The potential of gene-knockdown for controlling varroa mites

Lead Research Organisation: University of Aberdeen
Department Name: Inst of Biological and Environmental Sci

Abstract

Honey bees are incredibly important to humans through their pollination of flowers of both wild and farmed plants. It is estimated that very third mouthful of food we eat is reliant to some extent on honey bees. When there was an accidental overuse of insecticides in an area of China that killed all the bees and other pollinating insects, the apple trees had to be hand pollinated that year. Taking into account higher wages in the UK, such a scenario here would make apples an absolute luxury item. Unfortunately, honey bee numbers are seriously declining year on year with areas in Europe and the USA suffering bee losses over 30% every year – this is not sustainable. The exact reasons for honey bee declines are not known for certain, but are likely due to several factors. One of the most important factors is the parasitic mite Varroa destructor that sucks the blood from bees and transmits serious viral diseases.

Control of Varroa using insecticides is becoming less effective as the Varroa have become resistant to the handful of available insecticides. Developing insecticides that kill Varroa but leave the bee unharmed is very difficult as they are relatively closely related to each other, unlike the scenarios of developing pesticides to kill fleas on cats or tapeworms in pigs. There is an urgent need to develop a Varroa-specific, environmentally friendly insecticide or some method of overcoming the Varroa’s resistance mechanism to the registered insecticide.

We have recently developed a method to knockdown individual and specific genes in the Varroa that will not affect honey bees or, indeed any other animals. In this project we aim to construct pieces of double-stranded RNA (dsRNA) that effectively kill the mite, but do not affect the bees. By scouring large databases of all the Varroa genes will identify pieces of genes that can be effectively and safely targeted. These dsRNA constructs will be fed to larval and adult bees to assess their effectess as a proof-of-concept before embarking on hive treatments. Additionally, we will identify the genes involved in resistance to a specific insecticide and investigate whether resistant mites become susceptible to the insecticide when we have knocked down these genes

Technical Summary

Honey bee numbers are declining to an unsustainable degree threatening global food security if not rectified. Though the cause of the bee crisis is multifactorial, it is accepted that the ectoparasitic mite Varroa destructor, plays a central role largely through its interaction with serious bee viruses. Developing new acaricides that target Varroa but are harmless to honey bees is difficult due to the relative close relatedness of these two arthropods. One new approach would be gene-knockdown by double -stranded RNA-interference. This approach would be Varroa-specific and benign to the honey bee.

We have demonstrated dsRNAi in Varroa by a method amenable to relatively high throughput screening of dsRNA constructs. Using Varroa transcriptome databases and a preliminary genome we will identify genes expected to be lethal to Varroa but benign to bees based on sequence divergence. Such dsRNA constructs will be tested for gene knockdown by qRT-PCR and mite mortality. Using a reiterative screening process dsRNA constructs with high lethality will be selected. Taking the lead construct, we will test different administration routes of the dsRNA to larval and adult bees and assess subsequent gene knockdown and mortality in mites. Concentrations of longer dsRNA pieces and shorter interfering RNAs will be assessed in bee and mite tissues over time by qRT-PCR. Additionally, we will use Illumina HiSeq2000 paired-end sequencing of fluvalinate-susceptible and resistant mite populations to identify genes potentially involved in fluvalinate resistance. If these are detoxifying genes (cytochrome P450 monooxygenases, esterases and glutathione transferases) we will knock down the specific gene(s) in an attempt to resensitize such mites to fluvalinate.

Planned Impact

Honey bee offer critical pollination services to crop production and the natural fauna across the globe. So whilst being rather glib, it is true to state that our research has the potential of benefitting all humankind. The major impact would be to improve the control of Varroa, but during the research process towards that goal we would be gaining insight of dsRNAi gene knockdown that could be used in other spheres of pest control.

