| In recent years, the industry of crustacean aquaculture, especially shrimps and crabs, has experienced a rapid expansion in China, which has developed into a main way to increase the peasant income in some areas. So, the quality control and monitoring should be strengthened in the process of aquatic breeding, and the water pollution should be strictly controlled, while antibiotics and other chemical substances should be ensured that the breed was supported in a pool of quality of security. It was guaranteed the product quality from the crab and the shrimp, represented the pollution-free production of aquatic products, for a sustained and healthy development in our country. However, with the development of agriculture, industry and sewage effluent discharg was allowed. The water of culture was polluted and the pollution was created in breed aquatics itself. The quality of cultivating environment was in degradation. It caused much unhealthful influence on the cultivating of the fresh crabs and shrimps. The disease in breeding was more and more serious and expanding, and it was the serious damage to many manufacturer and breeder. Simultaneously, industry, agriculture and human activities have resulted in cadmin (Cd) and copper (Cu) pollution. The crustaceans having economic value have been pressed by Cd and Cu in growth and development. So it stunts the further growth of the aquaculture industry. Whereas, studying the effect of Cd and Cu in waterbody to shrimps and crabs, it is important to comprehend the damage in aquaculture breeding from gene expression level, to understand damage to biology body and to crustacean toxicology.The freshwater crab Sinopotamon yangtsekiense belongs to Crustaceae, Decapoda, Potamidae and Sinopotamon. Sinopotamon is representative of the most widely distributed endemic freshwater crabs in our country, and the variety and numbers are the largest in the Yangtze River and its tributaries such as wide area of high mountains and hills, plains and other different ecological zones. According to national standards for fishery of China, the freshwater crab S. yangtsekiense was exposed at different Cd concentrations (5 mg/L,0.5 mg/L,0.05 mg/L,0.005 mg/L Cd) and 0.1 mg/L copper from 24 to 96 hours. On the one hand we observed that the highest Cd level was found in gill after 96 h using atomic absorption spectrophotometry (AAS). The order of accumulation is gill>hepatopancreas >heart. The metal concentration was as high as 75±3.8μg/g in gill, and it was 70±2.9μg/g in hepatopancreas. But the amount of Cd accumulated in heart of the crab was much lower. It was observed that the highest Cd level was found in heart after 96 h, and the metal concentration was 8±0.9μg/g. Furthermore, the amount of Cd accumulated in these tissues showed an increase with increasing duration. The baseline amount of Cu was higher in S. yangtsekiens. It was up to 20.8±1.2μg/g in gill and hepatopancreas, and wasn’t detected in heart. It showed a little different from control in these tissues when combined to Cd and Cu. It was observed that the highest Cd level was 101.2±3.4μg/g in gill after 96 h, and it was 121.3±3.1μg/g in hepatopancreas. But the amount of Cd accumulated in heart of the crab was still much low. The amount of Cd accumulated in gills, heart and hepatopancreas of the crab showed an increase with an increase in the treatment concentration. On the other hand, RNA was extracted from the tissues of gill, heart and hepatopancreas in S. yangtsekiense. It aims at evaluating the induction of metallothionein (MT) synthesis in gill, heart and hepatopancreas of S. yangtsekiense.β-actin of S. yangtsekiense was isolated for the first time, and it was upload into GenBank, its accession NO.GQ415418. A phylogeny study based on (3-actin gene sequences was performed in seven species of crabs. The results implied that Scylla serrata to be more closely related to the sand crab Portunus pelagicus than any other species. S. yangtsekiense to be farther related to Gecarcinus lateralis, Neohelice granulate. Furthermore, Eriocheir sinensis to be farther related to P. pelagicus. The phylogenetic trees constructed by neighbor joining (NJ) and maximum likelihood (ML) supported the same topology. S. yangtsekiense was singleton branch, far away from other marine crabs, so should belong to potamidea. Subsequently, metallothionein andβ-actin genes were amplified using reverse transcription PCR (RT-PCR). Metallothionein gene expression induced by Cd and Cu was analyzed using real-time quantitative polymerase chain reaction. The results showed its expression levels were correlated to duration time and increased with cadmium concentrations in water, whilstβ-actin was expressed at a relatively constant level. The MT transcript levels in gill and hepatopancreas were higher than that in heart. The highest value is 22.5 folds in hepatopancreas, but it was changed slightly on treatment with 0.05mg/L and 0.005 mg/L Cd. The effect of low concentration Cu itself on RQ levels of SYMT was a slight elevatory, but the interactive effect of combined Cd and Cu was significantly different from single Cd at P<0.05. Therefore, Cu could enhance the effect of Cd on expression of metallothionein in S. yangtsekiense (SYMT), which showed cooperative effect. The RQ levels of 48 h treatment were similar to those of 96 h treatment exposed to the same concentration. After Cd exposure, SYMT levels persistently increased, and showing a clear dose response and significant elevation in experimental concentration rage, suggesting that RQ levels of SYMT were suitable a biomarker for evaluating Cd pollution. |
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