RNA-seq Analysis of Hepatic Response to Handling and Confinement Stress in Rainbow Trout

Fish under intensive rearing conditions experience various stress conditions, which have negative impacts on survival, growth and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and production efficiency. In this study, we focused on hepatic response to handling and confinement stress in rainbow trout due to their relevance in aquaculture production. Our objective was to identify differentially expressed transcripts (DETs) in liver in response to handling and confinement stress using RNA-seq. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted for a total of 491,570,566 reads. These reads were mapped onto the previously generated stress reference transcriptome, and 316 DETs were identified. Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (r = 0.94). Several gene ontology terms including transcription factor activity and metabolic process especially carbohydrate metabolism were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to the reference pathways in KEGG database.