| The prophenoloxidase (proPO) activating system, present in invertebrates and considered to be an important innate immune mechanism for protection against infection and injury has been well characterized in several arthropod species. Biochemical studies of this enzyme system in Procambarus clarkii hemocyte lysate fractions were carried out. Three types of phenoloxidase were observed: Catecholase, Cresolase, and Laccase. Catecholase and cresolase activities have been reported in other invertebrates. However, laccase activity, originally described to be important in cuticle escleotization and exoskeleton hardening, has not been previously reported in crustacean hemocytes (blood cells). Microbial compounds such as lipopolysaccharides (LPS) of Gram-negative bacteria, and zymosan (fungal cell walls) were able to activate the proPO system extracted from crayfish hemocytes. Activation of proPO resulted in the formation of the active enzyme, phenoloxidase. This activation was mediated by an endogenous serine protease, because the addition of a serine protease inhibitor restricted the activation of the system by exogenous trypsin and zymosan. Activation of the proPO system by LPS seems to require the presence of the complete molecule, namely polysaccharide and lipid A. As zymosan A and LPS were able to activate the proPO system extracted from lysed hemocytes, these microbial compounds were tested on live hemocytes to observe their effect on cellular activation. Flow cytometry was used to assess cellular activation of live hemocytes from the white river crayfish Procambarus zonangulus. Cell activation resulted in cell size reduction and death. The activation patterns observed in this study were comparable to the action of the tested microbial compounds in mammalian systems, which implies a similar immune response. |
