Study On The Effects And Molecular Mechanisms Of Extreme Temperature And Salinity Stressors On Hybrid Grouper (Epinephelus Fuscoguttatus ♀× Epinephelus Lanceolatus(?))

Global warming and frequent extreme climate events have seriously affected the safety of marine ecosystems.However,the effects and molecular mechanisms of environmental factors such as high temperature and low salinity on marine organisms are still unclear.In the present study,the extreme high temperature(HT1 1,33℃;HT2,36℃),the extreme low salinity(LS,17)and combined stressors(HT1+LS and HT2+LS)were set up to explore the effects and molecular mechanisms on the hybrid grouper(Epinephelus fuscoguttatus ♀× Epinephelus Lanceolatus ♂)by transcriptome and proteomics techniques.The data obtained from this project provide a strong foundation for understanding the negative effects and mechanisms of extreme temperature and salinity events on aquaculture.The main results are as follows:(1)Changes of physiological and biochemical indexes:the enzymatic activities of superoxide dismutase(SOD),catalase(CAT)and glutathione reductase(GR)in liver of the hybrid grouper under high temperature and combined stress for 3 d were more obvious than those under low salt stress compared with the control,while the activities of glutathione peroxidase(GSH-PX)were significantly increased under the high temperature and combined stressors for 14 d.High temperature significantly inhibited the activities of SOD and CAT,while low salt significantly induced the expression levels of SOD and CAT mRNA.There were significant differences of GSH-PX and GR mRNA expression levels in the combined exposure group.Heat shork protein 70(HSP70)mRNA was highly induced in the LS group,and there was no difference between the high temperature group and the control,and ATP2B1 mRNA expression level was significantly up-regulated in the all experimental groups.The enzyme activities of antioxidant related molecular were significantly induced under the stress,and HSP70 mRNA was highly expressed under the low salt treatment.The results indicated that the antioxidant system of the grouper was activated under the stress of extreme temperature and salinity.(2)The characteristics of transcriptome in the hybrid grouper livers:a full length transcriptome database of the grouper livers was constructed by PacBio isoform sequencing(Iso-Seq)technique,and 49333 genes were annotated.Among which,the length of 43%(21384)genes were over 3000 bp.The results of KEGG pathway enrichment showed that the top 10 pathways were related to immune system or disease in the treatment groups,which suggest that high temperature stress may greatly decrease the ability of fish to be infected by pathogenic microorganism.(3)The characteristics of proteome in the hybrid grouper livers:a proteome database of the grouper liver was obtained using Tandem Mass Tag(TMT)techenique,and 4772 proteins were annotated.The differentially expressed proteins are mainly involved in the pathways of post transcriptional modification,lipid transport and metabolism,and signal transduction.ApoA1 and CYP3A40 proteins were markedly increased in the different treatment groups,which suggest that high temperature and low salinity may lead to energy shortage and then mobilize the metabolism of lipids and sterols to make for the energy deficiency..(4)Histopathological changes and intestinal microbial diversity:the results of Hematoxylin-eosin(HE)staining and Oil Red O staining of liver sections showed that the hepatocytes of the grouper were coagulated,and the cells were steatosis under the stress of high temperature.The epithelial cells proliferated in the gills,the intestinal wall became thinner and the structure was destroyed in the groups of high temperature and low salt treatment under the stress of low salt and combined stress for 14 d.The gill fragments were necrotic and exfoliated,the length of intestinal villi became shorter and part of intestinal villi were broken and damaged in the combined treatment group.The abundance and diversity of intestinal microflora decreased for 3 d and 14 d exposure,which showed a positive correlation with the exposure time.Nautella and Ruegeria significantly increased in the combined exposure for 3 d,which showed a positive correlation with the exposure time for 14 d.The abundance of Escherichia-Shigella species was markedly lower than that of the control,while the abundance of Halomonas and beneficial bacteria Lactobacillus was significantly decreased.The results showed that extreme temperature and salinity caused tissue damage and decreased the microbial abundance and diversity in the hybrid grouper.