Unravelling the impact of the mite Varroa destructor on the interaction between the honeybee and its viruses

我们的项目将专注于蜜蜂、Varroa和病毒之间相互作用的基本机制。我们提出了以下假设，这些假设将在本项目中得到验证。H-1。Varroa的引入导致了与DWV相关的新病毒株的出现，这些病毒因其能够由Varroa传播而被选中，并对蜜蜂具有高度致病性。H-2.Varroa抑制蜜蜂的抗病毒防御，如天然免疫和/或RNAi反应的信号通路，这些防御通路对DWV/VDV1是活跃的，从而导致病毒复制和病理的增加。H-3。蜜蜂的基因型是在抗病毒反应、DWV相关病毒复制水平和畸形翅膀疾病发展方面存在差异的关键决定因素。工作计划与瓦罗阿无关的蜂群将来自英国无瓦罗阿的地区(天空岛和/或科隆塞)，并将受到瓦罗阿的侵扰。这些感染了Varroa的蜜蜂和不感染Varroa的对照蜜蜂将为研究蜜蜂、Varroa与其病毒(特别是DWV和VDV-1)之间的分子相互作用以及蜜蜂基因型对与病毒和Varroa相互作用的影响提供一个模型系统。我们将使用高通量的伊鲁米纳测序技术分析用于实验感染的尘螨、有瓦罗瓦的蜜蜂群体和对照的无瓦罗阿的蜂群中的病毒多样性。我们将使用qRT-PCR检测和量化已鉴定的病毒株(DWV、VDV-1或重组体)，包括它们的负链RNA复制中间产物，在单个蜂蛹和相关的瓦罗螨中。。我们将使用全基因组微阵列转录图谱来识别蜜蜂途径和基因(特别是参与抗病毒防御的信号通路)，这些途径和基因在暴露于Varroa的蛹中与未接触Varroa的蛹中表达的差异，以确定Varroa是否对先天免疫有影响。所选基因的水平将通过qRT-PCR在大量的个体蜂蛹中进行量化。我们将比较病毒来源的siRNAs水平，并使用‘Illumina’高通量测序技术分析来自无Varroa和Varroa感染的蜂群的蜜蜂以及Varroa螨类中病毒特异性siRNAs的多样性。将使用基于qPCR的检测方法对大量单个蜜蜂蛹中的病毒特异性siRNAs进行定量。我们将对本研究中使用的每一只蜜蜂(蛹)进行基因分型(将对病毒多样性、基因表达和siRNA研究进行测试)。将进行多变量分析以确定：(A)Varroa的存在是否加强了与高病毒水平和病理相关的DWV相关病毒(DWV、VDV-1或DWV-VDV-1重组体)的传播和复制；(B)RNAi反应是否控制蜜蜂中的病毒(DWV样)，以及Varroa是否影响蜜蜂的抗病毒RNAi反应；(C)是否有抗病毒信号通路参与对DWV的反应，以及Varroa是否影响这种抗病毒反应；以及(D)个体蜜蜂的基因型、抗病毒反应和对Varroa的敏感性之间是否有联系。我们将使用RNAi(鸡蛋显微注射实验)来测试候选基因的作用。这些结果将使用一系列数学方法进行分析，以建立不同基因类型的蜜蜂、Varroa和病毒之间相互作用的模型。我们的结果将有助于发展蜜蜂群体对变形翅病反应的预测模型和对其他病毒病原体反应的更一般模型，将有助于改进蜜蜂健康监测和诊断，并将为合理培育抗病原菌的蜜蜂提供信息。

Our project will focus on fundamental mechanisms underlying interactions between honeybees, Varroa and viruses. We proposed the following hypotheses, which will be tested in this project. H-1. Introduction of Varroa has resulted in the emergence of new strains of DWV-related viruses, which have been selected for their ability to be transmitted by Varroa and are highly pathogenic to the honeybees. H-2.Varroa suppresses honeybee antiviral defences, such as the signalling pathways of innate immunity and/or RNAi responses, which are active against DWV/VDV1, thus leading to increased virus replication and pathology. H-3. Honeybee genotype is a key determinant of differences in the antiviral response, in the levels of replication of DWV-related viruses, and in deformed wing disease development. WORKPLAN The genetically related Varroa-free honeybee colonies will be sourced form the Varroa-free regions of the UK (Isle of Sky and/or Colonsay) and will be infested with Varroa. These Varroa-infested and the control Varroa-free honeybees will provide a model systems for studying molecular interactions between honeybees, Varroa and their viruses (in particular DWV and VDV-1) and the effect of honeybee genotype on interaction with viruses and Varroa. We will analyse virus diversity using high-throughput Ilumina sequencing in the mites used for experimental infestation, Varroa-infested honeybee colonies, and the control Varroa-free colonies. We will detect and quantify the identified virus strains (DWV, VDV-1, or recombinants), including their negative-strand RNA replication intermediates, in individual bee pupae and associated Varroa mites using qRT-PCR. . We will use the whole-genome microarray transcriptional profiling to identify the honeybee pathways and genes (in particular signalling pathways involved in antvirus defence), which are differentially expressed in Varroa-exposed pupae compared to Varroa-free pupae in order to determine whether Varroa has an effect on innate immunity. The levels of the selected genes will be quantified in large number of individual bee pupae by qRT-PCR. We will compare the levels of virus-derived siRNAs and analyse diversity of the virus-specific siRNAs using 'Illumina' high-throughput sequencing in the honeybee form the Varroa-free and Varroa-infested colonies, as well as in the Varroa mites. Quantification of the virus-specific siRNAs in large number of individual honeybee pupae will be carried out using qPCR-based detection approach. We will genotype each individual honeybee (pupae) used in this study (for which virus diversity, gene expression and siRNA studies will be tested). A multivariate analyses will be carried out to identify: (a) Whether the presence of Varroa enhances the transmission and replication of DWV-related viruses (DWV, VDV-1 or DWV-VDV-1 recombinants) associated with high virus levels and pathologies; (b) If RNAi response controls viruses (DWV-like) in the honeybees and whether Varroa compromises honeybee antiviral RNAi response; (c) Whether of antivirus signalling pathways are involved in response to DWV, and whether Varroa affect this antiviral response; and (d) whether there is a connection between the genotype of individual honeybees, antivirus response and susceptibility to Varroa. We will test the roles of candidate genes using RNAi (egg microinjection experiments). These results, will be analysed using a range of mathematical approaches to develop the models of the interaction between honeybees of different genotypes, Varroa and viruses. Our results will allow the development of predictive models of honeybee colony response to deformed wing disease and more general models of the response to other viral pathogens, will contribute to improvements in the monitoring and diagnosis of honeybee health, and will inform the rational breeding of pathogen-resistant honeybees.