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In the last few decades, marine hypoxia has become one of the major ecological concerns in the world, because of the increase of excessive anthropogenic input of nutrients and organic matter into coastal seawater [1]. Benthic communities are the most sensitive parts of the coastal ecosystem to eutrophication and resulting hypoxia [2]. High production in stratified waters results from nutrient enrichment and can cause hypoxic or anoxic bottom waters because of the subsequent deposition of algal biomass [3]. Marine organisms are directly affected by hypoxia at various levels of organization and behavioural, biochemical and physiological responses to limited availability of oxygen have been well studied in fish and marine invertebrates [4]. Most of the invertebrate species that inhabit the intertidal zone, and especially sedentary ones, have developed mechanisms for surviving twicedaily oxygen deprivation at low tide. Depression of metabolic rate can be considered as one of the most important adaptations for hypoxia endurance [5,6]. Many marine molluscs do indeed show reversible protein phosphorylation to limit the activity of many enzymes and functional proteins during anoxia [5,7]. The same response to hypoxia has already been The molecular response to hypoxia stress in aquatic invertebrates remains relatively unknown. In this study, we investigated the response of the Pacific oyster Crassostrea gigas to hypoxia under experimental conditions and focused on the analysis of the differential expression patterns of specific genes associated with hypoxia response. A suppression subtractive hybridization method was used to identify specific hypoxia up-and downregulated genes, in gills, mantle and digestive gland, after 7-10 days and 24 days of exposure. This method revealed 616 different sequences corresponding to 12 major physiological functions. The expression of eight potentially regulated genes was analysed by RT-PCR in different tissues at different sampling times over the time course of hypoxia. These genes are implicated in different physiological pathways such as respiration (carbonic anhydrase), carbohydrate metabolism (glycogen phosphorylase), lipid metabolism (delta-9 desaturase), oxidative metabolism and the immune system (glutathione peroxidase), protein regulation (BTF3, transcription factor), nucleic acid regulation (myc homologue), metal sequestration (putative metallothionein) and stress response (heat shock protein 70). Stress proteins (metallothioneins and heat shock proteins) were also quantified. This study contributes to the characterization of many potential genetic markers that could be used in future environmental monitoring, and could lead to explore new mechanisms of stress tolerance in marine mollusc species.Abbreviations GPx, glutathione peroxidase; HIF-1, hypoxia-inducible factor-1; HSP, heat shock protein; MT, metallothionein; SSH, suppression subtractive hybridization.

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.

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The fluctuating thermal nature of the marine environment induces physiological changes in ectotherms that require molecular and gene expression adjustments [1]. Comparative gene expression studies can be used to characterize these adjustments and lead to a better understanding of organismal responses to environmental change. Gene expression datasets can be clustered into groups of genes that represent different compartments of cellular function, and changes in the expression of genes from these clusters can be used to formulate hypotheses as to how different tissues and whole organisms respond to particular biotic or abiotic stresses. Few studies have addressed changes in gene expression in response to temperature variation on marine organisms. Alterations in gene expression have been observed in fish acclimated to constant temperatures and then exposed to daily temperature fluctuations [2] or to a strong heat stress [3]. However, few molecular investigations have focused on the thermal stress response in marine invertebrates [4,5], particularly in the context of global changes and the potential effects on marine invertebrates [6,7].The Pacific oyster Crassostrea gigas is a eurythermic bivalve mollusc that colonizes most of the western coast of Europe. This species prefers sheltered estuarine waters, where it is found in intertidal and shallow subtidal zones. Within their geographic range, oysters typically experience and respond to seasonal temperatures ranging from 4 to 24°C [8]. In the coldest regions inhabited by C. gigas, such as Brittany, Groups of oysters (Crassostrea gigas) were exposed to 25°C for 24 days (controls to 13°C) to explore the biochemical and molecular pathways affected by prolonged thermal stress. This temperature is 4°C above the summer seawater temperature encountered in western Brittany, France where the animals were collected. Suppression subtractive hybridization was used to identify specific up-and downregulated genes in gill and mantle tissues after 7-10 and 24 days of exposure. The resulting libraries contain 858 different sequences that potentially represent highly expressed genes in thermally stressed oysters. Expression of 17 genes identified in these libraries was studied using real-time PCR in gills and mantle at different time points over the course of the thermal stress. Differential gene expression levels were much higher in gills than in the mantle, showing that gills are more sensitive to thermal stress. Expression of most transcripts (mainly heat shock proteins and genes involved in cellular homeostasis) showed a high and rapid increase at 3-7 days of exposure, followed by a decrease at 14 days, and a second, less-pronounced increase at 17-24 days. A slow-down in protein synthesis occurred after 24 days of thermal stress.Abbreviations CTSL, cathepsin L; EST, expressed sequence tag; HYPK, Huntingtin-interacting protein K; HSP, heat shock protein; LDH, lactate dehydrogenase; MTA-1, metastasis-associated protein 1; SSH, suppression substractive hybridization.

BackgroundReconstructing the history of divergence and gene flow between closely-related organisms has long been a difficult task of evolutionary genetics. Recently, new approaches based on the coalescence theory have been developed to test the existence of gene flow during the process of divergence. The deep sea is a motivating place to apply these new approaches. Differentiation by adaptation can be driven by the heterogeneity of the hydrothermal environment while populations should not have been strongly perturbed by climatic oscillations, the main cause of geographic isolation at the surface.Methodology/Principal FindingSamples of DNA sequences were obtained for seven nuclear loci and a mitochondrial locus in order to conduct a multi-locus analysis of divergence and gene flow between two closely related and hybridizing species of hydrothermal vent mussels, Bathymodiolus azoricus and B. puteoserpentis. The analysis revealed that (i) the two species have started to diverge approximately 0.760 million years ago, (ii) the B. azoricus population size was 2 to 5 time greater than the B. puteoserpentis and the ancestral population and (iii) gene flow between the two species occurred over the complete species range and was mainly asymmetric, at least for the chromosomal regions studied.Conclusions/SignificanceA long history of gene flow has been detected between the two Bathymodiolus species. However, it proved very difficult to conclusively distinguish secondary introgression from ongoing parapatric differentiation. As powerful as coalescence approaches could be, we are left by the fact that natural populations often deviates from standard assumptions of the underlying model. A more direct observation of the history of recombination at one of the seven loci studied suggests an initial period of allopatric differentiation during which recombination was blocked between lineages. Even in the deep sea, geographic isolation may well be a crucial promoter of speciation.