Effects Research Of Environmental Factors And Drug Residues On The Production Of ROS By The Coelomocytes Of Apostichopus Japonicas

Due to the lack of adaptive immunity, sea cucumber Apostichopus japonicas relies mainly on the innate immune to defend themselvs against the non-self substances such as bacteria, fungi and foreign body. Coelomocyte phagocytosis is of great significance to sea cucumber immunity. And the key of phagocytosis is to secrete lysosomal enzymes and reactive oxygen species. Reactive oxygen species(ROS) play an important role in eliminating pathogen such as bacteria, fungi and large particulate matters.In this paper, using the method of chemiluminescence, the process of coelomocytes of sea cucumber to produce reactive oxygen species, which is stimulated by yeast cells, was studied. Sea cucumber coelomocyte optimal chemiluminescence reaction system was determined: the coelomocyte concentration in 106/mL, yeast concentration in 0.5 mg/mL and Luminol concentration in 2.5*10-4 mol/L. The experimental data showed that, environmental temperature could influence coelomocytes, and ideal testing temperature was around 17 °C. After the injection of LPS to sea cucumbers, ROS production during in vitro phagocytosis of yeast cells by coelomocytes was increased at the beginning, came to the peak value at 9h, and then declined.Here, the effects of environmental factors such as water temperature, salinity and pH on the production of ROS by the coelomocytes of sea cucumber Apostichopus japonicas during phagocytosis were studied. Sea cucumbers were maintained under different environmental conditions after being temporarily kept for one week, coelomic fluid was extracted at different time points and then ROS production during in vitro phagocytosis of yeast cells by coelomocytes was measured by the luminol chemiluminescence method. The results showed that the ROS production in sea cucumbers which were held in 6°C and 26°C was dramatically increased from the first day and the fifth day respectively compared with the control group(17°C). Coelomocytes of the sea cucumbers cultured at 25 and 35 of salinity generated much more ROS than the control group, while the same went for sea cucumbers kept at 16 and 40 of salinity on the first day. Low pH also dramatically enhanced the production of ROS during phagocytosis. In conclusion, water temperature, salinity and pH can influence the production of ROS by the coelomocytes of sea cucumber during phagocytosis. What’s more, this report lays a foundation for the further study of the physiological and biochemical processes of adaptation of Apostichopus japonicas to different environments as well as the innate immune mechanisms in invertebrates.Aquatic animal antibacterial counter drugs have been widely applied in aquaculture. But due to the abuse of antibiotics, the ecological environment and human health had been faced with great potential safety hazard. In this experiment three groups were set up, which were injected with furazolidone, chloramphenicol and erythromycin respectively for 3 consecutive days. Then their coelomic fluid was extracted in different time points and ROS production during in vitro phagocytosis of yeast cells by coelomocytes was measured by the luminol chemiluminescence method. The results showed that during the whole experiment process, furazolidone and erythromycin both depressed the production of ROS by coelomocytes in the resting state. The ROS production in sea cucumbers which were injected with erythromycin was dramatically increased, and the maximum value appeared on the 15 th day. After the injection of furazolidone to sea cucumbers, their coelomocytes stimulated by yeast cells generated much more ROS at the beginning, then in the seventh day the value was lower than the control group, while it returned to normal levels in the 15 th day. In the whole process, ROS level during phagocytosis by coelomocytes of the sea cucumbers injected with chloramphenicol was improved except the first day.