Research Project: Managing Honey Bees against Disease and Colony Stress

Location: Bee Research Laboratory

2019 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 a bridge project for 8042-21000-277-00D which is currently under OSQR review. Along with direct outreach and engagement efforts to the general public and beekeepers at the public affairs and meetings ranging from county to national and international levels, Bee Research Laboratory (BRL) scientists responded to the field colony losses by identifying disease-causing parasites and pathogens in bee samples sent by beekeepers across the U.S., determining the impacts of pesticides and nutritional stress on bees, and providing guidelines for bee disease management. Through 17 total published peer-reviewed papers, the five BRL scientists (four Cat. 1 and one Cat. 4), identified and characterized a range of factors that influence virulence and pathogenicity of diseases in honey bees and developed innovative tools and strategies that reduce the impact of disease threats, pesticides, malnutrition, and other stress factors on bee health. Significant progress was made this year on all five objectives, all of which fall under National Program 305 (Crop Production), Action Plan Component II (Bees and Pollination), Problem Area A (Honey bees). Under Objective 1, BRL scientists made significant progress in developing genetic and genomic resources for examining the effects of biotic and abiotic stressors including Varroa mite, viruses, Nosema, bacteria, small hive beetle, pesticide residues, and malnutrition on bee health. BRL scientists initiated and led a collaborative research effort to estimate evolutionary relationships between the U.S. strains of Sacbrood virus (SBV) and epidemic SBVs strains circulating in the honey bee populations worldwide and provided the first documentation of the U.S. strains of SBV. As part of a collaborative effort, progress was made in producing a highly complete genome assembly of A. mellifera for studying genome function, mapping structural and functional genetic variation, and comparative genomics. Under Objective 2, BRL scientists made significant progress in developing innovative treatments for bee diseases. Progress was made by discovering the antiviral activity of extracts derived from the mycelia of several polypore mushroom species and evaluating the therapeutic potential of the fungal extracts for controlling virus infections in honey bees. Progress was made by genetically manipulating pathogens by using RNA interference (RNAi) technology to knock down the pathogen virulence factors that are critical for host invasion, thereby inhibiting replication of pathogens in honey bees and enhancing the overall fitness and lifespan of honey bees. Progress was also made by developing a cyclic polysaccharide-based approach to scavenge toxic compounds from honey bees and hives to boost their immunity against disease infections and reduce the effects of pesticide exposure thereby promoting their survival. Under Objective 3, BRL scientists made progress in understanding the influence of nutrition /gut microbiota /cold stress on honey bee development, immunity, metabolism, and survival. Progress was made by showing the involvement of the gut bacteria in the host’s nutrient metabolism, immune functions, and survivorship and providing a deeper understanding of the interconnections among gut bacteria, nutrition and immunity in honey bees. Under Objective 4, significant progress was made in defining the negative impacts of a sublethal doses of a neonicotinoid pesticides inlcuding imidacloprid, on olfactory orientation of honey bees. Significant progress was also made in elucidating molecular mechanisms involved in impaired learning performance in honey bees exposed to imidacloprid. Progress was made identifying how different neonicotinoids, and their doses, significantly affects important aspects of honey bee metabolic physiology. Under Objective 5, significant progress was made in gaining critical insights into the metabolism and feeding/excretory behaviors of the parasitic Varroa mite on bee hosts and laying the groundwork for developing effective method to mitigate detrimental effects by Varroa parasitism.

Accomplishments
1. Generation of critical insights into the metabolism and behavior of parasitic Varroa mites on honey bee hosts. The parasitic mite Varroa is the European honey bees’ most detrimental pest by feeding on the fat body and haemolymph of adult honey bees as well as larvae and pupae. Understanding the feeding behavior and nutritional requirements of the Varroa mites will help the proper design of strategies to control their activity and prevent them from causing damage. ARS scientists in Beltsville, Maryland, provide important insights into Varroa nutritional biology and behavior by analyzing the chemical composition of Varroa excreta and characterized behaviors of excreta deposition. This study also expands our understanding of the Varroa mite's digestion process and feeding behavior, thereby contributing to the development of effective management strategies for this important pest.

2. Genome sequencing and analysis of the small hive beetle, a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory. The small hive beetle is an invasive pest of bee hives. The beetles inhabit almost all honey bee colonies in their native range and exert significant pressures on beekeeping practices. ARS scientists in Beltsville, Maryland, sequenced and annotated the SHB genome, providing the first genomic resources for this species and for the Nitidulidae, a beetle family that is closely related to the extraordinarily species-rich clade of beetles known as the Phytophaga. Unique detoxification pathways and pathway members identified from this study can help identify which treatments might control this species even in the presence of honey bees, which are notoriously sensitive to pesticides. The study provides new insights into the genomic basis for local adaption and invasiveness in SHV and a blueprint for control strategies that target this pest without harming their honey bee hosts.

