Project description:Summer mortality of the Pacific oyster Crassostrea gigas is the result of a complex interaction between oysters, their environment and pathogens. Heredity appears to be a major factor determining the sensitivity of oysters to summer mortality, allowing resistant (R) and susceptible (S) lines to be produced. We conducted genome-wide expression profiling of R and S gonads during the 3-month period preceding a summer mortality event using a 9K cDNA microarray that we designed. This transcriptional analysis provides new indications to define markers for Quantitative Trait Loci searches and functional studies, and evaluates the potential role of each gene in the resistance to summer mortality For microarray analysis, R and S oysters were sampled four times (dates 1 to 4: May 9, May 25, June 6, and June 20, respectively). On each date, 3 replicates of 8 oysters were sampled from each line (R and S) and the gonads prepared for total RNA extraction. Furthermore, the entire tissues of 10 wild oysters were collected, pooled and homogenized to constitute a single total RNA sample for use as a reference in all slide hybridizations and RT-PCR analysis. For microarray hybridizations, 5µg of total RNA were directly labeled by reverse transcription and then purified using the Direct ShipShot Labeling kit (Promega). This reaction was performed for each of the 24 gonad samples, with Cy5 (red) incorporation. The reference sample was Cy3-labeled (green) in 24 separate tubes following the same protocol. The 24 Cy3-labeled cDNAs were next pooled, and then divided once more into 24 samples to obtain a homogeneous reference. Equimolar amounts of cDNA samples and cDNA reference labeled with Cy5 and Cy3, respectively, were SpeedVac evaporated and mixed into a single pool with the hybridization buffer (ChipHyb™ hybridization buffer, Ventana Discovery, Tucson, AZ, USA). They were then co-hybridized on the same microarray slide, in a Ventana hybridization station (Ventana Discovery, Tucson, AZ, USA). The data submitted here correspond to the mean of the three replicates for each line and each date, representing 8 samples : gonad_resistant_date1 gonad_sensitive_date1 gonad_resistant_date2 gonad_sensitive_date2 gonad_resistant_date3 gonad_sensitive_date3 gonad_resistant_date4 gonad_sensitive_date4

Project description:Summer mortality of Crassostrea gigas is the result of a complex interaction between oysters, their environment and pathogens. A large genetic basis and a high heritability were demonstrated for the observed variation in resistance to summer mortality, which offered the possibility to develop lines of oysters that were resistant (R) or susceptible (S) to summer mortality. Previously, genome-wide expression profiling of R and S oyster gonads highlighted reproduction and antioxidant defense as constitutive pathways that operate differentially between these two lines. Here, we show that signaling in innate immunity also operates differentially between these lines and we postulated that it is at the main origin of their difference of survival in the field. From the already published microarray data, we employed an ANOVA analysis that reveals a specific “immune” profile at the date preceding the mortality. In addition, we conducted a microarray profiling of two other tissues, gills and muscle, that also showed an over-representation of the immune genes (46%) among the selected genes. Eleven genes were pinpointed to be simultaneously differentially expressed between R and S lines in the three tissues. Among them, ten are related to “Immune Response”. The kinetics of their mRNA levels appeared clearly different between lines and suggests that in environment, R oysters had the capacity to modulate signaling in innate immunity whereas S oysters did not. This study enhances our understanding of the complex summer mortality syndrome and provides candidates of interest for further functional and genetics studies. For microarray analysis, R and S oysters were sampled three times (dates 1 to 3: May 25, June 6, and June 20, respectively). On each date, 3 replicates of 8 oysters were sampled from each line (R and S) for three tissues (gonad, muscle and fills) and all the samples prepared for total RNA extraction. Furthermore, the entire tissues of 10 wild oysters were collected, pooled and homogenized to constitute a single total RNA sample for use as a reference in all slide hybridizations and RT-PCR analysis. For microarray hybridizations, 5µg of total RNA were directly labeled by reverse transcription and then purified using the Direct ShipShot Labeling kit (Promega). This reaction was performed for each of the 18 samples, with Cy5 (red) incorporation. The reference sample was Cy3-labeled (green) in 18 separate tubes following the same protocol. The 18 Cy3-labeled cDNAs were next pooled, and then divided once more into 18 samples to obtain a homogeneous reference. Equimolar amounts of cDNA samples and cDNA reference labeled with Cy5 and Cy3, respectively, were SpeedVac evaporated and mixed into a single pool with the hybridization buffer (ChipHyb™ hybridization buffer, Ventana Discovery, Tucson, AZ, USA). They were then co-hybridized on the same microarray slide, in a Ventana hybridization station (Ventana Discovery, Tucson, AZ, USA). The data submitted here correspond to the mean of the three replicates for each line and each date, representing 18 samples.

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