PHENOLOXIDASE ACTIVITY IN THE HEMOLYMPH AND HEMOCYTES OF THE MARINE MUSSEL MYTILUS-EDULIS

Cytocentrifuge preparations of mussel haemocytes were tested cytochemically, using both light and electron-microscopy, for the presence of phenoloxidase activity which was demonstrated by incubation with the substrate dihydroxyphenylalanine. The blood-cells were also tested for peroxidase activity using the substrate diaminobenzidine. Both enzyme techniques produced a reaction product in the cytoplasmic granules of the eosinophilic cells which varied in its intensity within and between haemolymph samples. The activity was demonstrated to be the result of two distinct enzymes within the same cell type using the following criteria: differential inhibition; contrasting percentages of phenoloxidase and peroxidase-positive cells within pooled samples; variation in numbers of positive cells over time in a seasonal survey; differential localisation of the reaction products in different size granules as seen ultrastructurally. Activation of the phenoloxidase enzyme was demonstrated biochemically using a 96-well microplate reader; pre-incubation of the cells with 0.025% zymosan-supernatant produced a ten-fold increase in activity as compared with those preincubated in phosphate buffered saline or 0.1% zymosan. The results are discussed in relation to the possible significance of the presence of phenoloxidase in the defence cells of Mytilus edulis and the role of its activation in vivo.