Research Project: Managing Honey Bees against Disease and Colony Stress

Location: Bee Research Laboratory

2020 Annual Report

Objectives
The overarching goal of the Bee Research Laboratory (BRL) is to provide beekeepers and regulators practical advice for maintaining sustainable honey bee populations for pollination and hive products. BRL will use integrated laboratory and field approaches to develop novel management strategies for 1) diagnosing and mitigating disease, 2) reducing the impacts on bees of pesticides and other environmental chemicals, and 3) improving bee health through better nutrition. Objective 1: Exploit genetic analyses of parasites, pathogens and bee immunity (including gut microbe interactions) to improve diagnosis and management of bee diseases. [NP305, Component 2, Problem Statement 2B] Subobjective 1.A: Manipulate honey bee immune responses toward disease. Subobjective 1.B: Conduct metagenomic analyses to identify novel pathogens and pathogen webs important for bee disease. Subobjective 1.C: Exploit bacterial gut symbionts to defend honey bees against disease. Objective 2: Use genomic information to develop novel controls for mites and other parasites, e.g., controls based on RNAi strategies or that target parasite vulnerabilities. [NP305, Component 2, Problem Statement 2B] Subobjective 2.A: Produce genome-led control strategies against Varroa mites. Subobjective 2.B: Develop gene-based control strategies for the gut parasite Nosema ceranae. Subobjective 2.C: Reduce the impacts of mite-transmitted viruses on bee health. Objective 3: Determine the impacts of physiological stress on worker and queen development and longevity, including that caused by overwintering and unbalanced diets. [NP305, Component 2, Problem Statement 2A] Subobjective 3.A: Determine the impacts of nutritional components on behavioral development, immune response and susceptibility to disease. Subobjective 3.B: Determine the effects of dietary fatty acids (FAs) on honey bee colony survival. Subobjective 3.C: Improve queen fecundity and longevity through better nutrition. Objective 4: Identify key impacts of in-hive and environmental pesticides on bee health, including sub-lethal effects and interactions with bee pathogens. [NP305, Component 2, Problem Statement 2B] Subobjective 4.A: Define synergisms between chemical exposure and disease. Subobjective 4.B: Determine whether pesticide exposure increases oxidative stress in honey bees, and if so, develop means to reduce the potential negative effects of oxidative damage to honey bees. Subobjective 4.C: Determine the effects of pesticides on honey bee basal metabolic rate (BMR). Objective 5: Develop and test hive-level treatments against mites and other bee threats. [NP305, Component 2, Problem Statement 2B] Subobjective 5.A: Develop best practices for resource availability, mite control, and colony health for migratory commercial beekeepers. Subobjective 5.B: Develop colony management strategies for improved queen health.

Approach
Research at the Bee Research Laboratory (BRL) focuses on using microbiological, genomic, physiological, and toxicological approaches to improve the management of bee diseases and parasites, with a mission to develop innovative tools that can be used by beekeepers to build and maintain healthy bee populations. Integrated laboratory and field approaches will be used to develop novel management strategies for 1) diagnosing and mitigating disease, 2) reducing the impacts on bees of pesticides and other environmental chemicals, and 3) improving bee health through better nutrition.

