NATURAL PRODUCTS FOR HONEY BEE IMMUNITY & DISEASE RESISTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1022150
• Grant No.: 2020-67013-31861
• Cumulative Award Amt.: $414,224.00
• Proposal No.: 2019-06113
• Project Start Date: Jul 1, 2020
• Project End Date: Jun 30, 2024
• Grant Year: 2020
• Program Code: [A1113]- Pollinator Health: Research and Application

Recipient Organization
AGRICULTURAL RESEARCH SERVICE
RM 331, BLDG 003, BARC-W
BELTSVILLE,MD 20705-2351

Performing Department
Bee Research Laboratory

Non Technical Summary
The goal of this project is to screen infectedhoney bees with 50+ naturally occurring chemical compounds to identify safe and effective medicines for Deformed wing virus, Nosema ceranae, Nosema apis and Lotmaria passim. These four pathogens are several of the many factors causing domestic and global honey bee decline and overwinter colony loss. Despite the importance of these four agents, beekeepers have no registered and scientifically verified medicines to combat infection. Novel bee medicines that control virus and parasite levels at the colony level will increase hive efficiency, reduce total hive management costs, check the rapid escalation of pollination fees paid by almond growers and other crop producers, and contribute to domestic crop security. Screening of these compounds will advance basic honey bee science by providing factual insight into classes of compounds that may or may not be palatable in sugar water, that may or may not have acute toxicity, and that may or may not impair the honey bee gut microbiome.By focusing on natural products, we hope to reduce the use of synthetic antibiotics and pesticides in bee hives and reduce the regulatory burden for licensing new products. This will decrease costs and increase diversity of food, especially the many specialty crops that require insect pollination. Our ultimate goal is to help hobbyist, sideliner, and commercial beekeepers keep their bees healthy and disease free.

Animal Health Component

50%

Research Effort Categories

Basic

20%

Applied

50%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
211	3010	1130	60%
205	3040	1060	15%
205	1212	1101	25%

Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants; 205 - Plant Management Systems;

Subject Of Investigation
1212 - Almond; 3040 - Honey, honey products, and apiary products; 3010 - Honey bees;

Field Of Science
1060 - Biology (whole systems); 1101 - Virology; 1130 - Entomology and acarology;

Keywords

apis mellifera

colony collapse disorder

natural products

pollination

varroa

Goals / Objectives
The goal of this three-year project is to screen infected European honey bees with 50 naturally occurring chemical compoundsto identify safe and effective medicines for Deformed wing virus, Nosema ceranae, Nosema apis and Lotmaria passim. These four pathogens are several of the many factors causing domestic and global honey bee decline and overwinter colony loss. Despite the importance of these four agents, beekeepers have no registered and scientifically verified medicines to combat infection. The seven objectives of the project are listed below. Novel bee medicines that control virus and parasite levels at the colony level will increase hive efficiency, reduce total hive management costs, check the rapid escalation of pollination fees paid by almond growers and other crop producers, and contribute to domestic crop security. Screening of these compounds will advance basic honey bee science by providing factual insight into classes of compounds that may or may not be palatable in sugar water, that may or may not have acute toxicity, and that may or may not impair the honey bee gut microbiome. By focusing on natural products, we hope to reduce the use of synthetic antibiotics and pesticides in bee hives and reduce the regulatory burden for licensing new products. This will decrease costs and increase diversity of food, especially the many specialty crops that require insect pollination.Develop prioritized list of 50 candidate compounds to screen.Perform 7-day laboratory bee cup trials to screen infected bees with selected compounds at three concentration levels.Assess compound safety & toxicity by recording bee mortality daily during cup trials.Conduct 14-day field 'mark & recapture' trials to assess efficacy in natural environment.Assess compound efficacy by measuring Deformed wing virus, Nosema, and Lotmaria transcript levels among trial and control conditions.Assess 20 compounds on Lotmaria using cell culture assay.Replicate results found to be promising in initial bee cup and mark & recapture trials using a secondary bee cup and mark & recapture trial using one or more concentrations.

