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

Lead Research Organisation: University of Warwick
Department Name: Warwick HRI

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description Identified a single virulent strain of DWV, the major viral pathogen of honeybees.
Defined how transmission by Varroa contributes to virulence.
Developed in vitro methods to study and recapitulate the Varroa/pupae/virus interaction.
Developed in vitro methods to analyse the virus further.
Exploitation Route Exploit the methods we have developed to design and test therapies for honeybee viruses.
Sectors Agriculture, Food and Drink,Environment
 
Description DWV biology and pathogenesis
Amount £610,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 11/2014 
End 10/2017
 
Description EASTbio PhD. studentship
Amount £75,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2016 
End 09/2020
 
Description Follow on Fund
Amount £165,588 (GBP)
Funding ID BB/M013685/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2015 
End 04/2017
 
Title DWV by needle 
Description Recapitulated the change in DWV virus population (level and diversity) by in vitro transmission using direct inoculation. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact Makes the study of virus therapies possible in the absence of Varroa. Allows tests of the time course of virus replication and virulence to be readily conducted in the incubator. 
 
Title MosaicSolver 
Description Software for analysis of next generation sequence datasets 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Publication Graham R. Wood, Eugene V. Ryabov, Jessica M. Fannon, Jonathan D. Moore, David J. Evans and Nigel Burroughs (2014) MosaicSolver: a tool for determining recombinants of viral genomes from pileup data. Nucleic Acids Research doi: 10.1093/nar/gku524 
 
Title Bee array data 
Description Microarray dataset of gene expression in four stratified groups of honeybee pupae - with four individual samples, sampled individually (!), in each stratified group. Control, no varroa, varroa-associated and low virus, varroa-associated and high virus 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Publication in PLoS Pathogens - Ryabov et al., 2014 
URL http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1285/
 
Title Bee microarray probes 
Description 60-mer oligonucleotidide array based on Apis mellifera transcriptome (OGS1) and Apis mellifera fungal and viral pathogens (Agilent ID: 027104, SurePrint G3 Custom GE 8x60K). 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Publication in PLoS Pathogens - Ryabov et al., 2014 
URL http://www.ebi.ac.uk/arrayexpress/arrays/A-MEXP-2251/
 
Description Bowman 
Organisation University of Aberdeen
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Collaborative studies on coordinated control of Varroa - this was our idea and implementation
Collaborator Contribution Expertise in Varroa and varroaicides
Impact Pending article in Scottish Beekeeper magazine for October 2015. Not listed elsewhere as not yet published.
Start Year 2014
 
Description Highet 
Organisation Scottish Government
Department SASA (Science and Support for Scottish Agriculture)
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Coordinated Varroa treatment study in Arran - we initiated the study and the implementation.
Collaborator Contribution SASA provide contacts in the beekeeping community and with bee inspectors.
Impact Joint article for October 2015 Scottish Beekeeper.
Start Year 2014
 
Title Strain of Deformed wing virus (DWV) 
Description Unique sequence of a highly virulent strain of deformed wing virus 
IP Reference 1306812.7 
Protection Patent application published
Year Protection Granted
Licensed Commercial In Confidence
Impact The identification of this virus forms the basis for both the analysis of the molecular basis of pathogenesis and the is the target for novel therapies designed to improve the health of honeybee colonies.
 
Title MosaicSolver 
Description Next generation sequence analysis software 
Type Of Technology Software 
Year Produced 2014 
Impact Publication : Graham R. Wood, Eugene V. Ryabov, Jessica M. Fannon, Jonathan D. Moore, David J. Evans and Nigel Burroughs (2014) MosaicSolver: a tool for determining recombinants of viral genomes from pileup data. Nucleic Acids Research doi: 10.1093/nar/gku524 
URL http://nar.oxfordjournals.org/content/early/2014/08/12/nar.gku524.full.pdf+html
 
Title NGS diversity 
Description Development of a method - implemented in Excel and Matlab - to analyse genetic diversity in next generation sequencing datasets 
Type Of Technology Software 
Year Produced 2014 
Impact Publication in PeerJ ... independently listed. 
URL https://peerj.com/articles/645/
 
Description TV and radio 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview for South Korean TV on honey bee viruses
Interview for Spanish TV/video media about honey bee viruses

Both were documentaries on honey bee viruses - interview length was 3-5 hours each.
Year(s) Of Engagement Activity 2016,2017
URL http://one.lavanguardia.com
 
Description Talks - many 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Talks
Evening and weekend talks to beekeeping association in England, Scotland and Wales - total number of talks over 2013-2017 is at least 30. Audiences range from ~20 to ~200. Total reached well in excess of 500.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
 
Description Talks at National/International beekeeping events 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Talk sparked questions afterwards and resulted in further invitations to talk, newsletter articles and collaborations

Collaborations, newsletter articles
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Talks to beekeeping associations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Very extensive Q&A from interested groups

Sponsorship was provided, though this wasn't the primary reason for the presentation.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description Talks to commercial beekeepers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk resulted in extensive Q&A and supplementary invitations, article in newsletters etc.

Beefarmers agreed to provide letters of support for further research
Year(s) Of Engagement Activity 2013,2014,2015