Evaluation of haemolymph phenoloxidase activity from the grub of Zophobas morio as a predictor of immune response.

In insects, enzyme phenoloxidase plays a critical role in cuticular sclerotisation and defensive functions. In the present investigation, haemolymph phenoloxidase activity from the grub of Zophobas morio was attempted to evaluate as a reliable predictor of insect's immunological response. Among the various substrates tested, L-DOPA was chosen as an appropriate substrate due to its high oxidation. The optimum pH and temperature for haemolymph PO activity was found to be 8 and 30 °C, respectively. The optimum substrate concentration of L-DOPA was found to be 7.5 mM for subsequent PO enzymatic characterisation. Among the various chemical inhibitors and copper chelators, PO activity was significantly reduced in the case of PMSF and thiourea. Preincubation of haemolymph with non-self-molecules showed enhancement of PO activity in the case of LPS from Serratia marcescens. In addition, exogenous proteases like α-chymotrypsin enhanced the PO activity of haemolymph and an increase in PO activity was demonstrated when haemolymph was preincubated with the anionic detergent, SDS and cationic detergent, cetyl pyridium chloride. Alteration of PO activity was observed under agonising conditions of starvation, ligation and microplastics injection at different time intervals. Interestingly, there were no correlation between PO and insect defence under live challenge of microbes. SDS protein profile revealed a significant increase in the 85 kDa and 55 kDa polypeptides in all the experiments over control after 24 h, 48 h and 96 h. Mass spectrophotometric analysis of the polypeptides revealed their homology to antimicrobial peptides for 55 kDa protein and 85 kDa protein. A significant increase in 85 kDa polypeptide was observed in the haemolymph of the grubs after 72 h in the case of starved and microplastics injected groups only. These results demonstrated that PO may not be a reliable benchmark of immunological response in this insect.