肠道共生菌促进球孢白僵菌杀蚊的分子机制

Anopheles mosquitos serve as vectors of malaria and many other human pathogens of public health importance, mosquito vector control is the key to block the disease transmission. Continuous emergence and rapid spread of insecticide-resistant mosquitoes strongly challenges the fight against mosquito-borne diseases, in particular malaria, new methods to control adult mosquito are urgently needed. Recent studies have shown that the entomopathogeni fungus Beauveria bassiana holds promise as biological insecticide to control both insecticide-susceptible and insecticide-resistant adult mosquito, and significantly reduce malaria transmission intensity. Furthermore, emerging data suggest that mosquito gut microbiota play important roles in regulation of the insect host’s immune system, and interaction with pathogens. We have recently found that the gut microbiota facilitate Beauveria bassiana to kill Anopheles mosquito. The new phenomenon and its associated molecular mechanisms have not been reported. In this study, we will plan to determine the dynamic gut microflora of the adult mosquitoes after infected by Beauveria bassiana using 16S rRNA gene based pyrosequencing and culture-dependent methods, and further identify the gut bacteria that play a key role in assisting Beauveria bassiana to kill mosquito, and elucidate the related mechanisms. Whole transcriptome sequencing (RNA-seq) analysis will be used to comprehensively analyze the changes and mechanisms of sytemic immune and gut epithelial immune response to Beauveria infection, dissect the correlation between dysbiosis of the gut microbiota and mosquito immune changes by fungal infection, and elucidate the melecular mechanisms by which gut microbiota facilitate Beauveria bassiana to kill Anopheles mosquito. This project will help deepen our understanding of the tripartite interactions between gut microbiota, mosquito and fungal pathogen, providing the foundation and new perspectives to control mosquito and prevent mosquito borne disease transmission.

按蚊是疟疾等多种疾病的传播媒介，蚊虫防治是阻断疾病传播的主要措施。由于蚊虫抗药性的产生，急需新方法防治蚊虫和阻断疟疾传播。近来研究表明，球孢白僵菌在成蚊的生物防治和阻断疟疾传播上有广阔的应用前景。肠道共生菌在按蚊免疫和病原菌感染中发挥重要的作用。我们最近发现肠道共生菌在白僵菌感染按蚊中起到促进作用，这一新现象及其相关的分子机制未见报道。本项目以肠道共生菌-按蚊-白僵菌三者互作为模型，通过16S rDNA测序和体外分离培养的方法解析白僵菌感染按蚊后肠道菌群结构的变化，揭示在白僵菌杀蚊中起关键作用的肠道菌群与作用机制；通过比较转录组分析肠道共生菌对白僵菌感染引起按蚊系统免疫和肠道免疫应答的影响和调控机制，解析白僵菌感染按蚊后免疫变化与肠道菌群失衡的内在联系和分子机制，阐明肠道共生菌加速白僵菌杀蚊的分子机理，为研发蚊虫生物防治新策略提供理论依据，为揭示病原真菌与按蚊互作的分子机理提供新的视角。

蚊虫传播多种疾病，严重威胁人类的生命健康。蚊虫控制是阻断蚊媒传染病的重要措施。病原真菌通过昆虫体壁侵染，在蚊虫生物防治和阻断疾病传播上具有巨大优势和广阔的应用前景。病原真菌在与宿主昆虫进攻与防御的军备竞赛中进化出多种致病因子和毒素，成为真菌战胜昆虫的有力“武器”。近年来研究表明，肠道微生物在调节宿主免疫稳态和影响肠道病原感染中发挥重要作用，对宿主的健康和病原感染的结局产生重要的影响。然而，肠道菌群和昆虫病原真菌之间的相互作用未知。本项目以肠道共生菌-按蚊-白僵菌三者互作为模型，研究肠道菌在球孢白僵菌感染按蚊中的作用；研究白僵菌侵染过程中按蚊肠道菌群结构的变化动态，揭示在白僵菌杀蚊中起关键作用的肠道细菌；进而深入解析白僵菌感染引起肠道菌群紊乱以及操控肠道细菌加速杀蚊的分子机理。围绕项目的目标和研究计划，我们全面完成了项目申请时提出的各项指标。重要结果、关键数据及其科学意义概述如下：首次发现肠道细菌能够加速病原真菌侵染致死蚊虫，成为真菌杀虫的“帮凶”。当真菌通过体壁侵染蚊虫后，有肠道菌的蚊子死亡速率显著快于无肠道菌的蚊虫。研究发现，真菌侵染导致肠道菌群失调、肠道细菌数量显著增多，但肠道菌群多样性指数显著降低，一些有益肠道菌的丰度显著下降或消失，条件致病菌如丰度显著增加，优势菌群发生易位，造成肠道菌群失衡。随后，条件致病菌粘质沙雷氏菌(Serratia marcescens)在中肠过度增殖，并突破肠道屏障转移到蚊虫血腔，造成系统性感染，从而加速蚊虫死亡。进一步研究发现，真菌感染后显著抑制肠道双氧化酶和抗菌肽的表达，导致肠道免疫失调，进而造成肠道菌群紊乱。其中，中肠Duox基因的下调是由真菌分泌的毒素—卵孢子素介导的。该项目研究首次揭示了体壁入侵的病原真菌通过招募肠道细菌促进杀虫的新机制，为理解肠道微生物的作用和病原真菌致病机理提供新视角，为发展新型害虫生防制剂提供新的思路。