昆虫水虻肠道微生物抑制人畜共患致病菌的机制研究

Hermertia illucens (L.), also named black soldier fly, can convert livestock and poultry manure efficiently. Larvae can be used for protein source feed and the manure residues can be used as organic fertilizer. This technology, turning waste into treasure, has a broad development prospects. Previous studies have found that the intestinal microbes of black soldier fly larvae can inhibited several common zoonosis pathogens in livestock and poultry manure, but the mechanism is unclear..In this study, the methods of high-throughput sequencing, efficient purification and antimicrobial detection of active substances were used to analyze the genome of intestinal microflora at different time in the conversion of pig manure from sterile black soldier fly larvae to determine the groups and abundance of microorganisms and the changes of key gene expression and its relationship with inhibition of pathogenic bacteria. To find out the key role of cultivable intestinal bacteria and active substances, revealing the direct mechanism of black soldier fly larvae intestinal microbial in pathogens inhibition.To obtain the intestinal bacteria which can stimulate intestinal immunity, the cultivable intestinal bacteria were added into the aseptic larvae and then analyze the transcriptome of different time of the larvae midgut immune gene. The metabolomics method will be used to obtain the new regulatory factor that can stimulate the immune gene dual oxidase gene DUOX. In the further work, silence of the key genes such as DUOX will be to determine the effect on the population, quantity of black soldier fly intestinal microbes and pathogen..It is the first and foremost to reveal the direct and indirect mechanism of the inhibition of the common zoonosis pathogens by the black soldier fly larvae intestinal microbes and provide theoretical basis for the application of intestinal probiotics in livestock and poultry breeding.

昆虫水虻能高效转化畜禽粪便，幼虫可作蛋白饲料，残渣可作有机肥，变废为宝，发展前景广阔。前期研究发现，水虻肠道微生物能清除畜禽粪便中几种常见人畜共患致病菌，但机制不清楚。本研究通过高通量测序、活性物质高效纯化和抗菌检测等技术，对建立的无菌水虻转化猪粪不同时间肠道微生物宏基因组进行分析，确定水虻肠道微生物类群、丰度和活性物质关键基因表达变化与抑制致病菌关系；找出抑制致病菌起关键作用的肠道微生物及活性物质，揭示水虻肠道微生物抑制致病菌直接机制；在无菌水虻中加体外培养的肠道菌，对不同时间水虻中肠免疫基因进行转录组分析，获得能激发水虻肠道免疫的肠道菌；利用代谢组学方法，获得能够激发免疫基因双氧化酶基因DUOX的新调节因子，进一步对水虻DUOX基因进行沉默，确定沉默后对水虻肠道菌种群、丰度和致病菌的影响。首次揭示水虻肠道菌抑制人畜共患致病菌的直接和间接机制，为肠道益生菌在畜禽养殖中的应用提供理论依据。

昆虫水虻能高效转化畜禽粪便，幼虫可作蛋白饲料，残渣可作有机肥，变废为宝。前期研究发现，水虻肠道微生物能清除畜禽粪便中几种常见人畜共患致病菌，但机制不清楚。本项目在前期研究的基础上进行了如下研究：1. 水虻肠道微生物抑制人畜共患致病菌的直接机制；2. 水虻肠道微生物抑制人畜共患致病菌的间接机制，在此基础上建立水虻肠道微生物与水虻免疫系统联合抑制人畜共患致病菌的模型。取得的创新性结果如下：1. 水虻幼虫转化能够消减猪粪中的人畜共患致病菌，例如金黄色葡萄球菌、沙门氏菌、大肠杆菌和志贺氏菌; 2. 对转化猪粪的水虻幼虫的肠道菌群进行多样性分析; 3. 从转化猪粪的水虻幼虫肠道中获得10株能够在离体条件和水虻幼虫体内稳定拮抗金黄色葡萄球菌的肠道微生物。其中拮抗效果最好的菌为B. velezensis D2406。B. velezensis D2406基因组中与抗菌相关的次生代谢物基因簇发现D2406菌株能够分泌或代谢出多种具有抗菌活性的天然产物，其中Fengycin、Macrolactins、Surfactin、Bacilysin和Paenibactin对S. aureus等革兰氏阳性菌具有强的抑制作用。这些次级代谢产物可能是水虻幼虫在转化猪粪过程中起抑制金黄色葡萄球菌的物质。.4. 发现2,5-二甲基吡嗪能够通过触杀和熏蒸作用显著抑制金黄色葡萄球菌等人畜共患致病菌。综上，发现抑制人畜共患致病菌的关键水虻肠道微生物B. velezensis D2406和活性物质2,5-二甲基吡嗪，揭示了水虻肠道微生物抑制人畜共患致病菌的直接机制。.5. 研究显示通过RNAi干扰水虻的免疫基因：Duox和TLR3，增加了水虻肠道微生物的丰度，但减少了与病原菌抑制相关的肠道有益菌Providencia 和Dysgonomonas的丰度，对人畜共患病原菌抑制减弱，推测这些肠道细菌通过激活免疫基因Duox和TLR3的表达，调节肠道微生物的组成和结构从而抑制病原菌，从而揭示水虻肠道微生物抑制人畜共患致病菌的间接机制。.在以上基础上建立了水虻肠道微生物与水虻免疫系统联合抑制人畜共患致病菌的模型，通过揭示水虻肠道微生物消除人畜共患致病菌的机制，对水虻和微生物联合无害化利用畜禽粪便等有机废弃物，保障人类健康具有重要的科学意义。