The Study On Melanization Immune Responses In Silkworm,Bombyx Mori

The immune defense is entirely dependent on innate immunity in arthropods.when pathogens invade, arthropods can only rely on the innate immune for broad-spectrum response. Whether innate immunity developed is a decisive factor for measuring whether arthropods have a strong disease resistance. Melanin deposited on the surface of intrusion pathogens, which is an important performance of immune response in arthropods. Melanization immune is the sign of immune response, which can only be directly observed for immune response in arthropods. Therefore, from melanization immune to study innate immune mechanisms of insects have long been concentrated on, especially in fruit flies, mosquitoes and other insects.The silkworm is economically important beneficial insects, but the silkworm is vulnerable to the invasion of a variety of pathogens, often resulting in huge losses on the production. Therefore, studying and revealing innate immune mechanisms of silkworms has an important theoretical value to improve disease resistance of silkworms and prevent the occurrence of silkworms disease. At the same time, silkworm as an important lepidoptera molecular model, studying and revealing innate immune mechanism of silkworms is conducive to promoting correlated study in other lepidopteran insects, and providing a new way to prevention and cure lepidopteran pests.Currently, the reports about the innate immunity of the silkworm mainly focus on antimicrobial peptides, pattern recognition protein, signaling pathway and so on. But the reports about melanization immune of silkworm are rarely. This article explored genes participating in melanization immune of silkworm and so on from four aspects, that is, comparing gene expression differences between disease black silkworm and normal silkworm, immune induction in vitro, immune induction of living body and the influence of related substances on the growth of bacteria. The main findings are as follows:1. Screening candidate genes participating in melanization immune of silkwormFluorescence quantitative PCR (qPCR) analysis of differences in expression level of melanin metabolic pathways related genes between normal ripe silkworms and black disease silkworms, the results showed:expression level of BmYellow-f、BmTan、BmDat had no significant difference in two type of silkworms; expression level of BmPAH of black disease silkworms was1/8times of that of normal ripe silkworms; compared with expression level of BmTH、BmBlack、BmDDC、BmYellow、BmEbony of normal ripe silkworms, expression level of these genes of black disease silkworms had a significant increase (P<0.001).2. The experiment for melanization immune of humour in vitroWe used three kinds of important substances molecules from cell walls of three kinds of microorganisms, that is, mannan from fungi (yeast cells), peptidoglycan (PGN) from Gram-positive bacteria and lipopolysaccharide (LPS) from Gram-negative bacteria, to induce melanization immune of silkworms humour in vitro. The results showed that: three kinds of substances can induce melanization immune of silkworms humour; the speed for melanization immune of silkworms humour induced by LPS was faster than that induced by mannan and PGN, and the degree was also stronger.The experiment of protease inhibitors showed that:inhibitors of DDC activity (carbidopa) can inhibit LPS-induced melanization immune of silkworms humour. This shows that melanization immune of silkworms humour indeed need DDC activity.3.Immune inductionWe injected silkworms with E. coli (Gram-negative bacteria) and Bacillus subtilis (Gram-positive bacteria) to induce immune response of silkworms. Semi-quantitative RT-PCR analysis of melanin metabolism-related genes, BmPAH、BmTH、BmDDC、 BmYellow、BmEbony、BmTan、Bm Dat and phenol oxidase in blood cells of injected silkworms, showed that:expression level of BmPOl decreased and expression level of BmPO-1S、BmPO-2S had no obvious relationship with invasion; expression level of BmTH、BmDDC、BmTan had an upward trend; expression level of BmPAH、BmDat had an downward trend, which was broadly consistent with the results of qPCR; only when Gram-negative bacteria invaded silkworms, tiny expression level of BmYellow were detected.4.Antibacterial analysisCompared with control, the growth of E. coli from medium added with phe or tyr had no significant difference and the color of medium didn’t change. When medium was added with dopa or DA, the number of colonies of E. coli was significantly less than the control, and the growth of E. coli was suppressed. The medium added with dopa or DA obviously darken. These showed that phe and tyr in the air can not be derivatized melanin and had little or no role in the killing of E. coli. On the other hand, dopa and DA can derivatized melanin in the air and has a significant role in the killing of E. coli.In summry, we speculate that:melanin metabolism related gene, BmPAH、BmTH、 BmDDC、BmYellow、BmTan、BmDat were related with immune response of silkworms.; the produced melanin is mainly DA melanin, but also a small amount of dopa melanin during immune response of silkworms; the intermediates of melanin metabolism, phe, tyr, dopa, DA and NBAD may be related with immune response of silkworms.