Molecular characterization of TNF-β and IFN-γ in yellowfin seabream (Acanthopagrus latus, Hottuyn, 1782) and their immune responses to density stress during transport

Abstract

The inflammatory cytokines TNF-β and IFN-γ are important mediators of the vertebrate inflammatory response and coordinators of the immune system in regard to NF-κB signalling pathways. In this study, the TNF-β and IFN-γ genes of yellowfin seabream, Acanthopagrus latus were identified, and the multiple sequence alignments, evolutionary relationships and gene expressions of the two genes were also determined. AlTNF-β contained a 762 bp open reading frame (ORF) encoding 253 amino acids, while AlIFN-γ contained a 582 bp ORF encoding 193 amino acids. An amino-acid sequence alignment analysis showed that these proteins have highly conserved transmembrane structural domains among teleosts. Moreover, AlTNF-β has a close affinity with TNF-β of yellowfin seabream while AlIFN-γ has a high evolutionary correlation with A. regius and Sparus aurata. In addition, the mRNAs of AlTNF-β and AlIFN-γ are widely expressed in various tissues. AlTNF-β is highly expressed in gill and intestinal tissues, and the mRNA levels of AlIFN-γ are higher in spleen, skin, and gill tissues than in other tissues. Under transportation density stress, the mRNA level of AlTNF-β was significantly elevated in the intestine of the high-density group, while AlTNF-β transcription in the gills did not vary significantly among the density groups. Furthermore, AlIFN-γ expression was increased in liver, intestinal, and gill tissues under high transportation density. The results of this study show that TNF-β and IFN-γ expression in yellowfin seabream is greatly affected by density stress. The density of 125 per bag for 4–5 cm fry or 1200 per bag for 1–2 cm fry is most suitable for the transportation of live fish. These results might provide a reference for further studies on the immunomodulatory response process and auxiliary function of immune stress of TNF and IFN genes in fish under density stress.