巴氏新小绥螨感染球孢白僵菌的体液免疫应答机制

Utilization of spreading the entomopathogenic fungi by natural enemies to improve the biological control efficacy is an important “web-based coordinated control approach”. However, the fungi also negatively affected the natural enemies as well as they infected the target pest. Therefore, it is needed to understand the immune and defense by the natural enemies against the entomopathogenic fungi. Insect humoral immune has gradually attracted broad attention because of its important physiological functions in the process of insect resistance to harmful exogenous stimuli, while these researches are limited in natural enemies. This study focuses on the humoral immune response of biological control agents, Neoseiulus barkeri. A series of advanced techniques including survival analysis, scanning electron microscope, qPCR, RNAi, western blot and immunofluorescent labeling are utilized to study the immunoregulation network of N. barkeri infested by Beauveria bassiana; the temporal expression pattern of the genes of PPO, Toll and MAPK pathway are analyzed; ployclonal antibody preparation and expression localization are systematically analyzed to clarify the molecular mechanism of humoral immune response in this predatory mite against infection by B. bassiana. The results will theoretically provide the knowledge to explain humoral immune response mechanisms in biocontrol natural enemies, and meanwhile provide new insights in the coordinated utilization of the predatory mites with the entomopathogenic fungi.

利用生防天敌携带病原真菌协同控害是促进生防产品高效利用的重要模式。然而生防菌作用于靶标害虫的同时也会对天敌造成不利影响，因此亟需了解并增强天敌自身对病原真菌的免疫与抵抗。尽管体液免疫在昆虫抵御外源刺激过程中的生理调控作用备受关注，但天敌捕食螨的免疫应答机制仍不清楚。本项目以商品化天敌巴氏新小绥螨为对象，基于转录组、蛋白组和修饰组分析，综合运用分子克隆、RNAi、Western blot、免疫荧光标记等技术，系统开展巴氏新小绥螨体液免疫调控网络研究；解析其受球孢白僵菌感染后PPO、Toll和MAPK通路相关基因的表达模式；对其免疫响应关键基因进行功能验证及表达定位，明确其体内抗菌肽及MAPK磷酸化修饰水平。研究旨在阐明巴氏新小绥螨应答球孢白僵菌感染的体液免疫分子机制，完善天敌昆虫（螨类）免疫应答研究基础，为利用巴氏新小绥螨携带球孢白僵菌作为“生物导弹”联合防控靶标害虫害螨提供理论支撑。

捕食螨联合昆虫病原真菌形成“生物导弹”在小型吸汁型害虫害螨防治中具有重大应用潜力。本项目聚焦捕食螨对球孢白僵菌的侵染响应机制这一科学问题，以巴氏新小绥螨及其猎物二斑叶螨为主要对象，从二者对球孢白僵菌的耐受性差异、Toll信号通路的应答模式差异、Toll通路对下游抗菌蛋白的功能分析等方面开展研究。结果表明，巴氏新小绥螨对球孢白僵菌的耐受性较高，并主动摄食球孢白僵菌孢子，而二斑叶螨对球孢白僵菌高度敏感，二者差异显著。巴氏新小绥螨更偏好捕食球孢白僵菌侵染的猎物。共筛选获得巴氏新小绥螨Toll通路基因10条，其表达量在不同发育阶段存在显著性差异，且在球孢白僵菌侵染后的多个时间段显著性上调表达，而二斑叶螨Toll通路基因对白僵菌的响应较弱，无明显应答反应。沉默巴氏新小绥螨Toll通路基因NbSpz-3、NbMyd88-1、NbCactus、NbDorsal后均会导致巴氏新小绥螨对球孢白僵菌的耐受性显著性降低。筛选7条对球孢白僵菌侵染具有明显应答反应的抗菌蛋白基因，发现NbSpz-3、NbToll-4、NbMyd88-1、NbCactus、NbDorsal对7条下游基因具有明显调控作用。且RNAi NbLysozyme-1/4/5/6后均会导致巴氏新小绥螨对球孢白僵菌的耐受性降低。研究结果阐明巴氏新小绥螨体液免疫Toll信号途径的调控模式，揭示了巴氏新小绥螨与二斑叶螨对球孢白僵菌耐受性差异的内在调控机制，丰富了植绥满的体液免疫理论，为菌螨协同应用提供了理论支撑。