Research Project: Using Genetics to Improve the Breeding and Health of Honey Bees

Location: Honey Bee Breeding, Genetics, and Physiology Research

2021 Annual Report

Objectives
Meaningful contributions towards enhancing the economic value of the nation’s commercially managed honey bee populations can be achieved through identifying, characterizing and breeding more robust bees. The proposed 5-year plan focuses on synergistic projects (Fig.1) that capitalize on genetic and breeding approaches with the following objectives: Objective 1: Identify and evaluate traits, strains and stocks for improved honey bee health. Sub-objective 1A: Understand the mechanisms of viral transmission and resistance or tolerance to reduce impacts of infection through selective breeding. Sub-objective 1B: Evaluate genotype-dependent nutrient efficiency in commercial honey bee stocks. Sub-objective 1C: Evaluate genotype-dependent nutritional stress resistance in commercial honey bee stocks. Sub-objective 1D: Characterize the impact of genetically based variation in vitellogenin -- the primary honey bee storage protein with roles in immune function, oxidative stress resistance and lifespan -- on colony and reproductive (queen and drone) health and productivity. Sub-objective 1E: Identify and characterize genetic and physiological mechanisms of pesticide resistance in honey bees. Objective 2: Characterize genetic, physiological and behavioral aspects of important traits, strains and stocks. Sub-objective 2A: Examine patterns of genetic diversity and loci under selection in United States honey bee breeding populations, with a focus on stocks exhibiting high VSH activity. Sub-objective 2B: Elucidate the interaction between individual and social immune defenses. Sub-objective 2C: Improve understanding of the biology of the VSH trait. Objective 3: Conduct traditional breeding or marker-assisted selection of honey bees. Sub-objective 3A: Ascertain the effect of inbreeding on genetic diversity across the honey bee genome to support breeding and maintaining health of breeding populations. Sub-objective 3B. Assess genetic diversity at the sex locus of commercial breeding populations of honey bee stocks developed by USDA, ARS HBBGPL. Sub-objective 3C: Determine the potential usefulness of a simple hygiene assay as a selection tool to predict VSH-based mite resistance in honey bee colonies. Objective 4: Develop management tools for improving honey bee health. Sub-objective 4A: Identify and characterize genetic differences in honey bee response to introduced dsRNA, and test for correlations with viral infection and resistance. Sub-objective 4B: Improve understanding of the flight activity of Russian honey bees during almond pollination. Sub-objective 4C: Evaluate the efficacy of a microalgae platform to improve honey bee colony performance and health. Sub-objective 4D: Determine the sublethal effects of fungicides on honey bee health. Sub-objective 4E: Assess sustainability of Varroa control methods.

Approach
Honey bee health is threatened by parasites, pathogens, poor nutrition and pesticides. Breeding robust bees with improved resistance (or tolerance) to threats could mitigate these problems. The project combines diverse approaches and techniques to seek and exploit genotype-dependent responses of honey bees to biotic-, nutrition- and pesticide-related stressors. The project improves understanding of genetic diversity across U.S. commercial stocks, enabling both marker-assisted selection and conservation of genetic resources. This will enhance the effectiveness of contemporary breeding programs. Varroa destructor (hereafter, Varroa) is the greatest threat to bee health worldwide. The project builds on past successes by improving selection efficiency for resistance to Varroa and for elevated colony performance, promoting adoption by beekeepers. Investigations target relationships between genetic diversity across stocks, immune responses, and treatment effectiveness against Varroa, viruses, and other related biotic threats. This is critical because of recent beekeeper reports of miticide- (amitraz-) resistant Varroa. Given the threat from Varroa, the plan outlines novel (Sub-objectives 2B, 4A) and continuing (Sub-objectives 2C, 3C, 4B) research on breeding and management related to Varroa-resistant honey bees. In addition, we also initiate a suite of new studies addressing the negative impact of stressors whose prevalence has increased across managed honey bees in the past decade. These studies will assess differences in genotype-dependent responses to viruses and other pathogens (Sub-objectives 1A, 2B), poor nutrition (Sub-objectives 1B, 1C, 1D, 4C), and pesticides (Sub-objectives 1E, 4D, 4E). The project seeks to improve nutrient assimilation efficiency through breeding. Similarly, genotype-dependent differences in bee responses to pesticides will be targeted for breeding less susceptible bees and reducing queen failures. Biomarkers identified as useful for signaling emerging health threats will be verified, benefitting beekeepers by allowing for rapid corrective intervention. These approaches will capitalize on novel sequencing technologies to examine many of these issues at a higher level of resolution across the honey bee genome (Sub-objectives 2A, 3A, 3B).

