Effect Of Copper Stress On The Immune Response Of Red Swamp Crayfish, Procambarus Clarkii

In recent years, with the rapid development of the industry and the agriculture, the heavy metals pollution of water become seriously and it causes a serious threat to the survival and reproduction of the aquatic. " China Fishery Ecological State of the Environment" pointed out that copper excessive levels of copper pollution was still increasing in coastal waters, rivers and lakes.("Chinese names Republic of the Fishery Water Quality Standards" provides Cu2+≤0.01 mg/L). Copper is a essential micronutrients of the crustaceans. It is not only a phenoloxidase and superoxide dismutase activity center factor, but also an essential component of hemocyanin. However, high concentrations of copper will produce toxic effects on crustacean. Research shows that: the crustaceans live in the aquatic environment of variety factors, and they have been evolved a strong resistance of the immune system to recognize or resist environmental stress in the evolutionary process. Therefore, the studies of copper contamination have a great significance.Procambarus clarkii is subordinated to Crustacea, Decapoda, Cambaridae and it commonly known as crayfish. With a wide distribution, strong resistance, and low requirements of the environmental conditions, so it is applied as an animal model in immunological study and an indication of environmental pollution in recent years. Immunology indices are closely related with the physiological status, metabolism, nutrition and disease of the shrimp, especially when the shrimp affected by external factors occurring physiological or pathological changes, it will be reflected in the immune parameters. Thus, in order to research the heavy metals impact on Procambarus clarkii, detecting the change of immune-related indicators is the most direct and effective.In this study, taking the Procambarus clarkii as the research object and combining the immunology, toxicology and pathology methods. By testing the immune factors and the influence of immune organs which exposure to different concentrations of copper, it can provide a theoretical basis using immunological methods to monitor the water pollution. Meanwhile, this study also enriches the knowledge of crustacean immunology. The results and conclusions are as follows:1 The accumulation and distribution of copper in the Procambarus clarkiiBy measuring the lethal concentration of the Procambarus clarkii, with reference to the " China Fishery Ecological State of the Environment" and the relevant literature on the basis set the copper concentration gradient. Taking the single factor gradient test, the Procambarus clarkii exposure to 0.0,0.5,1.0,3.0,5.0 and 10.0 mg/L. After 24 h,48 h, 72 h and 96 h, we measured the distribution of copper in hepatopancreas, gill and muscle. The results show that:all tissues, the copper accumulation in the tissues with the increase of copper concentration and increased induction time, that copper accumulation with obvious time-dependent and dose-dependent. In this study, the copper accumulation has obvious organizational differences. gills> hepatopancreas> muscle, which may be induced by higher copper concentration and shorter exposure time. The highest accumulation of Copper in the gill, hepatopancreas and muscle were 22.57μg/g,11.84μg/g,1.56μg/g. With the increase of the copper concentration, increased faster in hepatopancreas, gill follow, the muscle increase slowly. Therefore, in higher copper concentration, hepatopancreas is the main place of copper accumulated and the hepatopancreas plays a major role in copper metabolism.2 The effect on the immune factors of the Procambarus clarkii exposure to the copperIt was found that crayfish blood cells were divided into three types:clear cell, small granule cells and large granular cells. The no granular cells were blue and the nucleus were stained deeply after stained by Giemsa stain; small granulocytes and large granular particles were red, and the nuclear were blue. After 96 h exposure experiment the concentration gradient within the blood cells were lower than the control group, but with the increase of copper concentration and exposure time, the treatment group did not have significant changes in blood cell morphology. Crayfish blood cells of Staphylococcus aureus and phagocytic index phagocytic activity have similar trend, with the increase of copper concentration and exposure time, phagocytic activity (PP) and phagocytic index (PI) decreased gradually. The PP of treatment group, which exposure to 0.5 mg/L copper concentration, was significantly increase in the early exposure time. Throughout the experimental period. The blood cells of the PP and the PI,which exposure to 3.0,5.0 and 10.0 mg/L copper concentration, was significantly lower than the control group (P< 0.05). The results showed that low concentrations of copper in the short term can improve the immunity of crayfish, a high concentration of copper or low concentrations of copper for a long time can reduce, the immunity of crayfish, and increase the susceptibility of pathogenic microorganisms.3 Effect of the enzyme activity of serum, tissues and organs of the immune-related in Procambarus clarkii exposure to the copperIn this study, after exposure to different concentration of copper, we measured the content of serum's phenoloxidase (PO), antibacterial activity, lysozyme activity and serum, liver and pancreas and gills of superoxide dismutase (SOD), acid phosphatase (ACP), peroxide Catalase (CAT), and malondialdehyde (MDA). The results showed that: compared with the control group,1.0,3.0,5.0 and 10.0 mg/L respectively in 72h and 48h after exposure to the copper, serum's Ua and Ub viability decreased significantly (P< 0.05); the PO activity of the serum is time-dose-dependent, after 96h experiment, the PO activity of the serum of treatment groups were significantly lower (P< 0.05); The SOD and CAT activity in hepatopancreas and gill had significant changes (P< 0.05) and the activities of two enzymes in hepatopancreas were higher than gills; exposuring to low copper concentration in a relatively short time can activate the SOD and the CAT. With time prolonging the low concentration of copper showed a suppression effect, while high concentrations of copper treatment showed an inhibitory effect; The activity of MDA in hepatopancreas changed more significantly than in gill and serum, The activity of MDA after exposure to low concentrations of copper in a relatively short period of time was lower than the control group. With time prolonging and the increasing of copper concentration, the MDA activity appears a increasing trend, and the change of MDA activity and SOD activity showed a negative correlation, so using of the immune system of crayfish to forecast heavy metal pollution of water have practical and theoretical significance.4 Effect of copper on microstructure of hepatopancreas in Procambarus clarkiiAfter exposured to copper, the hepatopancreas tuble of Procambarus clarkii was significantly thicker, the simple columnar cells was swelling, the ductus cavity became thin and the basement-membrane of hepatopancreas tuble appeared corrugated arch; the number of hemocyte in hemocoel increased obviously and they crowded together in the breakage of basement-membrane in hepatopancreas tuble; Exposured to 5.0mg/L Cu2+,part of the columnar cells have been denaturated and vacuoles emerged between the basement-membrane and columnar cell; Exposured to 10.0 mg/L Cu2+, the simple columnar cells have been denaturated and even disintegrated. Cell debris appeared in the ductus cavity, the hemocyte in the hepatopancreas increased and between the basement-membrane and the simple columnar cells appeared large vacuoles. The results indicated that high concentrations of copper caused an obvious damage on microstructure of hepatopancreas in Procambarus clarkii.In summary, the immune response mechanism of Procambarus clarkii which exposured to the copper is the water-borne copper mainly absorbed by gills then to the hemolymph, and last mainly accumulated in the gill and hepatopancreas. When the copper along with the blood lymphatic circulation arrived throughout the body, it affected the body's related immune index, and then affected the tissue organ and the individual. As the structure, the nature and function of various immune factors were different, the changes of their activity were various when exposured to the copper, it can be used as the biological indicators to monitor the water environment pollution of copper. With the increase of Cu2+ concentration, crayfish hepatopancreas structures have irreversible damage, thereby affecting the growth of crayfish and even lead to death.The study preliminary revealed the immune response mechanisms of Procambarus clarkii exposured to the copper. This not only enriched the content of crustacean immunology, but also provided the reference for the healthy breeding of the Procambarus clarkii and the warning of early water environment pollution of copper, at the same time provides a basis for other heavy metals in immunology, toxicology research.