Melanization reaction products of shrimp display antimicrobial properties against their major bacterial and fungal pathogens

•Two PmPOs display a substrate specificity towards monophenols and diphenols.•Two PmPOs play important role in shrimp defense against Fusarium solani infection.•Shrimp melanization reaction products exhibited antimicrobial activities.

Melanization is a rapid defense mechanism in invertebrates. The substrate specificity of phenoloxidases (POs) and the role of melanization reaction products were investigated in the black tiger shrimp, Penaeus monodon. Two PmPOs (PmproPO1 and PmproPO2) were found to display a substrate specificity towards monophenols and diphenols, and exhibit relatively weak activity against 5,6-dihydroxyindole (DHI). Systemic infection of the PmproPO1/2 co-silenced shrimp with the fungus, Fusarium solani, led to a significantly increased mortality, suggesting an important role of PmproPOs in shrimp’s defense against fungal infection. Using l-DOPA, dopamine or DHI as a substrate, the melanization reaction products exhibited in vitro antimicrobial activities towards Gram-negative bacteria (Vibrio harveyi and Vibrioparahaemolyticus) and Gram-positive bacteria (Bacillus subtilis), whereas the lower effect was detected against the fungus (F. solani). SEM analysis revealed the morphological changes and damage of cell membranes of V. harveyi and F. solani after treatment with shrimp melanization reaction products. Together, these findings demonstrate the crucial functions of the proPO system and the importance of melanization reaction products in the shrimp’s immune defense.