Effects Of Acute Heat Stress On Liver And Gill Tissue Damage,Apoptosis And Immune Response Of Pikeperch (Sander Lucioperca)

Pikeperch(Sander Lucioperca)is an important sub-cold water economic fish.In recent years,extreme high temperature in summer has seriously affected the growth of pikeperch in culture ponds,and even caused mass death.In this study,with 23℃ as the control group,acute heat stress(30℃,32℃ and 34℃)and recovery experiments(R30℃,R32℃ and R34℃)were performed on pikeperch,respectively.The histomathological and ultrastructural changes of liver and gills of pikeperch were observed.The indexes of oxidative stress,apoptosis index and the activities of key enzymes of apoptosis were detected.The relative expressions of genes related to apoptosis mitochondria and death receptor pathway and immune-related genes such as Toll-like receptors and cytokines were determined.The correlation between P53,Bax gene expression and Bax/Bcl-2 ratio,and the correlation between H2O2 content and TNF-α,IL-1β gene expression were analyzed.The purpose of this study was to explore the effects and mechanisms of acute heat stress on liver and gill tissue damage,apoptosis and immune response of pikeperch.The contents of the study are as follows:1 Effects of acute heat stress on liver oxidative stress and histology of pikeperchAccording to the results of our previous research and pre-experiment,23℃ was used as the control group,and the target temperature of acute heat stress were set at 30℃,32℃ and 34℃ respectively.720 juvenile pikeperch(average weight 1.7±0.3g)were randomly assigned to 12 water tanks in the circulatory system,and each stress temperature was set with three repeats.Water temperature was increased from 23℃ at 1℃/h to the target temperature,sampled after holding the stress for 2h,and then decreased to 23℃ at-1℃/h and sampled after 48h of recovery.Some of the liver and gill samples were sectioned,HE stained and observed,and changes in gill ultrastructure were observed by transmission electron microscopy.The other part of the samples were used for physiological and biochemical analysis.The results showed that the liver and gill of pikeperch had obvious tissue damage under acute heat stress,including gill lamellae swelling,epithelial dissection and rapture,liver congestion,vacuolar degeneration,and nuclear number significantly decreased with the increase of heat stress temperature.The most severe damage occurred at 34℃,including vascular rupture in the liver,hemocyte infiltration,severe vacuolization,and fusion of gill lamellae in individual gill filaments.Acute heat stress caused mitochondrial swelling,endoplasmic reticulum and Golgi apparatus deformation,cytoplasmic vacuolation in gill cells,and cytoplasmic membrane outgrowth to form vesicles and cell disintegration to generate regulatory vesicles were seen in the 34℃ group.Among the oxidative stress indicators,H2O2,MDA,SOD,CAT,GPX and GST were significantly increased with increasing heat stress temperature,except for CAT and GST activities in gills,which were significantly decreased.After 48 h of recovery,liver and gill tissues basically returned to normal state,and liver oxidative stress index and gill GPX activity were still significantly higher than those of the control group,but gill CAT activity was significantly higher in the R34℃ group.The above results indicated that acute heat stress caused oxidative stress and tissue damage in the liver and gills of pikeperch,and the oxidative stress was counteracted by elevated antioxidant enzyme activities.The timely cooling recovery contained and reversed the damage to the liver and gills of pikeperch,but it was not possible to return to the normal state in the short term.2 Effects of acute heat stress on the expression of immune-related genes in the liver and gill of pikeperchThe heat stress protocol was the same as experiment 1,and the effects of acute heat stress on the expression of genes related to innate immunity in liver and gill of pikeperch were analyzed by real-time quantitative PCR.The results showed that the mRNA of liver TOLL-like receptor pathway-related genes(TLR1,TLR2,MyD88,IRAK1 and IRAK4)were significantly down-regulated in the heat stress group compared with the control group,reaching the lowest values