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Grass carp (Ctenopharyngodon idella) is the largest economic fish in freshwater culture in China, which is predisposed to infectious diseases under high temperature. Under the background of global warming, the industrialization of the Pearl River Delta region has led to aggravated thermal pollution, which has increasingly serious impacts on the aquatic ecological environment. This will result in more frequent exposure of grass carp to overheated water temperatures. Previous studies have only identified the regulatory genes of fish that respond to pathogens or temperature stress, but the transcriptional response to both is unknown. In this study, the histopathological analysis showed heat stress exacerbated spleen damage induced by Aeromonas hydrophila. The transcriptional responses of the spleens from A. hydrophila lipopolysaccharide (LPS) - injected grass carp undergoing heat stress and at normal temperatures for 6, 24, and 72 h were investigated by mRNA and microRNA sequencing. We identified 28, 20, and 141 differentially expressed (DE) miRNAs and 126, 383, and 4841 DE mRNAs between the two groups after 6, 24, and 72 h, respectively. There were 67 DE genes mainly involved in the cytochrome P450 pathway, antioxidant defense, inflammatory response, pathogen recognition pathway, antigen processing and presentation, and the ubiquitin-proteasome system. There were 5 DE miRNAs involved in regulating apoptosis and inflammation. We further verified 17 DE mRNAs and 5 DE miRNAs using quantitative real-time PCR. Based on miRNAs and mRNAs analysis, continuous heat stress will affect the antibacterial responses of grass carp spleens, resulting in aggravation of spleen injury. Together, these results provide data for further understanding of the decreased tolerance of fish to pathogen infection in persistent high-temperature environments. • Continuous heat stress exacerbates spleen damage induced by Aeromonas hydrophila. • Continuous heat stress impaired the antioxidant and immune functions of grass carp. • HSPs are induced to persist after co-treatment with LPS and heat stress. • MicroRNAs mediate apoptosis and inflammation in the damaged spleen of grass carp. [ABSTRACT FROM AUTHOR]

Abstract: In this study the gene encoding HSP70 was isolated from Phascoloma esculenta by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of cDNA (2520 bp) consists of a 5′-terminal untranslated region (UTR) (125 bp), a 3′-terminal UTR (421 bp) with a canonical polyadenylation signal sequence (AATAAA), a poly (A) tail, and an open reading frame (ORF) (1974 bp). The predicted molecular mass and isoelectric point for HSP70 is 71.6 kDa and 5.15, respectively. BLAST analysis showed that P. esculenta HSP70 gene shared high similarity. Classical HSP signature motifs, ATP/GTP-Binding Site Motif A, Bipartite Nuclear Targeting Sequence, the cytosolic HSP70 could be expressed in Escherichia coli BL21. After purification, the recombinant pET-HSP70 protein was used to produce the polyclonal antibody in mice and the specificity of the antibody was confirmed by Western blot analysis. Fluorescent real-time quantitative PCR analysis showed that expression of Hsp70 in sipuncula was increased significantly after exposure to 10 mM Zn for12 h, Cd for 24 h, Cu for 48 h, and was exposure to 37 °C for 24 h sea water. [Copyright &y& Elsevier]