Progress Report
This is the final report for project 8042-21000-290-00D which was terminated on February 9, 2020. Substantial and impactful results were obtained over the five years of the project which falls under National Program 305 (Crop Production), Action Plan Component II (Bees and Pollination), Problem Area A (Honey bees). Essentially all milestones were “Met” or “Substantially Met.” During this period, our Disease Diagnostic Service responded to field colony losses by identifying disease-causing agents in bee samples sent by beekeepers across the U.S. These determinations included over 100 positive identifications of American foulbrood annually, helping in the control of this regulated infectious disease. We determined the impacts of pesticides and nutritional stress on bees, and subsequently provided beekeepers with advice and guidance for building and maintaining healthy bee populations for pollination. This was alongside with direct outreach and engagement efforts to the general public and beekeeping community at the public affairs and meetings ranging from county to national and international levels. Through 124 total published peer-reviewed papers and five invention disclosures, the six Bee Research Laboratory (BRL) scientists (five Cat. 1 and one Cat. 4) generated large sets of genetic data and publicly accessible platforms that have allowed for the development of diagnostic and detection tools, produced new knowledge relating to mechanisms in underlying complex diseases, facilitating the development of novel therapeutic strategies for bee ailments caused by various factors, and leading to the formulation of the best practice guidelines for effective bee disease and pest management. Under Objective 1 (Exploit genetic analyses of parasites, pathogens and bee immunity including gut microbe interactions to improve diagnosis and management of bee diseases), BRL scientists developed genetic and genomic resources for examining the effects of biotic and abiotic stressors including parasitic Varroa mite, viruses, Nosema, bacteria, small hive beetle, pesticide residues, and malnutrition on bee health and identified key factors that are possibly responsible for driving widespread colony collapse. Furthermore, BRL scientists made significant progress towards identifying and elucidating new emerging pathogens such as Varroa destructor virus-1 (VDV1) in the U.S. bee population and then developed a novel tool to monitor the spread and prevalence of the pathogens nationwide. BRL scientists also showed that the destruction of gut bacteria by the antibiotic treatment could increase bees’ vulnerability to disease infection, thereby providing new evidence that such treatment not only leads to the antibiotic resistance but also damages gut microbial communities, thereby disrupting normal immune function that results in more complex diseases in honey bees. In addition, BRL scientists demonstrated that an early perturbation in the gut microbial populations of young adult bees could cause microbial imbalance and might increase the host’s susceptibility to disease infections, providing a cautionary tale regarding the arbitrary use of probiotics in animal health management. In collaboration with colleagues at the University of Maryland, BRL scientists conducted a national survey of colony losses among beekeepers in the U.S. This gathered information on annual colony losses improves our understanding of the forces shaping the viability of the pollination industry and drives best management practices to promote the health of honey bees, as well as allowing for the mitigation of risks to honey bees. BRL scientists played a major role in developing HoloBee, a database of pathogen sequence data associated with honey bees, which is available for public use and is now established in USDA’s AgDatabaseCommons and is helping researchers worldwide. Under Objective 2 (Use genomic information to develop novel controls for mites and other parasites, e.g., controls based on RNAi strategies or that target parasite vulnerabilities), BRL scientists developed innovative treatments for bee diseases using state-of-the-art technology termed RNA interference (RNAi) to genetically knockdown both pathogen virulence factors and host-based suppressors linked to immune function, thereby inhibiting the replication of pathogens and enhancing the overall fitness of honey bees. BRL scientists also developed an innovative detoxification strategy to scavenge toxic compounds from honey bees and their hives. This was done in order to reduce the effects of pesticide exposure and to boost their immunity against disease infections which helped to promote their survival. Furthermore, BRL scientists discovered the antimicrobial and antioxidant properties of natural products and highlighted the therapeutic potential of the extracts derived from the mycelia of polypore mushroom species for controlling virus infections in honey bees. Moreover, BRL scientists led and teamed with national and international colleagues to conduct a genomic study of Varroa mites, the most detrimental pest to honey bees, to explore complex interactions of the Varroa -virus with honey bees and to reveal the depth of the honey bees’ vulnerabilities in relation to Varroa mites. The generated genomic and transcriptomic resources have been utilized by the bee research community to study biology and management of the Varroa mite. Similarly, the bee industry has used these findings to breed Varroa resistant traits to improve honey bee health. Under Objective 3 (Determine the impacts of physiological stress on worker and queen development and longevity, including that caused by overwintering and unbalanced diets), BRL scientists made significant progress in understanding the influence of nutrition and gut microbiota on honey bee physiology, development, and immune defense to infectious diseases. BRL scientists established a state-of-the-art metabolic chamber to investigate the various impacts of physiological stress on both bee energy consumption and metabolism. The study showed for the first time that lipid, and not sugar metabolism may cause the energetic stress in disease infected honey bees. This finding is important because the lipid content of young adult bees is an important factor in determining an adult worker bee’s behavioral development. BRL scientists provided the first evidence that low sperm viability is linked to poor colony performance and that temperature extremes are a potential causative factor of the reduced sperm viability. BRL scientists also identified key genetic changes in queen and worker larvae that are responsible for healthy queen development. These results offer new insights into the high rate of queen failures in the U.S. as well as the timing of queen development and the nutritional and energetic needs of healthy queens. Under Objective 4 (Identify key impacts of in-hive and environmental pesticides on bee health, including sub-lethal effects and interactions with bee pathogens), BRL scientists investigated the interactive effects of chemical and disease stress on bee health and illustrated the combined effects of a neonicotinoid pesticide and mite stress on the decline of bee health and their populations. Using field-realistic exposure to in-hive and environmental pesticides, BRL scientists showed the combined impacts of chemical and disease stress on the expression of genes expected to determine honey bee queen health and longevity. This consequently led to the identification of biomarkers that can be used to diagnose, monitor disease, or measure a stress response in honey bees. By collaborating with university colleagues, BRL scientists found that pesticide residues in the hive were associated with colony mortality, leading to the development of much-needed recommendations to mitigate problems associated with pesticide exposure. Furthermore, BRL scientists provided evidence of the synergistic effects of a microsporidian parasite Nosema and Deformed wing virus (DWV) in honey bees under different environmental conditions, suggesting that the synergistic effect of two pathogens can lead to interrelated problems that influence on honey bee health. Such findings provide new insight into our understanding of worldwide honey bee colony losses. Under Objective 5 (Develop and test hive-level treatments against mites and other bee threats), BRL scientists developed treatments against Varroa mites by testing novel acaricides and disease-control compounds including phytochemicals, low-toxicity chemicals, food additives, etc. under both laboratory and field conditions. Combined with Foundation-funded research, the BRL is leading the search for the next generation of miticides and developed a novel method to mitigate the detrimental effects induced by synergistic interactions of pesticide and virus infection. BRL scientists, in collaboration with ARS scientists in Baton Rouge, Louisiana, performed genetic analysis of the small hive beetle (SHB) and underlined the potential differences between SHB and its honey bee hosts. This work generated SHB genetic resources that are made publicly available and suggests various mechanisms of SHV infestation for future biology research and control of this species. BRL scientists, in collaboration with ARS scientist colleagues in Tucson, Arizona, also undertook studies to examine how several key factors affect the population growth of the small hive beetle and concluded that temperature and food availability play a critical role in the SHB development and survivorship. This study has important implications for the development of an accurate simulation model of SHB dynamics to forecast outbreaks, particularly in an already variable field environment.

