Phenoloxidase specific activity in the red swamp crayfish procambarus clarkii

Abstract

The prophenoloxidase (proPO) system is considered an important mechanism of innate defence in arthropods. This enzymatic cascade has been studied in crustaceans such as the crayfishes Astacus astacus and Pacifastacus leniusculus and is located inside the haemocytes. An initial characterisation of this system in the commercially important red swamp crayfish Procambarus clarkii is described. The P. clarkii proPO system was activated by trypsin and also by zymosan A. This activation was calcium ion dependent. The calcium ion concentration also a#ected the background activation of the system and at 5 mM was highest as measured by the ability of phenoloxidase to oxidise L-3,4-dihydroxyphenylalanine. The e#ect of calcium ions appears to be related to the activation of an endogenous serine protease, but other calcium ion-dependent factors can also a#ect proPO activation. Lipopolysaccharides (LPS), glycolipids found in the outer leaflet of the outer membrane of Gram-negative bacteria, were also able to activate the proPO system in P. clarkii after a significant lag time of 25 to 30 min. However, LPS derivatives (deacylated LPS, lipid A and â-D-GlcNAc-[1]6]-D-GlcNAc) were not able to activate the enzymatic cascade in P. clarkii. Activation of the proPO system in other crayfishes by LPS has been shown to be mediated by serine protease-like enzymes. The observed e#ect of LPS and LPS derivatives on the activation of the P. clarkii proPO system suggests that a protease activity triggered by these molecules may be mediated through the recognition of a ‘‘complete’’ LPS molecule (polysaccharide and lipid A). The intermolecular recognition of LPS by a putative endogenous serine protease zymogen might explain the lag time observed in proPO activation.