Transcriptome analysis gene expression in the liver of Coilia nasus during the stress response

The estuarine tapertail anchovy (Coilia nasus) is widely distributed in the Yangtze River, the coastal waters of China, Korea, and the Ariake Sound of Japan. It is a commercially important species owing to its nutritional value and delicate flavor. However, Coilia nasus is strongly responsive to stress, this often results in death, which causes huge losses. In this study, we used next-generation sequencing technologies to study changes in gene expression in response to loading stress and the mechanism of death caused by loading stress in Coilia nasus.

Using next-generation RNA-seq technologies on an Illumina HiSeq 2000 platform, we assembled a de novo transcriptome and tested for differential expression in response to stress. A total of 65,129 unigenes were generated, the mean unigene size and N50 were 607 bp and 813 bp, respectively. Of the assembled unigenes, we identified 2,990 genes that were significantly up-regulated, while 3,416 genes were significantly down-regulated in response to loading stress. Pathway enrichment analysis based on loading stress-responsive unigenes identified significantly stress related pathways. "Metabolism" and "immunity" were the two most frequently represented categories. In the "metabolism" category, "glucose metabolism" and "lipid metabolism" were major subclasses. The transcriptional expression of rate-limiting enzymes in "glucose metabolism" and "lipid metabolism" was detected by RT-qPCR, all were significantly increased after stress. Apoptosis associated proteins tumor necrosis factor alpha (TNF-α), caspase 9, cytochrome c and caspase 3 in the stress group were significantly elevated, moreover, liver injury indicators (Alanine aminotransferase, ALT, and aspartate transaminase, AST) were also significantly elevated, which indicates that loading stress induced liver injury.

This study provided abundant unigenes that could contribute greatly to the discovery of novel genes in fish. The alterations in predicted gene expression patterns reflected possible responses to stress. Loading stress may induce liver injury through the mitochondrial apoptosis pathway, which was activated by TNF-α. Taken together, our data not only provide information that will aid the identification of novel genes from fish, but also shed new light on the understanding of mechanisms by which physical stressors cause death in fish.