Primary Hematocyte Cultivation and House-keeping Gene Detection of Chinese Mitten Crab (Eriocheir sinensis)
YANG He, YUE Wu-Cheng, HOU Xin, WANG Jun, CHEN Xiao-Wen, WU Jia-Min*, WANG Cheng-Hui*
Key Laboratory of Freshwater Fisheries Germplasm Resources, Ministry of Agriculture/National Demonstration Center for Experimental Fisheries Science Education/Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai 201306, China
Abstract：Culture of hemocytes in vitro has been preliminarily studied in many aquatic crustaceans, but different researchers have not reached a broad consensus in the study of the key culture conditions, such as medium type, serum concentration and osmotic pressure. Culture of hemocytes in vitro is of great importance for further molecular study in Chinese mitten crab (Eriocheir sinensis). In order to investigate the optimal culture condition in vitro, such as medium, osmotic pressure and serum concentration, cultivation efficiencies of hemocytes in Chinese mitten crab from 5 different culture medium (1 × L-15, 2 × L-15, 3 × L-15, Sf-900 and TC-100 medium, respectively), osmotic pressures (345, 569, 803, 991 and 1215, mOsm/kg, respectively) and serum concentrations (0%, 5%, 10%, 15% and 20%, respectively) were analyzed. In addition, this study also detected the relative gene expression in the cultured hemocytes. The results showed that the TC-100 medium was the best culture medium according to morphological profiles and survival rate of cultured hemocytes. Furthermore, the best cultivation conditions were determined under the condition of osmotic pressure of 991 mOsm/kg and 0% bovine serum, which showed the best morphological profile and more than 50% of survival rates after 10 d cultivation. Expressions of the 3 house-keeping gene (ubiquitin-conjugating enzyme E2b (UBE), ubiquitin/ribosomal S27 fusion protein gene (S27) and β-actin) of Chinese mitten crab were steadily detected in hemocytes in continuously cultured for 10 consecutive days. The results have revealed that the medium and the key culture conditions could better cultivate hemocytes of Eriocheir sinensis, and the cultured cells could be used in gene function experiments. This study provides a new potential technical method for the study of gene function of Eriocheir sinensis.
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