Project Methods
Objective 1. Develop List of 50 Candidate Compounds Deriving a prioritized list of 50 candidate compounds is the first, most challenging, and most important project task. It is the most important because the likelihood of discovering a safe and effective compound is highly dependent on the prior probability of success. We seek to enrich our list of candidates by identifying natural compounds that are generally recognized as safe by the U.S. FDA and are potential immune stimulants, anti-viral, or anti-parasite in the honey bee. Products in this candidate stream will derive from the following sources and will be further filtered based on cost (a projected maximum cost of $5/treatment). Co-PD William Collins will lead this objective. Our direct targets for candidates include:Natural products already undergoing testing at the Bee Research Laboratory, including plant nectar products {Palmer-Young, 2017 #9008}.Plant resins (and compounds derived from plant resins) identified by team member William Collins.Natural products proposed by community members and tested under Material Transfer and Research Agreements (e.g., polypore mushroom extracts developed by Fungi Perfecti and natural products proposed by Strong Microbials).FDA GRAS database and lists of natural extracts vetted against peer-reviewed literature to identify those with known antiviral and anti-parasitic activity.Compounds known from the insect literature to be antimicrobial.Objective 2. Laboratory Bee Cup Trials (Three Concentrations) To start, in spring 2020 Bee Research Lab technicians will establish a feeder array of 20 honey bee colonies. They will do this by splitting mature overwintered colonies. After splitting, the technicians will measure each colony for the presence of Deformed wing virus, Nosema, and Lotmaria . Other than this spot-check, the technicians will maintain the colonies with minimal interference. Bee cup trials will begin in early summer 2020. Specifically, technicians will collect adult worker honey bees from established field colonies that are positive for all three disease targets (Deformed wing virus, Nosema, and Lotmaria). In the laboratory, technicians will anesthetize the collected bees with CO2, count 30 bees each into individual bee cups, and then feed with a maintenance diet of sucrose diluted in water (1:1) via a plastic pipette bulb. On the next day, technicians will introduce treatments into the sugar water via the sucrose feeder. The research plan is to screen compounds in the parts per million range for three concentrations: 1 ppm, 10 ppm & 100 ppm. We plan to test 25 compounds in the first project year and 25 compounds in the second project year. This makes for a project goal of 50 compounds. Each trial takes one calendar week (seven days). We anticipate processing five compounds in each trial week. In our experience, this plan requires a minimum of eight summer weeks to process 25 compounds if all goes well. Allocating several down weeks to account for poor weather, lost trials, and other unforeseen issues, we anticipate carrying out cup procedures for 25 successful trials in ten to twelve weeks during summer 2020 and again in summer 2021.Objective 3: Measure Safety & Toxicity Reducing pathogen load is just one aspect of an effective honey bee medicine. It must also be safe and non-toxic. We will assess safety and acute toxicity by survival. Specifically, we will count bee mortality daily during the mature bee cup trials. Compounds found to cause any level of bee mortality even at the highest doses will be downgraded. We will still measure survivors for disease load for all treatments.Objective 4: Parallel Mark and Recapture Field Trials The mark and recapture studies will have the same research design as the mature bee cup trials. That is, 3 concentrations of each compound times five candidates making 15 randomized and marked bees sets per trial. These 15 sets plus one control will be placed into 16 conditions. Bees aged 0-24 hours will be painted with a two-color system using Pro Painter non-toxic paint pens. Painted bees will be housed in plastic cups, while being fed each compound at doses of 1, 10, or 100 ppm. After 48 hours, cohorts of 30 bees in 16 conditions (5 compounds at three concentrations of medicine each, plus one control set fed only sucrose) will be released into one of 24 'host' colonies. After living in these colonies for 10 days, bees with markings will be collected via a modified insect vacuum, counted, and then saved for genetic analyses. 20 bees from each group will then be assayed for disease loads as in the cup studies above.Objective 5: Measure Treatment Disease Load using Quantitative PCR At the end of each seven-day cup trial, a technician will collect and flash freeze 20 live surviving bees from each cup for measurement of pathogen load. The flash freeze enables the technician to store the bees for later analysis. Perhaps even for fall and winter, to allow summer to be devoted to treatment trials. The technician will measure pathogen load by performing quantitative PCR. We will assess pathogen load as cycle threshold number for primer sets specific to Deformed wing virus (strains A and B), Nosema ceranae, and Lotmaria passim normalized to cycle threshold number for honey bee RpS5 transcript {Traynor, 2016 #5664}. In total we plan on 2,920 genetic screens for disease (87,600 bees) in 2020 and 2021. This is 480 cup treatments + 480 * 2 = 960 mark-recapture treatments + 20 spot checks = 1,460 assays each year.Objective 6: Lotmaria Cell Growth In addition to the live bee trials, Dr. Schwarz has developed a method to culture cell lines for the trypanosomatid gut parasite Lotmaria passim. This enables efficient, low-cost, and high-throughput in vitro screening of natural compounds against Lotmaria. Dr. Schwarz will treat his cell line cultures with 20 of the natural products that we are testing in live bees. These trials will take place using a strain of Lotmaria passim isolated from Beltsville, MD. Liquid cultures will be infused with 1, 10, and 100 ppm of active ingredients. After three days of growth, extracts will be taken for DNA extraction and L. passim will be assayed using real-time quantitative PCR {Schwarz, 2016 #5409}. He will also assess cell growth rate and morphology using colorimetric assays he has developed (see Ft. Lewis College SOW). These assays may be more controlled and sensitive than the live bee cup procedure and have the additional benefit of being able to determine the minimum effective dose for any compound that may inhibit biological activity of this protozoan parasite. Of note, this project will be paying only for Dr. Schwarz's time. He and Fort Lewis College will pay for the media and other reagents required to perform the screens.Objective 7: Perform Secondary Screen of Promising Compounds For all the natural products that show promise in terms of disease loads in the initial screen, we will repeat the mature bee cup procedure and mark-recapture trials at the indicated dose or at additional doses. We will expand the screen to perform both a replicate of the seven-day trial (again measuring at day seven) and to perform a field trial for thirty days to assess mortality (e.g., {Li, 2016 #5990}. Ideally, we will have at least four compounds to test in secondary screens, leading to strong candidates for trials at the colony level by year three.

