Location: Bee Research Laboratory
Project Number: 8042-21000-291-036-I
Project Type: Interagency Reimbursable Agreement
Start Date: Oct 1, 2020
End Date: Sep 30, 2021
Objective:
Since becoming a major pest of Apis mellifera honey bees in the late 1980s, Varroa mites continue to pose a serious threat to the US industry. Along with direct impacts on bees, Varroa mites vector Deformed wing virus (DWV), the most damaging of the honey bee RNA viruses.
The primary purpose of this agreement is to use a newly developed (thanks in part to past USDA-APHIS support) rapid screening tool to assess novel bee medicines. The USDA-ARS Bee Research Laboratory in Beltsville, MD has partnered with other USDA agencies to identify and mitigate the threats of novel and emergent disease agents on honey bees and other pollinators. The overall aim is to reduce the impacts of disease on honey bees. The USDA-ARS Bee Research Laboratory has assembled a team of scientists committed to studying ways to combat viruses and other honey bee disease agents. Prior challenges include tedious screens of potential antiviral treatments involving colonies of bees and imprecise methods for infecting bees. A screening technique for honey bee viruses was recently perfected that will allow one technician to screen approximately 10 compounds per week. The ultimate goal of this project is to identify novel medicines that reduce the impacts of viruses and other pathogens in honey bees. Objective 1 will be to validate a recent screening technique based on injecting bees with viruses labeled with Green fluorescent protein (GFP) against older, more costly, methods. Objective 2 will focus on screening 30-40 proposed compounds for bee safety and virus inhibition. Objective 3 will be to confirm that virus reduction in the injection assays also occurs with bees fed medicines directly. This experiment will be carried out in August 2021, with the four most-promising candidates.
Approach:
For Objective 1, frames of sealed honey bee worker brood will be collected from colonies with low levels of the mite Varroa destructor. Early stage (white-eyed or pale pink-eyed) pupae will be harvested from comb brood cells, incubated on Whatman paper in Petri dishes for 3 hr +33oC and 85% relative humidity and then injected with viral inoculum in PBS (generally 12 x 105 or 12 x 106 GE of cloned DWV-GFP per µl but this will be optimized for the trials. Bees will be scored for fluorescence (from the GFP reporter) at 24 and 48 hours to determine the most quantitative screening time. Once conditions are established, candidate treatments will be provided via the same injections in a final solution from 1 to 10 ppm immediately prior to injection. Briefly 8 µl of viral inocula (with or without drugs) or PBS control will be given using disposable insulin syringes with 31G needles. Viral levels will be confirmed by qPCR using in-house standard assays and the control gene b-actin. As promising treatments emerge, they will be become the focus for cup exposure to bees to establish effects on survival and viral reduction in a realistic delivery mode. Again, bees will be monitored daily for survival and then assessed after 8 days for virus levels. Results will be summarized by standard descriptive statistics (ANOVA) by treatment and by logistics survival analyses for the cup experiments.