Progress Report
This report documents progress of the second year for project 6050-21000-016-00D (Using Genetics to Improve the Breeding and Health of Honey Bees), which began in March 2020. Progress was made by ARS scientists at Baton Rouge, Louisiana in research objectives that fall under National Program 305, Component 2, Bees and Pollination. The goal of this research is to enhance the economic value of the nation’s commercially managed honey bee populations through identifying, characterizing and breeding more robust bees while concurrently informing management practices. ARS scientists at Baton Rouge, Louisiana research regarding identification and evaluation of traits, strains and stocks for improved honey bee health (Obj. 1) progressed with specific developments in experiments related to viral infection, nutrigenomics and susceptibility to pesticides. Progress was made by ARS scientists at Baton Rouge, Louisiana in understanding resistance and tolerance traits associated with viral infection and how honey bees stocks and genotypes differentially respond to Deformed wing virus, Israeli acute paralysis virus and Chronic bee paralysis virus across levels of biological organization (cellular, individual and colony levels) (Obj. 1A). This work involves several projects including collaborative efforts with Louisiana State University, University of Minnesota, University of Alberta, and University of Olomouc (Czech Republic). Progress has also been made by ARS scientists at Baton Rouge, Louisiana toward the development of a novel RNAi delivery system to mitigate honey bee pathogens (in-house) and on initial field trials of chemical-based viral treatments (collaboration with Louisiana State University). In assessments of nutritionally regulated traits for potential future breeding efforts, different honey bee genotypes were assessed by ARS scientists at Baton Rouge, Louisiana for efficiency of food conversion (Obj 1B), proteomic responses to rich and poor nutrient conditions (Obj 1C), and production of the key storage protein vitellogenin (Obj 1D). Results suggest that genotype-dependent nutritional responses are present, with promising implications for honey bee breeding efforts and tailored approaches to diet and health. To aid in understanding the genetic variation in pesticide detoxification capabilities (Obj. 1E), an extensive literature review by ARS scientists at Baton Rouge, Louisiana produced a large database on honey bee pesticide toxicity. ARS scientists at Baton Rouge, Louisiana collaborating with University of Minnesota and Louisiana State University improved understanding of the influences that environmental factors, the nest environment (e.g. amount of propolis deposition) or broader apiary conditions, impart on pesticide exposure and sensitivity. Developments were made by ARS scientists at Baton Rouge, Louisiana in the characterization of genetic, physiological and behavioral aspects of important traits, strains and stocks (Obj. 2). Samples have been sequenced by ARS scientists at Baton Rouge, Louisiana for follow-up work to our earlier large-scale genomic sequencing effort examining genetic diversity across seven commercial honey bees stocks in order to expand those initial results and conduct more detailed analysis on specific genomic regions (Obj 2A). Additional analyses by ARS scientists at Baton Rouge, Louisiana include a collaboration with ARS researchers in Stoneville, Mississippi involving the construction of a honey bee pangenome from research lines and commercially relevant populations and subsequent validation with current and historical samples. ARS scientists at Baton Rouge, Louisiana collaborative work with researchers at the University of Puerto Rico Rio Piedras and Florida International University involves genome assembly of African honey bees (Apis mellifera scutellata), and Puerto Rican honey bees as a representative hybrid population with the aim of characterizing genetic diversity in ancestral and derived populations. A focus on information specifically to mitigate Varroa mites and support various resistance traits in honey bees continued with ARS scientists at Baton Rouge, Louisiana research aimed at identifying upregulated genes in mite-infested pupae targeted for removal by bees performing Varroa sensitive hygienic (VSH) behavior (Obj 2B). ARS scientists at Baton Rouge, Louisiana efforts to support traditional breeding or marker-assisted selection of honey bees progressed (Obj. 3). Genetic diversity at the complementary sex-determiner (csd) locus was assessed by ARS scientists at Baton Rouge, Louisiana in Pol-line and Hilo bees and was determined to be comparable to other selected stocks. ARS scientists at Baton Rouge, Louisiana in collaboration with University of Missouri, a global standardized nomenclature system