Progress 07/01/20 to 06/30/24

Outputs
Target Audience:This research was focused on novel management tools for honey bee health, with the aim of benefiting both U.S. agricultural food production and the global scientific community. Stakeholders, including commercial and hobby beekeepers, require novel treatments for disease and that was the focus of this project, with them as our primary audience. There was an effort as well to help commercialize products derived from this research and those efforts will continue by USDA-ARS. We have a long history motivated the entire research process and we spoke with beekeepers ab out the incoming results at state and national meetings multiple times during the project. Using natural products to boost bee immunity while tackling overwintering and queen losses will give beekeepers new tools and sustainable solutions for disease and stress management. This, in turn, will reduce pesticide application, improve bee colony survival, and increase profitability of beekeeping operations. Changes/Problems:Our major changes were budgetary, as we tripled the number of candidate genes to screen we invested in human power to help run and analyze a greatly expanded set of experiments. We also shifted after year two to focus on antiviral treatments exclusively, rather than gut parasites, as we felt we had good candidates in hand for those and yet no viable antiviral treatment is on the market. What opportunities for training and professional development has the project provided?This project helped fund two postdoctoral fellows at the USDA-ARS, two undergraduate technicians (summer 2022 and 10 hrs/week during the 2023-2024 school year, and visiting scientists from China and the US. We also hosted a total of seven STEM students from local High Schools reflecting under-represented minorities in science, leading to numerous regional science fair successes and one student presenting and receiving a category (Microbiology) award at the ISEF science fair in Texas (2023). How have the results been disseminated to communities of interest?Four talks for the American Beekeeping Federation and American Honey Producers Annual meetings (two each), talks at the Association of Apicultural Professionals American Bee Research Conference (2 x), talks at eight state beekeeping meetings, and at two Maryland County Beekeeping meetings. Publications in peer-reviewed journals and trade journals (Bee Culture magazine and the monthly research/extension column by JDE "Found in Translation", www.beesfoundintranslation.org), talks on three podcasts, and numerous opportunities to correspond with beekeepers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the end we purchased and tested 147 natural compounds over the course of this project, using both 5-7 day assays with live bees injected with a newly developed cloned virus andscreening of pupae and adult bees with a natural cocktail of viruses. These tests resulted in nearly ten plant compounds we are actively testing in field colonies, with field trials of paired colonies (n = 24, 20, and 22 colonies in the summers of 2022, 2023, and 2024, respectively). Insights from these trials resulted in a technology transfer with USA-ARS and a filed patent in April 2023, and we will continue these efforts by seeking commercial partners for product development. We did not conduct mark-recapture studies extensively and instead were able to monitor the effects of natural products on the health of older, microbially normal bees by narcotizing and raising these bees in plastic arenas, fulfilling the primary benefit of mark-recapture experiments. We found three plant compounds that were effective in reducing Lotmaria parasite loads after testing over 20 candidates.

