王中, 张伟杰, 刘雷, 冷晓飞, 高洪涛, 江会杰, 常亚青
In order to investigate the effect of black mouth disease pathogen infection on phagocytosis of sea urchin Strongylocentrotus intermedius, the coelomocytes density, apoptosis rate, cell necrosis rate and phagocytosis related immune indices, including acid phosphatase activity (ACP), reactive oxygen species (ROS), total antioxidant capacity (T-AOC) and phagocytosis related immune gene expression were determined 0, 1, 6, 12, 24 and 48 h post pathogen stress in the sea urchin damaged by piercing the mouth membrane with a needle into the body cavity and then exposed to black mouth disease pathogen fluid (102 CFU/mL). The single phagocyte contribution value of several immune parameters was calculated by using the proportion of effective phagocyte. The density of phagocytes in coelomic fluid was shown to be decreased by 80%, and the apoptosis rate of phagocytes to be increased to 60.92% 1 h post stress. There were different relative trends of ACP activity, ROS level and TAC in coelomocytes after stress. However, the average contribution of single phagocyte showed a consistent trend of first increase and then decrease, with the maximal expression level of C3-pre and Clec4g genes at 1 h post stress, increased by 3 times, 6 times, 7 times, 4 times and 7 times, respectively. The apoptosis rate showed a downward trend, and the necrosis rate gradually was increased to 63.98%. C3-pre and Clec4g genes, as well as the single phagocytemean relative expression of ACP, ROS and T-AOC was decreased to the level before stress from 6 h to 24 h post stress, although the density of phagocytes gradually recovered. The maximal expression level of Caspase-8, as about 2 times as that at 0 h stress, was observed at 48 h post stress. It was found that the phagocytosis of the sea urchin was enhanced by the increase in ACP activity, ROS， T-AOC, and up regulation of phagocytic related immune genes at the beginning of infection. Meanwhile up-regulation of apoptosis rate of phagocytes and cell regeneration were also found to maintain the number of phagocytes. At the late stage of the invasion of pathogens, phagocytes failed to clear all the pathogen, the above phagocytosis related indices gradually were decreased, and the necrosis rate of phagocytes so significantly increased that the sea urchin became diseased.
