Effect Of High Temperature Stress On Physiology And Biochemistry,HSC70 Gene Cloning And Expression Of Pikeperch (Sander Lucioperca)

The pikeperch?Sander lucioperca L.?belonges to Percidae.It is one of the most important economical cold-water fishes.Its stress reaction is stronger in the summer.In order to investigate the mechanism of high temperature stress in pikeperch,four temperature treatmens 28?,32?,34?and 36?and control group of 23?were carried out.The activities of antioxidant enzymes SOD?superoxide dismutase?,CAT?catalase?and GPX?glutathione peroxidase?,the MDA contents and histological damage in the liver of pikeperch were analyzed.We also detected the effects of temperature stress on the number of blood cell and the content of several serum biochemical components.In addition,molecular cloning and gene expression analysis of HSC70 gene in pikeperch were also implemented.These studies surely provide basic knowledge on the mechanism of heat stress of pikeperch.1 Effects of high temperature stress on antioxidant enzymes and histological damage in the liver of pikeperch?Sander Lucioperca?Effects of high temperature stress on the activities of antioxidant enzymes?SOD,CAT and GPX?,the MDA contents and histological damage in the liver of pikeperch?Sander lucioperca L.??107±5g?were investigated.Results showed that high temperature exercised a great effect on the activities of antioxidant enzymes.The activities of SOD,CAT and GPX in each group first increased and then decreased.There were significant differences in SOD,CAT and GPX activities between 28?and23?control group?P<0.05?.In 34?group,SOD activity reached to the highest and was 4.2-fold than control group?23??.In the meantime,high temperature caused an increase of liver CAT activity by 1.7-3.5 fold compared with 23?control group.CAT activity of the treatment groups reached the highest?3.5-fold compared with 23?control group?at 28?group firstly and then declined from 32?to 36?compared with 28?group.The GPX activity in liver reached the highest?5.8-fold compared with23?control group?at 28?group and decreased then.Except for 36?group,there were significant differences between 23?control and other groups in GPX activity?P<0.05?.In addition,MDA contents of all treatment groups were significantly higher than that of 23?control group?P<0.05?.The results of histological damage in the liver of pikeperch associated with different temperature indicate that the cytoplasm of parenchymatous hepatic cells remains unchanged in 28?and 32?group.However,when the temperature increased to 34?and 36?,hepatic cells displayed increasing vacuolation,in which vacuoles increased in both number and size,and nuclei were displaced toward the cell periphery with the increased temperature.This study found that from 23?to 32?,the increase of temperature has certain effect on pikeperch antioxidant system,but the high temperature?34?and 36??will cause the serious change of liver antioxidant enzyme activities and liver injury.2 Effects of high temperature stress on blood physiology and biochemistry in pikeperch?Sander Lucioperca?This study research the effects of high temperature stress on the number of RBC?Red blood cell?,Hb?hemoglobin?,WBC?White blood cell?,PCV?packed-cell volume?,potassium-ion?K⁺?,sodion?Na⁺?,calcium-ion(Ca²⁺)and the content of several serum biochemical components in the blood of pikeperch.The results showed that the high temperature stress has some influences on blood cells and serum biochemical components in pikeperch in different degrees.There was no significant difference in the number of RBC and PCV between 23?control group and 28?,32?groups?P>0.05?,when the temperature increased to 34?and 36?groups,the number of RBC and PCV increased significantly?P<0.05?.Except for 28?group,there were significant differences between 23?control and other groups in the number of WBC?P<0.05?.The number of Hb in treatment groups were significantly higher than that of the 23?control group?P<0.05?except for 28?and 32?groups.Compared with 23?control group,the content of ALT and AST in 28?and 32?groups increased significantly?P<0.05?.The content of ALB decreased with the increasing temperature in treatment groups.During high temperature stress,the contents of CHOL and TG decreased with the increasing temperature in treatment groups?P<0.05?.The content of HDH decreased with the increasing temperature in treatment groups.There was a significant difference between the 28?,32?groups and 23?control group?P<0.05?.The LDH content increased with the increasing temperature in treatment groups,when the temperature reached 34?and 36?,there was significant differences in the content of LDH between 34?,36?groups and 23?control group?P<0.05?,the other treatment groups had no significant difference with 23?control group?P>0.05?.The high temperature stress has no significant effect on the level of Ca²⁺?M²⁺and Na⁺?P>0.05?in the blood of pikeperch.3 Molecular cloning and gene expression analysis of cognate heat shock proteins?HSC70?in pikeperch?Sander lucioperca?The full-length cDNA sequence of heat shock cognate 70?HSC70?was isolated from pikeperch?Sander lucioperca L.?.The cDNA is 2208 bp,which contains an 83 bp5?-UTR,a 172 bp 3?-UTR including the polyA tail,and a 1953 bp open reading frame?ORF?that encodes 650 amino acids.The pikeperch HSC70 protein sequence contains three typical motifs including DLGTTSV?aa 10–18?,IFDLGGGTFDVSIL?aa197–210?,and IVLVGGST-RIPKIQK?aa 334–348?.Like other fish HSC70 protein sequences,the pikeperch HSC70 protein sequence also contains an HSP70 EEVD motif?aa 647–650?and three C-terminal tetrapeptide sequences?GGMP,aa 615–618,aa619–622,and aa 627–630?.Phylogenetic analysis based on amino acid sequence shows that pikeperch HSC70 is the most similar?98%?to HSC70 of Siniperca chuatsi,indicating that this protein is a member of the heat shock protein HSP70 family.Fluorescent real-time quantitative RT-PCR showed that HSC70 is expressed in all tested tissues of pikeperch,including brain,heart,liver,spleen,muscle,bowel,stomach,and gill.The HSC70 expression level in liver and brain increased significantly during heat stress,suggesting that the liver and brain are more sensitive to heat stimulation than other pikeperch tissues.HSC70 mRNA expression reached a maximum at 34?in gills,muscle,and the heart while it reached a maximum level at 36?in the other five tissues.This study constructs a pikeperch heat stress model,research the liver damage,blood biochemical and expression of HSC70 gene.This study is helpful to explain the high temperature stress mechanism of pikeperch.