Thus, the subsequent ability of the knockdown shrimp to oxidize a range of phenolic substrates was then evaluated. Using L-tyrosine as a monophenolic substrate the PO activity of PmproPO1- and PmproPO2-silenced shrimp were found to be significantly and equally decreased to around a 59% residual activity, some 1.7- to 1.8-fold lower than that of the control shrimp injected with GFP dsRNA or NaCl alone (Fig. 1B). The ability of two shrimp POs to oxidize diphenolic substrates was assessed using L-DOPA or dopamine as the substrate, where a significant decrease in the PO activity was observed in both the PmproPO1- and PmproPO2- silenced shrimp (Fig. 1C and D). With L-DOPA as the substrate a residual hemolymph PO activity of 5.5% and 7.4% was observed in the PmproPO1- and PmproPO2-silenced shrimp, respectively (Fig. 1C). Using dopamine as the substrate, a similar residual hemolymph PO activity of 8.5% and 10.3% was detected in the PmproPO1- and PmproPO2-silenced shrimp, respectively (Fig. 1D). It has previously been reported that DHI is an intermediary product that is essential for melanin formation and exhibits a broad-spectrum antimicrobial activity (Zhao et al., 2007, 2011). In this study, the substrate specificity of the two P. monodon POs against DHI was evaluated as above and a 61.4% residual PO activity was observed in the PmproPO1-silenced shrimp and this was not significantly different to the numerically smaller residual PO activity of 45.7% in the PmproPO2-silenced shrimp, indicating that DHI is a relatively poor substrate for shrimp PmPOs (Fig. 1E). Taken