| The pikeperch(Sander lucioperca)belonges to Perciformes,Percidae.It is a sub-cool-water fish with great aquaculture potential.High temperature in summer is the main cause of massive death of pikeperch in its breeding ponds.In this study,the damages of gills of Sander lucioperca under heat stress were observed by light microscopy and scanning electron microscopy.The activities of antioxidant enzymes and malondialdehyde content were determined in gills of pikeperch.The transcriptome of the gills of pikeperch under thermal stimulation and the significant differentially expressed genes were anlyzed.The expression levels of antioxidant enzymes and HSP family genes were determined under different heat stimulation time.The above results explored the heat stress mechanism of pikeperch and evaluated the effect of thermal stimulation on the pikeperch and enriched the basic data of heat stress physiology research of pikeperch.The specific research is as follows:1 Effects of different heat stimulation temperatures on the tissue structure and antioxidant enzymes of pikeperchIn order to explore the effects of heat stress on gills of pikeperch,100 fish individuals were exposed to 28?,31?,34?,36? heat stress for 2 h respectively,with 23? as control group.The gills were collected for paraffin sections preparation and hematoxylin eosin(HE)stain was used for general histological observations.Enzyme activity of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPX)and malonaldehyde(MDA)were analyzed.When exposed to 28?,congestion of secondary filament appeared.While in 31? group,the secondary gill lamellae of pikeperch became curly and shorter,and epithelial cells appeared hydropic degeneration at the same time.In 34? group,the deformation of gills lamella was aggravated with a little fusion.The epithelial cells became more edema and obvious swelling,and the mucous cells were hypertrophy and hyperplasia between the small gill pieces.In 36? group,a lot of fusions and serious deformations appeared in the gills,which arranged disorderly,and a large number of epithelial cells deteriorated and fell off,and the filament mucous cells proliferated at the same time.When treated temperature increased,SOD,CAT,GPX activity and MDA in gills of pikeperch presented the similar trend,first increased and then decreased,with significant drops in 36? group dropped.This indicates that after heat stress,ROS increased and lipid peroxidation increased.Due to the large amount of ROS and H2O2 produced in the body,the body also increased the synthesis of GPX and CAT.When exposed to 36?,the stress intensity has exceeded the adaptability of pikeperch,and the antioxidant system function cannot be performed normally.This study indicated that the gill injuries and metabolic disorders were gradually aggravated with the increases of heat stress.2 Different thermal stimulation time on the damage of pikeperchTaking 23? as the control group,the test water temperature just rose to 30? as Oh,and samples were taken at Oh,3h,6h,12h,24h and 48h.Light microscopy and scanning electron microscopy were used to observe the damages of gills tissue of pikeperch.The results showed that the epithelial cells of the gill lamellaes were observed in the Oh and 3h test groups;the bending deformation of the gill lamellaes was observed in the 24h group;the obvious gill lamellaes fusion was observed at 48h.In this study,it was found that the degree of gills damage continued to increase with the prolongation of thermal stimulation time from to 48h after 30? thermal stimulation.There were grade II injury in gills of pikeperch at 48h,and no grade III injury was found.3 Effects of different heat stimulation time on enzyme activities related to antioxidant system in pikeperchThe materials used were the same as experiment one.A total of six enzyme including the total superoxide dismutase(T-SOD),glutathione peroxidase(GPX),catalase(CAT),glutathione(GSH),glutathione reductase(GR),total antioxidant capacity(T-AOC)and malondialdehyde(MDA)content in the pikeperch were detected under different time thermal stimulation.The results showed that the activity of T-SOD and CAT in gills of pikeperch first increased and then decreased.The activity of T-SOD increased with the increase of thermal stimulation time before 6 h,and it reached peak in 6 h group.But when the stimulation reached 12h,the T-SOD activity decreased and was lower than the control group at 48h.The CAT activity was significantly different from the control group at 3h(P<0.05),and it reached the maximum at 12h,and then decreased to the level of no significant difference comparing with the control group(P>0.05).The changing models of GPX and GSH activity in pikeperch were similar,and reached the maximum at 24h and 12h,respectively,which were significantly higher than that in the control group(P<0.05),and then decreased to no significant difference with the control group(P>0.05).GR activity at both of 12h and 24h showed no significant difference comparing with the control group(P>0.05).At other time points,the GR activity of the experimental group was significantly higher than that of the control group(P<0.05).The content of MDA in the gills of pikeperch continued to increase with the stimulation time increasing and it reached the maximum at 48h.T-AOC activity was significantly different from the control group only at 6 h(P<0.05).In conclusion,oxidative stress was produced in gills of pikeperch when it was heat stimulated,and the state of stress increased with increasing time of heat stimuation.4 transcriptome analysis of gills of pikeperch under thermal stimulationTaking 23? as the control group,the experiment group was heat stressed at 30? for 6 hours.6 samples of gills tissue from each fish in each group were taken for transcriptome sequencing.A total of 65.85Gb clean data were obtained,and the Q30 base percentage was above 93.45%.After assembly,a total of 59,275 Unigenes were obtained.The results of Nr function annotation showed that there were 3362(18.01%)unigene matching up with the functional gene sequence of Lates calcarifer,indicating that the relationship between the Sander lucioperca L.and the Lates calcarifer was close.The COG,GO and KEGG annotations show that 4,797?10,798 and 11,305 unigenes successfully completed the COG,GO and KEGG functional classifications,respectively.The results of differentially expressed genes showed that compared with the normal group,a total of 252 genes expressed significant changes under heat stress.Among which,191 genes were up-regulated and 61 genes were down-regulated.A total of 15 differentially expressed genes were successfully analyzed by the KEGG pathway.The KEGG pathway enrichment analysis showed that multiple pathways were involved in the heat stress response of pikeperch,which involved the most lipid metabolism,a total of 6;followed by amino acid metabolism(4)and carbohydrate metabolism(3)and folding,sorting and degradation.Six genes were selected from differentially expressed genes for Real-time PCR.The expression of HSC70,GATM and ATP-binding genes was significantly increased,and the expression of CPS,HUNK and NHSL genes was significantly decreased.The changing models of six differential genes expression were the same as that in transcriptome sequencing.This indicated that the transcriptome sequencing results in this study were reliable.5 Effects of different heat stimulation time on the expression of antioxidant related genes and HSP family genes in gills of pikeperchThe materials used were the same as experiment one.Q-PCR was used to detect the expression of antioxidant related genes and HSP family genes in gills of pikeperch under different heat stimulation time.The results showed that the changing models of Cu-Zn SOD and Mn SOD genes were similar,except that there was no significant difference between the 6h group and the control group(P>0.05),and the other groups were significantly higher than the control group(P<0.05).The expression of CAT gene was significantly higher than the control group at 3h(P<0.05)and rose to the maximum at 48h.The expression of GPX1 and GPX3 genes were significantly higher than the control group at Oh(P<0.05),while GPX7 was slightly delayed,and significantly higher than the control group at 3h(P<0.05),then the change was not obvious,and reached peak at 48h.Although the expression of HSP90 and HSC70 genes fluctuated greatly,they were significantly higher than the control group(P<0.05).The expression of HSP70 gene began to decrease after rising to the peak at 6h,and was lower than that of the control group at 48h.The mRNA expression of the above genes in the experimental group was significantly higher than the control group. |
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