Effects Of Nitric Oxide On The Immune Activity Of Chinese Mitten Crab Eriocheir Sinensis

After several different types of micro-organisms were injected in vivo and pathogens were co-incubated with hemocytes in vitro, we detected the NOS activity of Chinese mitten crab (Eriocheir sinensis) and changes of NO concentration. Results showed that the inducible NOS existed in Chinese mitten crab hemocytes and its product of NO was related with immunity. NOS inhibition tests exhibited that EDTA and L-NAME significantly inhibited the NOS activity induced by pathogenic bacteria, but the SMT had no significant inhibition. Thus, we speculated that the structure of inducible NOS in Chinese mitten crab may be more similar to mammalian constitutive NOS.Under the same concentration of exogenous NO, it had no significant inhibitory effect on bacterial growth when it was used alone, but the growth of bacteria was suppressed remarkably when exogenous NO was co-existed with hemocytes, therefore, we concluded that NO can significantly improve hemocytes to inhibit the bacteria growth. In order to further explore the effect of NO on immune activity of Chinese mitten crab, firstly we injected SNP into the crab body at a certain concentration, and tested the changes in the immune-related indicators after sampling at different time. The next, we co-incubated hemocytes with SNP for 2 hours in vitro, then detected hagocytic activity of hemocytes and the bacterial adhesion to hemocytes. Finally, we injected SNP into the crab body, and then detected effects of SNP on the expression of antimicrobial peptides crustins mRNA by semi-quantitative RT-PCR method. Research data demonstrated that exogenous NO significantly affected the hemocytes count (DHC) of different types. For example, granulosa cells reduced and small granule cells and non-granule cells increased in a certain period. Exogenous NO could induce some immune-related enzyme activities. For example, the phenol oxidase and alkaline phosphatase activity were increased significantly by SNP at 100μmol/L, and acid phosphatase and lysozyme activity were enhanced by SNP at 100μmol/L and 500μmol/L, however, many enzyme activity was inhibited by SNP at 1000μmol/L. Bacterial adhesion to hemocytes was strengthened by SNP at more than 500μmol/L. Phagocytosis activities were increased by SNP at 20μmol/L, but this effect was not observed when SNP concentration increased more than 500μmol/L. A short-term expression of antimicrobial peptides crustinIII was induced by SNP at 100μmol/L. CrustinIII expression of crab body increased after 12h, but it returned to the normal level (control group) after 24h. In the meanwhile, the expression of crustin I had no significant change.In our research, we explored the characteristics of hemocytes NOS and systematically studied the effect of NO on the immune activity of crustacean represented by Chinese mitten crab. In general, NO have positive and negative effects on crustacean immune, depending on the concentration of NO. Exogenous NO at appropriate concentration could strengthen immune defense of Chinese mitten crab to microble.