Insect immulectins are involved in various aspects of the innate immunity, including encapsulation, melanization, and phagocytosis. Although the silkworm Bombyx mori immulectin (BmIML) has been reported previously, the ligand and functions of BmIML have not been investigated. Here, we show the dual roles of BmIML in cuticular melanization and immunity of B. mori. BmIML recognizes carbohydrates in a Ca2+-dependent manner and binds to Gram-negative and Gram-positive bacteria, fungi, and yeast. BmIML was expressed in the fat body during infections and localized to the hemocytes of silkworms. Additionally, BmIML was expressed in the epidermis during the prepupal stage and localized to the cuticle of silkworms. After treatment with E. coli, dopa, dopamine, or tyrosine injections, BmIML production was induced in the fat body but not in the epidermis of silkworms. Treatment with BmIML RNAi resulted in the arrest of pupal cuticular melanization. Therefore, we concluded that BmIML is involved in pupal cuticular melanization and innate immunity responses of silkworms, suggesting dual roles for BmIML.

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.

Melanization is a unique defence mechanism in arthropods involved in wound healing and pathogen encapsulation. Phenoloxidases (PPOs) are key enzymes of melanization, which mediate the enzymatic conversion of tyrosine to eumelanin. A serine-protease (SP) cascade, similar to the blood-clotting cascade of vertebrates, proteolytically activates prophenoloxidases to phenoloxidases. This proteolytic activation is tightly controlled by serpins and other melanization inhibitors. Melanization has been regarded as one of key immune responses against malaria parasites in mosquitoes. The ookinete melanization of both the simian malaria parasite, Plasmodium cynomolgi, and of the rodent parasite, Plasmodium berghei, prevent parasite development in the human malaria vector, An, gambiae. However, the recent studies revealed a melanization response regulated by Serpin-2 and two C-type lectins (CTL4 and CTLMA-2) was shown to result in ookinete melanization but did not affect the development of the natural human parasite P. falciparum in the mosquito. Instead of melanization, TEP1/APL1/LRIM1 complement-like pathway has been identified as major immune response that regulate parasite loads in the natural association of An. gambiae and P. falciparum. The studies by me and my colleagues revealed another melanization response independent on Serpin-2. Genome analyses of mosquitoes revealed a large expansion of the PPO, SP, and serpin genes potentially involved in the melanization pathway. This expansion was devoted to existence of at least two distinct SP-Serpin regulation modules in controlling separate melanization responses, tissue and hemolymph melanization, in the mosquito, Aedes aegypti. Tissue melanization regulated by Serpin-2 has role in melanotic tumor formation, but not in ookinete melanization. Hemolymph melanization regulated by Serpin-1 and a couple of SPs was activated by the infection of various pathogens and is involved in anti-malarial defense against the avian malaria parasite, P. gallinaceum. A new type of regulator, CLSP2, negatively modulate this hemolymph melanization. Cross-talk between hemolymph melanization and complement-like pathway will be discussed.

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The fungal transformant also shows mammal fibrinolytic activity, by which the transformant can be used pharmaceutically. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.

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.