The dynamic immune responses of Mandarin fish (Siniperca chuatsi) to ISKNV in early infection based on full-length transcriptome analysis and weighted gene co-expression network analysis.

Mandarin fish (Siniperca chuatsi) been seriously harmed by infectious spleen and kidney necrosis virus (ISKNV) in recent years, but the early immune response mechanism of infection is still unknown. Here, we performed RNA sequencing on the spleens of mandarin fish infected with ISKNV at 0, 12, 24, 48, and 72 h post-infection (hpi) using short-read Illumina RNA sequencing and long-read Pacific Biosciences isoform sequencing to generate a full-length transcriptome. The immune responses of mandarin fish infected with ISKNV at the molecular level were characterized by RNA-seq analysis and weighted gene co-expression network analysis (WGCNA). A total of 26,528 full-length transcript sequences were obtained. There were 2,729 (1,680 up-regulated and 1,112 down-regulated), 1,874 (1,136 up-regulated and 738 down-regulated), 2,032 (1,158 up-regulated and 847 down-regulated), and 4,176 (2,233 up-regulated and 1,943 down-regulated) differentially expressed genes (DEGs) in mandarin fish at 12, 24, 48, and 72 hpi, compared with uninfected fish, respectively. A total of four modules of co-expressed DEGs identified by WGCNA were significantly positively correlated to the four time points after infection, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the immune-related DEGs in all these modules were mainly enriched in Phagosome, Endocytosis, Herpes simplex infection, and Cytokine-cytokine receptor interaction pathways. Further analysis showed that oher signaling pathways, including CAMs, NOD-like receptor and ER protein processing, Intestinal immune network for IgA production, TLR pathway, and Apoptosis significantly enriched in four modules corresponding to 12, 24, 48, and 72 hpi respectively, had specifically participated in the immune response. Hub genes identified based on the high-degree nodes in the WGCN, including CAM3, IL-8, CCL21, STING, SNX1, PFR and TBK1, and some DEGs such as MHCI, MHCII, TfR, STING, TNF α, TBK1, IRF1, and NF-kB, BCR, IgA and Bcl-XL had involved in dynamic molecular response of mandarin fish to ISKNV infection. In sum, this study provides a set of full-length transcriptome of the spleen tissue of mandarin fish for the first time and revealed a group of immune genes and pathways involved in different temporal responses to ISKNV infection, which has implications for resource conservation and aiding the development of strategies to prevent virus early infection for mandarin fish.

Characterization of mandarin fish (Siniperca chuatsi) IL-6 and IL-6 signal transducer and the association between their SNPs and resistance to ISKNV disease.

In fish, interleukin-6 (IL-6) is a very important immune-regulatory cytokine that plays a polyfunctional role in inflammation, metabolism, regeneration, and neural processes. IL-6 signal transducer (IL-6ST) is a specific receptor for IL-6 and expressed mainly in immune cells and hepatocytes. In this study, the complete cDNA and genomic DNA sequences of mandarin fish (Siniperca chuatsi) IL-6 and IL-6ST genes were identified and analyzed. Quantitative real-time PCR showed that IL-6 and IL-6ST were chiefly expressed in the immune organs. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the expression levels of IL-6 were significantly up-regulated after 6 h and 24 h in the head kidney and spleen, respectively (p < 0.01), the peak value for both reached at 72 h, IL-6ST increased significantly after 120 h with a peak at 168 h in the head kidney (p < 0.01) and improved markedly at 168 h in the spleen (p < 0.01). Besides, IL-6 and IL-6ST have been identified 3 and 8 single nucleotide polymorphisms (SNPs), respectively. Statistical analysis showed that one SNP locus (1625C/T) in the coding region of IL-6 was significantly related to the resistance of mandarin fish against ISKNV. The 1625C→T locus in the coding region of IL-6 is a synonymous mutation; compared with the susceptible group, the frequency of allele T in the disease resistance group was significantly higher, which may be due to the rare codon produced by the mutation affecting translation. The involvement of IL-6 and IL-6ST in response to ISKNV infection in mandarin fish clearly indicate that the role of SNP markers in IL-6 was associated with the ISKNV resistance, which was demonstrated for the first time in our results. Thus, the current study may provide fundamental information for further breeding of mandarin fish with resistance to ISKNV infection.