Accomplishments
1. Illustration of genetic diversity of Deformed wing virus (DWV) in the U.S. bee population. DWV is the most widespread viral pathogen in honeybee colonies. In association with the parasitic Varroa mite, it is one of the leading causes of honey bee colony losses. ARS scientists in Beltsville, Maryland, provided the first evidence of the presence of a virulent strain of DWV in the U.S. bee population and demonstrated that DWV populations in U.S. bee populations are genetically distinct. This work highlights the complexity of DWV infection in honey bees and in turn, can inform the design and development of therapeutic strategies for honey bee diseases.

2. A new tool to track pathogenic viruses in the honey bee. Deformed Wing Virus (DWV) is the most widespread viral pathogen in honeybee colonies. ARS scientists in Beltsville, Maryland, developed a DWV-based vector containing a reporter gene that produces green fluorescent protein and demonstrated that the DWV-based vector could be used to track the virus in the honey bee. This new tool will be used for the development of effective vaccines for fighting against infectious viral diseases and maintaining honey bee health.

3. Characterization of caging stress on honey bees. Caging or non-hive environments affect the performance of honey bees. ARS scientists in Beltsville, Maryland, conducted a study to monitor and characterize changes in the physiology, dietary behaviors, and genetics of caged bees with and without pesticide exposure. The result shows that while one pesticide, neonicotinoid imidacloprid, has little effect on honey bee dietary behavior, the deprivation of natural hive conditions could result in oxidative stress to honey bees. This study provides new insights into how caging stress affects honey bees and increases the knowledge necessary for maintaining bee health.

