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.

在集约化养殖条件下，鱼类会经历多种压力状况，这些状况对鱼的存活、生长和鱼片品质产生负面影响。识别和表征压力反应背后的分子机制，将有助于开发旨在提升动物福利和生产效率的策略。在本研究中，我们聚焦于彩虹鳟鱼在养殖生产中的处理和限制性压力的肝部反应，因其与水产养殖生产的关联性。我们的目标是利用RNA-seq技术识别肝脏在处理和限制性压力下的差异表达转录本（DETs）。从五个水族箱中每箱八条鱼的肝脏中提取总RNA，其中三个水族箱的鱼经历了处理和限制性压力，两个为对照组。将六个个体的总RNA等量混合，以创建五个RNA-seq文库，这些文库在Illumina HiSeq 2000的一个通道上进行测序。共进行了三次测序，总共有491,570,566个读段。这些读段被映射到先前生成的压力参考转录组，并识别出316个DETs。从这些DETs中选择了21个进行qPCR验证RNA-seq方法。RNA-seq和qPCR识别的基因表达变化倍数高度相关（r = 0.94）。在这些DETs中，包括转录因子活性和代谢过程（特别是碳水化合物代谢）在内的多个基因本体术语得到了富集。通过将DETs映射到KEGG数据库中的参考通路，涉及到处理和限制性压力响应的通路被涉及。