3. Characterization of honey bee host immune responses to the association of parasitic Varroa and Deformed wing virus. The parasitic mite Varroa in association with Deformed wing virus (DWV) has often been implicated in colony losses and is responsible for the death of millions of honey bee colonies worldwide. However, to date, the underlying mechanisms that facilitate the interactions between the bee, Varroa mite, and virus have not been fully explained. ARS scientists in Beltsville, Maryland, identified that honey bees could mount rapid and immediate immune responses to threats, however, the immune system slowed down in its normal function two days post the Varroa infestation, leaving the bee vulnerable to expansive viral replication. The critical insights into the defense response upon Varroa and DWV challenges generated in this study enhance our understanding of complex honey bee diseases and have many applications for the future development of effective honey bee disease management strategies.

4. Genome sequencing and characterization of the U.S. strains of Sacbrood virus. Sacbrood virus (SBV) is the most widely distributed of all the bee viruses and has evolved the greatest number of variant strains currently circulating in bee populations around the world, posing a significant risk for future honeybee emergent disease events. ARS scientists in Beltsville, Maryland, provided the first complete genome sequencing of U.S. strains of SBV, estimated the evolutionary relationships between the U.S. strains of SBV and epidemic SBV strains worldwide, and then characterized host responses to SBV infection. The genomic resources generated from this study can serve as a background for the geographic origin of the U.S. strains which would allow us to monitor the emergence of new adaptive mutations of SBV in the future. This study contributes to a better understanding of the evolution and pathogenesis of SBV infection in honey bees, and has important epidemiological relevance.

5. Designing and development of a novel strategy to control honey bee Nosema disease. Nosemosis, or Nosema disease of honey bees is caused by the microsporidia parasite N. ceranae and has often been implemented in honey bee colony losses worldwide. So far, the only approved treatment against Nosemosis is the antibiotic fumagillin. However, this product is no longer available for sale, creating an urgent need for new therapeutic options. ARS scientists in Beltsville, Maryland, employed a new technology, termed RNA interference (RNAi) which is a method to shut down expression of any gene in a wide range of organisms, and explored the possibility of silencing the expression of a N. ceranae virulent gene for controlling Nosema disease in honeybees. The study demonstrated that knocking down the gene encoding Polar Tube Protein 3 (PTP3) which is essential in the invasion of host cells in Nosema-infected bees, could suppress pathogen replication, enhance host immune responses and improve the overall health of the infected bees. This study identified a new therapeutic target in Nosema disease treatment, holding great promise in developing the effective treatment for controlling diseases in honey bees.

6. Demonstration of RNA interference (RNAi) pathway as an important infection strategy for the microsporidian parasite, Nosema ceranae. RNA interference (RNAi) is a natural mechanism for silencing gene expression and is used by many different organisms to regulate the activity of genes. While the protein-coding gene Dicer which is the key enzyme of the RNAi pathway is lost in most microsporidian genomes, it is present in honey bee microspodia parasite Nosema ceranae. ARS scientists in Beltsville, Maryland, identified Dicer as an important regulator for N. ceranae proliferation and showed that silencing the Dicer gene by RNAi method could lead to significant reduction of N. ceranae spore loads in infected bees. This study improves our understanding of the virulence traits and biologics of pathogens and parasites of honey bees, which in turn is a prerequisite for the future development of effective honey bee disease management strategies.

7. Generation of new evidence showing that the adverse effects of gut bacteria disruption by antibiotics on honey bee health could be rescued by the addition of pollen to diets. Gut bacteria play vital roles in the food digestion and immune functions of their animal hosts. And in turn, the food can drive changes in the composition and diversity of gut microbes. ARS scientists in Beltsville, Maryland, showed that antibiotics disrupt gut bacteria, leading to decreased lifespan, altered nutritional metabolism, and suppressed immunity in honey bees. Further, ARS scientists in Beltsville, Maryland, discovered that the pollen in the bees’ diet reverses adverse effects caused by the antibiotic treatment by improving honey bees' metabolic and immune functions. This study provides a deeper understanding of the interconnections among gut bacteria, nutrition and immunity in honey bees and highlights the importance of pollen in sustaining healthy honey bee populations.

