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.

集约化养殖环境下的鱼类会遭遇多种胁迫条件，这些胁迫会对其存活率、生长性能及鱼肉品质产生负面影响。解析胁迫响应背后的分子机制，将有助于开发提升动物福利与生产效率的相关策略。本研究聚焦于虹鳟在操作与约束胁迫下的肝脏响应，因其与水产养殖生产密切相关。本研究的目标是利用RNA测序（RNA-seq）技术，鉴定虹鳟肝脏中响应操作与约束胁迫的差异表达转录本（differentially expressed transcripts, DETs）。我们从5个各投放8尾鱼的养殖缸的单尾鱼肝脏中提取总RNA，其中3个养殖缸的鱼接受操作与约束胁迫处理，剩余2个为对照缸。每个养殖缸取6尾鱼的总RNA进行等量混合，共构建5个RNA测序文库，随后在Illumina HiSeq 2000的单个测序泳道中完成测序。本次测序共开展3轮，累计获得491,570,566条测序读段（reads）。将这些测序读段比对至此前构建的胁迫参考转录组，最终鉴定得到316个差异表达转录本。选取21个差异表达转录本用于实时荧光定量PCR（qPCR）验证，以确认RNA测序结果的可靠性。RNA测序与实时荧光定量PCR所得到的基因表达倍数变化呈现高度相关性（相关系数r=0.94）。本研究通过富集分析发现，多个基因本体（Gene Ontology, GO）条目在差异表达转录本中显著富集，包括转录因子活性、代谢过程（尤其糖类代谢）相关条目。将差异表达转录本比对至京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）的参考通路数据库，进一步明确了参与操作与约束胁迫响应的相关通路。