at 32℃ or 34℃.Gill TLR1,TLR2 and MyD88 expression were significantly down-regulated at 30℃,and IRAK1 and IRAK4 mRNAs were not significantly down-regulated.The mRNA of TOLL-like receptor pathway was significantly up-regulated in both 32℃ and 34℃ heat stress groups,reaching the highest value at 34℃.The expression of liver and gill cytokines TNF-α,IL-1β,IL-7,C3,IL-8 and IL-10 were significantly increased in pikeperch under acute heat stress.Liver H2O2 content with TNF-α(r=0.91,P<0.01)and IL-1β gene expression(r=0.69,P<0.01),and gill H2O2 content with TNF-α(r=0.68,P<0.01)and IL-1β(r=0.80,P<0.01)gene expression were significantly positively correlated.After 48 h recovery at 23℃,liver TOLL-like receptor-related gene expression increased significantly,but was still significantly lower than the control level,while some of the gill TOLL-like receptor genes were significantly down-regulated,even lower than the control group.After the same recovery,the expression of liver and gill cytokine-related genes were still significantly higher than the control levels.The results indicated that acute heat stress inhibited the liver TOLL-like receptor pathway in pikeperch,and ROS(H2O2)mainly induced the expression of liver inflammatory factors.However,heat stress activated the innate immune and inflammatory responses in gills through TOLL-like receptor pathway and ROS to induce non-infectious immunotoxicity.And C3 may be involved in the inflammatory and antioxidant responses of tissues under heat stress and in tissue repair during recovery.3 Effects of acute heat stress on liver and gill apoptosis and signal transduction of pikeperchThe heat stress protocol was the same as experiment 1,and the effects of acute heat stress on apoptosis and signal transduction-related gene expression in the liver of pikeperch were analyzed by TUNEL,Caspase enzyme activity and real-time quantitative PCR.The results showed that with the increase of acute heat stress temperature,the number of apoptosis-positive cells in the liver and gills of pikeperch increased significantly,and the apoptosis index of liver cells increased significantly.Liver Caspase-9 and Caspase-3 activities were significantly increased at 34℃,but gill Caspase-9 and Caspase-3 activities did not change significantly.The expression of apoptotic mitochondrial pathway-related genes(P53,Bax,Apaf-1,Caspase-3,Caspase-9)in liver and gill cells under acute heat stress increased significantly with increasing temperature,and liver P53 gene expression with Bax gene expression(r=0.81,P<0.01)and Bax/Bcl-2 ratio(r=0.92,P<0.01),and gill P53 gene expression with Bax gene expression(r=0.89,P<0.01)and Bax/Bcl-2 ratio(r=0.81,P<0.01)were significantly positively correlated.Liver death receptor pathway(TNFR1,TRAF2,Caspase-8,Fas and FADD)mRNAs reached their lowest levels at 30℃,after which TNFR1,Fas and FADD mRNAs were significantly upregulated only at 34℃.Gill death receptor pathway genes were significantly down-regulated at 30℃,but significantly higher than controls at 32℃ and 34℃.After 48 h of recovery,apoptosis-positive cells in liver and gill were still significantly higher than the control group,while Caspase-9 and Caspase-3 activities had returned to the control level.Meanwhile;the expression of most genes of the mitochondrial apoptotic pathway remained significantly higher in liver and gill than in the control group,and the expression of Bcl-2 was significantly higher in the liver R34℃ group.The gene expression of liver death receptor pathway remained significantly lower than that of the control group,while the gill partial death receptor gene expression was significantly down-regulated,even lower than that of the control group.Our above results suggest that under moderate heat stress,pikeperch first reduced the adverse effects of heat stress on liver and gill tissues by inhibiting the outer pathway,increasing anti-apoptosis and anti-oxidation.However,both intrinsic and extrinsic pathways are involved in apoptosis under severe heat stress and may lead to liver necrosis.During the recovery period,pikeperch rapidly reduced the gene expression of apoptosis even below the control level to reduce apoptotic damage to liver and gill and continued to clear damaged cells through the mitochondrial pathway.