Review Publications
Evans, J.D., McKenna, D., Scully, E.D., Cook, S.C., Dainat, B., Egekwu, N.I., Grubbs, N., Lopez, D.L., Lorenzen, M., Reyna, S.M., Rinkevich Jr, F.D., Neumann, P., Huang, Q. 2018. Genome of the small hive beetle (Aethina tumida, Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory. Gigascience. 7(12):1-16. https://doi.org/10.1093/gigascience/giy138.
Huang, S.K., Ye, K.T., Huang, W.F., Ying, B.H., Su, X., Li, L., Li, J.H., Chen, Y., Li, J.L., Bao, X.L., Hu, Z.Z. 2018. Influence of food and Nosema ceranae infection on the gut microbiota of Apis cerana workers. mSystems. https://doi.org/10.1128/mSystems.00177-18.
Li, J., Heerman, M.C., Evans, J.D., Li, W., Rodriguez-Garcia, C., Hoffman, G.D., Zhao, Y., Huang, S., Li, Z., Hamilton, M.C., Chen, Y. 2019. Pollen reverses decreased lifespan, altered nutritional metabolism, and suppressed immunity in honey bees (Apis mellifera) treated with antibiotics. Journal of Experimental Biology. 222:jeb202077. https://doi.org/10.1242/jeb.202077.
Li, J., Wang, T., Evans, J.D., Rose, R., Zhao, Y., Li, Z., Li, J., Huang, S., Heerman, M.C., Rodriguez-Garcia, C., Banmeke, O.A., Brister, R., Cao, L., Hamilton, M.C., Chen, Y. 2019. The phylogeny and pathogenesis of Sacbrood virus (SBV) infection in European honey bees, Apis mellifera. Viruses. 11(1):61. https://doi.org/10.3390/v11010061.
Li, Z., He, J., Yu, T., Chen, Y., Huang, W., Huang, J., Zhao, Y., Nie, H., Su, S. 2019. Transcriptional and physiological responses of hypopharyngeal glands in honey bees (Apis mellifera L.) infected by Nosema ceranae. Apidologie. 50(1):51-62. https://doi.org/10.1007/s13592-018-0617-8.
Li, Z., Yu, T., Chen, Y., Heerman, M.C., He, J., Huang, J., Nie, H., Su, S. 2019. Brain transcriptome of honey bees (Apis mellifera) exhibiting impaired olfactory learning induced by a sublethal dose of imidacloprid. Pesticide Biochemistry and Physiology. 156:36-43. https://doi.org/10.1016/j.pestbp.2019.02.001.
Posada-Florez, F., Sonenshine, D.E., Egekwu, N.I., Rice, C., Lupitskyy, R., Cook, S.C. 2018. Insights into the metabolism and behavior of the honey bee ectoparasitic mite, varroa destructor, from quantitation and chemical analysis of their waste excretions. International Journal for Parasitology. 146(4):527-532. https://doi.org/10.1017/S0031182018001762.
Rodriguez-Garcia, C., Evans, J.D., Li, W., Branchiccela, B., Li, J., Heerman, M.C., Hamilton, M.C., Martin-Hernandez, R., Higes, M., Chen, Y. 2018. Nosemosis control in European honey bees Apis mellifera by silencing the gene encoding Nosema ceranae polar tubule protein 3. Journal of Experimental Biology. 221:jeb184606. https://doi.org/10.1242/jeb.184606.
Zhao, Y., Heerman, M.C., Peng, W., Evans, J.D., Rose, R., Hoffman, G.D., Simone-Finstrom, M., Li, J., Li, Z., Cook, S.C., Su, S., Rodriguez-Garcia, C., Banmeke, O.A., Hamilton, M.C., Chen, Y. 2019. The dynamics of deformed wing virus titer and host defensive gene expression after varroa mite parasitism in honey bees, Apis mellifera. Insects. 10(1):16. https://doi.org/10.3390/insects10010016.
Brown, K., Olendraite, I., Valles, S.M., Firth, A., Chen, Y., Guerin, D., Hashimoto, Y., Herrero, S., De Miranda, J., Ryabov, E. 2019. ICTV Virus Taxonomy Profile: Solinviviridae. Journal of General Virology. 100(5):736-737. https://doi.org/10.1099/jgv.0.001242.
Brown, K., Olendraite, I., Valles, S.M., Firth, A., Chen, Y., Guerin, D., Hashimoto, Y., Herrero, S., De Miranda, J., Ryabov, E. 2019. ICTV virus taxonomy profile: Polycipiviridae. Journal of General Virology. 100(4):554-555. https://doi.org/10.1099/jgv.0.001241.
Stamets, P.E., Naeger, N., Evans, J.D., Han, J., Hopkins, B., Lopez, D.L., Moershel, H., Nally, R., Sumerlin, D., Sheppard, W.S. 2018. Extracts of polypore mushroom mycelia reduce viruses in honey bees. Scientific Reports. 8(1):13936. https://doi.org/10.1038/s41598-018-32194-8.
Wallberg, A., Bunikis, I., Petterson, O.V., Mosbech, M., Childers, A.K., Evans, J.D., Mikheyev, A.S., Robertson, H.M., Robinson, G., Webster, M. 2019. A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds. BMC Genomics. 20:275. https://doi.org/10.1186/s12864-019-5642-0.
Maori, E., Navarro, I., Boncristiani, H., Seilly, D.J., Rudolph, K., Sapetsching, A., Lin, C., Ladbury, J.E., Evans, J.D., Heeney, J. 2019. A secreted RNA binding protein forms RNA-stabilizing granules in the honeybee royal jelly. Molecular Cell. 74(3):598-608.e6. https://doi.org/10.1016/j.molcel.2019.03.010.
Huang, Q., Li, W., Chen, Y., Retschnig-Tanner, G., Yanez, O., Neumann, P., Evans, J.D. 2019. Dicer regulates Nosema ceranae proliferation in honeybees. Insect Molecular Biology. 28(1):74-85. https://doi.org/10.1111/imb.12534.