The potential of gene-knockdown for controlling varroa mites

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 effectiveness 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

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.

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.