Effects Of Heat Stress On Transcriptomic Profile,Oxidative Stress Status And Apoptosis In Liver Of Pikeperch(Sander Lucioperca)

The pikeperch(Sander lucioperca)belongs to Perciformes,Percidae,Sander.It is a sub-cool-water fish with great aquaculture potential.In recent years,the high temperature in summer has seriously threatened the culture of pikeperch.In our previous study,the effects of heat stress on pikeperch liver under different temperatures were investigated,however,the transcriptional expression profile and physiological and biochemical responses of the liver under different heat stress times are still unknown.In this study,pikeperch were treated with consistent heat stress(29??,0-48?h),followed by anlysis of transcriptomic profiles of 29??(0?h)group,detection of oxidative stress status,observation of tissure damages,examination of the apoptosis of liver tissue under different heat stress time.In addition,the expression levels of antioxidant enzyme genes and HSPs in pikeperch were also determined under heat stress.Our results will provide basic data for further analysis on the molecular mechanisms of heat stress response in pikeperch.The researches are as follows:1 Transcriptome analysis in liver of pikeperch under heat stressThe pikeperch was heat stressed at 29? in increments of +1 ? per hour from23?,and 6 pikeperch liver tissue were sampled for transcriptome sequencing.A total of 46.57 Gb clean reads were obtained,and the Q30 base percentage was above93.59%.After assembly,a total of 100,871 unigenes were obtained,and Nr function annotations showed that 23,648 unigenes were successfully annotated,with 5,474,12,286,and 13,036 unigenes mapped the COG,GO,and KEGG databases,respectively.There were 3613(16.54%)unigene matching up with the functional gene sequence of Larimichthys crocea,indicating Sander lucioperca L.were most closed with Larimichthys crocea.Compared with the control group,403 differentially expressed genes(DEGs)were detected,of which 278 genes were up-regulated and 125 genes were down-regulated.Eight stress-related genes were selected from DEGs for q RT-PCR and the results showed that the expression of STAT1,LDH-A,RHEB and HES-1B genes were significantly increased,and the expression of LFNG,PIK3R1,GDF2 and HMGB2 genes were significantly decreased.The expression patterns of these genes were consistent with the RNA-Seq results,indicating that the RNA-Seq results in this study are reliable.The GO and KEGG annotation of DEGs showed that many DEGs were enriched in some pathways,including protein processing in endoplasmic reticulum(ER),insulin signaling pathway,and immune-related pathways in pikeperch under heat stress.These results indicate that pikeperch is mainly resistant to high temperature by activating protein processing in ER,insulin signaling pathway and immune-related pathways,thereby protecting itself from damage.2 Effects of heat stress on oxidative stress and tissue damage in liver of pikeperchPikeperch were heat stressed at 29 ? for 48 h,livers were sampled at 0 h,3 h,6h,12 h,24 h and 48 h under stress.The parameters of oxidative stress and liver damage were measured,and liver histology was observed under different heat stress time.The results showed that compared with the control group,there was no significant pathological change in the experimental group at 29 ?(0 h).However,there were some pathological changes in liver of pikeperch,such as hypochromatosis and the vacuolar degeneration of cells at 3 h,6 h,12 h,and 24 h groups.In addition,compared with the control group,the liver of pikeperch showed the most serious pathological changes,such as increased hypochromatosis and vacuole degeneration,and obscure boundaries among hepatic cells at 48 h.When the water temperature rose from 23 ?(control)to 29 ?(0 h),the glutathione peroxidase(GPx)activity and glutathione(GSH)content in liver of pikeperch were decreased.The total antioxidant capacity(T-AOC)activity increased significantly,while total superoxide dismutase(T-SOD)and catalase(CAT)activity,and malondialdehyde(MDA)content did not change significantly.At 3 h of heat stress,T-AOC activity decreased.At 3 h of heat stress,the activities of T-SOD and CAT,and the content of GSH significantly increased(P <0.05),and thereafterdecreased.At 6 h of heat stress,the activities of GPx and T-AOC increased and then decreased.There was no significant difference in MDA content at 0-3 h of heat stress,but the MDA content at 6 h group increased significantly(P <0.05),and then decreased with the duration of heat stress.In addition,compared with the control group,the ALT and AST activities of the high temperature group were significantly increased at 0 h and 3 h,respectively(P <0.05),but decreased with the duration of heat stress.3 Analysis of pikeperch liver apoptosis under heat stressThe heat-stressed method was the same as experiment 2.The Td T-mediated d UTP Nick-End Labeling(TUNEL)method was used to detect the apoptosis in pikeperch liver,and the expression levels of apoptosis-related genes were determined.The results showed that the liver nuclei in the control group were mostly blue,and the brown nuclei were fewer.The apoptosis of liver in pikeperch was significantly aggravated after heat stress,the liver cell apoptosis index increased significantly at each time point under heat stress(P <0.05).Compared with the control group(23 ?),q RT-PCR results showed that the m RNA expression levels of p53,Bax,Apaf-1,Caspase9,and Caspase3 in liver of pikeperch were increased significantly under heat stress(29 ?,0 h),and afterwards their expression levels decreased with duration of heat stress.4 Effects of heat stress on HSPs and antioxidant enzyme gene expression in pikeperch liverThe heat-stressed method was the same as experiment 2.The expressions of HSPs and antioxidant enzyme genes in pikeperch liver were measured by q RT-PCR under different heat stress time.The results showed that,compared with the control group,the m RNA expression levels of HSP70 and HSP90? in liver of pikeperch were significantly increased at 0 h under heat stress,but their expression levels were significantly decreased with the duration of heat stress(P <0.05).In the early stage of heat stress,the m RNA expression levels of HSC70,GRP78 and GRP94 increased significantly(P <0.05),all reached the peak at 3 h of heat stress,and then their overall expression levels decreased.The m RNA expression levels of Mn-SOD and GPx7 increased significantly under heat stress(29 ?,0 h),but their overall expressionlevels decreased with the duration of heat stress(P <0.05).The m RNA expression of CAT and GPx1 increased significantly in the early stages of heat stress,reaching peaks at 3 h and 6 h,respectively.The GPx3 m RNA expression level increased significantly and reached peak at 3 h of heat stress.The Cu/Zn-SOD m RNA expression level decreased at 0 h of heat stress,afterwards increased and reached peak at 6 h.In summary,the pikeperch can activate the antioxidant defense system and HSPs expression to protect cells from oxidative damage under short-term heat stress.Consistent heat stress can cause oxidative stress to pikeperch liver,which leads to liver tissue damage and apoptosis.