Transcriptome profiling of selectively bred Pacific oyster Crassostrea gigas families that differ in tolerance of heat shock

Abstract

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 degrees 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.

Fig. 1

Summary of experimental design. a Gills from two oyster families characterized by high (white circles) or low survival (hatched circles) of a 1 h, 43°C heat shock were collected before and at 1, 3, 6, and 24 h after a non-lethal heat shock (40°C, 1 h) in an experiment that was repeated three times to yield 60 samples. Each circle represents a sample that contained gill of three oysters. b One sample of cy3-labeled antisense RNA was hybridized to a single microarray slide (eg, a single-color microarray experiment). Slides were hybridized in batches of ten, as shown. c A subset of ESTs was studied using real-time quantitative polymerase chain reaction (RT-QPCR). The original samples of total RNA were used as template. Samples collected after 3 h recovery of heat shock were omitted

Fig. 2

Time-course clusters (95% confidence intervals) for ESTs in which transcription level differed among sampling times in gill of heat-shocked (40°C, 1 h) oysters before and at 1, 3, 6, and 24 h after heat shock. Data were clustered using the software SSClust (Ma et al. 2006). The vsn-transformed data for each sampling time are presented relative to the average for all sampling times for a given EST

Fig. 3

Average (±SEM) microarray-generated vsn-adjusted signal data and RT-QPCR-generated relative concentration data (target concentration relative to elongation factor 1; Log2RC) for heat shock protein genes and genes putatively related to growth and reproduction measured in gill before and at 1, 6, and 24 h after heat shock (40°C, 1 h). Each bar represents three replicates of two families (six samples total) with either low survival (hatched bars) or high survival (white bars) after heat shock. Asterisks above or between individual sampling times indicate significant (P<0.05) differences between family types at that time or between sampling times, respectively

Fig. 4

Average (±SEM) microarray-generated vsn-adjusted signal and RT-QPCR-generated relative concentration data (target concentration relative to elongation factor 1; Log2RC) for genes putatively encoding antioxidant enzymes, genes involved in lipid synthesis and genes involved in cellular immunity measured in gill before and at 1, 6, and 24 h after heat shock (40°C, 1 h). Each bar represents three replicates of two families (six samples total) with either low survival (hatched bars) or high survival (white bars) after heat shock. Asterisks above or between individual sampling times indicate significant (P<0.05) differences between family types at that time or between sampling times, respectively

Fig. 5

Average (±SEM) microarray-generated vsn-adjusted signal data and RT-QPCR-generated relative concentration data (target concentration relative to elongation factor 1; RC or Log2RC) for genes involved in cellular immunity measured in gill before and at 1, 6, and 24 h after heat shock (40°C, 1 h). Each bar represents three replicates of two families (six samples total) with either low survival (hatched bars) or high survival (white bars) after heat shock. Asterisks above or between individual sampling times indicate significant (P<0.05) differences between family types at that time or between sampling times, respectively