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.
Organisations
- University of Warwick (Lead Research Organisation)
- Scottish Government (Co-funder)
- Department for Environment Food and Rural Affairs (Co-funder)
- Natural Environment Research Council (Co-funder)
- Wellcome Trust (Co-funder)
- UNIVERSITY OF ABERDEEN (Collaboration)
- Science and Advice for Scottish Agriculture (Collaboration)
Publications
Moore J
(2011)
Recombinants between Deformed wing virus and Varroa destructor virus-1 may prevail in Varroa destructor-infested honeybee colonies.
in The Journal of general virology
Vanbergen A
(2013)
Threats to an ecosystem service: pressures on pollinators
in Frontiers in Ecology and the Environment
Wood GR
(2014)
MosaicSolver: a tool for determining recombinants of viral genomes from pileup data.
in Nucleic acids research
Wood GR
(2014)
Error correction and diversity analysis of population mixtures determined by NGS.
in PeerJ
Paxton R
(2015)
How are pests and diseases affecting bee pollinators?
Gusachenko ON
(2020)
Evidence for and against deformed wing virus spillover from honey bees to bumble bees: a reverse genetic analysis.
in Scientific reports
Gusachenko O
(2020)
Green Bees: Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism
in Viruses
Gusachenko ON
(2021)
First come, first served: superinfection exclusion in Deformed wing virus is dependent upon sequence identity and not the order of virus acquisition.
in The ISME journal
Description | We studied the virus population in honey bees. We demonstrated that the most important pathogenic virus of honey bees (deformed wing virus; DWV) essentially exists at either low levels and high population diversity, or high levels and very low population diversity. We further demonstrated that it is the route of transmission of the virus that defines which of these two infection outcomes is realised. If transmitted orally, through feeding, it is essentially harmless, only growing to low levels. In contrast, if transmitted by the Varroa mite, it rapidly grows to pathogenic levels and causes overt disease. We further demonstrated that we could recapitulate this infection outcome by simply injecting virus using a syringe. Using this approach we demonstrated the same changes in virus levels and diversity. This formally confirms that it is the transmission method NOT the virus per se that defines the outcome of infection/exposure. To achieve these results we developed a number of novel approaches. The most important of these was the introduction of unique genetic tags to the genetic material of the virus. This was necessary to discriminate the input virus from endogenous virus that is present in every honey bee. We produced numerous different virus variants containing different genetic tags enabling is to conduct virus competition studies. The formal term for this type of experimental system is a "reverse genetic system" which allows the recovery of virus from biologically cloned cDNA. It is a key approach to facilitate further studies of the biology of the virus. We additionally studied the response of the honey bee to high or low levels of DWV. We did this focussing on two specific aspects of the bee - the changes in host gene expression and the immune response of the bee. Whilst these studies are largely descriptive - we simply recorded what we observed - they will be useful in future understanding of virus pathogenesis and (potentially) control. |
Exploitation Route | The reverse genetic system will be useful to understand the structure/function of the virus genome and to further investigate the complicated relationship between different strains of the virus that may compete during coinfections. We have conducted some of these studies in BBSRC-funded follow-on work. Our understanding of the fundamental role of Varroa on the levels of the virus reinforces the importance of Varroa mite control for beekeepers. In the absence of this control virus levels have the potential in individual bees to increase by 5 orders of magnitude. Our analysis of the immune response of the bee to the virus may be useful in the development of novel antiviral strategies to control this virus. However, they also hint that such control may be rather difficult to achieve. |
Sectors | Agriculture Food and Drink Environment |
Description | Our understanding of the virus population in honey bees and the Varroa mite has significantly influenced our development of advice to beekeepers (commercial and amateur) on how to best manage colony health. These have been disseminated in numerous talks to beekeepers at annual or regional conventions and in evening talks. They are also contained on my beekeeping website - see https://theapiarist.org/when-to-treat/ this webpage has been accessed over 28,000 times since I wrote it and still forms the basis for regular talks to international and national beekeeping association. |
First Year Of Impact | 2015 |
Sector | Agriculture, Food and Drink,Environment |
Impact Types | Societal Economic |
Description | DWV biology and pathogenesis |
Amount | £610,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
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 |
Start | 09/2016 |
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 |
Start | 09/2015 |
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 |
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 | Science and Advice for Scottish Agriculture |
Country | United Kingdom |
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 | GB1306812.7 |
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 | Multiple articles on a website for beekeepers |
Form Of Engagement Activity | A magazine, newsletter or online publication |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | I maintain a website for beekeeping which regularly discusses honey bee health. In particular there is a focus on the rational combination of science and practical beekeeping to improve honey bee health. The website was started in late 2014 and involves about 50 posts per year. 2017 access statistics are ~145,000 'real' page views (i.e. not robots or search engines) from ~75,000 visitors spread across ~120 countries. 2018 statistics show about 4,000 visitors per week visit the site. ~66% of site visits are from the UK and USA. My writing results in many invitations to speak at national beekeeping events. Access statistics show a significant increase from 2014 (~12,000), 2015 (~38,000), 2016 (~71,000) to 2017 (~145,000). Several beekeeping associations reproduce the content attributed to thrown members. |
Year(s) Of Engagement Activity | 2014,2015,2016,2017,2018 |
URL | http://www.theapiarist.org |
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 - evening talks and weekend National conventions |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Numerous talks to beekeeping associations - Perth and District, Nairn, Peebles, Edinburgh and Midlothian, South Devon Beekeepers, Welsh Beekeeping Association and others - either in evenings or weekends. The purpose of the talks is to mix science and practical beekeeping to improve honey bee colony health. Evening talks might be attended by 30-50 people, weekend talks (like the Welsh beekeepers convention) attract an audience in excess of 300. Most of the talks are associated with extensive Q&A sessions and follow up correspondence. In 2020/21 over 30 talks were delivered online (during lockdown) to a combined total audience of well over 3000. Many beekeeping associations reported larger audiences to my talks than to any others they had run during the winter. Several talks involved EventBrite booking and audience numbers were capped. Feedback - written and verbal - was excellent and many attendees reported planning or actual changes in policy and practice. |
Year(s) Of Engagement Activity | 2017,2018,2019,2020,2021 |
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 |
Description | The Apiarist website |
Form Of Engagement Activity | Engagement focused website, blog or social media channel |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | This website is a science-rich site for beekeepers. It carries weekly posts of relevance to practical beekeeping. In 2018 access statistics showed approaching 4000 pages a week were being read, over 200,000 a year by over 100,000 visitors. Visitors were from over 50 countries. Some posts generated comments from visitors, with ~500 comments/discussions over the 2018. Readership (visitor numbers - not bots etc) has been increasing year on year, 17,000 in 2015, 34,000 in 2016, 75,000 in 2017 and 102,000 in 2018. 2019 total page views = >235,000 by >107,000 visitors 2020 total page views = >367,000 by >180,000 visitors 2021 page views currently up by 40% on 2020 weekly/monthly numbers The website generates numerous invitations to speak at beekeeping associations and conventions. Many of these talks are on the science underlying practical beekeeping. Many audiences report changes in their beekeeping and improvements in bee health as a result of the information in my talks and on the website. Several articles and posts have been reproduced on beekeeping association sites around the world and in printed beekeeping magazines. A specific article on CBPV was written in May 2020 and has received >1200 unique views from beekeepers around the world since then. |
Year(s) Of Engagement Activity | 2014,2015,2016,2017,2018,2019,2020,2021 |
URL | https://www.theapiarist.org |