Agglutinin-mediated phagocytosis-associated generation of superoxide anion and nitric oxide by the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii

Hemocyte mediated phagocytosis is one of the vital components of innate defence mechanisms in crustaceans and this phagocytic process is aided by serum agglutinins. However, literature on agglutinin mediated opsono-phagocytosis is unclear in the case of Macrobrachium rosenbergii hemocytes. Further, very few studies in the case of superoxide anion generation and none with regard to nitric oxide generation during phagocytosis exist among crustaceans. We investigated the occurrence of agglutinins in the serum and the role of serum agglutinins in mediating phagocytosis by the hemocytes. We show that the prawn serum possesses agglutinins that function as opsonins during phagocytosis of HB RBC by the hemocytes. Hemagglutination-inhibition assays revealed the specificity of serum agglutinins for N-acetylated hexoses, namely GalNAc, GlcNAc and ManNAc, with a higher affinity for ManNAc. In addition, ManNAc was able to inhibit the phagocytic response (by about 60%) of the hemocytes against serum pretreated HB RBC, wherein the serum was previously treated with ManNAc. We next investigated the ability of the hemocytes to generate superoxide anion and nitric oxide during HB RBC phagocytosis and results show generation of both these free radicals. In addition, there was an enhancement in generation (75% increase) of these free radicals during agglutinin mediated opsonophagocytosis, when compared to buffer treated targets and interestingly this enhanced generation was inhibited by ManNAc (27% for superoxide anion and 36% for nitric oxide), an inhibitory sugar for phagocytosis. Inhibition of phagocytosis induced superoxide anion generation by DPI (53%), sodium azide (56%) and tropolone (61%), reveals the possible involvement of NADPH-oxidases, peroxidases and probably phenoloxidases, respectively, in the generation of superoxide anion. Similarly, decrease in nitric oxide generation in the presence of l-NIO (47%) during phagocytosis lends support to the role of nitric oxide generation during cellular immune processes. These findings thus suggest a role for superoxide anion and nitric oxide in the innate defense mechanism, namely phagocytosis, in Macrobrachium rosenbergii.