were identified and assigned to linkage groups V, VI, VII and IX. Analysis of single full-sib families revealed differential QTL segregation between families. QTL for the two-recorded traits presented very similar locations, highlighting the interest of further study of their respective genetic controls. These QTL show substantial genetic variation in resistance to summer mortality, and present new opportunities for selection for resistance to OsHV-1.

Combining two-stage testing and interval mapping strategies to detect QTL for resistance to bonamiosis in the european flat oyster Ostrea edulis.

We have identified quantitative trait loci (QTL) in the flat oyster (Ostrea edulis) for resistance to Bonamia ostreae, a parasite responsible for the dramatic reduction in the aquaculture of this species. An F2 family from a cross between a wild oyster and an individual from a family selected for resistance to bonamiosis was cultured with wild oysters injected with the parasite, leading to 20% cumulative mortality. Selective genotyping of 92 out of a total of 550 F2 progeny (i.e., 46 heavily infected oysters that died and 46 parasite-free oysters that survived) was performed using 20 microsatellites and 34 amplification fragment length polymorphism primer pairs. Both a two-stage testing strategy and QTL interval mapping methods were used. The two-stage detection strategy had a high power with a low rate of false positives and identified nine and six probable markers linked to genes of resistance and susceptibility, respectively. Parent-specific genetic linkage maps were built for the family, spanning ten linkage groups (n = 10) with an observed genome coverage of 69–84%. Three QTL were identified by interval mapping in the first parental map and two in the second. Good concordance was observed between the results obtained after the two-stage testing strategy and QTL mapping.

Study of Atrazine Effects on Pacific Oyster, Crassostrea gigas, Haemocytes

Shellfish farming is an important economic activity around the world. This activity often takes place in areas subjected to various recurring pollutions. The recrudescent use of herbicides in agriculture including atrazine implies pollutant transfer towards aquatic environment in estuarine areas. Harmful effects of such substances on animals in marine environment, particularly on cultured bivalves, are poorly documented. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate and concentrate contaminants within their tissues. Moreover, development of techniques allowing effect analysis of such compounds on bivalve biology may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, influence of atrazine on defence mechanisms was analyzed in Pacific oysters, Crassostrea gigas. Atrazine was tested in vitro and in vivo on oyster haemocytes, and its effects were analyzed by flow cytometry. Haemocyte viability, cell cycle and cellular activities were monitored. Atrazine induced no significant effect in oyster under tested conditions except for peroxidase activity.

Population genomics shed light on the demographic and adaptive histories of European invasion in the Pacific oyster, Crassostrea gigas.

Crassostrea gigas originated from the Pacific coast of Asia, but was introduced into several European countries in the early 1970s. Natural populations have now spread across the length of the western seaboard of Europe. To elucidate the demographic and selective processes at play during this rapid expansion, genome‐scan analysis was performed on different populations. High diversities and low differentiation were observed overall, but significant genetic differentiation was found among newly established populations and between the newly established northern group and a nearly panmictic group composed of southern European populations and a population from Japan. Loss of genetic diversity was also seen in the north, likely caused by founder events during colonization. The few strongly supported outlier loci revealed a genetic structure uncorrelated with the north/south differentiation, but grouping two samples from the Danish fjords (northern group) and one from the Dutch Scheldt estuary (southern group) with the one from Japan. These findings might reflect the following: (i) parallel adaptation to similar environmental pressures (fjord‐like environment) within each of the two groups or (ii) a footprint of a secondary introduction of an alternative genomic background maintained by multifarious isolation factors. Our results call for a closer examination of adaptive genetic structure in the area of origin.