for honey bee csd alleles was developed and made publicly available in the Hymenopteramine database. (Obj. 3B). Allelic data for csd are currently being used by ARS scientists at Baton Rouge, Louisiana to inform breeding decisions for Pol-line and Hilo stocks. Modernization of the established stock identification assay for Russian honey bees progressed, identifying approximately 200 potentially informative markers to be applied using microfluidics technology. This platform has the strong potential to be used for additional marker-assisted selection assays and csd sequencing going forward. An assay using the chemical ecology that regulates expression of hygienic behavior was tested (Obj 3C), and results indicated that the assay, developed by collaborators from the University of North Carolina at Greensboro, may require further refinement before it can be effectively used as a selection tool for VSH. Work is also ongoing by ARS scientists at Baton Rouge, Louisiana to identify molecular markers related to expression of the trait of VSH, including approaches using candidate genes (in-house), whole-genome sequencing and marker discovery using both gene expression and sequence information via “eQTL” (with the University of Missouri). Breeding for productive, Varroa-resistant bees continues in a public-private partnership in which bees selected by the Unit for Varroa sensitive hygiene form much of the founding population for a new stock, called Hilo Bees. ARS scientists at Baton Rouge, Louisiana research in collaboration with the University of Minnesota and a commercial beekeeper cooperator has continued to clarify the role of propolis in honey bee immunity and its potential benefits in beekeeping management, and to breed bees with improved health, founded on social immunity. Progress was also made by ARS scientists at Baton Rouge, Louisiana in projects related to the development of management tools for improving honey bee health (Obj. 4). As an initial assessment of potential genotypic differences in responsiveness to RNAi-based treatments, the robustness of RNAi pathway response was evaluated by ARS scientists at Baton Rouge, Louisiana for Russian, Pol-line and Saskatraz honey bee stocks (Obj 4A). Evaluation of a new nutritional supplement, ARS scientists at Baton Rouge, Louisiana in collaboration with a commercial beekeeping operation, was conducted to evaluate the use of microalgae as an alternative nutrition source for bees (Obj 4C). Results indicate that this preliminary formulation is comparable to current commercial products, at least with respect to colony survival rates. Additional collaborative efforts with North Carolina State University and beekeeper stakeholders characterized the overall effectiveness of and the metabolic responses to natural and artificial diets used by commercial beekeepers as well as novel microalgae-based diets developed in-house. From a toxicological perspective, preliminary experiments have begun with regard to chlorothalonil toxicity (Obj. 4D). Both technical grade and formulated product yielded very little mortality and no effects on longevity in tests performed in the lab. Experiments are underway by ARS scientists at Baton Rouge, Louisiana to study if chlorothalonil synergizes insecticide toxicity or acts as an inhibitor of detoxification, and how the lab results translate to colony-level exposures. Work on amitraz resistance in Varroa mites (Obj. 4E) has seen significant strides. A second year of resistance testing was able to be completed by utilizing a network of collaborators, and samples are being prepared for genomic analyses. Collaboration with a USDA-ARS scientist (Beltsville, Maryland) has shown that changes in the physical or chemical properties of amitraz do not explain control failures, which are most likely due to amitraz resistance in Varroa mites. This work has led to additional collaborations on this subject with The Ohio State University, Michigan State University and Bee Informed Partnership to greatly expand the scope of the resistance monitoring program. In subordinate projects, research was conducted by ARS scientists at Baton Rouge, Louisiana on the influence of propolis deposition on insecticide sensitivity and detoxification activity in honey bees, and to determine the presence of pesticides in propolis collected from colonies across different landscapes. Two longitudinal field trials of two years each are yielding information about the biotic and abiotic health threats to honey bees in commercial beekeeping operations. These trials were conducted by ARS scientists at Baton Rouge, Louisiana in collaboration with Louisiana State University. Progress was also made in collaboration with North Carolina State University and University of Pennsylvania to clarify the genetic determinants of queen quality.

