Background: Grass carp (Ctenopharyngodon idella) is one of the most widely cultivated fishes in China. High stocking density can reportedly affect fish growth and immunity. Herein we performed PacBio long-read single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing to evaluate the effects of high stocking density on grass carp transcriptome. Results: SMRT sequencing led to the identification of 33,773 genes (14,946 known and 18,827 new genes). From the structure analysis, 8,009 genes were detected with alternative splicing events, 10,219 genes showed alternative polyadenylation sites and 15,521 long noncoding RNAs. Further, 1,235, 962, and 213 differentially expressed genes (DEGs) were identified in the intestine, muscle, and brain tissues, respectively. We performed functional enrichment analyses of DEGs, and they were identified to be significantly enriched in nutrient metabolism and immune function. The expression levels of several genes encoding apolipoproteins and activities of enzymes involved in carbohydrate enzymolysis were found to be upregulated in the high stocking density group, indicating that lipid metabolism and carbohydrate decomposition were accelerated. Besides, four isoforms of grass carp major histocompatibility complex class II antigen alpha and beta chains in the aforementioned three tissue was showed at least a 4-fold decrease. Conclusions: The results suggesting that fish farmed at high stocking densities face issues associated with the metabolism and immune system. To conclude, our results emphasize the importance of maintaining reasonable density in grass carp aquaculture.

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.