Location: Honey Bee Breeding, Genetics, and Physiology Research
2024 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
In work related to identification and evaluation of traits, strains and stocks for improved bee health (Objective 1), progress has been made with respect to viral infection (1A) and nutritional responses (1C, 1D). Efforts toward selective breeding for honey bees that are resistant to viral infection (1Ai) continued to focus on drone-based selection in collaboration with a commercial beekeeping operation. Analyses determined that drone responses are consistent within a colony and assessed age-based responses, allowing for increased efficiency in breeding progress. Current work aims to confirm the relationship between drone responses and their sisters (worker honey bees) and offspring to fully assess the viability of drone- based selection. Additional work examined the relationship between viral infection and breeding for disease and mite-resistance via hygienic-based traits (i.e. behavior where adult bees remove sick or mite-infested larvae and pupae from the hive) (1Aii). Genomic sequencing of viral loads of samples collected as part of a large effort in five different geographically distinct beekeeping operations provided insight into viral dynamics in mite-resistant versus mite-susceptible honey bee colonies. Virus work as part of subordinate projects indicated that caged bees show a preference for sugar syrup contaminated with deformed wing virus (DWV), which has implications for viral transmission at floral sources.
To examine how different genetic stocks respond to a resource dearth, colonies from two different stocks (Russian and Italian honey bees) were fit with pollen traps that reduce the amount of incoming pollen have access to, thus compromising their nutrition. Their temperament, colony resources, and colony population were sampled weekly to see if effects of nutritional deprivation are consistent across the genetic backgrounds of colonies. Gene expression analyses were completed from Year 1 of the study, confirming that previously identified aggression genes are differentially regulated in response to the nutritional stress. Year 2 has shown that there is a genetic component to nutritional deprivation in terms of resulting bee temperament, but that the nutritional component overwhelms the genetic component in regard to nosema spore load.
Progress was also made in the characterization of the impact of genetically based variation in vitellogenin (Vg)—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 (1D). Measurements of lines produced via bi-directional selection for high and low Vg were taken including overwintering success, spring buildup and productivity. An additional selection program for Vg allelic variants has begun as part of subordinate international collaborative work. Other collaborative work with the University of Minnesota continued exploring the relationship between Vg expression and swarming behavior.
In collaboration with the Tucson research unit, research was completed examining honey bee stock comparisons in different climates with implications for climate change. Mite-resistant (Pol-line and Russian) and Italian honey bee stocks were compared in variable-temperature cage experiments (200 bees per cage) with respect to food consumption, thermoregulation, gene expression, and lifespan, in three experiments over two years. The Italian stock bees consumed more syrup and pollen on average than the mite-resistant stocks, but the mite-resistant stocks maintained higher cluster temperatures and had median lifespans 8 days longer. Model results indicated that, to maintain the same colony size as the mite-resistant stocks, Italian stock colonies would need about 13% more sealed brood to offset reduced worker lifespans. These differences among bee stocks likely influence colony-level productivity and health, and showed the importance of experimental replication.
Research has continued characterizing genetic, physiological and behavioral aspects of important traits, strains and stocks (Objective 2). Work investigating genetic diversity across the honey bee genome (2A) resulted in the first honey bee pangenome reference tool and its use in the analysis of commercial and research-relevant breeding lines. Research combining laboratory and field assessments of colony and larval susceptibility to disease has indicated that larvae that respond with a stronger physiological immune response are more likely to be from hygienic colonies, suggesting that this immune signal alerts adult bees to remove them (2B). Additional developments include establishment of a “working group” with members of the Russian Honey Bee Breeders Association, academia and non-profits to assess how the traits of the Russian honey bees have changed since their release by the Unit, investigation of barriers to wider adoption of the stock by beekeepers, and development of strategies to recruit new breeders to the association. A case study on a stakeholder’s commercial beekeeping operation using mite-resistant and susceptible stocks showed a high potential for economic gain for using mite-resistant honey bees. Locational differences between apiaries had a greater impact than bee stock in honey production, queen loss was high in both stocks, and the Varroa-resistant bee stock had less Varroa than the non-resistant one. Collaborative work also began with Louisiana State University to determine how selection for mite resistance has affected behavioral development in Russian and Pol-line stocks.