Publications

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:This project aims to provide the first antiviral medicines for beekeepers, addressing a major cause of honey bee colony losses. Target audiences are scientists, bee industry leaders, commercial vendors, and US regulators. Changes/Problems:We have focussed heavily on viruses in this project, in part at the expense of Nosema work, since viruses continue tobe the primary unaddressed issue for honey bee health. What opportunities for training and professional development has the project provided?We have employed one postdoctoral (ORISE) fellow thanks to these funds and temporarily employed one graduate student (Lindsey Markowitz) who was then able to secure National Science Foundation funding to continue her work in the lab. This system has also been a platform for three high school students from the Charles H. Flowers STEM program for under-represented minorities, and these projects have helpedwith one college acceptance (Harvard University, Nana Safo-Mensa), and one first-place Regional Science Fair project (Alriana Buller-Jarett, also a 4th-place Microbiology award at the International Science and Engineering Festival, Dallas, TX). How have the results been disseminated to communities of interest?ONe national and six state-level beekeeping meetings in the past 12 months, along with talks for the USDA/EPA 'State of the Sciuecne' 2022 workshop for pollinators, and several interactions/updates for theUS Government Pollinator InteragencyWorking Group. What do you plan to do during the next reporting period to accomplish the goals?Weare preparing for an ambitious field trial to reduce viral loads and improve colony health, followed by laboratory follow-up and hopefully beekeeper demonstrations in the fall of 2023

Impacts
What was accomplished under these goals? Goal 1. We screened 112 compounds and developed a new assay using honey bee pupae that was published and will help others test for bee medicines Goals 2 and 3. We have identified 6 compounds that seem safe for bees and have antiviral properties, and did a preliminary field trial in 2022 with two of these. While the trial did not resultin lower virus levels in the fiekld, we showed that the compounds were safe for bees and were consumed with sugar water feeders. We are planning, with the help of a no-cost extension from NIFA, to screen two different candidates in the fields in 2023. Goal 5 and 6, we focussed primarily on viruses and have data for > 3000 bees for one compound (thymol), trying to optimize effectiveness, we also have published a short paper showing the best compounds against Lotmaria. We were not able to screen for efficacy against Nosema. Goal 7, we are repeating the mark-recapture and full-colony field trials in July-August 2023, with new compounds that showed the most promise in the past year.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Palmer-Young, E.C., Ryabov, E.V., Markowitz, L.M., Boncristiani, D., Grubbs, K.E., Pawar, A., Peterson, R, Evans, J.D. Host-driven temperature dependence of Deformed wing virus infection in honey bee pupae. Commun Biol 6, 333 (2023). https://doi.org/10.1038/s42003-023-04704-6
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Palmer-Young, E. C., Markowitz, L. M., Grubbs, K., Zhang, Y., Corona, M., Schwarz, R., . . . Evans, J. D. (2022). Antiparasitic effects of three floral volatiles on trypanosomatid infection in honey bees. Journal of Invertebrate Pathology, 194, 107830. doi:https://doi.org/10.1016/j.jip.2022.107830

