Research Project: Genetics and Breeding in Support of Honey Bee Health

Location: Honey Bee Breeding, Genetics, and Physiology Research

2020 Annual Report

Objectives
The overall goal of this research is to develop and use genetic resistance or tolerance of honey bees to biotic agents, and to devise management strategies to improve the quality of queen honey bees. This will enhance the economic value of the nation’s honey bees for pollination, honey production, overwintering and hazard resistance. Over the next five years we will focus on interrelated projects with the following objectives: Objective 1: Identify and evaluate traits, strains, and stocks for improved honey bee health, e.g., improved immunity, tolerance, or resistance to Varroa and tracheal mites, the fungi Nosema and chalkbrood, and viruses. Sub-objective 1A: Evaluate the potential for viral resistance. Sub-objective 1B: Establish and characterize genetically and functionally distinct lines that differentially respond to Nosema. Sub-objective 1C: Evaluate the potential of worker brood to suppress reproduction by Varroa. Sub-objective 1D: Determine the effect of Varroa infestations on honey bee behavior. Sub-objective 1E: Evaluate immune and stress related responses in selected honey bee stocks. Objective 2: Characterize genetic and physiological aspects of important traits, strains, and stocks and their interaction with biotic and abiotic stressors. Sub-objective 2A: Characterize the chemical ecology of VSH to aid the development of a practical selection method. Sub-objective 2B: Understand the genetic basis of Ascosphaera apis (chalkbrood) resistance observed in RHB larvae. Sub-objective 2C: Genomic sequencing of multiple stocks of honey bees. Sub-objective 2D: Differential responses to Nosema in honey bees. Objective 3: Conduct traditional and marker-assisted breeding and develop management tools for improved bees. Sub-objective 3A: Develop and use improved methods to evaluate Varroa-resistant phenotypes. Sub-objective 3B: Determine management methods using ionizing radiation to increase honey bee colony fitness. Sub-objective 3C: Evaluate autogrooming as a common resistance mechanism towards tracheal and Varroa mites. Sub-objective 3D: Use traditional and marker-assisted selection programs to improve honey bees with VSH-based resistance. Funding will be used in support of Objective 3: Conduct traditional and markerassisted breeding and develop management tools for improved bees. Objective 4: Improve knowledge of the biology, physiology, genomics, and behavior of pests (i.e. mites and the small hive beetle) that may be useful for improving their management. Sub-objective 4A: Elucidate the Nosema infection process. Sub-objective 4B: Understand effects of resistance level on Nosema infection across honey bee life stages. Sub-objective 4C: Identify new methods to limit SHB population growth in honey bee colonies.

Approach
The health and availability of honey bees (Apis mellifera) have been diminished by multiple biological problems, most notably the parasitic mite Varroa (V.) destructor. Other important threats include fungi (Nosema spp. and chalkbrood), viruses, small hive beetles and tracheal mites. Some of these interact and result in mortality described as colony collapse disorder (CCD). The major goal of this project is to mitigate these threats by finding and selecting for genetically based traits of honey bees that confer resistance to the biological problems. Scientists also will improve management techniques and describe pest biology related to control. Most research targets V. destructor. Efforts that build on our prior work with Varroa-resistant honey bees include using knowledge about Russian Honey Bees to find new traits that enhance resistance, and to improve selection methods. Honey bees with Varroa sensitive hygiene are undergoing traditional and molecular-markerassisted selection. Marker-assisted selection would be a valuable tool to overcome the difficulty of phenotyping this trait, and increase the capacity to select and breed Varroa-resistant bees. Novel traits for Varroa resistance also are being sought. New selection of honey bees will target Nosema ceranae and Deformed Wing Virus because these pathogens are thought to contribute significantly to colony losses. Research with Nosema, viruses, tracheal mites and chalkbrood will culminate in development of molecular markers for resistance factors. Management research will expand promising preliminary findings about queen and drone health associated with the irradiation of comb in mating colonies. The products of this research -- knowledge and technology -- will strengthen integrated pest management strategies for controlling honey bee pests and diseases. This in turn should lead to better profitability for beekeeping and crop pollination.