Beneficiaries in Commercial Sector: Though the work is fundamental it is highly applied. The proposed work is 50% funded by a commercial partner who has plans for exploiting the expected deliverables. Should control of Varroa be achieved by our approach, this would have a knock-on benefit to all those industries reliant on honey bees (pollination services: soft fruit and top fruit growers; commercial honey producers). We would expect that our approach may be taken up by parties interested in controlling other pests by gene-knockdown. Further, our approach of gene-knockdown to establish exactly which detoxifying genes in insecticide resistance is likely to be of interest to the general insecticide / pesticide industry.

Beneficiaries in Policy Making: It is still unclear what the regulatory issues are in the UK and Europe regarding taking dsRNA from the lab into the field. We know this has been addressed by the Environmental Protection Agency (EPA) in the USA where in silico proof of the absence of 20bp stretches in the honey bee genome is required, but this needs to be addressed in the UK and Europe. This project, together with the industrial partner, allows such policy making to be advanced.

Benefits in the Wider Public: The general public have a great affinity for honey bees and there is widespread awareness and concern about the plight of the honey bee among the wider public. General interest in our work on gene knockdown in Varroa has been demonstrated by the very many pieces that have been broadcast and published internationally (television, radio, broadsheet & tabloid newspapers and websites). The project would show this concern is being clearly addressed. Hobbyist beekeepers both individually and via their local Beekeepers Associations (registered charities) are keenly interested in our work as demonstrated by the great number of contacts that have been made to us.

Publications

10 25 50
 
Description Several potential gene targets for controlling Varroa by gene-knockdown were identified and tested. We demonstrated that neural targets were amenable to gene knockdown which may well constitute the most lethal targets. The three best candidate targets were further tested singly, in combination and when administered as a single entity and proved successful in lab and small glasshouse trials.
Exploitation Route Together with an industrial partner we are pursuing the development of our findings as a potential commercial varroa control agent via applications to the EU's SME Instrument funding programme. To this end, we have filed patent protection on our findings.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Our findings have been used to file two patents on the use of gene-knockdown. The first patent application involves the use of gene-knockdown on controlling varroa. Together with our industrial partner, we aim to build on our findings to develop a practical and commercial application for gene-knockdown in varroa control. We have discussed the necessary information needed to register such a "medicine" with the European Medicines Agency and have applied or EU funding to do so. The second application involves the broader use of some of our findings that offer improved efficacy of gene-knockdown and can be used in both pest control, but also medical and veterinary healthcare.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Scottish Government Bee Health Strategy
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact Dr Ewan Campbell was invited to sit on the Science and Advice for Scottish Agriculture (SASA) and the Scottish Beekeepers Association's consultation meetings to formulate the Scottish Government's Bee Health Strategy.
 
Description CASE partner to cat flea studentship (BB/L502467/1)
Amount £33,400 (GBP)
Funding ID CASE partner contribution to BB/L502467/1 
Organisation Zoetis 
Sector Private
Country United States
Start 02/2014 
End 02/2014
 
Description DEFRA - Veterinary Medicines Directive
Amount £120,000 (GBP)
Organisation Department For Environment, Food And Rural Affairs (DEFRA) 
Sector Public
Country United Kingdom
Start 04/2014 
End 10/2018
 
Description EMC Scottish Crucible Nanoparticles
Amount £3,800 (GBP)
Organisation Royal Society of Edinburgh (RSE) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2014 
End 08/2015
 
Description EMC Scottish Food and Drink Federation
Amount £2,991 (GBP)
Organisation Scottish Food and Drink Federation 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2014 
End 08/2015
 
Description EU-FP7 KBBE
Amount € 314,627 (EUR)
Funding ID SMARTBEES, Grant Number 613960 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 11/2014 
End 10/2018
 
Description KTN Biosciences BBSRC CASE (cat flea)
Amount £93,520 (GBP)
Funding ID BB/L502467/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2014 
End 02/2018
 
Description KTN Biosciences BBSRC CASE (varroa artificial culture)
Amount £96,000 (GBP)
Organisation ICT KTN Scholarship 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 06/2019
 