Accomplishments
1. Commercial breeding improves resistance to parasites in honey bees. ARS scientists at Baton Rouge, Louisiana suggests the parasitic varroa mite is responsible for nearly 50% of annual honey bee colony losses. Widespread breeding for resistance to this mite is a complex process, which has deterred adoption by stakeholders. ARS scientists at Baton Rouge, Louisiana worked collaboratively with beekeepers in Hilo, Hawaii to develop a strategic breeding program designed to encourage use of mite-resistant honey bee lines developed in coordination with ARS to the beekeeping industry. The collaboration resulted in an independent breeding operation in Hilo, Hawaii and mass production of queens from that population. This effort shows that breeding for mite resistance can be implemented at a commercial scale. The roadmap established by this collaboration provides a precedent for other interested beekeeping stakeholders to develop their own breeding operations. Ultimately, breeding for desirable traits, particularly parasite resistance, will improve honey bee health and have a direct impact on global food security.

2. Honey bee genetics influence bee’s response to nutrition. ARS scientists at Baton Rouge, Louisiana believes malnutrition is a major factor underlying honey bee colony declines with poor nutrition continually listed as a top cause of annual colony death. Currently beekeepers provide supplements to improve bee health, but how these diets may differentially help certain bee stocks based on genetic differences is unknown. ARS scientists at Baton Rouge, Louisiana tested the influence of honey bee genetic variation on physiological responses to natural and artificial bee diets. Additional research with ARS scientists at Houma, Louisiana examined how different stocks infected with virus differentially forage for pollen and nectar. Results of each study indicated that stock-dependent nutritional responses are present in honey bees, which has promising implications for new breeding efforts and tailored approaches to diet and health in a changing global climate.

Review Publications
Saelao, P., Simone-Finstrom, M., Avalos, A., Bilodeau, A.L., Danka, R.G., De Guzman, L.I., Rinkevich Jr, F.D., Tokarz, P.G. 2020. Genome-wide patterns of differentiation within and across U.S. commercial honey bee stocks. BMC Genomics. 21:1-12. https://doi.org/10.1186/s12864-020-07111-x.
Black, T.E., Fofah, O., Dinges, C., Ortiz-Alvarado, C.A., Avalos, A., Ortiz-Alvarado, Y., Abramson, C.I. 2021. Effects of aversive conditioning on expression of physiological stress in honey bees (apis mellifera). Neurobiology of Learning and Memory. 178:107363. https://doi.org/10.1016/j.nlm.2020.107363.
Gerdts, J., Roberts, J., Simone-Finstrom, M., Ogbourne, S., Tuccie, J. 2021. Genetic variation of Ascosphaera apis and colony attributes do not explain chalkbrood disease outbreaks in Australian honey bees. Journal of Invertebrate Pathology. 180:107540. https://doi.org/10.1016/j.jip.2021.107540.
Ricigliano, V.A., Dong, C., Richardson, L.T., Donnarummar, F., Williams, S.T., Solouki, T., Murrary, K.K. 2020. Honey bee proteome responses to plant and cyanobacteria (blue-green algae) diets. ACS Food Science and Technology. 1:1-10. https://doi.org/10.1021/acsfoodscitech.0c00001.
