Molecular Regulation Mechanism Of Phenoloxidase-induced Melanin Synthesis In The Mealworm Beetle, Tenebrio Molitor Larvae

The melanization reaction induced by activated phenoloxidase (PO) in arthropods is important in the multiple host innate immune reactions, leading to the sequestration and killing of invading microorganisms. This reaction ought to be tightly controlled because excessive formation of quinones and systemic excessive melanization are deleterious to the hosts. However, molecular mechanism of how PO-induced melanization reaction is regulated in vivo has remained to be determined. Here, we demonstrate the biochemical evidence that Tenebrio molitor Sp(a|¨)tzle processing enzyme (Tm-SPE), which is a key enzyme to process pro-Sp(a|¨)tzle to the cleaved Sp(a|¨)tzle in Toll pathway, converted 79 kDa Tm-pro-phenoloxidase (Tm-proPO) to 76 kDa, 46 kDa and 27 kDa PO fragments without showing any melanin synthesis. However, co-incubation of Tm-proPO, active form of Tm-SPE and Tenebrio clip-domain serine protease homologue 1 (Tm-SPH1) zymogen, but not Tm-SPH2, generated high molecular melanization complexes consisting of 76 kDa Tm-PO and active form of Tm-SPH1. This complex synthesized strong melanin on the surface of Gram-negative and -positive bacteria and then induced strong bactericidal effects. These results demonstrate that Tenebrio Toll pathway and proPO activation cascade are sharing common serine protease cascade for the activation of these two major innate immune responses. However, PO-induced local melanization reaction is requiring additional regulatory Tm-SPH1 protein for controlling specific activation of proPO cascade because systemic melanization could also cause fatal damage to hosts.