Progress Report
This is the final report of a bridge project (6050-21000-015-00D) that ended when a new Project Plan (6050-21000-016-00D; “Using Genetics to Improve the Breeding and Health of Honey Bees) was initiated in March 2020. Progress was made in research objectives that fall under National Program 305, Objective 2, Bees and Pollination. ARS researchers at Baton Rouge, Louisiana, continued research on genetics and genomics of honey bees. Analysis of the genetic diversity of seven commercial honey bee stocks found strong genetic similarity among the populations except for Pol-line bees, a stock which has undergone strong selective pressure in a breeding program, and Hilo Bees, a commercial stock derived from Pol-line. Diversity at the complementary sex-determiner locus (csd) was assessed in Pol-line and Hilo bees. Allelic data are currently being used to inform breeding decisions for both stocks. ARS researchers at Baton Rouge, Louisiana, worked to identify molecular markers related to expression of the trait of varroa sensitive hygiene, including approaches using candidate genes (in-house), whole-genome sequencing and marker discovery (in collaboration with the Institut National de la Recherche Agronomique, France) and both gene expression and sequence information via “eQTL” (in collaboration with the University of Missouri). Mite-associated viruses received significant attention. Progress was made by ARS researchers at Baton Rouge, Louisiana, in understanding resistance and tolerance traits associated with viral infection and how different honey bees stocks and genotypes differentially respond to Deformed wing virus, Israeli acute paralysis virus and chronic bee paralysis virus. This work involves several projects including collaborative efforts with Louisiana State University, University of North Carolina at Greensboro, and University of Olomouc (Czech Republic). The use of gamma irradiation was found to reduce levels of some viruses of bees reared in irradiated combs, but irradiated comb did not regulate Varroa populations and did not affect survival and productivity of field colonies. Efforts to mitigate varroa mites are ongoing. ARS researchers at Baton Rouge, Louisiana, tested “social apoptosis”, or brood fragility, as a mechanism for Varroa resistance in selected stocks of honey bees and suggested that this could be a potential factor particularly in the Russian stock. The impacts of Varroa mite parasitism on flight activities and survival of drones has also been measured with the data currently undergoing analysis. Breeding for productive, Varroa resistant bees continues in the Hilo breeding program, a public-private partnership in which bees selected by the Unit for Varroa sensitive hygiene form much of the founding population. The chemical ecology that regulates expression of hygiene underpinning Varroa resistance is being explored with collaborators from the University of North Carolina at Greensboro, who developed a simple assay for evaluating response of bees in field colonies to chemical stimuli related to Varroa sensitive hygiene. ARS researchers at Baton Rouge, Louisiana in collaboration with the University of Minnesota and commercial beekeeper cooperators 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 made for research in regard to other parasites and pests. Assessment of prevalence and abundance of Acarapis woodi, Acarapis dorsalis and Acarapis externus in honey bee colonies is ongoing. Progress was also made by ARS researchers at Baton Rouge, Louisiana, in the discovery of feeding behavior of Tropilaelaps mites. Feeding of these mites on unsealed brood caused multiple injuries and significantly reduced survival of bees that were infested during their development. Management strategies for small hive beetles were also evaluated. The use of screen bottom boards does not promote an increase of small hive beetle populations in honey bee colonies, and thus can still be used to manage Varroa mite population. Major insecticide target sites in the small hive beetle genome were also identified. ARS researchers at Baton Rouge, Louisiana, began research in anticipation of the new Project Plan. An initial screen of three honey bee stocks was completed regarding the influence of honey bee genotype on the efficiency of food conversion, finding that some stocks have more variable responses as compared to others, an important consideration for future breeding goals. ARS researchers at Baton Rouge, Louisiana, evaluated the use of microalgae as an alternative nutrition source for bees and indicated that in controlled, caged settings a microalgal diet performs just as well as a pollen-based diet based on several metrics. Additional research by ARS researchers at Baton Rouge, Louisiana, has begun, to develop a novel RNA interference-based delivery system to mitigate honey bee pathogens. ARS researchers at Baton Rouge, Louisiana, examined various insecticide-related issues. Baseline data were established for a nationwide assessment of the resistance of Varroa mites to amitraz in commercial beekeeping operations. ARS researchers at Baton Rouge, Louisiana, documented that some Varroa mite populations do demonstrate resistance to amitraz that is associated with treatment failures, however resistant populations appear to be related to specific operations and amitraz use patterns and not necessarily geographic area. Investigations began into substrates that can be used as reliable surrogates of insecticide detoxification by esterases, and the relationship of esterase activity inhibition on insecticide toxicity. In a corollary project, ARS researchers at Baton Rouge, Louisiana, conducted research 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. In subordinate projects, two large-scale longitudinal field trials of two years each are yielding information about the biotic and abiotic health threats to honey bee productivity and survival in commercial beekeeping operations. These trials were conducted by ARS researchers at Baton Rouge, Louisiana, in collaboration with Louisiana State University and the University of Minnesota. Progress was made in collaboration with North Carolina State University and University of Pennsylvania to clarify the genetic determinants of queen quality.

