| Copper is a trace element that is essential for the growth and development of animals,and it is an essential nutrient for crustaceans to synthesize hemocyanin.As an indigenous species in China,Eriocheir sinensis is one of the most important economic crabs species in China.However,at present,the underlying molecular mechanism of the effect of Cu2+ on E.sinensis is still poorly understood,and this study explored the effects of Cu2+ on the physiological and biochemical of E.sinensis as well as the underlying molecular mechanism.1.To investigate the toxicity mechanism of Cu2+ against E.sinensis,the determination of LC50 of Cu2+ stress E.sinensis was firstly performed in this study.The results showed that 24h LC50,48h LC50,72h LC50,96h LC50 of E.chinensis in cope with Cu2+ were 3.824 mg/L,2.779 mg/L,1.941 mg/L,1.666 mg/L,respectively.Then acute toxicity test was performed to observe the effect of Cu2+ on the microstructure of hepatopancreas and gill tissues in E.sinensis.Experimental groups were divided into four groups with the concentrations of 0 mg/L,0.1 mg/L,0.2 mg/L and 0.4 mg/L.The results showed that the low concentration（0.1mg/L）of Cu2+ could cause damages to hepatopancreas and gill tissues.With the increase of Cu2+ concentration,the toxicity showed an upward trend,which caused serious pathological damage to hepatopancreas and gill tissues of E.sinensis.Hepatopancreatic tissues showed that a large number of vacuoles appeared in the cells,the basal membrane was separated by a single layer of columnar epithelium in hepatic tubules to form a cavity,and even cell debris appeared in hepatic tubules.Gill tissues showed that the branchial lobes were irregularly thickened,and the epithelial layer was severely damaged and even appeared disintegrated.2.To investigate the effect of Cu2+ on physiological aspects of E.sinensis and whether the normal level can be achieved after recovery,the metabolic and immune related enzyme activities of E.sinensis were detected in this experiment.The results of metabolism related enzymes showed that CL viability,G6PD viability,LPS viability and PFK content all decreased significantly compared with the control group,and all of them,except G6PD,showed a continuous decreasing trend with time in a timedependent manner.MT content increased gradually with time and was significantly higher than that of control at 96h.TCH content was higher in each experimental group than in the control group at 96h,in the low concentration group at 24h,in the medium concentration group at 96h,and in the high concentration group,although all were higher than the control group but not significantly different.The results of immune related enzyme activities showed that the concentrations of each experiment in which ATP content,GSH activity,and GST activity decreased,and all showed significant differences compared with the control group.LZM viability at 24h was significantly increased in the high concentration group compared to the control group and was again significantly decreased after 96h,with no significant change in the low concentration group.The MT content in the 24h low concentration group was significantly decreased compared with that in the control group,with no significant change at both 48 h and 96 h.The SOD content in the low concentration group at 24h significantly decreased compared with the control group and then returned to nearly the level of the control group,and the high concentration group showed a trend of gradually decreasing with time,and was significantly lower than the control group at 96h.After 14 days of recovery from acute copper ion stress,the body cholesterol metabolism function was still severely impaired,the glucose metabolism function was basically restored to a normal level,the antioxidant system was still not restored,the innate immune system was still under stress,and the body ATP content was basically up to a normal level.Taken together,even though E.sinensis did not recover to a healthy state after Cu2+stress for 14 days,the basic energy requirements required for growth could already be met.3.To investigate the effect of Cu2+ stress on the transcriptomics of E.sinensis,the transcriptomic analysis of the treated E.sinensis gill tissues and hepatopancreatic tissues was performed in this experiment,which was validated in combination with real-time fluorescence quantification.The results showed that a total of 13153 DEGs were identified in gill tissue in the 0.2mg/L group,of which 682 were upregulated and 12471 were downregulated,and a total of 13534 DEGs were identified in the 0.4mg/L group,of which 812 were upregulated and 12722 were downregulated.Among them,the pathways of glutathione metabolism,lysosome pathway,phagosome pathway and ABC transporter pathway showed that Cu2+stress seriously affected the expression of immune related genes in E.sinensis,and then did harm to its innate immunity.A total of 650 DEGs were identified in the 0.2mg/L group with 290 upregulated and 360 downregulated in the liver tissue,and a total of 975 DEGs were identified in the 0.4mg/L group with 434 upregulated and 541 downregulated in the liver tissue.Among them,the glycolysis gluconeogenesis pathway,fat digestion and absorption pathway and cholesterol metabolism pathway had a high impact,which indicated that Cu2+seriously affected the expression of genes related to glucose metabolism and lipid metabolism in E.sinensis,and then caused harm to its normal growth and development.In the present study,molecular insights into the toxicity of some E.sinensis for Cu2+were provided.However,more in-depth studies on DEGs are also needed to better understand the mechanisms underlying the response of aquatic organisms to copper ion toxicity.4.To investigate whether the Cu2+ contration in cultured ponds cause toxicity to E.sinensis,three crab culture ponds of E.sinensis farmers in Pukou District of Nanjing city were randomly selected for this experiment,and continuous and timed site-specific detection of Cu2+ was performed.The results showed that the average values of Cu2+contents in the bottom mud of three ponds were 33.1 mg/kg,32.1 mg/kg,33.9 mg/kg,and no Cu2+ were detected in cultured water,indicating that the levels of Cu2+ in water were all below the safe detection limit（0.04 mg/L）.Farmers should reduce Cu sulfate use and remove silt at the end of each farming cycle. |
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