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

我们的项目将集中研究蜜蜂、瓦螨和病毒之间相互作用的基本机制。我们提出了以下假设，将在本项目中进行检验。的h。瓦螨的引入导致了与dwv相关的新毒株的出现，这些毒株之所以被选中，是因为它们能够通过瓦螨传播，并且对蜜蜂具有高度致病性。2。Varroa抑制蜜蜂的抗病毒防御，如先天免疫和/或RNAi反应的信号通路，这些信号通路对DWV/VDV1具有活性，从而导致病毒复制和病理增加。H-3。蜜蜂基因型是抗病毒反应差异的关键决定因素，在dwv相关病毒的复制水平和畸形翅膀疾病的发展中。工作计划基因相关的无瓦螨蜂群将从英国无瓦螨地区（天空岛和/或科隆萨）采购，并将被瓦螨感染。这些被瓦螨侵染和未被瓦螨侵染的蜜蜂将为研究蜜蜂、瓦螨及其病毒（特别是DWV和VDV-1）之间的分子相互作用以及蜜蜂基因型对病毒和瓦螨相互作用的影响提供一个模型系统。我们将使用高通量illumina测序分析用于实验侵染的螨虫、被瓦螨侵染的蜜蜂群体和未被瓦螨侵染的蜜蜂群体的病毒多样性。我们将使用qRT-PCR在单个蜂蛹和相关瓦螨中检测和量化鉴定的病毒株（DWV、VDV-1或重组），包括它们的负链RNA复制中间体。我们将使用全基因组微阵列转录分析来鉴定蜜蜂途径和基因（特别是涉及抗病毒防御的信号通路），这些途径和基因在暴露于瓦罗亚的蛹中与未暴露瓦罗亚的蛹中表达差异，以确定瓦罗亚是否对先天免疫有影响。所选基因的水平将通过qRT-PCR在大量个体蜂蛹中量化。我们将比较病毒衍生sirna的水平，并使用“Illumina”高通量测序在无瓦螨和感染瓦螨的蜂群以及瓦螨中分析病毒特异性sirna的多样性。将使用基于qpcr的检测方法对大量单个蜜蜂蛹中病毒特异性sirna进行定量分析。我们将对本研究中使用的每只蜜蜂（蛹）进行基因分型（为此将测试病毒多样性、基因表达和siRNA研究）。将进行多变量分析，以确定：(A)瓦罗亚的存在是否会加强与高病毒水平和病理相关的DWV相关病毒（DWV、VDV-1或DWV-VDV-1重组病毒）的传播和复制；(b) RNAi反应是否控制蜜蜂体内的病毒（dwv样），以及Varroa是否损害蜜蜂抗病毒RNAi反应；(c)是否有抗病毒信号通路参与对DWV的应答，以及Varroa是否影响这种抗病毒应答；(d)个体蜜蜂的基因型、抗病毒反应和对瓦螨的易感性之间是否存在联系。我们将使用RNAi（卵子显微注射实验）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，以开发不同基因型蜜蜂、瓦螨和病毒之间相互作用的模型。我们的研究结果将有助于开发蜜蜂群体对变形翅膀疾病反应的预测模型，以及对其他病毒病原体反应的更一般模型，将有助于改善蜜蜂健康的监测和诊断，并将为合理培育抗病蜜蜂提供信息。

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.