非编码RNA调控蜜蜂预蛹抵御狄斯瓦螨与残翅病毒协同侵染及其机制的研究

The synergistic effects of the ectoparasitic mite Varroa destructor and deformed wing virus (DWV) are regarded as the key biotic threats to Apis mellifera colony health. In contrast, Apis cerana colonies are strongly resistant to them. Currently, however, the molecular mechanisms of the disease resistance of different honeybee species are not well understood. Non-coding RNAs have been proved to be generally involved in biological processes such as the disease resistance, while related researches in honeybees are still in the infancy. Based on the preliminary results of phenotypic differences of two honeybee species after V. destructor infestation and of the rapid proliferation of DWV in honeybee prepupae, we will quantify DWV titers with and without mite mediation using established V. destructor and DWV artificial infection and qPCR. We will also identify, screen and compare the key non-coding RNAs in different honeybee species to show the differential expression patterns. Molecular network of lncRNA-miRNA-mRNA will be constructed to analyze the regulatory mechanisms. The selected key non-coding RNAs will finally be validated by artificial larva feeding and microinjection. In this project, we will reveal the different resistance mechanisms between A. mellifera and A. cerana to the synergistic infections of V. destructor and DWV from a new perspective of RNA-mediated genetic information network. This research is of importance on providing theoretical basis for disease control of honeybees.

狄斯瓦螨与残翅病毒（DWV）协同侵染对西方蜜蜂健康危害极为严重，而东方蜜蜂则表现出较强抗性，当前对不同蜂种间差异性抗病分子机制，尤其是非编码RNA（ncRNA）的调控作用认识尚显不足。在前期试验发现不同蜂种被侵染个体发育的显著表型差异和DWV在蜜蜂预蛹期迅速扩增现象的研究基础上，本项目拟通过对蜜蜂预蛹接种狄斯瓦螨与DWV，采用qPCR定量分析比较DWV在有、无狄斯瓦螨介导下对不同蜂种蜜蜂预蛹的侵害程度，借助RNA-seq及生物信息学分析手段，鉴定、筛选并对比同一蜂种不同处理及不同蜂种相同处理间差异表达的关键ncRNA，探明ncRNA在不同蜂种和不同处理间差异表达模式，构建lncRNA-miRNA-mRNA互作分子调控网络以分析其机制，并加以验证。本项目将从RNA介导遗传信息表达网络的角度在分子水平上探究不同抗性蜂种对狄斯瓦螨和DWV协同侵染的抵御机制，继而为蜜蜂疾病的有效防控提供理论依据。

狄斯瓦螨是全球西方蜜蜂健康的最大生物威胁，其不仅对宿主个体发育和行为造成直接影响，更为重要的是以携播病毒等方式间接作用于蜂群，并可抑制蜜蜂的免疫系统从而引发病毒性疾病的爆发。本项目围绕狄斯瓦螨及其所携播的病毒与不同抗病力宿主（东方蜜蜂和西方蜜蜂）间的相互作用关系，从多角度开展研究，主要取得以下成果：1）蜜蜂体内的内源性病毒对宿主危害有限，而狄斯瓦螨所携播的外源性以色列急性麻痹病毒和残翅病毒分别是封盖后幼虫/蛹死于大幼虫期或蛹期的关键致病因子，且幼虫化蛹阶段（预蛹期）很可能是残翅病毒的快速增殖期；2）群体抗病力更强的东方蜜蜂个体表现出更低的病毒耐受性和更高的死亡率，脆弱个体孕育强壮群体的观念对于蜜蜂抗病育种具有重要启示；3）首次鉴定分析了狄斯瓦螨的长链非编码RNA和环状RNA，加深了对其基础分子生物学的认知，有助于建立特征性靶标从而开发出高效防控方法；4）狄斯瓦螨在其原始宿主东方蜜蜂群内呈现出丰富的遗传多样性，而新宿主西方蜜蜂群内仅存在一种单倍型，且不同蜂种来源的狄斯瓦螨繁殖能力差异巨大，导致螨群体间的基因单向流动和基因渗入现象，这可能对原宿主群构成新的潜在威胁；5）从宿主和病毒两方面证实，宿主被寄生后的生理反应及狄斯瓦螨所携带的残翅病毒载量均会影响螨的繁殖。项目以狄斯瓦螨、残翅病毒、蜜蜂作为研究载体，不仅为寄生虫–病毒–宿主间互作关系提供了新见解，且对于养蜂生产中开展蜜蜂抗病育种和疾病防控工作具有指导意义。