Arnaud Tanguy

Molecular identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas

Abstract The 70-kDa heat shock protein (Hsp) family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. We sequenced 2 genes encoding an Hsp70 and an Hsc70 in the Pacific oyster Crassostrea gigas. The Cghsc70 gene contained introns, whereas the Cghsp70 gene did not. Moreover, the corresponding amino acid sequences of the 2 genes presented all the characteristic motifs of the Hsp70 family. We also investigated the expression of Hsp70 in tissues of oysters experimentally exposed to metal. A recombinant Hsc72 was used as an antigen to produce a polyclonal antibody to quantify soluble Hsp70 by enzyme-linked immunosorbent assay in protein samples extracted from oysters. Our results showed that metals (copper and cadmium) induced a decrease in cytosolic Hsp70 level in gills and digestive gland of oysters experimentally exposed to metal. These data suggest that metals may inhibit stress protein synthesis.

A cDNA Microarray for Crassostrea virginica and C. gigas

The eastern oyster, Crassostrea virginica, and the Pacific oyster, C. gigas, are species of global economic significance as well as important components of estuarine ecosystems and models for genetic and environmental studies. To enhance the molecular tools available for oyster research, an international group of collaborators has constructed a 27,496-feature cDNA microarray containing 4460 sequences derived from C. virginica, 2320 from C. gigas, and 16 non-oyster DNAs serving as positive and negative controls. The performance of the array was assessed by gene expression profiling using gill and digestive gland RNA derived from both C. gigas and C. virginica, and digestive gland RNA from C. ariakensis. The utility of the microarray for detection of homologous genes by cross-hybridization between species was also assessed and the correlation between hybridization intensity and sequence homology for selected genes determined. The oyster cDNA microarray is publicly available to the research community on a cost-recovery basis.

Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.

Molecular identification of differentially regulated genes in the hydrothermal-vent species Bathymodiolus thermophilus and Paralvinella pandorae in response to temperature.

BackgroundHydrothermal vents and cold seeps represent oases of life in the deep-sea environment, but are also characterized by challenging physical and chemical conditions. The effect of temperature fluctuations on vent organisms in their habitat has not been well explored, in particular at a molecular level, most gene expression studies being conducted on coastal marine species. In order to better understand the response of hydrothermal organisms to different temperature regimes, differentially expressed genes (obtained by a subtractive suppression hybridization approach) were identified in the mussel Bathymodiolus thermophilus and the annelid Paralvinella pandorae irlandei to characterize the physiological processes involved when animals are subjected to long term exposure (2 days) at two contrasting temperatures (10° versus 20°C), while maintained at in situ pressures. To avoid a potential effect of pressure, the experimental animals were initially thermally acclimated for 24 hours in a pressurized vessel.ResultsFor each species, we produced two subtractive cDNA libraries (forward and reverse) from sets of deep-sea mussels and annelids exposed together to a thermal challenge under pressure. RNA extracted from the gills, adductor muscle, mantle and foot tissue were used for B. thermophilus. For the annelid model, whole animals (small individuals) were used. For each of the four libraries, we sequenced 200 clones, resulting in 78 and 83 unique sequences in mussels and annelids (about 20% of the sequencing effort), respectively, with only half of them corresponding to known genes. Real-time PCR was used to validate differentially expressed genes identified in the corresponding libraries. Strong expression variations have been observed for some specific genes such as the intracellular hemoglobin, the nidogen protein, and Rab7 in P. pandorae, and the SPARC protein, cyclophilin, foot protein and adhesive plaque protein in B. thermophilus.ConclusionOur results indicate that mussels and worms are not responding in the same way to temperature variations. While the results obtained for the mussel B. thermophilus seem to indicate a metabolic depression (strong decrease in the level of mRNA expression of numerous genes) when temperature increased, the annelid P. pandorae mainly displayed a strong regulation of the mRNA encoding subunits and linkers of respiratory pigments and some proteins involved in membrane structure. In both cases, these regulations seem to be partly due to a possible cellular oxidative stress induced by the simulated thermal environment (10°C to 20°C). This work will serve as a starting point for studying the transcriptomic response of hydrothermal mussels and annelids in future experiments in response to thermal stress at various conditions of duration and temperature challenge.

Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality

Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.