Work to develop traditional breeding and marker-assisted selection (MAS) strategies in honey bees (Objective 3) continues. We conducted research on semen storage and viability, a critical logistic limitation to the application of genomic-based MAS (3A). Direct tests of viability were promising in lab, and efforts continue to examine effective viability in the field. Collaborative studies with Colorado State University included a proof of concept for MAS using metabolic rate since the trait is largely determined by a small set of markers. Results showed that for traits associated with few markers (1-3) integration and use of MAS is possible. Additional collaboration with Breeding Insight continues for more complex traits and panels to reduce the turnaround time for genotyping towards the effective application of MAS in honey bees.
Development of management tools to improve bee health (Objective 4) has progressed in several areas. Work continued to test the utility of microalgae diet nutritional supplements for beekeepers to provide to maintain or encourage colony growth during periods of dearth (4C). A study in cooperation with a commercial beekeeping operation found that colonies supplemented with feed containing the microalgae spirulina had improved brood production and thermoregulation prior to almond pollination relative to unfed controls. Based on these findings, manufactures of honey bee nutrition supplements have developed a commercial product available at the end of July. Progress continued on the development of an edible therapeutic based on genetic engineering of blue-green algae that activates the antiviral response of honey bees and has shown to reduce DWV replication and increase survival of honey bees exposed to the virus in the laboratory.
A study was initiated on the effects of colony boxes that stimulate propolis deposition (antimicrobial plant resins collected by the bees) on Varroa infestation in honey bee colonies (4E). Preliminary evidence suggests that a propolis-enriched environment does reduce mite infestation loads, as suggested by previously published work, but that it only does so in colonies that are susceptible to mites. Russian honey bees and Pol-line honey bee colonies maintained low mite levels regardless of increased propolis in the nest environment. This work is being continued, but shows support for the value of mite resistant honey bees and using alternative equipment that can easily be integrated into operations to reduce impacts of Varroa. A new collaborative, subordinate project to evaluate the use of these propolis-enriched hive boxes with the Michigan State University and a cooperating commercial beekeeper investigated if propolis improves health of honey bee colonies during and post-blueberry pollination.
Research to develop integrated pest management tools for beekeepers to use against the fungal pathogen Ascosphaera apis, which causes chalkbrood, are ongoing in both the field and laboratory. Larger scale laboratory and field experiments include collaborations with University of Florida and University of Minnesota. Work to identify effects of other stressors and tools to combat them include a
collaboration with Mississippi State University to quantify the temporal, directional and epidemiological patterns of
drift (i.e. bees returning to the wrong colony. Additional collaborations with Southern University have been established to test how honey bees of different genetic stocks are impacted by industrialization along the Mississippi corridor.
In collaboration with the Tucson, Arizona research unit, a longitudinal study on queen health in a commercial beekeeping operation was completed. We explored gut microbiota, host gene expression, and pathogen prevalence in honey bee queens overwintering in a warm southern climate. Biologically older queens had larger microbiotas, particularly enriched in Bombella and Bifidobacterium. Both DWV-A and B were highest in the fat body tissue in January, correlating with colony Varroa levels and worker DWV titers. High viral titers in queens were associated with decreased Vg expression, suggesting a potential trade-off between immune function and reproductive capacity. Overall, our findings highlight the intricate interplay between pathogens, metabolic state, and immune response in honey bee queens.
Accomplishments
1. Novel feed additives improve artificial diets for honey bees.. Beekeepers rely on providing honey bee colonies with supplemental diets so that they can be productive, especially going into winter and to fulfill pollination services. ARS researchers at Baton Rouge, Louisiana found that microalgae-based feed additives are an effective and sustainable feed additive for managed honey bees involved in agricultural pollination. A series of laboratory experiments and trials in commercial beekeeping operations have been completed showing that the microalgae-based diets improve colony size and that the nutritional content is similar to that of pollen. The beekeeping industry has begun translating our research into commercial products including a new microalgae-containing bee food product released in July 2024. Further optimization of bee diets will improve feed sustainability and agricultural pollination efficiency by supporting larger, healthier honey bee colonies.
