Transcriptomic analysis of the immune response mechanism in the intestine and spleen of black rockfish following bacterial infection

Chengbin Gao

Nearly 50% of global aquaculture production is contributed by China. The emergence of diseases is increasing and have caused immense economic losses to the aquaculture industry. Black rockfish (Sebastes schlegelii) is a commercially important maricultured species in east Asian countries, but the rapid expansion of black rockfish aquaculture has been accompanied by outbreaks of bacterial disease. Although transcriptomic sequencing has been used to investigate the immune system and thereby discover more efficient measures of disease prevention and control in aquaculture species, there remain limitations on our understanding of the regulatory role of non-coding RNAs in the immune response of in teleost fishes and of the interaction between the mucosal immune response in the intestine and the traditional immune response in the spleen. To this end, the current research project aimed firstly to explore the immune response of black rockfish at different time points (0 h, 2 h, 12 h and 24 h) following bacterial challenge, through profiling the expression of circular RNA (circRNA), long non-coding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA) by high-throughput sequencing, and constructing circRNA- and lncRNA-related regulatory interaction networks. Furthermore, I investigated the relationship between the intestine and spleen in the immune response after bacterial challenge and explored how the interaction of these two tissues contributed to the local immune environment. A total of 1364 mRNAs, 17 miRNAs and 1584 lncRNAs exhibited significantly differential expression in the black rockfish spleen following challenge with Aeromonas salmonicides.Three lncRNA-miRNA-mRNA networks were respectively established at 2, 12 and 24 h post bacterial infection, as well as three circRNA-miRNA-mRNA networks. These networks were enriched in several immune-related signaling pathways and significantly induced by bacterial infection, which suggested that they play important roles in the immune regulation during bacterial infection in black rockfish. Among the established networks a NLRC3/novel-264/LNC_00116154 pathway was of particular interest because of the specific function roles of NLRC3 genes in fish species. Analyses of the intestine-spleen transcriptome of black rockfish found a total of 251 and 208 differentially expressed immune genes in the intestine and spleen, respectively, with 45 genes differentially expressed in both organs. According to the signaling pathways in which these jointly expressed immune genes were involved, a hypothesis of the potential regulatory pathway in the intestine-spleen axis was proposed. According to this hypothesis, after bacterial infection, complement C3 and macrophage mannose receptor 1 genes are regulated by related miRNAs and circRNAs to recognize bacterial antigens on the epithelial cells of the intestine with the signals present to dendritic cells and transited to the spleen through MHC class I genes to activate T and B cell receptor signaling pathways. Finally, IgA productions were generated and transited to the intestinal lumen by the vascular system for the immune response. Finally, a comparative study was undertaken of the evolution of NLRC3 subfamily genes in black rockfish, 13 other fish species and three species of higher vertebrates. Different patterns of gene expansions and contractions were observed among species, and one and two whole-genome duplication events were observed in two members of family Sebastidae (black rockfish and honeycomb rockfish Sebastes umbrosus, respectively. Subsequently, 179 copy numbers of NLRC3 genes were found in black rockfish and 166 in honeycomb rockfish. Phylogenetic analyses corroborated the conservation and evolution of NLRC3 orthologues between Sebastidae and other fish species. Finally, differential expression analyses provided evidence of the immune roles of NLRC3 genes in black rockfish during bacterial infections. In conclusion, this study successfully constructed RNA immune regulatory networks and identified potential regulatory mechanisms in the intestine-spleen immune axis of black rockfish during bacterial infections, and provided new insights into the evolution of NLRC3 genes, especially in the immune system of the Sebastidae family. Although further research is required to further develop these findings, they provide the basis for a better understanding of the immune response of black rockfish and may help with the development of new treatment and control options for bacterial diseases in this species. Keywords: Black rockfish, Intestine, Spleen, Immune response, Interaction network, Transcriptomic analysis, NLRC3.

Transcriptomic analysis of the immune response mechanism in the intestine and spleen of black rockfish following bacterial infection

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