4. Development of an evaluation system for testing natural products as novel medicines for honey bees. Bee disease management that focuses on natural products, which are chemical compounds or substances produced by plants and living organisms, will lead to a reduction in the use of pesticides and antibiotics in bee hives. ARS scientists in Beltsville, Maryland, successfully developed an efficient system that allows for rapid and accurate testing of natural products for treating honey bee diseases. The natural products that are safe, effective and low-cost will lead to honey bee medicines and in turn contribute to the long-term profitability of the beekeeping industry.

5. Elucidation of Deformed wing virus (DWV) – Varroa vector interactions. DWV and its vector, the parasitic mite Varroa, have been implicated in the deaths of millions of honeybee colonies worldwide. Varroa vectoring has led to selection of at least three DWV variants (A, B, or C) circulating in honey bee populations. However, it is not clear if replication of all DWV types occur in Varroa mites which is critical for the prediction of the viral disease epidemics. ARS scientists in Beltsville, Maryland, conducted a study to demonstrate for the first time that DWV-A, the most predominant variant of the DWV, is vectored by the Varroa mite suggesting that the control of the Varroa mite population is an effective method of controlling DWV in honey bees. This study expands our understanding of the DWV-Varroa interactions and contributes significantly to the development of a model system to predict disease events, thereby safeguarding bee health.

6. Development of an RNA interference (RNAi)-based strategy for controlling honey bee chalkbrood disease. Chalkbrood disease is caused by a fungal pathogen that kills larval bees. RNA interference (RNAi) is a natural mechanism to silence gene expression and is used by many different organisms to defend against pathogenic infections. ARS scientists in Beltsville, Maryland, demonstrated that decreasing the expression of a gene that is specific for the causative agent of chalkbrood disease using RNAi-based strategies could reduce germination rates of this fungal pathogen. This study demonstrates that RNAi technology holds much promise as a therapeutic strategy for honey bee disease treatment.