Progress 07/01/21 to 06/30/22

Outputs
Target Audience:honey beekeepers and industry partners who are able to carry new be treatments to market Changes/Problems:We have had inconsistent results with some compounds (most notablythymol), with viral control in some trials and not in others, we are determining if this is a result of compound availability or (we think more likely) bee host effects What opportunities for training and professional development has the project provided?IN FY22, the grant has helped train one volunteer high school student, one PhD student, and one postdoctoral scholar. How have the results been disseminated to communities of interest?We have presented results from this grant at the past two annual meetings of the American Honey Producers Association and American Beekeeping Federation, as well as several state beekeeping meetings (via Zoom) in Fy22 and the 'Bee College' in May, 2022. What do you plan to do during the next reporting period to accomplish the goals?We are finishing with additional live-bee cage trials December 2022-April 2023 and plan to release the results for field testing in the summer of 2023

Impacts
What was accomplished under these goals? We accomplished goals 3-6 in FY22, and are nearly complete with goal 7

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Palmer-Young, E. C., L. M. Markowitz, K. Grubbs, Y. Zhang, M. Corona, R. Schwarz, Y. Chen, and J. D. Evans. 2022. Antiparasitic effects of three floral volatiles on trypanosomatid infection in honey bees. Journal of Invertebrate Pathology 194:107830.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Palmer-Young, E. C., R. S. Schwarz, Y. Chen, and J. D. Evans. 2022. Can floral nectars reduce transmission of Leishmania? PLoS Neglected Tropical Diseases 16:e0010373.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Palmer?Young, E. C., R. S. Schwarz, Y. Chen, and J. D. Evans. 2022. Punch in the gut: Parasite tolerance of phytochemicals reflects host diet. Environmental Microbiology 24:1805-1817.
• Type: Other Status: Published Year Published: 2022 Citation: Evans, J.D. (2022) Found in translation:Herbal medicine for bees? Bee Culture Vol. 8:20-21 .

Progress 07/01/20 to 06/30/21

Outputs
Target Audience:This project served beekeepers and those in the pollination industry by demonstrating the potential for safe virus medicines for honey bees, and pushing the science of identifying new honey bee drugs forward. Talks were given at four state beekeeping organizations (virtually) and for the American Honey Producers Association. Scientists were also targeted via several pere reviewed publications and two research talks ONe supported postdoctoral scientist delivered research talks to two university audiences Changes/Problems:We had a serious setback at the start of the grant with COVID and were able to get back on track by the end of the current reporting year, while also dealing with COVID by working individually in the lab. What opportunities for training and professional development has the project provided?Two postdoctoral scientists were supported as well as one undergraduate assistant. These individuals were given on-campus work status during COVID-19 thanks to the time-dependency of this grant, and we were able to work safely together to advance the goals. How have the results been disseminated to communities of interest?Yes, we have reached out for the past year to several beekeeping groups and also have kept potential industry partners informed. What do you plan to do during the next reporting period to accomplish the goals?With the screening system in hand, we are testing combinations of different antivirals and are prea[spring for a second field season inJune 2022, for screening the best candidates.

Impacts
What was accomplished under these goals? We developed a rapid screening system for testing chemicals in honey bee pupae, using reporter viruses that indicate the levels of virus replication. We then screened 37 compounds for their impacts on Deformed wing virus. ONgoing work has focussed on three of these compounds (provisional patent submitted in April, 2022) and a larger screening of 40+ new candidates, focused on plant extracts used in traditional medicine.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Evans, J.D., Banmeke, O., Palmer?Young, E.C., Chen, Y. and Ryabov, E.V. (2022) Beeporter: Tools for high?throughput analyses of pollinator?virus infections. Molecular Ecology Resources, 22, 978-987.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Boncristiani, D.L., Tauber, J.P., Palmer-Young, E.C., Cao, L., Collins, W., Grubbs, K., Lopez, J.A., Meinhardt, L.W., Nguyen, V. and Oh, S. (2021) Impacts of Diverse Natural Products on Honey Bee Viral Loads and Health. Applied Sciences, 11, 10732.