Project : USDA ARS

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

Location: Honey Bee Breeding, Genetics, and Physiology Research

2022 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 third year for project 6050-21000-016-000D (Using Genetics to Improve the Breeding and Health of Honey Bees), which began in March 2020. Progress was made 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 also informing management practices. For research related to identifying and evaluating traits, strains, and stocks for improved bee health (Obj 1), developments across several projects were made and findings often highlighted the continued need to integrate bee genotype with external factors (environment, social, etc.) to fully evaluate traits. Projects done in collaboration with stakeholders provide key examples, such as a recent analysis of performance of the mite-resistant Pol-line population, developed in Baton Rouge, Louisiana, in commercial beekeeping. The study also identified major determining factors that lead to colony death during migratory beekeeping, such as viral infections. A series of collaborative studies with ARS researchers in Houma, Louisiana, and Beltsville, Maryland, examined how different stocks respond to viral infections in the laboratory. Results from each study highlighted the importance of evaluating the interaction between genetic background (bee stock) and viral strains, as there are variable outcomes when different bee stocks and different types of viruses interact (Obj 1A). This was evident in terms of how viral infections spread throughout bee tissues after infection and how viral infections alter diet and foraging choices. In other viral work, a new collaboration was developed with a stakeholder and conducted bi-directional selection for viral susceptibility based on drone responses to Deformed wing virus (Obj 1A). This breeding effort combined with ongoing work with collaborators from the University of Alberta should lead to identifying genetic markers associated with antiviral resistance. Complementarily, additional projects include development of novel RNAi-based delivery systems to mitigate the effects of honey bee viruses that combine nutritional supplementation with antiviral treatments. Other drug-based antiviral field testing is being conducted with collaborators at Louisiana State University. Projects in Obj 1 also advanced our knowledge of how different genetic stocks responded to periods of nutritional stress. Colonies of three different stocks of bees (Italian, Pol-line, or Russian) were evaluated in two different geographic locations (Louisiana and Arizona) under nutritionally restricted conditions in collaboration with ARS researchers in Tucson, Arizona. Results indicated that geographic location plays a large factor in stock-related performance metrics (Obj 1C). Basic research into nutritional mechanisms has provided a pathway to improve honey bee health and resiliency through a new breeding program for enhanced vitellogenin (Vg) production. Vg is a key storage protein in honey bees that is associated with longevity and overwintering success. Initial bi-directional selection for high and low Vg lines has been completed and continued selection is ongoing to develop these lines further (Obj 1D). In addition, assessment of more than 150 queens from over 10 different stakeholders have been examined with a focus on pesticide sensitivity finding both within and between stock variation. Enzymatic activity evaluation and DNA sequencing efforts are ongoing using bees from the 6 stocks tested (Obj 1E). Efforts toward characterizing genetic, physiological, and behavioral aspects of important traits, strains, and stocks (Obj 2) was made via enhanced and strengthened collaborative efforts. A honey bee pangenome from research lines and commercially relevant populations is being made to catalog genetic diversity in the U.S. (Obj 2A). Baton Rouge is leading this effort as part of a collaborative project with ARS units in Stoneville, Mississippi; Beltsville, Maryland; and Hilo, Hawaii. Component reference genomes are currently being generated as part of the development process. Together with complementary population sequencing, the project will result in the most complete catalogue of genetic variation within U.S. honey bee populations. For one specific stock, the Russian honey bee population, genetic diversity data analysis has been conducted showing healthy breeding populations and will be used to inform breeding decisions for the Russian honey bee stock managed by the Russian Honey Bee Breeders Association. A collaboration with the University of Illinois resulted in a gene network analysis that was used to refine genetic markers associated with traits of interest. While much focus has been on characterizing genetic aspects of traits, recent progress on identifying behavioral aspects related to parasite resistance in stocks, highlighted our need to further evaluate our stocks. One defense that the original host of the damaging, invasive parasitic varroa mite uses to prevent it from killing its colonies is a behavior called social apoptosis. It is a defense where mite-infested pupae rapidly die and thus prevent the mite from becoming a colony-wide threat. Research found that Russian honey bee pupae infested with mites have decreased survival suggesting they may exhibit this trait as one of their mechanisms of mite resistance (Obj 2B). Work related to traditional breeding and marker-assisted selection of honey bees (Obj 3) continued to develop. To quantify the effects of constrained breeding on population health, colonies were setup, monitored, and bred for subsequent generations to study effects on inbreeding depression focused on targeted areas of reduced genome diversity (Obj 3A). One genomic area of interest has been the complimentary sex determiner (csd) locus. Genetic analysis of csd of the mite resistant Russian honey bee population was completed, providing general characterization of the overall health and strength of the breeding population (Obj. 3B). Work with the University of Missouri to identify molecular markers directly correlated with the Varroa Sensitive Hygiene (VSH) trait has continued. The project implements candidate genes (in-house) analysis and “eQTL”, an approach that uses gene expression and whole genome sequencing to discover markers with a high degree of certainty. Breeding for productive, Varroa-resistant bees has also continued in a public-private partnership in which bees selected by the Unit for VSH formed much of the founding population in the development of a stock called Hilo Bees. Research in collaboration with the University of Minnesota, conducted in a commercial beekeeping operation, has shown the benefits of propolis in beekeeping management with colonies kept in rough boxes with more propolis having more stable immune responses and larger populations in almonds. Lastly, projects involving development of management tools for improving honey bee health (Obj 4) advanced. Significant developments were made toward evaluating the efficacy of microalgal diets for improving honey bee health (Obj 4C). A large-scale field trial testing a microalgae-based artificial diet was conducted in a commercial beekeeping operation located in California. Colony performance, health biomarkers and thermoregulatory behavior was monitored in hives fed microalgae or a commercial protein supplement and unfed hives were used as a control. Hives fed microalgae produced more brood relative to the protein supplement at one of the apiary sites tested. In general algae-fed hives showed improved performance relative to unfed hives. Overall nutritional supplementation improved brood area thermoregulation. In collaboration with scientists at the University of North Carolina at Greensboro, further research aimed to determine the utility of artificial microalgal based diets for honey bees focusing on fine-scale measures. Results indicated that microalgae have potential as sustainable bee feed additives and health-modulating natural products. Metabolomics-guided diet development could eventually help tailor feed interventions to achieve precision nutrition in honey bees. Additional work with collaborators at University of Olomouc, Czech Republic explored alternative algal species for supplemental diets in honey bees. Experiments have been conducted to determine the sublethal effects of fungicides on colony health (Obj 4D). Both technical grade and formulated chlorothalonil showed synergism, antagonism, or no effect on insecticide sensitivity. This project expanded to include collaboration with Mississippi State University to test effects on the royal jelly fed by nurse bees to developing queen larvae to fully assess any sublethal effects of fungicide exposure on the full queen developmental process. In addition, colony level experiments are assessing the effects of chlorothalonil on queen health and colony performance and colonies are being monitored through spring of FY 23. Work on amitraz resistance in varroa mites (Obj. 4E) is also a major research focus and a major concern for the beekeeping industry as cases of resistance have been noted in the field. Expansion of the amitraz resistance monitoring network in 2021 allowed for tests to be performed on 426 colonies across 34 apiaries managed by 24 beekeepers. A survey on amitraz use patterns and colony management was conducted in collaboration with researchers at Auburn University to identify factors that lead to amitraz resistance in varroa mites. Genomic analyses are being conducted to identify what genes are involved in amitraz resistance in varroa mites. DNA tests to detect resistance in pooled collections of varroa mites are being developed in collaboration with researchers at University of Maryland and University of Valencia in Spain.

