Bee Viruses: The Evaluation of Flowering Plants in Horizontal Transmission and Conditions Promoting Viral Replication
Pollinating insects are important for food security and ecosystem function, providing over $200 billion annually in pollination services. Recent honeybee declines have underlined the importance of native pollinators and their ability to provide effective pollination services. However, native bees are also affected by multiple pressures including pathogens, pesticide use and poor nutrition. RNA viruses, once considered to be specific to European honey bees, are among the suspected threats to native bumble bees. However, very little is known about how these viruses are transmitted and their affect on bumble bees. Filling these knowledge gaps is critical for making management recommendations that will lessen the risk of virus infection to important crop pollinators. The purpose of this project is to examine the transmission and effects of RNA viruses among bumble bees. A pilot experiment was conducted to test inoculation methods and determine effects of deformed wing virus on bumble bees. Bees orally inoculated with sucrose mixed with 4 different concentrations of purified deformed wing virus (DWV) exhibited similar mortality rates as control bees fed sucrose only. To test for differences in infection levels between treatments, I will quantify viral loads of the bees using RT-qPCR.
This research has two main objectives: 1) To examine the synergistic effects of pesticide exposure and nutrition on viral infection and 2) To evaluate the role of flowering hedgerow plant species in viral transmission. Results of the proposed research will inform agricultural management recommendations to lessen viral transmission and infection among important crop pollinators. To accomplish objective 1, I conducted a pilot experiment to a) test inoculation protocols, b) determine the concentration of deformed wing virus (DWV) isolate to be used in later experiments and c) examine the effects of DWV infection on bumble bees. Results will not only serve to inform later experiments, and provide valuable data on an understudied topic: RNA virus infection in bumble bees.
To accomplish objective 1, purified deformed wing virus (DWV) isolate was prepared at the University of Maryland by collaborator Humberto Bonchristiani. Five commercial bumble bee colonies were obtained and tested for 3 RNA viruses (black queen cell virus BQCV, Israeli acute paralysis virus IAPV, and deformed wing virus) upon arrival. Of the 5 colonies, all were infected with black queen cell virus and 4 were infected with deformed wing virus. The results of this preliminary testing provide evidence that commercial bumble bee colonies may be contributing to RNA virus spread. To determine the concentration of DWV isolate to be used in later experiments, I conducted a pilot experiment in which I tested four different concentrations of virus inoculum on bumble bee workers.
One hundred bumble bee (Bombus impatiens) workers were transferred to individual containers and assigned to one of 5 treatments: 4 different concentrations of DWV and a control. After a 5-hour period without food, each bee was fed 10 ul of an inoculum containing DWV and 50% sucrose. The control bees only received 10 ul of 50% sucrose. All bees were given pollen and 30% sucrose ad libitum for 14 days (December 5th-December 19th). Mortality and morbidity were recorded. After 14 days, all surviving bees were transferred to -80°C. No differences in mortality were found between treatments. These results may be due to the oral method of inoculation. In a recent publication, bumble bees inoculated with DWV by injection had significantly higher mortality rates than control bees injected with a control solution (Graystock et al., 2015a ). For honey bees, inoculation by injection mimics virus transmission by the Varroa mite (Varroa destructor) whereby the ectoparasite acts as a vector, transmitting RNA viruses directly to bee’s hemolymph. However, bumble bees are not known hosts of the Varroa mite and transmission most likely occurs orally through the use of shared floral resources (Singh et al., 2010; Graystock et al., 2015b; McMahon et al., 2015). Therefore, observed effects of deformed wing virus in bumble bees inoculated by injection may not be realistic. In January, using colonies and equipment I already purchased, I will test differences in effects of my DWV inoculum on bumble bees when either fed orally or injected. Results will provide methodological information to ensure realistic yet effective methods for virus inoculation in bumble bees. Results of these pilot experiments are also defining the dependent variables I will measure in future experiments. Since mortality may not be a symptom of deformed wing virus in orally inoculated bumble bees, sub-lethal effects such as learning/foraging behavior should be examined.
In January, I will analyze the viral loads of the bees used in the pilot experiment using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results will provide data on the amount of DWV necessary to cause an infection in bumble bees and the variation of viral infection I can expect among individuals.
Using results from the pilot experiments, I will conduct a larger experiment in January/February with micro colonies were I will test the synergistic effects of pesticide exposure and nutrition on viral infection (Objective 1). In preparation for this experiment, I have obtained pollen from beekeeper/collaborator Charles Mraz of Champlain Valley Apiaries. To create experimental diets of both high and low pollen diversity, the pollen was sorted by color.
In July-September 2016, I will evaluate the role of flowering hedgerow plant species in viral transmission (Objective 2). Plants will be purchased from Northeast Pollinator Plants of River Berry Farm, a company specializing in providing pollinator-friendly perennials selected for New England and New York State regions (www.northeastpollinator.com). Experiments testing virus transmission across multiple plant species will begin in late summer/early autumn 2016.
Graystock, P., Meeus, I., Smagghe, G.U.Y., Goulson, D. & Hughes, W.O.H. (2015a). The effects of single and mixed infections of Apicystis bombi and deformed wing virus in Bombus terrestris. Parasitology.
Graystock, P., Goulson, D. & Hughes, W.O.H. (2015). Parasites in bloom: flowers aid dispersal and transmission of pollinator parasites within and between bee species. Proceedings of the Royal Society B: Biological Sciences, 282, 20151371.
McMahon, D.P., Fürst, M.A., Caspar, J., Theodorou, P., Brown, M.J.F. & Paxton, R.J. (2015). A sting in the spit: widespread cross-infection of multiple RNA viruses across wild and managed bees. Journal of Animal Ecology, 84, 615-624.
Singh, R., Levitt, A.L., Rajotte, E.G., Holmes, E.C., Ostiguy, N., Vanengelsdorp, D., Lipkin, W.I., Depamphilis, C.W., Toth, A.L. & Cox-Foster, D.L. (2010). RNA viruses in hymenopteran pollinators: Evidence of inter-taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species. PLoS ONE, 5, e14357.
Impacts and Contributions/Outcomes
At the early stages of this research, I aim to bring awareness to the beekeeping community that ‘honey bee’ viruses also infect wild bees and that good beekeeping practices that reduce pathogens in managed honey bees can benefit wild bees living near apiaries by reducing the risk of pathogen spillover. Thus, this January, I am presenting at 3 different events: the Bennington beekeeping club meeting, the Vermont Beekeeping Association meeting, and at the Vermont Grazing and Livestock Conference. At each of these venues, I plan to present Vermont’s results from the 2015 National Honey Bee Survey and highlight the viral load data. As evidence that these same viruses infect wild pollinators, I will present preliminary data from my 2015 field survey of bumble bees in VT. Bumble bees caught near apiaries were more likely to be infected with DWV, BQCV, and IAPV than bumble bees caught far from apiaries (Alger unpublished). I will also present my future plans to investigate 1. How pesticide and nutrition may influence viral loads and 2. How viruses may transmit between bee species through the use of shared flowering resources.
University of Vermont
109 Carrigan Drive
Burlington, VT 05405