Studies On The Stress Effect Of Sulfide On Adult Chinese Mitten-handed Crab, Eriocheir Sinensis Female

As a toxicant, sulfide may influence the health, survival, productivity and distribution of aquatic organisms. Under natural conditions it is produced by the anaerobic decomposition of organic materials in the sediments. However, the toxicity of sulfide to the Chinese mitten-handed crab, E. sinensis, which is economically important crustacean, has not been fully evaluated. Hence, an attempt was made to study the toxic effect of sulfide on crab at adult stage, including the acute toxic effect, effects on respiration metabolism, antioxidant ability, ovary mature and cellular changes. The purpose is to provide more basic information for aquatic animal toxicology and references for establishing the system of stress evaluation in crustacean, which could boost the sustainable development of crustacean aquaculture.The results of acute toxicity to female adult E. sinensis showed that the 24h-, 48h-, 72h-, and 96h- LC50 of sulfide were 12.51, 6.73, 5.66, 3.09 mg/L respectively. The safe concentration of sulfide was 0.31 mg/L.Based on the result of 96h- LC50, three treatments of sulfide (control, 0.10-, 0.33- and 1.0-mg/L) were designed in acute stress experiment and sampled at 3h, 6h, 12h and 24h. There are six treatments (LSI ([S] 0.1mg/L pH 6.5), MS1 ([S]0.33 mg/L pH 6.5) ,HS1 ([S]1.0mg/L pH6.5) , LS2 ([S] 0.1mg/L pH 8.5) ,MS2 ([S]0.33 mg/L pH 8.5) and HS2 ([S] 1.0mg/L pH 8.5)) and one control group in chronic stress experiment and sampled at 4d, 10d, 20d and 30d.Results showed that the CCO activity in gill turned down with the increase of sulfide concentration in acute stress and the it decreased gradually with time elapsed. So the energy was produced by anaerobic metabolic routes, which could lead to the elevation of glucose and lactate levels in haemolymph. Compared to the control, the level of oxyhemocynin in haemolymph decreased by chronic sulfide exposure, and then lowered the oxygen-carrying capacity in haemolymph.As to the antioxidant ability, the activity of superoxide dismutase (SOD) in hepatopancreas and gill were significantly increased after lower concentration of ambient sulfide exposure in short time, which exhibited the phenomenon of "poison-intoxication-effect". With time elapsed, the intoxication phenomenon was diminished the SOD activity gradually decreased. However the SOD activity was decreased by higher concentration of ambient sulfide exposure, which could depress the ability of scavenging superoxide anion radicals and depressed the ability of immunity at some degree. Furthermore, the accumulation of malondialdehyde (MDA) in haemolymph was increased with ambient sulfide increased and with time elapsed, which exaculate lipid peroxidation.In present study, ELISA was used to measure the ovarian lipovitellin concentration. Results have showed that ovarian lipovitellin concentration and ovarian total lipovitellin content in all treatments were lower than those in control at all intervals. A significant decrease(P<0.05) of GSI was registered in HS at 30d, and a significant decrease (P<0.05) of lipovitellin content of ovary was apparent in MS and HS at 10d and 20d, and HS1 at 30d comparing with the control. The difference of ovarian total lipovitellin content between the control and the other treatments was increased gradually with time elapsed. Ovarian total lipovitellin content significantly decreased in HS1 after 10d and in MS and HS at 20d, and ovarian total lipovitellin content in all treatments were significantly lower than that in control at 30d. The toxicity of sulfide was aggravated by the low pH. Results also showed that cholesterol content in hepatopancreas of E.sinensis female turned down in HS. There might be two reasons for the inhibition of ovary development of female E. sinensis by the sulfide stress. Sulfide stress disturbed the balance of energy metabolism, substance synthesis and accumulation so as to restrain the synthesis and accumulation of Lv and inhibit the growth of the ovary. On the other hand, cholesterol accumulation was decreased by sulfide stress, which inhibited the synthesis of hormone and resulted in the lag of ovarian development.Besides the physiological and chemical change caused by sulfide exposure mentioned above, there were still much more cellular changes. The experiment about effect of acute nitrite exposure on HSP70 (heat stress protein 70) expression in gills analyzed by Western-blotting showed that the expression of HSP70 rise at 3h and peaked at 6h of all the treatment. The expression of HSP70 was positively correlative to sulfide concentration. The expression of HSP70 turned down to the normal at 12h in LS, however, it was still much more than those in the control and LS at all intervals. Therefore, the heat stress protein could be expressed before the organism was seriously harmed and could be used as an early biomarker of physiological status evaluation.