Accomplishments
1. Detection of amitraz resistance and reduced miticide efficacy against parasitic mites. Varroa infestation is responsible for an overwhelming majority of honey bee colony losses in commercial beekeeping operations. Due to these dire consequences, beekeepers have used many types of chemicals to control Varroa populations. Amitraz is a widely used miticide to effectively control Varroa. However, anecdotal reports from beekeepers suggest Varroa may be becoming resistant to amitraz. ARS researchers at Baton Rouge, Louisiana, evaluated amitraz resistance in commercial beekeeping operations using two different methods, including one that can be easily conducted by beekeepers in the field. Significant amitraz resistance was only detected in 2 commercial beekeeping operations, so amitraz resistance appears to be rare. There was significant agreement with both tests, showing that monitoring for resistance could be implemented in a normal Varroa monitoring program. It appears that amitraz use alone cannot account for amitraz resistance in Varroa, so more data on differences in operational use needs to be collected. This research aims to establish a Varroa resistance monitoring program over the entire country in order to detect and mitigate miticide resistance in Varroa before it becomes widespread.

2. Microalgae as a nutritional supplement for honey bees. Honey bees face a number of threats that interact with and are exacerbated by malnutrition. Pollen is the bee’s primary source of proteins, amino acids and lipids, and is required for colony growth, which is crucial for beekeepers to be able to meet demands for pollination of crops. The use of artificial pollen substitute diets to support colonies during periods of reduced forage is a common management practice. These artificial diets may be deficient in essential macronutrients (proteins, lipids, prebiotic fibers), micronutrients (vitamins, minerals), and antioxidants. Therefore, improving the efficacy of pollen substitute diets can be considered vital to modern beekeeping. ARS researchers at Baton Rouge, Louisiana, evaluated the microalga Arthrospira platensis (commonly called spirulina), as a pollen substitute for honey bees. Nutritional analyses indicated that spirulina is rich in the essential amino acids and functional lipids common in pollen. Nutritional physiology and microbiome measures of bees fed spirulina closely matched those of bees fed a natural pollen diet. ARS researchers at Baton Rouge,Louisiana,current findings indicate that spirulina has significant potential as a pollen substitute or prebiotic diet additives to improve honey bee health. There is already significant interest in this work by the beekeeping community, as indicated by the major trade magazine (American Bee Journal) highlighting this study in a recent issue. More broadly, adapting beekeeping and broader livestock management practices with microalgae feeds could contribute to achieving objectives outlined in the United Nations sustainable development goals related to food security, sustainable water management, reversal of land degradation, and halting biodiversity loss. The long-term aim of this research is to characterize and develop microalgae as a sustainable feed source for honey bees that can be augmented via biotechnology to improve targeted aspects of bee nutrition and health.

3. Genomics of colony defensive behavior in honey bees. Breeding using genomic tools has not yet been adopted by the honey bee industry. In part, this is because some of the traits of highest interest for bee breeding are regulated by many genes making them challenging to characterize. In addition, honey bees are group living organisms, and many relevant honey bee traits are only measurable at the group level. Colony defensive behavior is an ideal example where many of these complications are evident. Furthermore, this trait is of particular interest to stakeholders as high defensiveness is undesirable from management and public health perspectives. By investigating the genomic structure of this clearly identifiable behavioral trait and using a novel population of gentle Africanized honey bees in Puerto Rico, ARS researchers at Baton Rouge, Louisiana, were able to identify a particular region in the genome contributing to reduced colony defensive behavior. These findings are scientifically relevant in that they provide a roadmap for the analysis of complex traits. Similarly, ARS researchers at Baton Rouge, Louisiana, findings have practical implications in that these same markers could lead to the development of a genetic monitoring panel that could contribute to existing breeding programs.

