入侵害虫红棕象甲与其肠道菌群协同互作的分子免疫机理

Growing evidence suggests that large quantity of microbes live in the gut of animal hosts and insects where they establish a symbiotic association with the host. It has been indicated that gut microbiota involved in many vital physical processes of the host including metabolizing indigestible compounds, supplying essential nutrients and promoting the development and regulation of the mocosal immune system. Therefore, the gut epithelium seemly tolerates the prolification of commensal bacteria to a certain extent to allow the beneficial interactions to happen. However, no study was conducted to reveal the role that gut microbiota play in the dispersion of an invasive pest. On the other hand, because of the conservative cell wall of commensal bacteria and pathogenic bacteria, it is seemly very difficult for gut cells to discriminate and activate the immune responses to eliminate the pathogenic bacteria. To our knowledge, it is still known little on how the gut epithelium tolerates and regulates the gut microbiota while retaining the ability to trigger an efficient immune response once infected with pathogens. The red palm weevil, Rhynchophorus feerugineus Olivier, is an invasive pest with high risk in China. R. feerugineus is also one of the most important pests of palms worldwide and its damage can eventually be lethal for the palm.In this project, we firstly would like to dissect the structure and function of the gut microbiota in this invasive pest by metagenomic methods.R. feerugineus host with only one species of gut bacteria is established to deeply elucidate its role for the dispersion of this pest. Thirdly, some new modern methods such as transcriptome with deep sequencing on the gut of this pest and RNAi are integrated to reveal the molecular immune mechanism undlying the toleration and regulation of gut microbiota. In the pests, the alteration of the gut microbiota has been determined to be linked to pest health and fitness disequilibrium. So the elucidation of the sicientific questions here will not only present novel evidence to answer the mechanism of biological invasion but also find some new targets to develop new ways of control strategy on this pest species.

肠道菌群与昆虫的生长发育、免疫和寄主利用等关键生理过程密切相关。从肠道菌群与宿主互作的角度来研究外来昆虫入侵扩散的机理以及昆虫如何耐受并调控其肠道菌群的机制等工作在国内外还尚未开展。红棕象甲Rhynchophorus ferrugineus Olivier是我国的一种高风险性外来入侵昆虫，是世界性的棕榈植物的毁灭性害虫。在前期已发现红棕象甲的肠道中生活有数种可培养细菌的基础上，本项目再结合宏基因组学测序技术揭示红棕象甲的肠道菌群的结构和功能，然后构建肠道无菌系和纯菌系昆虫模型并整合转录组学技术、RNAi技术等手段阐明肠道菌群对红棕象甲的生理影响以及宿主耐受肠道细菌的免疫互作分子机理。肠道菌群稳态的维持是动物正常生存的关键前提。因此，该研究不仅能从种间共生协同互作的角度为外来昆虫入侵扩散机理的阐释提供新的科学依据，同时也可为该害虫的综合治理技术开发提供新的科学思路和作用靶标。

红棕象甲Rynchophorus ferrugineus Olivier是一种严重为害我国棕榈科植物的外来毁灭性害虫。以其为研究对象，本项目阐明了肠道菌群对红棕象甲的生理影响以及PGRP家族基因和Relish基因在宿主耐受肠道菌群中的作用机理。研究发现，红棕象甲的肠道菌群主要由Enterobacteriaceae, Lactobacillaceae, Entomoplasmataceae和Streptococcaceae这四个科的细菌构成，其中一些细菌具有降解植物纤维素的活性，而且肠道菌群组成的改变能够显著地影响该害虫的营养代谢。 研究揭示RfPGRP-S1和RfPGRP-LB具有酰胺酶活性必需的五个保守氨基酸残基，这说明RfPGRP-S1和RfPGRP-LB具有酰胺酶活性，能够降解PGN。感染细菌后，RfPGRP-S1和RfPGRP-LB的表达量显著增高，说明RfPGRP-S1和RfPGRP-LB基因可能参与介导该害虫对细菌的免疫应答反应。体外功能验证发现，重组蛋白RfPGRP-S1和和RfPGRP-LB引起大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus的细胞发生聚集反应，添加Zn2+后，细菌的凝集现象更加明显，而且rRfPGRP-S1对S. aureus的生长具有明显的抑制活性，而RfPGRP-LB能够抑制E. coli的生长。这说明RfPGRP-S1和RfPGRP-LB是具有抑菌活性的免疫识别蛋白。RfPGRP-S1和RfPGRP-LB被分别沉默后，红棕象甲对血淋巴和肠道的感染大肠杆菌清除能力增强；在未感染的情况下，红棕象甲肠道菌群的总数量明显少于对照组，肠道菌群结构组成也发生了显著改变；RfPGRP-S1和RfPGRP-LB分别被干扰的试虫肠上皮中的相关抗菌肽的转录水平显著增高。这说明RfPGRP-S1和RfPGRP-LB不仅可能通过降解肽聚糖来避免肠道免疫的过度激活来维持其肠道菌群的健康稳态，而且可作为杀菌效应因子来控制病原菌的定殖和肠道菌群的增殖。当RfRelish被沉默后，红棕象甲的系统免疫和肠道局部免疫均被削弱，而且导致该害虫的肠道菌群数量增加、群落组成结构也发生明显的改变，这证实RfRelish介导的IMD pathway通过调控调控肠道免疫活性来维持红棕象甲与其肠道菌群之间的共生关系。