Location: Honey Bee Breeding, Genetics, and Physiology Research
2023 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
As part of identification and evaluation of traits, strains and stocks for improved bee health (Objective 1), efforts have continued to focus on viral infection (1A), nutritional responses (1B, 1C, 1D) and pesticide sensitivity (1E). The selective breeding effort to produce honey bees that are resistant to viral infection (1Ai) has shifted focus by using drone susceptibility to DWV inoculation as the target for selection. This has been done in coordination with a commercial beekeeping partner who has documented virus resistance in their population along with the Pol-line stock developed by the Unit. Investigation into colony-level responses and how breeding for disease and mite-resistance influences viral infection has also expanded through a collaborative study with commercial beekeeping partners. Data analysis is also ongoing to identify if the Hilo bee population, highly selected for Varroa Sensitive Hygienic (VSH) behavior, is distinct from stock that are susceptible to the parasitic Varroa mite across a geographic transect of five different environmental locations and a temporal transect of 3 years (1Aii and subordinate project). Other subordinate projects related to Obj 1A include determinations on how viral infection impacts bee vision and foraging choices, a follow-up to previous research that indicated that viral infection altered diet choice and type of resources that foraging bees collected.
The breeding potential for reduced susceptibility to pesticide exposure in honey bees was evaluated. Sensitivity to phenothrin, chlorpyrifos, and clothianidin was evaluated from 11 honey bee genetic lines. There was little variation in insecticide sensitivity (<3-fold) among these stocks, indicating minimal variation to select for insecticide resistant lines. Additional exploratory selection work was conducted through collaborative work with Agriculture and Agri-Food Canada. Data was curated and analyzed from the three year chalkbrood-stock experiment. Honey bee stock variation in chalkbrood infections correlated with hygienic behavior and in some stocks correlated with propolis production. Molecular work to determine the relationships between asymptomatic and symptomatic infections is ongoing.
Another project aimed to evaluate the potential of susceptibility to pesticide exposure could be a trait for breeding selection in honey bees. Sensitivity to phenothrin, chlorpyrifos, and clothianidin was evaluated from 11 honey bee genetic lines. There was little variation in insecticide sensitivity (<3-fold) among these stocks indicating minimal variation to select for insecticide resistant lines. Additional exploratory selection work was conducted under collaborative work with Agriculture and Agri-Food Canada. Data was curated and analyzed from the three year chalkbrood-stock experiment. Some stocks are more resistant to chalkbrood than others, this correlated with hygienic behavior and sometimes correlated with propolis production in this study. Molecular work to determine the relationships between asymptomatic and symptomatic infections is ongoing.
Progress has continued characterizing genetic, physiological and behavioral aspects of important traits, strains and stocks (Objective 2). In terms of genetic characterization, the Unit has led an inter-Agency collaboration to develop the first pangenome for honey bees while simultaneously applying this tool in the analysis of genetic diversity across research and commercial honey bee breeding lines (Obj 2A). This effort has also directly led to an international collaboration to develop a world-wide honey bee pangenome that incorporates genetic representation from all known honey bee subspecies.
Assessment of how social immunity or colony-level behavioral defenses interact with individual bee physiological defenses determined that there was no trade-off of among the defenses measures (grooming, resin collection, hygienic behavior, larval immunity), but that a colony’s level of hygienic behavior (tendency to remove dead or infected larvae and pupae) was positively associated with the immune response of larvae (2B). This finding provides further support for the hypothesis that larvae and pupae may be signaling to indicate their health status. Additionally, results indicate that selection for one social immune trait does not reduce a colony’s expression of another trait, so selection for multiple traits of resistance should be a high priority. Studies also described volatile chemicals released by Varroa-resistant stocks subject to hygienic uncapping (2C). Complementary research was developed as an international collaboration with researchers in Argentina to examine parallel selection programs for Varroa Sensitive Hygienic (VSH) behavior. Phenotypic data contrasting US and Argentinian populations and a model was developed to more fully capture the entire set of information gained during the phenotyping process. Efforts will eventually lead to an examination of the genetic architecture of the trait in the Argentinian population and assess how broadly distributed the VSH trait may be and the consistency of the underlying genetic architecture mediating it.
To examine the influence of nutritional status on colony behavior, Pol-line colonies were fit with pollen traps that reduce the amount of incoming pollen and thus nutrition colonies have access to. Their temperament, colony resources, and colony population were sampled weekly. Colonies that had pollen trapped had higher aggressive scores than colonies which were allowed to keep the pollen they collected. Continued work will focus on additional stocks to see if effects of nutritional deprivation are consistent across the genetic backgrounds of colonies.
