2024 ABRC Proceedings

Part 1

DOI: https://doi.org/10.55406/ABRC.4.24.1

The American Association of Professional Apiculturists (AAPA) hosted its annual meeting, the American Bee Research Conference (ABRC), in New Orleans, Louisiana in conjunction with the American Beekeeping Federation’s annual convention. This conference provides a unique opportunity for AAPA members to interact and exchange ideas between industry, academia and the beekeeping community. As an organization, AAPA consists of senior and junior scientists, students, beekeepers and inspectors who work with or study honey bees. At this year’s ABRC, we showcased a record number of presentations from bee researchers from the U.S. and Canada. Research topics included Pests, Pathogens and Beneficial Microbes; Breeding, Genetics and Evolution; Chemical Ecology, Behavior and Nutrition; Pesticides and Acaricides; and Beekeeping Management, Education and Outreach. The large number of presentations given by students and postdocs was noteworthy; the high quality of the research they presented is an asset for the AAPA. We were also thrilled to host two keynote speakers: Dr. Diana Cox-Foster and Dr. Reed Johnson. AAPA is pleased to share the abstracts of this year’s meeting with the readership of Bee Culture. We hope this information helps the beekeeping community learn about the latest research developments regarding honey bees in the U.S. and beyond. Thanks for reading and for participating in this year’s ABRC.

The editors of the 2024 ABRC Proceedings:
Izaak R Gilchrist
Margarita Lopéz-Uribe
Priyadarshini Chakrabarti
Brock Harpur

Keynote Speakers

Interactions Among Bee Species: How do honey bees affect other species of bees?
Cox-Foster, DL 1
1USDA-ARS Pollinating Insect Research Unit, Logan, Utah, USA
Over 4,000 species of bees are found in the U.S. and share the need for pollen and nectar. A major cause of poor bee health is lack of floral resources or “bee pasture”, which has been exacerbated by changing climate. Concerns about the impacts of honey bee apiaries on other bee species is causing some to restrict placement of honey bees. We did a three year study that asked how honey bees affect other species of bees using experiments in cages and in the field. Experiments in cages generated competition by restricting the bees over a set amount of floral resources. In this environment with limited floral resources, we found that reproduction and colony growth was equally negatively impacted for honey bees, bumblebees and solitary bees. On flowers, no aggressors were found. In forests, we examined the impact of honey bee apiaries on sentinel bumblebee colonies and sentinel solitary bees, as well as endemic native bees. For reproduction, floral interactions and disease/health, there was no evidence for competition by honey bees with other species. Our research does reveal the importance of defining the carrying capacity of a location. The list of factors impacting carrying capacity is expanded when other land uses and weather are considered.

Interactions between honey bees and pesticides in agriculture
Johnson, R1
1Department of Entomology, the Ohio State University, OH, USA
Pesticides are widely used in agriculture to control arthropod pests and plant diseases and are often applied together in “tank mix” combinations with multiple pesticide products and spray adjuvants. Testing for bee toxicity is performed as part of the pesticide registration process to ensure that a pesticide application will not harm bees, which is considered an “unreasonable adverse effect on the environment” under the Federal Insecticide Fungicide and Rodenticide Act (FIFRA). However, the risk assessment process for the environmental effects of pesticides includes a cost-benefit analysis and there may be situations where the benefit to crop production outweighs the risk to bees. With cost-benefit in mind, we performed laboratory testing with larval and adult honey bees using insecticides, fungicides and spray adjuvants, alone and in field-relevant combinations, that are commonly applied to almonds in California during bloom. The insecticides diflubenzuron and chlorantraniliprole demonstrated a toxic effect to bees and these effects were more pronounced when these insecticides were included in some tank mix combinations. Some spray adjuvant products also caused bee mortality, both alone and in tank mixes. While we demonstrated a risk to bees from insecticides and spray adjuvants included in bloom-time sprays, there is little evidence that there is a pest control benefit from these applications. The widespread use of neonicotinoid insecticides in corn seed treatments is another scenario where the risk to bees exposed to seed treatment dust during corn planting may not be justified as these products are largely ineffective at controlling pests in corn. The use of insecticides during soybean bloom, where they are commonly applied in a tank mix with fungicides, is another case where the risk to bees may outweigh benefits from insect pest control, particularly when pest populations are low and an insecticide application would not be justified. On the other hand, the risk to bees from fungicides applied alone may be low in many situations, but the benefit of disease control for growers is likely to be substantial. Bee researchers and beekeepers are likely to be more persuasive in arguments against pesticide use when they engage with agricultural researchers and growers to put pesticide risk to bees in a cost-benefit framework.

