Sessile inhabitants of marine intertidal environments commonly face heat stress, an important component of summer mortality syndrome in the Pacific oyster Crassostrea gigas. Marker-aided selection programs would be useful for developing oyster strains that resist summer mortality; however, there is currently a need to identify candidate genes associated with stress tolerance and to develop molecular markers associated with those genes. To identify candidate genes for further study, we used cDNA microarrays to test the hypothesis that oyster families that had high (>64%) or low (<29%) survival of heat shock (43°C, 1 h) differ in their transcriptional responses to stress. Based upon data generated by the microarray and by real-time quantitative PCR, we found that transcription after heat shock increased for genes putatively encoding heat shock proteins and genes for proteins that synthesize lipids, protect against bacterial infection, and regulate spawning, whereas transcription decreased for genes for proteins that mobilize lipids and detoxify reactive oxygen species. RNAs putatively identified as heat shock protein 27, collagen, peroxinectin, S-crystallin, and two genes with no match in Genbank had higher transcript concentrations in low-surviving families than in high-surviving families, whereas concentration of putative cystatin B mRNA was greater in high-surviving families. These ESTs should be studied further for use in marker-aided selection programs. Low survival of heat shock could result from a complex interaction of cell damage, opportunistic infection, and metabolic exhaustion.

We used qualitative complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) differential display analysis and real-time, quantitative PCR (RT-qPCR) to identify genes in the Pacific oyster Crassostrea gigas, whose transcription either changes in response to exposure to a pathogenic bacterium (Vibrio tubiashii) or varies between families known to differ in sensitivity to heat stress, before and at 12 and 36 h after bacterial exposure at a temperature of 25 degrees C. These conditions simulate those associated with summer mortality syndrome, a poorly understood cause of massive mortalities in cultured Pacific oysters in North America, Asia and Europe. Using 32 AFLP primer pairs, we identified 92 transcript-derived fragments that are qualitatively differentially expressed. We then cloned and sequenced 14 of these fragments, designed fragment-specific primers and quantified their transcription patterns using RT-qPCR. Most of the differences in transcription patterns between stress-tolerant and stress-sensitive families were evident before bacterial exposure, and genes that responded to bacterial exposure did so in parallel between stress-sensitive and stress-tolerant families. Blast searches of sequence databases revealed that these fragments represent genes involved in immune response as well as genes related to metabolic processes. Our data support the hypothesis that family level differences in resistance to stress in Pacific oysters are largely attributable to constitutive differences in gene transcription or 'general vigour' that are detectable before and maintained after infection, rather than being due to induced responses at the transcriptome level.

No abstract