蚊虫和虫生真菌相互作用的分子机制

One of new approaches for mosquito control is to use entomopathogenic fungi, which can be used as a safe and green alternative instead of most chemical insecticides. Mosquitoes transmit devastating diseases, such as malaria, Dengue virus, and West Nile virus. Increased incidences of chemical insecticide resistance compromise the ability to control vector-borne diseases. Two entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, could be reportedly used as biopesticides for the purpose of mosquito control. However, for industrial application of fungi, it is necessary to improve genetic characteristics to overcome the slow speed of kill and high dose of treatment by revealing the molecular mechanism of fungal pathogenicity. In Drosophila system, anti-fungal defense was reportedly dependent on Toll immune pathway. We have shown that major components of melanization pathway genes were regulated by Toll pathway in the mosquito Aedes aegypti. In addition, we found that an entomopathogenic fungus, B. bassiana, caused the abnormal cleavage of hemolymph prophenoloxidases (PPOs), which are the key enzymes of melanization. We propose that this abnormal cleavage of PPOs will inactivate the enzymes and eventually destroy the humoral immunity. The cleavage patterns of hemolymph PPOs will be compared between mosquitoes infected by pathogenic fungi and non-pathogenic fungi. Furthermore, it will be tested whether mosquito with the constitutively activated melanization by RNA interference- mediated depletion of melanization inhibitors will have an increased survival rate after the infection of an entomopathogenic fungus. These experiments will suggest a key role of melanization against fungal pathogens and reveal the suppression mechanism of host melaniztion by the pathogenic fungi. We will also analyze fat body transcriptome of A. aegypti infected by pathogenic and non-pathogenic fungus. Differential expressed gene repertoire will be analyzed as well as the implication of Toll pathway and melanization genes in the anti-fungi immunity will be revealed. This study will improve our understanding of interaction between mosquito and fungus in molecular aspect and also provide valuable information to construct highly effective and specific biopesticides.

杀虫剂的长期大量使用使媒介昆虫对杀虫剂产生了抗药性。研究发展绿色的新型生物杀虫剂具有重要的理论意义和广泛地应用前景。虫生真菌球孢白僵菌和金龟子绿僵菌被用于媒介昆虫和其他农林昆虫的生物防治，但仍存在不少弊端从而限制了其应用。对真菌生物杀虫剂的改造需要深入研究其作用的分子机制。本研究基于球孢白僵菌等昆虫病原体诱导蚊虫酚氧化酶原的异常剪切从而扰乱蚊虫的体液免疫反应，从昆虫免疫的角度率先探讨蚊虫病原体躲避、压制蚊虫黑化反应系统攻击的机制，明确蚊虫黑化反应在防御真菌侵染反应中的作用；深入研究蚊虫和病原体相互作用的分子机制。并使用高通量测序技术系统性地分析蚊虫在受真菌侵染时转录组的变化；通过差异表达基因的研究，揭示Toll免疫途径和黑化反应在蚊虫抗病原真菌防御中的关联作用，为进一步筛选抗真菌关键基因提供基础，有助于设计控制蚊虫的新思路，而且更有助于为设计开发新型生物农药提供理论基础。

媒介蚊虫传播的寨卡病毒等疾病对人类公共健康构成严重威胁。而研究发展绿色的新型真菌生物杀虫剂具有重要的理论意义和广泛地应用前景。虫生真菌球孢白僵菌和金龟子绿僵菌被用于媒介昆虫和其他农林昆虫的生物防治，但仍存在不少弊端从而限制了其应用。对真菌生物杀虫剂的改造需要深入研究其与宿主反应的互作分子机制。本研究基于球孢白僵菌等昆虫病原体诱导蚊虫酚氧化酶原的异常剪切从而扰乱蚊虫的体液免疫反应，从昆虫免疫的角度率先探讨蚊虫病原体压制蚊虫黑化反应系统攻击的机制，筛选参与抗真菌反应的关键基因，明确蚊虫黑化反应在防御真菌侵染反应中的作用；并使用高通量测序技术系统性地分析蚊虫在受真菌侵染时转录组的变化；通过差异表达基因的研究，揭示Toll免疫途径和黑化反应在蚊虫抗病原真菌防御中的关联作用。首先，我们表达纯化了酚氧化酶原(PPO3)活性蛋白，比较了其在不同激活方式下的活性。在此基础上制备了抗体，检测了虫生真菌球孢白僵菌菌感染后埃及伊蚊酚氧化酶原的异常剪切情况；同时测定蚊虫感染虫昆虫病原体后的转录组库，通过生物信息学分析结合实时定量PCR结果，获得了参与蚊虫抗真菌及细菌病原体的免疫基因群；揭示了黑化反应调节酶CLSP2在调节埃及伊蚊黑化反应及抗真菌免疫中的作用，敲除CLSP2后，提高了蚊虫对真菌感染的存活率。CLSP2在埃及伊蚊经球孢白僵菌和阴沟肠杆菌免疫刺激之后的表达量升高，同时CLSP2蛋白在埃及伊蚊受球孢白僵菌刺激后在血淋巴中发生剪切作用；而且其体外表达纯化Lectin结构域的蛋白能够凝集真菌细胞壁成分，并且能够结合荧光标记的球孢白僵菌，说明CLSP2具有凝集和结合活性。其次，利用高通量转录组测序技术探讨CLSP2与蚊虫免疫通路Toll途径和IMD途径的相互作用，发现CLSP2能够负调节蚊虫Toll途径和黑化途径主要免疫基因的表达。此外还能能够负调控PPO3在真菌免疫情况下血淋巴中的剪切作用。这些成果表明，CLSP2参与调节蚊虫黑化反应和抗真菌免疫反应，有助于进一步明确蚊虫的抗真菌免疫机制。最后还对蚊虫羽化后和吸血后基因动态表达进行了分析，聚焦于代谢调控，发现在羽化前由保幼激素（JH）控制，而蜕皮激素（20E）则在吸血后发挥重要作用。这些结果有助于我们进一步明确能量代谢途径、免疫与蚊虫生殖发育的密切关系。本研究有助于设计控制蚊虫的新思路，为开发新型生物农药提供理论基础。