Accomplishments
1. Improving artificial diets for honey bees. Honey bee colonies managed for agricultural pollination are highly dependent on human inputs, especially for supplemental nutrition. Hives are routinely fed artificial pollen substitute diets to compensate for insufficient pollen forage in the environment. ARS researchers in Baton Rouge, Louisiana, conducted a large-scale field experiment in collaboration with a commercial beekeeper to test the effects of different artificial diets on commercial honey bee colony performance. The results indicated that diet efficacy was correlated with essential amino acid ratios, which will help to inform industry regarding the development of improved bee feed. Additional work carried out by ARS scientists in Baton Rouge, Louisiana, in collaboration with University of North Carolina Greensboro applied metabolomic analyses to better understand the impact of novel microalgae-based artificial diets developed at the Baton Rouge location. The metabolomics results are useful to understand mechanisms underlying favorable growth performance and health characteristics in bees fed the microalgae diets. Metabolomics-guided diet development can help tailor feed interventions to achieve precision nutrition in honey bees and other livestock animals.

2. Testing and finding new traits of resistance to damaging mite pests in honey bees. Honey bee colonies face a variety of parasites and pathogens that result in significant annual losses by beekeepers and management costs. Breeding efforts for traits of resistance and a greater understanding of how resistant populations prevent damaging infestations, particularly from the damaging varroa mite, are of the utmost importance for the sustainability of the beekeeping industry. Research conducted by ARS researchers in Baton Rouge, Louisiana, in collaboration with Louisiana State University and a commercial beekeeper tested the functionality of mite-resistant Pol-line bees. The Pol-line stock expresses a high level of resistance to the varroa mite based on a trait called Varroa Sensitive Hygiene which results in the inability of mites to reproduce in a colony and transmit fewer viruses, as shown by this study. The Pol-line bees were found to be highly productive in the commercial migratory operation and were more successful through almond pollination than the control bees that the beekeeper would have normally relied on. While Pol-line rely on VSH behavior, other stocks like the Russian honey bees express multiple traits of resistance that are still being understood. Research has recently identified that Russian honey bees exhibit, social apoptosis, where varroa mite infested pupae die more quickly, and thus can prevent mites from successfully reproducing and producing offspring on pupae. These results shed light on how important it is to fully evaluate stocks to enhance resistance traits and also that breeding for multiple traits of resistance can provide robust support against parasites like varroa mites.

U.S. DEPARTMENT OF AGRICULTURE

Honey Bee Breeding, Genetics, and Physiology Research: Baton Rouge, LA