Specific progress related to Objective 3, with the goal of conducting traditional breeding or marker-assisted selection of honey bees has been made through subordinate projects. Allele-based breeding for predicted functional variants of the multifunctional gene vitellogenin began as a test case for neural network predicted marker assisted selection. Biomarkers for health and resilience have been identified and validation initiated through work with Agriculture and Agri-Food Canada and other Canadian partners.
Development of management tools to improve bee health (Objective 4) has progressed in several areas. Research has completed on effects of chlorothalonil on colony performance. Colonies treated with chlorothalonil experience higher rates of colony losses due to higher Varroa infestation. For an examination of how different stocks may be managed differently for pollination services (4B), the foraging rates of Russian, Pol-Line, and unselected stocks were compared in almond orchards in California using hive monitoring technology. Data was collected on honey bee flight activity, colony weight, adult bee populations, and pollen collection loads.
Amitraz resistant Varroa were subject to non-amitraz miticide applications to evaluate treatment success in collaboration with Auburn University (4E). This study also included the first evaluation of Varroa non-reproduction in amitraz resistant Varroa. Additionally, as part of a subordinate project, the bee toxicity of a potential new miticide to control Varroa was assessed. Research complementary to the broader goal of Obj. 4E was developed to examine the genetic mechanisms of resistance to control in the Varroa mite. The project aims to provide a US-wide catalogue of genetic variation of Varroa as a critical pest of honey bees while simultaneously conducting genome-wide association studies focusing on resistance to the primary mite control method (Amitraz). Results are expected to outline the key mutation(s) conferring resistance to this control method, opening a potential avenue to address this limitation.
A number of subordinate projects related to management tools also progressed. A final patent disclosure was submitted for “Engineered microalgae to improve honey bee pathogen resistance and nutrition.” Expanding on this work, an RNAi treatment against Varroa mites using engineered microalgae is currently being tested. Work to improve artificial diets for honey bees has begun by characterizing feeding stimulants in pollen with the aim of recapitulating those effects in artificial diets. This has major implications for the beekeeping industry, where the use of artificial diets has become commonplace due to climate change and agricultural intensification. Current research focused on evaluating the impacts of bacterial extracts on honey bee toxicity in collaboration with Johns Hopkins University. These bacterial extracts may be harmful to bees when it is the only food source they are fed, but bee mortality is reduced with presented with alternative food sources and bees learn to avoid food containing bacterial extracts. Additional studies include the effects of colony boxes that stimulate propolis deposition on Varroa infestation in 3 stocks of honey bees. Lastly, to improve reliability of honey bee queen insemination process, tests have been conducted to on the viability of honey bee semen stored for long periods of time under heat stress.
Accomplishments
1. Genetic mutation and beekeeper management predict mite resistance to miticide. The parasitic Varroa mite significantly impacts honey bee health and is the largest driver of colony mortality. Recently, reports indicate that chemical control of the mites by the most widely used treatment, Amitraz, has been inconsistent. How often and why the mites are developing resistance is key to improve treatment decisions. Mites from more than 1,382 colonies from 146 apiaries across 82 beekeeping operations have been sampled. Each beekeeping operation has its own resident Varroa population with Amitraz resistance levels likely to be driven by amitraz use patterns. Highly resistant Varroa tends to be the result of overuse (intensity and frequency) and overreliance on Amitraz as the sole measure of Varroa control. Evidence also indicates that Amitraz resistant Varroa do not move passively among beekeeping operations, even in close proximity and at high colony density. Through an international effort, a genetic mutation strongly associated with amitraz resistance in Varroa in the U.S. has been confirmed. This research is of importance to the beekeeping industry as the reduced efficacy of amitraz to control Varroa is increasing demand for alternative miticides and Varroa management practices. Results have also informed beekeepers of the need to change management practices by rotating types of miticide treatment to reduce the likelihood of resistance developing in their population.
2. Nutritional deprivation increases honey bee aggression and alters how they process food. Honey bee colonies are routinely provided with nutritional supplements during periods of drought and associated food shortages. In a changing climate, these events may increase in intensity and frequency, or occur at unexpected intervals. Understanding how bees respond to challenging nutritional times critically determines management strategies and breeding tactics. When colonies were deprived of pollen, they became more aggressive, overall, than colonies that had access to pollen resources. Temperament and aggression are sometimes used to select breeding stock in rearing operations, so these findings are important in light of breeding initiatives in our changing world. Additional research identified that honey bee stocks derived from Italian and Russian honey bees respond to nutritional deprivation in distinct ways. Russian honey bee workers appear to conserve individual nutritional stores at the expense of producing nutrient rich brood food components. Conversely, Italian honey bee workers continue production of brood food components at the expense of nutrient stores. This has potential implications for traits that are beneficial in a changing climate and informs beekeepers about when and how to use nutritional supplements based on the type of honey bee.
