Analysis Of Hemolymph Melanization And Melanin Metabolic Intermediate In Silkworm Larvae Induced By Exogenous Microorganisms

The silkworm, as an economic insects, plays an important role in our agricultural economy, but it is vulnerable to the invasion of a variety of pathogens that often causing huge economic losses to the state and farmers. Silkworm is an important mode of arthropod insects. Arthropods is not acquired immune defense system like mammals. They can only rely on the innate immune defense system of broad-spectrum response when there are xenobiotics infection. Therefore, the study of silkworm innate immune defense mechanisms have important theoretical implications for improving disease resistance and promoting the silkworm sericulture development. In addition, immune defense mechanism of silkworm not only is of great significance for promoting other lepidopteran insect-related research, but also for providing reference for the prevention and control of lepidopterous pests.Melanin can deposited on the surface of intrusion pathogens, which is one of the important immune response performance in arthropods. Whether melanization immune involved in the immune defense of arthropods has attracted people’s attention for a long time, scholars have conducted a lot study about insect blackening immune mechanisms, which focused on fruit flies and mosquitoes. 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 the silkworm blackening reaction in the immune defense process role based on the observation of silkworm larvae blood color changes in vitro, the analysis of silkworm larvae blackened blood induced by exogenous microbes, as well as microbial infection on larvae intermediate metabolism of melanin content in the blood and other experiments. The main findings are as follows: 1. Observation of silkworm larvae blood changes in vitroWool needle used in this study can blocked the contact of ambient air and needle tube material. Silkworm larvae blood in the hair needle can remain transparent in a long time. The blood color changed and the changes were observed in strong silkworm period in wool needle under the conditions of different temperatures (37℃, room temperature,4℃). The results showed that the blood color black speed of the4th instar larvae of first day is the slowest, that of the5th instar larvae of sixth day is the fastest, there is the characteristics that the blood color black speed is in accelerating gradually along with the larvae color black;at different temperatures, the formation of black blood is the fastest, deep blackening phenomenon becomes most obvious at high temperature37℃, followed is at room temperature, that is slowest at4℃, there is the characteristics that the apparent extent and speed of blood black is in decline with the increase of temperature.2. Blood melanization analysis induced by exogenous microbes of silkworm larvaeE. coli, Bacillus subtilis, B. bassiana were used as exogenous microbes, the blood color change of the5th instar larvae of sixth day and the6th instar larvae of third day were observed in wool needle with microbial:blood volume1:2,1:1and2:1. The results show that the blood color black speed is significantly faster when induced by exogenous microorganisms; the blood color black speed of five instar black blood is faster than that of fourth instar larvae; when induced by E-coli, the blood color black speed is the fastest and the phenomenon is most obvious, followed is B. subtilis, B. bassiana is the slowest; the blood color black speed is fastest when microbial:blood volume is1:23. Effect of silkworm larvae blood on microorganisms growth in vitroUsed the aforementioned microorganisms to reaction with blood of the4th instar larvae of third day, the5th instar larvae third day and the5th instar larvae sixed day for6h and24h and then statistics statistical growth status of microorganisms. The results show that growth status of microorganisms induce by the5th instar larvae of third day is the worst, followed is the5th instar larvae of third day, that of the4th instar larvae of third day is relatively good; the growth status of E. coli induced by larvae blood is worse than that of other microorganisms; the growth status of microorganisms with microorganisms:blood1:2is worse than with that2:1; the growth status of microorganisms reaction for6h is worse than that for24h.4. Effect of immune induction on blood dopa and dopamine levels in silkworm larvae bloodThe5th instar larvae of third day was induced by E. coli, the dopa and dopamine levels of larval blood reaction for6h and24h were detected by HPLC. The results show that there is a downward trend for dopa content after reaction for6h, but dopa content was significantly decreased after reaction for24h; there is no significant changes for dopamine levels after reaction for6h and24h.These results suggest that exogenous microorganisms can induce the occurrence of blackened blood for silkworm larvae; silkworm larvae blood blackening reaction can inhibit the growth of exogenous microbial, the stronger the blackening reaction, the better the effect of suppressing, that also suggest that blackening reaction of blood may play a immune defensive role for silkworm larvae; blackening immune of silkworm may be related to the melanin synthesis metabolism.