Description Scottish Beekeepers Association to be CASE partner to KTN Biosciences BBSRC CASE studentship
Amount £16,000 (GBP)
Organisation Scottish Bee Keepers Association 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 06/2019
 
Description Sparking Impact Award
Amount £14,988 (GBP)
Organisation University of Aberdeen 
Sector Academic/University
Country United Kingdom
Start 12/2014 
End 03/2015
 
Description National Bee Unit - varroa dsRNAi 
Organisation Fera Science Limited
Country United Kingdom 
Sector Public 
PI Contribution Our lab brings expertise on mites and ticks to the collaboration working on the honey bee mite Varroa destructor. Additionally, we bring molecular expertise to the collaboration.
Collaborator Contribution The National Bee Unit contribute expertise on honey bees and general apiculture to the collaboration. Also, the NBU has access to biosecure glasshouses in which some joint experiments can be conducted.
Impact Several papers and patents have arisen from the collaboration. Additionally, our collaboration has resulted in a >£500k project on artificially rearing varroa funded by DEFRA / Veterinary Medicine Directive.
Start Year 2009
 
Description Vita and BBSRC-LINK 
Organisation Vita (Europe) Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have developed the concept of using gene-knockdown in the control of varroa.
Collaborator Contribution Financial contribution.
Impact Patents.
Start Year 2012
 
Title Control of Varroa mIte Infestation 
Description Abstract: Nucleic acid agents for reducing or removing infestations of the Varroa destructor mite are described. Compositions comprising the nucleic acid agents and methods for controlling mite infestations using the nucleic acid agents and compositions are also disclosed. 
IP Reference WO2015001336 
Protection Patent application published
Year Protection Granted 2015
Licensed Commercial In Confidence
Impact This filed patent on using RNAi for controlling Varroa has formed the basis of an application to the EU under the SME Instrument programme to further develop the technology for future commercialisation by field-testing and gathering data for a regulatory registration dossier. This application is led by an industrial partner. Further work in this general area has led to filing of one further patent application to protect the IP (GB1500107.6 Enhanced RNAi mediated gene regulation", and subsequent PCT filing Jan 2016).
 
Description Media interest 
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 Media (as a channel to the public)
Results and Impact Bee health is of great interest to many people. I have given many newspaper, magazine, radio and television interviews on our research per se, but also on general bee health issues. These engagements with media have been international (e.g. BBC World Service, German Public Radio, Radio New Zealand, CNN World News, Aljazeerah World News) as well as local. It is difficult to guage the impact, but I received numerous emails from all over the world in days following interviews supporting our work, asking for clarification, and also offers of help.

see above
Year(s) Of Engagement Activity 2011,2012,2013,2014
 
Description School visits with bee observation hive 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We visit local schools (primary and secondary) with a portable observation hive (i.e. one with glass sides so all bee activity inside is visible) and discuss the role of bees in food production and bee biology. The visits sparked much enthusiasm among the pupils and a fascination over how vital insect pollinators are to their everyday lives and meals.

One primary school was so impressed with the bees that together we put a proposal together to secure funding and purchased a beehive, bee suits and ancillary equipment. The beehive is located at a local farm and the pupils visit several times a year together with the PDRA on this project. They are hoping to actually harvest some honey in 2015!
Year(s) Of Engagement Activity 2013,2014
 
Description Talking to local bee keepers associations across the country about varroa and modern approaches to control. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Over 50 invited/requested talks were given to beekeeper associations in Scotland, England and Wales to >2000 people. The talks were presented by both the PI and PDRA. These talks sparked questions and debates over modern approaches to varroa control.

In discussing the bee health crisis and how "research works" with beekeepers (i.e. stakeholders), I would discuss funding routes for research. Through this several local associations have investigated how to get involved in bee health research. I now have two BBSRC-CASE studentships where the CASE partners are groups of beekeeper associations with the required contribution. Many of the beekeepers association have now realized that researchers are more approachable and the associations can get involved in bee research.
Year(s) Of Engagement Activity 2012,2013,2014