Study On The Toxic Effect Of Copper Ions On Eriocheir Sinensis

Eriocheir sinensis is an important species of aquatic products in China,and it is one of the traditional economic crabs in China.Copper is an essential trace element in crustaceans.It plays a role in growth and development and immune regulation in Eriocheir sinensis.As copper sulfate is used in large quantities as aquaculture medicine and the growth and development of Eriocheir sinensis in natural waters may be under Cu²⁺stress.This study took Eriocheir sinensis as the research object,and explored the toxic effects of Cu²⁺in water bodies on crustaceans using research techniques such as slice observation,biochemical enzymology,and molecular biology.This study has important practical significance for the basic research on Cu²⁺poisoning mechanism of crustaceans.The main results are as follows:1.The effect of copper ions on the microstructure of hepatopancreas gills of Eriocheir sinensis.In the experiment,the 96h-lethal concentration(LC₅₀)of copper sulfate pentahydrate（Cu SO₄·5H₂O）to Chinese mitten crab was 22.164 mg/L,and in the toxicity test,five concentrations were set:0（control）,0.17 mg/L,0.69 mg/L,2.77mg/L and 11.08 mg/L.The Chinese mitten crabs were exposed for 5 days and 10 days,and hepatopancreas and gill tissues were collected.The results showed that the microstructure of the gills and hepatopancreas began to change when the copper ion was 0.69 mg/L.In 2.77 mg/L and 11.08 mg/L copper ion treatment,many vacuoles were observed in the gills tissue;the epithelial layer was severely damaged and disintegrated,and the gill lobes became thin.In hepatopancreas,intumescent of the monolayer columnar cells and the enlarged hepatic tubule lumen were also found.2.The effect of copper ions on the activity of ATPase in the gills of Eriocheir sinensis.The gill tissues of Chinese mitten crab exposed to different concentrations of copper inos were collected.Enzyme-linked immunoassay and fluorescent quantitative PCR were used to determine the activity of Na⁺/K⁺-ATPase and H⁺/Ca²⁺-ATPase and genes expression.The results showed that,after 5 and 10 days of exposure,the activities of Na⁺/K⁺-ATPase and H⁺/Ca²⁺-ATPase were the highest when the copper ion concentration was 0.69 mg/L,and they were significantly higher than the control group（P<0.05）.When the copper ion concentration is 2.77 mg/L and 11.08 mg/L,the activities of Na⁺/K⁺-ATPase and H⁺/Ca²⁺-ATPase significantly were decreased compared to 0.69 mg/L（P<0.05）.The activities of Na⁺/K⁺-ATPase and H⁺/Ca²⁺-ATPase after 5 days of exposure were higher than those after 10 days of exposure.Meanwhile,the changes in gene expression of nkcc,nak-atpase and ca-atpase were consistent with the changes in enzyme activity.3.The effect of copper ion on oxidative stress in the gills and hepatopancreas of Eriocheir sinensis.The hepatopancreas and gill tissues of Eriocheir sinensis were tested with different concentrations of copper,and the activity/content of SOD,CAT,GSH,T-AOC and MDA and the expression of related genes were determined by enzyme-linked immunosorbent assay and fluorescent quantitative PCR.The results showed that with the increase of Cu²⁺concentration,the activities of CAT,SOD and GSH in the hepatopancreas and gills increased first and then decreased,and the activities of T-AOC and MDA increased;the expression of sod,cat,gpx,gst,akt genes in the hepatopancreas and gills.It first increased and then decreased.The expression levels were highest in the 0.69 mg/L copper ion treatment group.The enzyme activity/content and gene expression trends of copper ion exposure for 5 days and 10 days were consistent.4.The effect of copper ion on non-specific immunity of Eriocheir sinensis hepatopancreasThe hepatopancreas of Eriocheir sinensis exposed to different concentrations of copper were coolected,and the activity of AKP,ACP,AST,ALT and genes expression were determined by enzyme-linked immunosorbent assay and fluorescent quantitative PCR.The results showed that at 5 and 10 days of exposure,the activity of ALT increased with the increase of copper ion concentration,and the activity of AKP,ACP and AST increased first and then decreased with the increase of copper ion concentration.After 5 and 10 days of exposure,the expression of lsz gene was the highest when the copper ion concentration was 0.17 mg/L;the m RNA level of mt and hsp70 were both highest when the copper ion concentration was 0.69 mg/L;the hsp90gene expression was upregulated firstly and then downregulated after copper ion exposure.In summary,copper ions have toxic effects on Chinese mitten crabs,which can cause changes in the tissue microstructure of the gills and hepatopancreas,leading to oxidative stress and activating non-specific immune response.Long-term exposure to high concentrations of copper ions will significantly inhibit the activity of ion transport enzymes in the gills;the activity of antioxidant enzymes and non-specific immunity.