Biochemical characterization of a hemolymph phenoloxidase and its endogenous inhibitor in the larvae of an invasive moth, Cydalima perspectalis Walker (Lepidoptera: Crambidae)

Abstract

• A phenoloxidase was purified from the hemolymph of C. perspectalis with the molecular weight of ~ 78.7. • The optimal values of 7 and 30–35 °C were evaluated for pH and temperature dependency of the purified PO. • Syntehtic and endogenous inhibitors significantly affected PO activity both in vitro and in vivo. Phenoloxidase system is a crucial component of insect innate immunity which contribute to oxidize phenols to quinones and to generate reactive oxygen and nitrogen intermediates. In the current study, a phenoloxidase (PO) was extracted by hemocyte lysate preparation and purified through ammonium sulfate precipitation, Sepharyl G-100, and DEAE-Cellulose fast flow columns. At the end of the purification process, an enzyme was purified with a specific activity of 0.462 U/mg protein, recovery of 40.47%, purification fold of 14.43 and molecular weight of ~78.7 kDa. The optimal activity was recorded at pH 7 while the optimal temperature was recorded at 30–35 °C, 35 °C and 25–35 °C, using L-dihydroxyphenylalanine, hydroquinone, and pyrocatechol, respectively. The highest V max of PO was obtained using L-dopa while the lowest K m value was gained using hydroquinone. Among used synthetic inhibitors of ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DTC), N, N,N0,N0-tetraacetic acid (EGTA) and triethylenetetramine hexaacetic acid (TTHA), EDTA and DTC inhibited more than 60% of the enzyme activity. Moreover, an endogenous phenoloxidase inhibitor (POI) was purified by twice processing of Sepharyl G-100 chromatography with the molecular weight of ~52 kDa. The IC 50 of POI was found 31.3 mg against the purified PO of C. perspectalis and led to a higher value of K m . Finally, larval injection by DTC and POI demonstrated significant inhibition of PO over the time of exposure. A comprehensive understanding of insect’s POs may better clarify the ways of their survival within infected areas and to potentially target them by specific and selective compounds.