2016 Annual Report for GNE15-094
Bee Viruses: The Evaluation of Flowering Plants in Horizontal Transmission and Conditions Promoting Viral Replication
Summary
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. This project focuses on deformed wing virus (DWV), a common RNA virus in bees, and examines its effects on bumble bees, whether pesticide exposure influences viral infection, and lastly, the role of plants in virus transmission between bee species.
Objectives/Performance Targets
This research has two main objectives: 1) To examine the effect of deformed wing virus (DWV) and how pesticide exposure influences viral infection in bumble bees and 2) To evaluate the role of flowering plant species in viral transmission between bee species. Due to several challenges over the past year, I have made some changes to the scope of the project. I found that virus-free bumble bee colonies are difficult to obtain and must be fed clean gamma-irradiated pollen in order to remain virus-free. Since bees must be fed a specific diet of clean pollen in the lab, I was unable to conduct the nutrition component of object 1. Instead, I plan to focus on the effect of pesticide exposure on viral infection. I will expand this objective to examine an understudied area of virus research: how viruses disseminate within a bumble bee colony and the effects of DWV on bumble bee fitness and offspring. To accomplish objective 1, two pilot experiments were conducted to a) determine the concentration of DWV isolate to be used for the inoculation of bumble bees as well as the sublethal effects of the virus and b) test the survivability of colonies fed imidacloprid inoculated sucrose to determine the amount I will use in later experiments. To address objective 2, a flight cage experiment was conducted using captive colonies and three species of flowering plants grown from seed in the greenhouse. I chose three plant species commonly used to increase bee forage in hay pastures throughout Vermont: white clover, red clover, and birds foot trefoil. In field surveys I conducted during 2014 and 2015, I found that all three of these plant species are common throughout my region and are highly visited by both bumble bees and honey bees.
Accomplishments/Milestones
OBJECTIVE 1
To accomplish objective 1, purified deformed wing virus 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, Israeli acute paralysis virus, and deformed wing virus) upon arrival. Of the five colonies, all 5 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. These results also presented many challenges, as I need virus free colonies for upcoming experiments. Despite these challenges, I conducted two pilot experiments to determine a. the effectiveness of the virus inoculum and b. effect of imidacloprid on bumble bee survivorship.
To test the virus inoculum on bumble bees, 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. Mortality and morbidity were recorded. After 14 days, all surviving bees were transferred to -80°C. Using RT-qPCR I analyzed two of the groups and found that bees fed the inoculum had higher DWV levels than the control group. Results will provide data on the amount of DWV necessary to cause an infection in bumble bees, the variation of viral infection I can expect among individuals and methodological information on inoculation protocols.
In a second pilot experiment, I tested the effect of different concentrations of imidacloprid (a commonly used neonicotinoid pesticide) on bumble bee survivorship and also tested whether exposure influenced the viral loads already present. This pilot experiment was necessary to ensure bees would experience only sublethal effects of imidacloprid in the larger future experiment while still surviving during the length of the experiment. Twenty bees were assigned to each of 4 treatments and a control. Treatment groups were fed pollen and 30% sucrose ad libitum inoculated with different concentrations of imidacloprid: 0.1, 1, 10, and 20 parts per billion (ppb) for 8 days. The control received 30% sucrose only. Sucrose consumption and mortality was measured for five days. Mortality did not differ between treatments. However, bees in the 20 ppb and 10 ppb group consumed significantly less sucrose (figure 1). In light of these important preliminary results, bees will be fed sucrose inoculated with less than 10 ppb imidacloprid in future experiments to ensure the bees eat and receive the pesticide exposure treatment. Since the bees arrived already infected with DWV and BQCV, I will use RT-qPCR to test bees to see if virus levels were affected by the pesticide exposure.
Using data from the pilot experiment, I plan to conduct a larger experiment in Spring 2017 with micro colonies where I will test the effects of pesticide exposure on DWV. In this experiment, I will also test how DWV affect colony development and how DWV spreads throughout a colony. In preparation for this experiment, I have arranged a shipment of bumble bees from a supplier who can provide virus-free bees. In order to maintain virus-free bees, I have obtained the necessary permits and the gamma irradiated pollen I will feed to the experimental bees.
OBJECTIVE 2.
To accomplish objective 2, testing for viral transmission between bee species through the use of shared floral resources, I conducted a series of experiments where I allowed honey bees infected with DWV to forage on arrays of flowering plants within a screened enclosure and later transferred these plants to two separate enclosures where non-infected bumble bee micro colonies were allowed to forage (figures 2 and 3). All bees and flowers were collected after each experiment and stored in -80°C. The experiment consisted of 4 parts where I explored how viral transmission between bee species through the use of shared floral resources is influenced by: 1) plant species, 2) plant diversity, 3) multiple exposures to infected plants, and lastly, 4) if direct contact or co-mingling is necessary for viral transmission by allowing bumble bees and honey bees to forage at the same time on the same plants. I am currently using RT-qPCR to test bees and plants for RNA viruses. So far, I have successfully detected both DWV and black queen cell virus (BQCV) on all plant species used in the experiment. Future lab work will test whether bees can become infected after visiting the infected flowers.
Impacts and Contributions/Outcomes
I aim to bring awareness to the beekeeping community that ‘honey bee’ viruses also affect 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. Over the past year, I have presented my research to a number of beekeeping and farming groups including the Bennington beekeeping club, the Vermont Beekeeping Association, and at the Vermont Grazing and Livestock Conference. At each of these venues, I presented Vermont’s results from the 2015 National Honey Bee Survey and highlighted the viral load data. I discussed how these same viruses affect wild pollinators and presented my future plans to investigate 1. How pesticide influences viral infection and 2. How viruses may transmit between bee species through the use of shared flowering resources. This past summer, I worked with the Bennington beekeeping club to hold a ‘bee pathogen workshop’ for small-scale beekeepers to gain hands-on experience identifying and monitoring honey bee pathogens. I have also presented my research at several other venues including the Bumble Bee Working Group Meeting in at the University of Sussex in the UK, the International Pollinator Conference at Penn State, and for the Garden Club of America in Lenox Massachusetts. Currently, the Vermont Pollinator Protection Committee is writing VT’s Pollinator Protection Plan (MP3). I presented my research at one of their meetings at the state house and I plan to keep the group updated with the results of these experiments as they work to write policy for the protection of Vermont’s pollinators. I expect my research will help drive decisions about the restriction of pesticide use as well as plant recommendations for pollinator habitat. I have three presentations scheduled in 2017 for local beekeeping organizations where I will highlight new results from the plant transmission experiments.