Spivak, M., Simone-Finstrom, M. 2019. Propolis. Int: Starr C. (eds) International Union for the Study of Scial Insects Congress. 1-3. https://doi.org/10.1007/978-3-319-90306-4_134-1.
Ricigliano, V.A., Sica, V.P., Knowels, S.L., Diette, N., Howarth, D.G., Oberlies, N.K. 2020. Bioactive diterpenoid metabolism and cytotoxic activities of genetically transformed Euphorbia lathyris roots. Phytochemistry. 179:1-9. https://doi.org/10.1016/j.phytochem.2020.112504.
Bilodeau, A.L., Avalos, A., Danka, R.G. 2020. Genetic diversity of the complementary sex-determiner (csd) gene in two closed breeding stocks of varroa-resistant honey bees. Apidologie. 51(6):1125-1132. https://doi.org/10.1007/s13592-020-00790-1.
Giordano, R., Donthu, R.K., Zimin, A.V., Julca Chavez, I.C., Gabalon, T., van Munster, M., Hon, L., Hall, R., Badger, J.H., Nguyen, M., Flores, A., Potter, B., Giray, T., Sato-Adames, F.N., Weber, E., Marcelino, J. A.P., Fields, C.J., Voegtlin, D.J., Hill, C.B., Hartman, G.L., Akraiko, Ta., Aschwanden, A., Avalos, A., Band, M., Bonning, B., Bretaudeau, A., Chiesa, O., Chirumamilla, A., Coates, B.S., Cocuzza, G., Cullen, E., Desborough, P., Diers, B., DiFonzo, C., Heimpel, G.E., Herman, T., Huanga, Y., Knodel, J., Ko, C., Labrie, G., Lagos-Kutz, D., Lee, J., Lee, S., Legeai, F., Mandriolo, M.,, Manicadi, G.C., Mazzoni, E., Melchiori, G., Micijevic, A., Miller, N., Nasuddin, A., Nault, B.A., O’Neal, M.E, Panini, M., Pessino, M., Prischmann-Voldseth, D., Robertson, H.M., Liu, S., Song, H., Tilmon, K., Tooker, J., Wu, K., Zhan, S. 2020. Soybean aphid biotype 1 genome: Insights into the invasive biology and adaptive evolution of a major agricultural pest. Insect Biochemistry and Molecular Biology. 120:103334. https://doi.org/10.1016/j.ibmb.2020.103334.
Hoffman, Gloria D., Corby-Harris, Vanessa L., Chen, Yanping, Graham, Henry, Chambers, Mona L., Watkins De Jong, Emily E., Ziolkowski, Nicholas F., Kang, Yun, Gage, Stepanhie L., Deeter, Megan E., Simone-Finstrom, Michael, De Guzman, Lilia, I. 2020. Can supplementary pollen feeding reduce varroa mite and virus levels and improve honey bee colony survival?. Experimental and Applied Acarology. 82:455-473. https://doi.org/10.1007/s10493-020-00562-7.
Milone, J.P., Rinkevich Jr, F.D., McAfee, A., Foster, L.J., Tarpy, D. 2020. Differences in larval pesticide tolerance and esterase activity across honey bee (Apis mellifera) stocks. Ecotoxicology and Environmental Safety. 206:111213. https://doi.org/10.1016/j.ecoenv.2020.111213.
Smith, R., Kraemer, F., Bader, C., Smith, M., Weber, A., Simone-Finstrom, M., Wilson-Rich, N., Oxman, N. 2021. A rapid fabrication methodology for payload modules, piloted for the observation of queen honeybee (Apis mellifera) in microgravity. Gravitational and Space Research. 9:104-114. https://doi.org/10.2478/gsr-2021-0008.
Ricigliano, V.A., Ihle, K.E., Williams, S.T. 2021. Nutrigenetic comparison of two Varroa-resistant honey bee stocks fed pollen and spirulina microalgae. Apidologie. 1-14. https://doi.org/10.1007/s13592-021-00877-3.