Effects Of Water Temperature On The Immune Responses Of Haemocytes In Chlamys Farreri Stimulated By Lipopolysaccharide

In this dissertation, variations in haemocyte count and haemocytes phagocytic activity of scallop (Chlamysfarreri) accustomed at different temperature (5℃,10℃,15℃,20℃and25℃) were investigated. The optical lipopolysaccharide (LPS) concentration used for stimulating the scallop was determined. Variations in total count, respiratory burst ability, intracellular lysozyme activity and intracellular phenoloxidase activity of haemocytes of scallops stimulated by lipopolysaccharide at the same temperature were researched. At last, the impact of both temperature and lipopolysaccharide challenge on the immune phagocytic activities of haemocytes was investigated. This work was consisted of the following three parts:Variations in total count and phagocytic activity of haemocytes in scollaps at different temperatures:at5℃,10℃and25℃the total number of haemocytes in scallops was significantly lower than that at15℃and20℃; both granular and hyaline haemocytes exhibited phagocytosis activity, however, the number of FM phagocytosed by haemocytes in scallops varied at different temperatures. The number of FM in a single haemocyte of scallops was1-2and1-12at5℃,10℃,25℃and15℃,20℃respectively.Variations in the number and enzyme activities of total haemocytes in scallops stimulated by LPS:at15℃, scallops were stimulated by LPS at concentrations of0.5,1,2,3and4mg/ml. The results suggested that scallops maintained regular at the concentrations of0.5,1and2mg/ml. The total haemocytes count (THC), respiratory burst activities (RBA), lysozyme (LSM) and phenol oxidase activities (PoA) were analysed after stimulating by LPS at concentrations of0.5,1.0and2.0mg/ml. The results indicated that THC were significantly up-regulated in all the LPS-treated scallops and the time course of up-regulation at1.0mg/ml reached the longest among the three concentrations, while the RBA did not show did not exhibit significant change.PoA of three groups all reached the top by2.69±0.19,3.86±0.23and2.11±0.21U/mg protein/min respectively at48h after stimulating by LPS at concentrations of0.5,1.0and2.0mg/ml. The LSM of haemocytes in scallops stimulated by LPS at concentration of0.5mg/ml did not exhibit significant change. While, the LSM of haemocytes in scallops stimulated by LPS at concentration of1.0and2.0mg/ml exhibited significant up-regulation at24,48and72h after the stimulation. The LSM of haemocytes in scallops stimulated by LPS at concentration of1.0mg/ml reached to4.86,5.17and5.56U/ml at24,48and72h respectively and the average activity of haemocytes in control group was2.36U/ml. These results indicated that the concentration of LPS at1.0mg/ml was the most appropriate concentration which would be applied in the stimulation experiments.Phagocytosis of haemocytes is the primary innate immune defense of scallop (Chlamys farreri). In this paper, the scallop was accustomed at5,10,15,20and25℃respectively, then injected with fluorescent microparticles (FM) labeled with phycoerythrin, lipopolysaccharide plus FM, or PBS. After that, haemocytes were collected at0,6,12,18,24,30,36,42and48h post injection hours, respectively, their phagocytic activity were measured by fluorescence microscope and flow cytometry (FCM). The results showed that haemocytes phagocytosis the FM since6h, the phagocytic rate rose first until18-24hours to reach the peak, then gradually declined. At5℃, the maximums of haemocyte phagocytic rate in the two treatment were8.66±0.26%and9.75±0.48%respective; at10℃they were14.00±0.22%and17.49±0.43%; at15℃, they were16.97±0.55%and23.13±0.57%; at20℃they were25.62±0.88%and34.16±0.97%; at25℃, they were8.97±0.25%and10.42±0.45%. Phagocytic rate in LPS-treated group was significantly higher than FM group. Moreover, at20℃, phagocytic rate was significantly higher than other groups, followed by a group of at15℃,10℃groups, and finally5℃and25℃groups, no significant difference between groups at15℃and20℃The results suggesting that, water temperature affect the phagocytic rate significantly and LPS can significantly enhance the phagocytic activity of scallop haemocytes.