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

Cytocentrifuge preparations of mussel haemocytes were tested cytochemi cally, using both light and electron-microscopy, for the presence of p henoloxidase activity which was demonstrated by incubation with the su bstrate dihydroxyphenylalanine. The blood-cells were also tested for p eroxidase activity using the substrate diaminobenzidine. Both enzyme t echniques produced a reaction product in the cytoplasmic granules of t he 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 cr iteria: differential inhibition; contrasting percentages of phenoloxid ase 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 s een ultrastructurally. Activation of the phenoloxidase enzyme was demo nstrated biochemically using a 96-well microplate reader; pre-incubati on of the cells with 0.025% zymosan-supernatant produced a ten-fold in crease in activity as compared with those preincubated in phosphate bu ffered saline or 0.1% zymosan. The results are discussed in relation t o the possible significance of the presence of phenoloxidase in the de fence cells of Mytilus edulis and the role of its activation in vivo.