Review Publications
Hao, Y., Chen, Y. 2019. Scientific outreach activities in the U.S. to support bees and other pollinators. Apiculture of China. 10:60-63.
Zhang, Y., Huang, S., Chen, Y. 2019. The role of migratory beekeeping in the U.S. agricultural economy. Apiculture of China. 12:16-18.
Alburaki, M., Karim, S., Lamour, K., Adamczyk Jr, J.J., Steward, S. 2019. RNA-seq reveals disruption in honey bee gene regulation when caged and deprived of hive conditions. Journal of Experimental Biology. https://doi.org/10.1242/jeb.207761.
Branchiccela, B., Castelli, L., Corona, M.V., Diaz-Cetti, S., Invernizzi, C., Jorge, D., Mendoza, Y., Santos, E., Zunino, P., Antunes, K. 2019. Impact of nutritional stress on honey bee colony health. Scientific Reports. 9(1):10156. https://doi.org/10.1038/s41598-019-46453-9.
Chen, X., Wang, S., Xu, Y., Gong, H., Wu, Y., Chen, Y., Hu, F., Zheng, H. 2019. Protective potential of Chinese herbal extracts against microsporidian nosema ceranae, an emergent pathogen of western honey bees, apis mellifera L. Journal of Asia-Pacific Entomology. https://doi.org/10.1016/j.aspen.2019.08.006.
Cook, S.C. 2019. Chemical and dose-dependent effects of two neonicotinoids on honey bee metabolism. Physiological Entomology. 10(1):18. https://doi.org/10.3390/insects10010018.
Kingan, S.B., Urban, J., Lambert, C.C., Baybayan, P., Childers, A.K., Coates, B.S., Scheffler, B.E., Hackett, K.J., Korlack, J., Geib, S.M. 2019. A high-quality genome assembly from a single, field-collected Spotted Lanternfly (Lycorma delicatula) using the PacBio Sequel II System. Gigascience. 8(10):1-10. https://doi.org/10.1093/gigascience/giz122.
Krongdang, S., Evans, J.D., Chen, Y., Chantawannakul, P. 2018. Comparative susceptibility and immune responses of Asian and European honey bees to the American foulbrood pathogen. Insect Science. 00:1-12. https://doi.org/10.1111/1744-7917.12593.
Li, A.Y., Cook, S.C., Sonenshine, D.E., Posada-Florez, F., Egekwu, N., Mowery, J.D., Gulbronson, C.J., Bauchan, G.R. 2019. Insights into the feeding behaviors and biomechanics of Varroa destructor mites on honey bee pupae using electropentography and histology. Journal of Insect Physiology. 119:103950.
Posada, F., Childers, A.K., Heerman, M.C., Egekwu, N., Cook, S.C., Chen, Y., Evans, J.D., Ryabov, E.V. 2019. Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa destructor in a non-propagative manner. Scientific Reports. 9(1):1244. https://doi.org/10.1038/s41598-019-47447-3.
Tauber, J., Collins, W., Schwarz, R., Chen, Y., Grubbs, K.F., Huang, Q., Lopez, D.L., Peterson, R., Evans, J.D. 2019. Natural product medicines for honey bees: Perspective and protocols. Insects. 10(10):356. https://doi.org/10.3390/insects10100356.
Tauber, J., Nguyen, P.T., Lopez, D.L., Evans, J.D. 2019. Effects of a resident yeast from the honey bee gut on immunity, microbiota and Nosema disease. Insects. 10(9):296. https://doi.org/10.3390/insects10090296.
Maeva, T., Rahul, R.V., Graul, M.L., Roberts, J.M., Sullivan, S.T., Liachko, I., Childers, A.K., Evans, J.D., Mikheyev, A.S. 2019. Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites. Communications Biology. https://doi.org/10.1038/s42003-019-0606-0.
Wang, L., Li, K., Sadd, B.M., Guo, Y., Zhuang, D., Chen, Y., Evans, J.D., Jie, W., Zhang, Z., Li, J. 2019. Gut microbial signatures associated with sexual maturity and oviposition behaviors in bumble bee queens. Insects. 4(6):e00631-19. https://doi.org/10.1128/mSystems.00631-19.
Watkins de Jong, E.E., Hoffman, G.D., Chen, Y., Graham, R.H., Ziolkowski, N.F. 2019. Effects of diets containing different concentrations of pollen and pollen substitutes on physiology, Nosema burden, and virus titers in the honey bee (Apis mellifera L.). Apidologie. 50:845-858. https://doi.org/10.1007/s13592-019-00695-8.
Wu, Y., Zheng, Y., Chen, Y., Wang, S., Chen, Y., Hu, F., Zheng, H. 2020. Honey bee (Apis mellifera) gut microbiota promotes host endogenous detoxification capability via regulation of P450 gene expression in the digestive tract. mBio. 13(4):1201-1212. https://doi.org/10.1111/1751-7915.13579.
Li, W., Wang, C., Huang, Z., Chen, Y., Han, R. 2019. Reproduction of distinct genotypes of varroa destructor on honeybee worker brood of the European honey bee apis mellifera and the Asian honey bee apis cerana. Insects. https://doi.org/10.3390/insects10110372.
Posada-Florez, F., Bloetscher, B., Lopez, D.L., Pava-Ripoll, M., Rogers, C.W., Evans, J.D. 2020. Aberrant cocoons found on honey bee comb cells are found to be Osmia cornifrons (Radoszkowski) (Hymenoptera: Megachillidae). Journal of Apicultural Research. https://doi.org/10.1080/00218839.2020.1740405.
Ray, A.M., Lopez, D.L., Iturralde Martinez, F.J., Galbraith, D.A., Rose, R., Vanengelsdorp, D., Rosa, C., Evans, J.D., Grozinger, C.M. 2020. Distribution of recently identified bee-infecting viruses in managed honey bee (Apis mellifera) populations in the USA. Apidologie. https://doi.org/10.1007/s13592-020-00757-2.
Ryabov, E., Christmon, K., Heerman, M.C., Posada-Florez, F., Harrison, R.L., Chen, Y., Evans, J.D. 2020. Development of a honey bee RNA virus vector based on the genome of Deformed wing virus. Viruses. https://doi.org/10.3390/v12040374.