Insect cuticular melanization is regulated by the prophenoloxidase (proPO)-activating system, which is a component of innate immunity. However, the differentiation between cuticular melanization and innate immunity is not well defined. Here, we demonstrate that the proPO-activating system regulates cuticular melanization in the silkworm pupae using a different mechanism. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating factor, tyrosine hydroxylase, proPOs, and immulectin, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism mediated by the priming of the proPO system.

Insect cuticular melanization is regulated by the prophenoloxidase (proPO)- activating system, which is also involved in the innate immune reaction. Here, we demonstrate how the differentiation of the proPO-activating system is regulated toward a cuticular melanization or innate immunity function in silkworm (Bombyx mori) pupae. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating factor, tyrosine hydroxylase, and porPOs, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism that is mediated by the priming of the proPO system.

In arthropods, an immune challenge triggers a serine protease cascade that leads to the activation of prophenoloxidase (proPO)-activating factors (PPAFs), which are also called proPO-activating enzymes (PPAEs) or proteinases (PAPs). PPAFs are activated by cleavage between their clip and serine protease domains. Once activated, PPAFs convert proPO to phenoloxidase (PO), which then catalyzes the production of quinones to form melanin. In this study, we identified a Bombyx mori PPAF(BmPPAF) that involves in the pupal melanization. In the fat body, expression of BmPPAF was detected on day 1 to 3 of the pupal stage. RNA interference (RNAi)-mediated BmPPAF knock-down inhibited pupal melanization, resulting in the delay of pupal melanization. Based on these results, we concluded that BmPPAF is involved in the melanization of pupal stage in silkworm metamorphosis.

곤충의 동종 내에서의 다양한 색상패턴발현과 연관된 유전자를 탐색하는 일은 다양한 각도에서 이루어질 수 있다. 본 연구에서는 동종내 색상다형현상을 보이는 무당벌레의 초시가 검은색과 적색 또는 황색으로 되어 있는 특징을 이용하여 멜라닌 색소 형성과 관련된 유전자에 의해 색상발현이 조절될 것이라 가정하고, 곤충의 체벽이나 날개의 경화 및 색소침적에 관여하는 phenoloxidase(PO) 유전자의 클로닝과 염기서열 분석을 수행하였다. Phenoloxidase의 다른 기능으로서 멜라닌 생합성과 관련된 두 가지 반응단계를 촉매작용을 한다. 곤충에 있어서는 일반적으로 불활성 상태인 prophenoloxidase로 존재한다고 알려져 있으며, 곤충의 혈구와 곤충류에서는 체액 내에서 발견이 되었다. 야외채집군에서 노란색 바탕에 검은 점이 전혀 없는 succinea2 개체와 검은색 바탕에 2개의 붉은 점을 가진 conspicua 개체를 사용하여서 실험하였다. primer는 무당벌레와 같은 목에 속하는 Tribolium castaneum과 유전적으로 연구가 많이 된 Drosophila melanogaster의 prophenoloxidase관련 유전자 서열을 바탕으로 design한 Forward primer와 oligodT를 reverse primer로 사용하여 PCR을 수행한 결과 발현된 것으로 sequencing한 결과를 비교분석하였다.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

The multicolored Asian ladybird beetle, Harmonia axyridis, is a generalist predator of aphids also, shows a high level of phenotype polymorphism in color pattern of elytra. Although, it is not sure about genetic information of color polymorphism, it has been confirmed that this phenomenon comes from their genetic traits. The color of H. axyridis elytra is mainly composed of black and red pigment. Phenoloxidase (PO) plays an important role in many insect physiological functions, i.e. sclerotization and pigmentation of cuticle and melanization of parasites. Following activation, PO catalyses the hydroxylation of tyrosine and subsequent oxidation of phenolic substance into quinines, which are further converted to melanin. However, the molecular bases of H. axyridis color pattern formation are almost unknown but it may be that the different pro-POs have different expression. In this study, total RNA samples from four each color pattern individuals, for example, succinea 1, succinea 2, conspicua and spectabilis was extracted. A cDNA enconding pro-PO was molecular cloned from each color pattern of H. axyridis and its putative amino acid sequence shared homology with pro-PO of other insects. We are pursuing to elucidate that their pro-PO sequence will be similar with those other insect PPO sequence. There are also regions of high sequence similarity, including putative activation site and two copper binding sites.