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

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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.