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

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.

To investigate the molecular mechanisms of honeybee antiviral defences, to determine the influence of Varroa on these processes and virus diversity, and to identify honeybee genetic markers associated with virus resistance we set up following objectives: O.1. To assess the effect of Varroa mites on virus diversity (DWV, VDV-1 and recombinants) and virus load in the honeybees. O.2. To analyse the influence of Varroa on the honeybee innate immunity (antiviral response), including signalling pathways and RNA interference. O.3. To dissect the influence of genotype variation in bees on the resistance to the immunosuppressive activity of Varroa and the generation of viral genetic diversity. Work summary - Varroa-free honeybee colonies sourced from Colonsay or Skye will be infested Varroa. These Varroa-infested and genetically related Varroa-free control honeybees will be used to study honeybee-Varroa-DWV interactions. - Virus diversity in honeybees and Varroa mites will be analysed using high-throughput Illumina sequencing. qRT-PCR tests for identification of individual components (in particular DWV, VDV-1 and recombinants thereof) will be devised. - Varroa-infested and -free honeybees will be tested for the presence of DWV-specific siRNAs. The diversity of siRNAs will be analysed by Illumina sequencing. - Microarray transcriptional profiling will be used to identify genes/signalling pathways involved in antivirus defence which are suppressed by Varroa. - The functions of the genes implicated in antivirus defence in the honeybees will be tested in RNAi experiments (egg injection with dsRNA). - Honeybee pupae from Varroa-infested and -free colonies will be subjected to multivariate analyses; individual pupae will be tested for the levels of viruses, expression of antivirus-defence genes, virus-specific siRNAs and will be genotyped. The results will be used to develop models of the interaction between honeybees of different genotypes, Varroa and viruses.

The project will focus on of the fundamental mechanisms underlying interaction between Varroa, honeybees and viruses. We anticipate that the project outcomes will include: - Sequences of pathogenic virus strains, in particular pathogenic strains of DWV-like viruses; - Mechanisms of antivirus response in honeybees; - Effect of Varroa on honeybee antivirus responses; - Genetic markers associated with higher resistance to viruses and Varroa infestation. This information will be essential for the development of novel approaches, which could reduce negative impact of Varroa an associated viruses and even reverse the honeybee decline. Therefore this project will have significant impact on the UK economy and wellbeing. Potential beneficiaries of this project include: Academic research community, beekeeping industry / community, as well as Food and Environment Research Agency (FERA) and government departments (DEFRA) and agricultural departments of the devolved UK governments). Academic Researchers in the fields of molecular virology, innate immunity and insect-pathogen interaction will benefit from the data on the role of recombination in emergence of novel strains of DWV; involvement of RNAi interference and innate immunity signalling pathways on antivirus immunity in honeybees; effect of Varroa on antivirus responses in honeybees; genetic markers associated with antivirus resistance in honeybees. Government agencies (FERA) and beekeeping industry / community will benefit from the data on the identity of the pathogenic virus strains and honeybee genetic markers associated with higher resistance to Varroa and viruses and the predictive models of honeybee response to viruses and Varroa. The novel sequence data can be used to develop diagnostic techniques for disease monitoring, which will inform the application of hygienic interventions for disease control. Beekeeping industry / community will also benefit from the data on genetic markers associated with higher resistance to viruses and Varroa infestation, which will be valuable for genetically-informed (artificial insemination) breeding programmes to create honeybees with higher resistance to these and related pathogens. Government departments will benefit from the predictive models of honeybee response to viruses and Varroa. These models will allow to make informed decisions on measures to control Varroa and viruses (i.e. application of restriction of honeybee and honeybee products movement to restrict spread of Varroa and pathogenic virus strains, regulation of monitoring and application of hygienic interventions). Sharing the project outcomes Sharing of the outcomes is an essential part of this project. To ensure that potential beneficiaries will be informed about the project outcomes and will have access to the project data we will use several specific ways of data sharing to target different categories of beneficiaries. Beekeeping industry / community will be informed done though the open access Project Website where summaries of the research outcomes will be posted during the course of the project. The website will be maintained after the completion of this project. The URL address of the Project Website and its pages dedicated to bee-keepers will be publicised in web site of The British Beekeepers Association (BBKA), and in the article in 'BBKA News'. We will also make presentations to local beekeepers associations to raise profile of research in honeybee health. Academic researchers, FERA, Govermnemt departments will have immediate access the data generated in the course of the project. The nucleic acid data and microarray gene expression data which will be accessible through the Project Website and the public databases (GenBank and GEO database, NCBI). The program codes will be available though the Project Website. Project outcomes will be released through conference presentations and publications in peer-reviewed journals.