Review Publications
Lopez-Uribe, M., Ricigliano, V.A., Simone-Finstrom, M. 2020. Defining pollinator health: assessing bee ecological, genetic and physiological factors at the individual, colony and population levels. Annual Review of Animal Biosciences. 8:296.
De Guzman, L.I., Simone-Finstrom, M., Cervancia, C., Tokarz, P.G., Frake, A.M. 2020. Tropilaelaps species identification and viral load evaluation of Tropilaelaps and Varroa mites and their Apis mellifera hosts in Palawan, Philippines. Journal of Invertebrate Pathology. 170:1-3.
Rinkevich Jr, F.D. 2020. Detection of amitraz resistance and reduced Apivar® efficacy in the Varroa mite, Varroa destructor, in commercial beekeeping operations. PLoS One. 1-12. https://doi.org/10.1371/journal.pone.0227264.
Rinkevich Jr, F.D., Bourgeois, A.L. 2020. Identification and assessment of insecticide target sites in the genome of the small hive beetle, Aethina tumida. BMC Genomics. 1-12. https://doi.org/10.1186/s12864-020-6551-y.
Ricigliano, V.A. Microalgae as a promising and sustainable nutrition source for managed honey bees. Archives of Insect Biochemistry and Physiology. 1-8. https://doi.org/10.1002/arch.21658.
De Guzman, L.I., Frake, A.M. 2019. A short communication on the invasive success of small hive beetles in honey bee colonies with screen and solid bottom boards. Journal of Apicultural Research. 34(4):279-283. https://doi:/10.17519/apiculture.2019.11.34.4.279
Acevedo-Gonzalez, J., Galindo-Cardona, A., Avalos, A., Whitfield, C., Rodriguez, D., Uribe-Rubio, J., Giray, T. 2019. Colonization history and population differentiation of the Honey Bees (Apis mellifera L.) in Puerto Rico. Ecology and Evolution. 9:1-8. https://doi.org/10.1002/ece3.5330.
Avalos, A., Fang, M., Pan, H., Ramirez Lluch, A., Lipka, A., Zhao, S., Giray, T., Robinson, G., Zhang, G., Hudson, M. 2020. Genomic regions influencing aggressive behavior in honey bees are defined by colony allele frequencies. Proceedings of the National Academy of Sciences. 1-7.
Ricigliano, V.A., Simone-Finstrom, M. 2020. Nutritional and prebiotic efficacy of the microalga Arthrospira platensis (spirulina) in honey bees. Apidologie. 51(2)1-13. https://doi.org/10.1007/s13592-020-00770-5.
Saelao, P., Borba, R.S., Ricigliano, V.A., Spivak, M., Simone-Finstrom, M. 2020. Honey bee microbiome is stabilized in the presence of propolis. 2020. Biology Letters. 16:1-5. https://doi.org/10.1098/rsbl.2020.0003.
Ricigliano, V.A., Anderson, K.E. 2020. Probing the honey bee diet-microbiota-host axis using pollen restriction and organic acid feeding. Insects. 11(5):1-14. https://doi.org/10.3390/insects11050291.
Mondet, F., Beaurepaire, A., Mcfee, A., Locke, B., Alaux, C., Blanchard, S., Danka, R.G., Le Conte, Y. 2020. Honey bee survival mechanisms against the parasite Varroa destructor: A systematic review of phenotypic and genomic research efforts. International Journal of Parasitology. 50:433-447. https://doi.org/10-1016/ijpara.2020.03.005
De Guzman, L.I., Rinderer, T.E., Burgett, D.M., Frake, A.M. 2019. Understanding trends in prevalence and abundance of Acarapis dorsalis and Acarapis externus in Apis mellifera colonies. Trends in Entomology. 15:85-94.