Elucidating the regulation of reproduction in Varroa mites: uncovering potential control strategies.

Insect pollination of crops is absolutely critical to food production with an estimated annual global value of $361 billion. Honey bees (Apis mellifera) are undergoing a serious bee health crisis threatening global food security. Huge annual losses of bee colonies in the US (45%, 34%, 40% in 2012/13, 2013/14 and 2015/16) are now being matched by similar losses in Europe. The status of bee health is recognised as critical with unsustainable losses of bees. Though the cause of the bee health crisis is multifactorial, it is generally accepted that the external parasite varroa mite (Varroa destructor) and its transmission of viral pathogens is one of, if not the, major cause of honey bee colony loss. Because of the impact on global food production, varroosis is arguably the most serious disease of livestock in any species.

Despite varroa's unquestionable significance, our understanding of its physiology could be considered rudimentary relative to its importance. Progress in varroa research is substantially hampered by the lack an artificial feeding and rearing system which would supply researchers with adequate numbers of consistent varroa year round and provide an ideal experimental system for studying varroa physiology, reproduction, disease transmission and the development and testing of new control strategies. We have recently developed an artificial feeding system for varroa that far exceeds any currently available. Subsequently, we succeeded in inducing varroa in the artificial feeding system to lay eggs by exposing them to the odour cues from either bee larvae or hormone-treated pupae, but not from standard pupae. These eggs hatched and developed through to adult varroa. We now need to capitalize on these initial findings and better understand the fundamentals of varroa reproduction to allow our artificial feeding system to fully evolve into an artificial rearing system.

To this end, the overall aim of the project is to understand the control of reproduction in varroa and assess if this information could be utilised as a control measure. By analysing the volatile compounds emanating from cohorts of larvae, pupae and juvenile hormone-treated pupae and applying a differential chemometrics approach, we will chemically characterize components(s) of the "Oviposition Kairomone" (OK). The effect of the larval volatiles or OK on induction of oviposition, the gender of the egg and cessation of egg laying in varroa will be studied by a combination of bioassay and chemical analysis. The role of juvenile hormone in the production of these bioactive volatiles or OK by bee larvae will be elucidated. We will investigate if the varroa detects this odour cue from the bee larvae with its legs and if this signal causes egg laying in varroa through induced sex hormone production (ecdysteroids) via a neuroendocrine factor. The optimum concentration of larval volatiles or OK required to induce varroa egg laying in the artificial rearing system will be determined by chemical analysis and bioassay and then a suitable method for delivery of that concentration range developed.

There are strains of bees in France, Sweden and Germany exhibiting a trait termed "Suppressed Mite Reproduction" by which varroa have reduced reproduction. By performing the oviposition bioassay and chemical analysis, we will test the hypothesis that altered levels of larval volatiles or OK are the basis for the reduced varroa reproduction on these bee strains. Finally, we will assess if larval volatiles or OK can induce varroa to lay eggs even when in an inappropriate scenario such as while on adult bees.

Annual losses of honey bee colonies are occurring to an unsustainable degree threatening global food security. Though the cause of the bee health crisis is multifactorial, it is accepted that the ectoparasitic mite Varroa destructor, plays a central role. Research into new strategies to control varroa are desperately needed.

Varroa research is significantly hampered by the lack of an artificial rearing system able to provide year-round and standardized varroa independent of the confounding variable of the bee larvae or pupae. We developed an artificial feeding system able to sustain varroa. The hurdle of being able to induce these varroa to lay eggs was recently overcome by exposing the varroa to volatile semiochemicals emanating from bee larvae or juvenile hormone-treated pupae, but not untreated pupae. The proposal aims to:

- characterize the bioactive component(s) in these volatiles;
- elucidate mechanism of the effect of these semiochemicals on varroa;
- optimize the delivery system of these semiochemicals to make a robust and reproducible artificial rearing system;
- elucidate the role these semiochemicals play in the trait of reduced reproduction of varroa on selected strains of bees in France, Sweden and Germany;
- determine if varroa exposed to these semiochemicals are forced into egg development and oviposition even when under unsuitable conditions such as on adult bees

An important tool in the work proposed is our oviposition bioassay. Approaches include GS-MS analysis of sampled headspace volatiles and chemometric analysis across cohorts of larvae, pupae and hormone-treated pupae. Induction of oogenesis and oviposition will be monitored by RT-qPCR of a suite of previously validated biomarker genes and the bioassay. The role of these semiochemcials on ecdysteroidogenesis in integumental tissue via a neuropeptide will be assessed by LC-MS/MS, enzyme immunoassay, RT-qPCR, gene knockdown of the neuropeptide, and short term culture.

Honey bees offer critical pollination services to crop production and natural flora across the globe. However, there is a serious health crisis threatening honey bees causing unsustainable colony losses every year. Though there are many causes of the bee health crisis, the ectoparasite Varroa destructor is recognised as being one of, if not the, major problem. Because of the vital importance of honey bees to food production and the seriousness of varroa, it is not an over statement that our research has the potential of benefitting all of humankind.

ACADEMIC BENEFICIARIES: Researchers on varroa will benefit from an artificial rearing system of varroa capable of maintaining and supplying varroa independent of requiring bees and being standardized by age, viral load and nutritional status. It will allow researchers to study the physiology of varroa in a more controlled manner than currently possible and perform investigations of the transmission of the vitally important deformed wing virus and other varroa-borne pathogens. To facilitate this knowledge exchange, we will run short hands-on courses. The semiochemical (odour cues) interaction between the immature bees and varroa will advance our understanding of the control of the induction and cessation of egg laying, egg number and gender of eggs. Elucidating the hormonal control of egg development and the neuroendocrine control of ecdysteroidogenesis will significantly advance our understanding in acari which lag behind the knowledge in insects. The project will allow the long-held hypothesis that the trait of Suppressed Mite Reproduction (SMR) that has arisen in several bee strains is due to changes in their semiochemical production to be finally tested.

INDUSTRIAL BENEFICIARIES: Current methods of varroacide testing either use whole colonies or a standardized test for pyrethroids lasting 48 hours. Our artificial rearing system will be of great benefit to the agrochemical industry who will be able to test potential control agents in a more standardized manner and on agents that act over several days or into the next generation. With refinement and further development it should be feasible to maintain long-term cultures of susceptible and resistant strains of varroa collected from field samples of known phenotypes or even induce resistance within the maintained colony. By identifying whether semiochemical production is the underlying cause of the Suppressed Mite Reproduction trait, commercial bee breeders would be able to breed strains selected on the chemical signature of the larval volatiles or the genetic marker for the SMR phenotype.

SOCIETAL BENEFICIARIES: Securing food production will benefit all of society. The general public have a great affinity for bees and there is widespread concern about the plight of the honey bee among the wider public. The project would show this concern is being addressed. The general public and beekeepers are interested in honey bees and research into them. We will present our findings to stakeholders and the public through talks and interactions with schools and the general media.