Substrate specificity of phenoloxidase-like activity in an ecoimmunological model species Lymnaea stagnalis

Ecoimmunological research on molluscs and other invertebrates frequently quantifies phenoloxidase (PO) activity to estimate the strength of the immune function. PO enzymes form different families whose relative roles in oxidative reactions are typically unknown. Understanding this could allow enzyme-specific assays with higher accuracy than in commonly used nonspecific assays. We tested the contribution of different PO enzyme families to haemolymph PO-like activity in Lymnaea stagnalis snails using substrates specific to enzymes detected in L. stagnalis transcriptome data (p-phenylenediamine, specific to laccases; L-tyrosine, specific to tyrosinases) and compared the reactions to those with a nonspecific substrate (L-dopa). We found laccase-like but no tyrosinase-like activity. However, reactions with L-dopa were the strongest, possibly due to other oxidative enzymes in snail haemolymph. Laccase-like activity is common in molluscs, and we propose the use of enzyme-specific assays in future ecoimmunological studies of this taxon. The lack of tyrosinase-like activity in L. stagnalis contradicts earlier transcriptome data, which calls for investigating the expression of PO enzymes in L. stagnalis at the proteome level.