3. Using advanced technologies to improve honey bee breeding and stock-specific selection. The Russian honey bee stock was the first population where use of a marker-based, genetic stock identification (GSI) assay was applied in breeding. Recent developments in sequencing have increased our ability to understand, evaluate and use genetic tools to advance breeding efforts. An updated stock identification panel using novel genetic variation would provide a more accurate discrimination tool and serve as a pilot for future marker panels. Using recent genomic data and historically preserved DNA, a new marker panel was developed using a microfluidic platform. The resulting assay outperformed the original GSI while simultaneously increasing efficiency and reducing cost. This novel approach is currently being finalized to incorporate in stakeholder breeding decisions. In addition, the method is inherently modular and has pioneered analytical approaches that can be directly implemented in trait-based or other marker-assisted selection panels.
4. New antiviral drug target identified in honey bees and field test completed. Honey bees contend with many parasites and pathogens that greatly impact colony productivity and survival. Viral infections, either transmitted by the parasitic Varroa mite or spread from bee to bee, cause both direct and subtle effects on bee behavior and lifespan. Despite the fact that honey bees are often co-infected with multiple viruses at both the individual bee and colony levels, there are no antivirals available for beekeepers. In collaboration with Louisiana State University, a compound was tested both in the laboratory and under field conditions that activates honey bee potassium ion channels. By regulating these channels, the drug increases a honey bee’s ability to fight infection of multiple viruses. Field tests feeding this drug to virus-inoculated colonies confirmed laboratory results and for the first time showed that pharmacological treatment against viruses is possible for honey bee colonies. This research identified a physiological target to focus on in honey bees that can be used to develop a drug treatment that could be approved to improve honey bee resilience to viral infection. As there are no treatments currently available against honey bee viruses, finding ways to reduce the impacts of virus in honey bees is a critical need to reduce potential virus spread to other bee species and increase the sustainability of the beekeeping industry.
Review Publications
Dostalkova, S., Kodrik, D., Simone-Finstrom, M., Petrivalsky, M., Danihlik, J. 2022. Fine-scale assessment of Chlorella syrup as a nutritional supplement for honey bee colonies. Frontiers in Ecology and Evolution. 10(1028037):1-12. https://doi.org/10.3389/fevo.2022.1028037.
Rinkevich Jr, F.D., Moreno-Marti, S., Hernandez-Rodriguez, C.S., Gonzalez-Cabrera, J. 2023. Confirmation of the Y215H mutation in the ß2-octopamine receptor in Varroa destructor is associated with contemporary cases of amitraz resistance in United States. Pest Management Science. pp.1-6. https://doi.org/10.1002/ps.7461.
Dickey, M., Walsh, E.M., Shepherd, T.F., Medina, R.F., Tarone, A., Rangel, J. 2023. Transcriptomic analysis of the honey bee (Apis mellifera) queen brain reveals that gene expression is affected by pesticide exposure during development. PLOS ONE. 18(4):e0284929. https://doi.org/10.1371/journal.pone.0284929.
Penn, H., Simone-Finstrom, M., De Guzman, L.I., Tokarz, P.G., Dickens, R.D. 2022. Viral species differentially influence macronutrient preferences based on honey bee genotype. Biology Open. 11(10):bio059039. https://doi.org/10.1242/bio.059039.
Fellows, C., Simone-Finstrom, M., Anderston, T., Swale, D. 2023. Potassium ion channels as a druggable target to inhibit viral replication in honey bees. Virology. 20(134):1-17. https://doi.org/10.1186/s12985-023-02104-0.
Ihle, K.E. 2023. Genetic stock affects expression patterns of the multifunctional gene Vitellogenin in honey bee workers. Journal of Apicultural Research. p. 1-3. https://doi.org/10.1080/00218839.2023.2166231.
Traniello, I.M., Bukhari, S.A., Dibaeinia, P., Serrano, G., Avalos, A., Ahmed, A.C., Sankey, A., Hernaez, M., Sinha, S., Zhao, S.D., Catchen, J., Robinson, G.E. 2023. Single-cell dissection of a collective behaviour in honeybees. Nature Ecology and Evolution. 2:1-25. https://doi.org/10.3389/finsc.2022.998310.
Avalos, A., Bilodeau, A.L. 2022. Russian honey bee genotype identification through enhanced marker panel set. Frontiers in Insect Science. 2:998310. https://doi.org/10.3389/finsc.2022.998310.
Nichols, B.J., Ricigliano, V.A. 2022. Uses and benefits of algae as a nutritional supplement for honey bees. Frontiers in Sustainable Food Systems. 6:1005058. https://doi.org/10.3389/fsufs.2022.1005058.