2. Edible treatments to improve honey bee virus resistance.. Honey bees are regularly infected with multiple viruses that reduce colony productivity and can lead to death of the colony. However, no antiviral treatments are currently available to beekeepers. A novel, RNA interference (RNAi)-based treatment has been developed using edible blue-green algae that have been genetically engineered to deliver therapeutic double-stranded RNAs (dsRNAs) to honey bees via feeding. Once consumed, the dsRNAs are released into the bee gut and trigger a sequence-specific RNAi response, targeting viral pathogens. Treatments targeting deformed wing virus—a notorious pathogen—suppressed viral infection and improved survival in honey bees. This design presents a versatile and sustainable therapeutic that can be directly incorporated into supplemental feeds for managed pollinators to mitigate viruses and support global food security.
3. Genomic advances improve global understanding of honey bee genetics.. Using improved sequencing methods and genome assembly methods the first honey bee (Apis mellifera) commercial and research pangenome has been developed. This tool unifies genetic variation from six key honey bee populations into a common reference allowing for the robust characterization of honey bee genetic diversity and the novel ability to identify and catalogue larger structural variants (e.g. indels, duplications, etc.). This novel tool represents a method by which to increase our understanding of genetic variation and improve breeding tools to allow the beekeeping industry to more rapidly and effectively select for specific traits. ARS researchers at Baton Rouge, Louisiana future work will focus on expanding the robustness of the tool by developing a pangenome that includes worldwide honey bee genetic representation, and will provide a foundation for all genomic work in honey bees.
Review Publications
Lang, S., Simone-Finstrom, M., Healy, K. 2023. Effects of honey bee queen exposure to Deformed wing virus-A on queen and juvenile infection and colony strength metrics. Journal of Apicultural Research. https://doi.org/10.1080/00218839.2023.2284034.
Rinkevich Jr, F.D. 2024. Temperature, strip age, exposure surface area affect the outcomes of testing for amitraz resistance in Varroa destructor. Journal of Apicultural Research. https://doi.org/10.1080/00218839.2024.2314420.
Shanahan, M., Simone-Finstrom, M., Tokarz, P.G., Rinkevich Jr, F.D., Read, Q.D., Spivak, M. 2024. Thinking inside the box: restoring the propolis envelope facilitates honey bee social immunity. PLOS ONE. https://doi.org/10.1371/journal.pone.0291744.
Ewert, A.M., Simone-Finstrom, M., Read, Q.D., Husseneder, H., Ricigliano, V.A. 2023. Effects of ingested essential oils and propolis extracts on honey bee (Hymenoptera: Apidae) health and gut microbiota. Journal of Insect Science. 23/6. https://doi.org/10.1093/jisesa/iead087.
Lu, R.X., Bhatia, S., Simone-Finstrom, M., Rueppell, O. 2023. Quantitative trait loci mapping for survival of virus infection and virus levels in honey bees. Infection, Genetics and Evolution. 116. https://doi.org/10.1016/j.meegid.2023.105534
Ricigliano, V.A., Mcmenamin, A., Martin, A.M., Adjaye, D.F., Simone-Finstrom, M., Rainey, V.P. 2024. Green biomanufacturing of edible antiviral therapeutics for managed pollinators. NPJ Sustainable Agriculture. 2:Article4. https://doi.org/10.1038/s44264-024-00011-7.
Gomes Viana, J., Avalos, A., Zhang, Z., Nelson, R., Hudson, M. 2024. Common signatures of selection reveal target loci for breeding across soybean populations. The Plant Genome. e20426. https://doi.org/10.1002/tpg2.20426.
Rinkevich Jr, F.D., Danka, R.G., Rinderer, T.E., Margotta, J., Bartlett, L., Healy, K. 2024. Relative impacts of varroa infestation and pesticide exposure and their effects on honey bee colony health and survival in a high-intensity corn and soybean producing region in northern Iowa. Journal of Insect Science. Vlume 25 Issue 3 Page 18. https://doi.org/10.1093/jisesa/ieae054.
French, S.K., Pepinelli, M., Conflitti, I.M., Higo, H., Common, J., Bixby, M., Walsh, E.M., Guarna, M.M., Pernal, S.F., Hoover, S.E. 2024. A systems approach to honey bee health. Current Biology. Volume 34 Page 1893-1903. https://doi.org/10.1016/j.cub.2024.03.039.
McMenamin, A., Weiss, M., Meikle, W.G., Ricigliano, V.A. 2023. Efficacy of a microalgal feed additive in commercial honey bee colonies used for crop pollination. ACS Agricultural Science and Technology. 3(9):701-834. https://doi.org/10.1021/acsagscitech.3c00082.