Pests, Pathogens and Beneficial Microbes

RNA-seq of bees infected by Nosema apis, N. ceranae or both species
Huang, WF; Huang, ZY
Our previous study showed that honey bee workers that were mixed-infected by both Nosema apis and N. ceranae showed significantly earlier mortality compared to single Nosema infections. We thus tried to determine the gene expression profiles of worker bees infected either with no nosema (Ctr), N. apis (Na), N. ceranae (Nc), or both (Mixed). Workers were inoculated with either species or mixed species by hand and then sampled on day eight. We then confirmed each bee’s infection status by PCR typing the content of each bee’s hindgut. Only these with the correct infection as originally planned were used for sequencing. RNA-seq was performed by BGI. We found significant differences in gene expression in workers with different pathogen infection. Mixed infection caused significantly more differentially expressed genes compared to other treatments. The significance of these findings will be discussed.

The dispersal phase is a prerequisite for successful reproduction in young varroa mites (Varroa destructor)
Sprau, L; Lee, SH; Cho, S; Traynor, K
The parasitic Varroa destructor travels and feeds on adult bees during the dispersal phase of its life cycle. It remains unclear whether this dispersal phase is critical for successful reproduction of mites post emergence from a bee cell. To answer this question, we collected mother and daughter mites just prior to emergence. Half were inserted back into freshly capped cells without a dispersal phase, while the other half were placed onto adult nurse bees for three days to experience a dispersal phase before being placed back into freshly capped cells. Eight to ten days post insertion, mite fecundity was assessed. Directly inserted daughter mites have approximately 7% their normal reproduction, while 93% are infertile or have delayed reproduction with immature offspring incapable of survival. In contrast, the mites allowed a three-day dispersal phase on worker bees, stabilized at a normal reproduction rate of 75%. This suggests that the dispersal phase is critical to normal reproduction, especially for the young daughter mites. The extent to which these mites need nutritional benefits from feeding on their hosts during the dispersal phase or whether it is simply a matter of time to activate their ovaries must be clarified in further studies.

Hidden in plain sight: Varroa aggregate on adult drones
Lamas, ZS; Krichton, M; Andrus-Lamas, N; Lamas, J; Hoang, T; Ryabov, EV; Evans, JD; Hawthorne, D
As an almost universal rule, parasites form aggregated distributions where a minority of hosts are responsible for harboring a majority of the parasite burden in any given population. For 30 years, Varroa destructor has been described as preferring nurse bees of its host Apis mellifera, while otherwise largely leaving Varroa’s distribution undescribed. As a result, disease transmission dynamics, sampling methodology and research focus has largely been worker centric. For the first time, we describe the intracolony dispersal patterns of Varroa on its western honey bee host. Varroa overwhelmingly distributes by sex and age cohorts, preferring young adult drones over any other cohort. We show Varroa form highly aggregated distributions on the drone cohort early in the season when infestation levels are low, and distribute broadly onto the worker cohort later in the season when infestation levels are high. In 1978, Anderson and May first proposed the theoretical framework that parasites would have little destabilizing effect on the host population until the degree of aggregation was low, or approached a Poisson distribution. The distribution patterns of Varroa are in alignment with their theoretical work, and maybe the underlying mechanism responsible for historical losses of honey bee colonies observed over the last decade.

Rates of Melissococcus plutonius infection in Michigan apiaries and strains associated with clinical European foulbrood disease
Fowler, P; Milbrath, MO
European foulbrood (EFB) is a serious disease of honey bees caused by the bacterium Melissococcus plutonius. Beekeepers in the United States have been burdened with this disease for over a century, but little is known about prevalence or strains circulating. Strains classified as “atypical” have shown high virulence in larval rearing assays when compared to some “typical” strains but how these strains impact the colony is poorly understood. Here we present two years of cross-sectional surveillance data from Michigan beekeepers which reveal high rates of infection, with many hives containing both typical and atypical strains. Many of these colonies were asymptomatic at the time of inspection suggesting widespread inapparent infections. Pathogen prevalence was highly seasonal, and disease was strongly associated with the presence of atypical strains. Whole genome sequencing reveals four dominant strain types with the most common being an atypical strain. Phylogenetic analysis reveals closely related strains in multiple operations and regions suggesting high rates of transmission within and between operations. These results suggest that EFB may be playing a more important role in hive health outcomes than previously thought, and more research in this area is warranted.

A novel neutralizing antibody therapy reduces Deformed wing virus loads in the western honey bee (Apis mellifera)
MacMillan, NJJ; Hause, BM; Feldon, A; Nordseth, T; Pitman, JL; Lester, PJ
The Deformed wing virus (DWV) is a major driver of high rates of colony loss of the western honey bee (Apis mellifera). There is currently no method to directly control the virus. Here, we demonstrate that a novel antibody therapy can reduce DWV loads in A. mellifera. Anti-DWV immunoglobulin Y (IgY) was raised in and collected from the eggs of chickens immunized with three recombinant DWV proteins, which was subsequently fed to adult bees. An ELISA demonstrated the anti-DWV IgY treatments oral bioavailability in A. mellifera. We then assessed viral loads in bees using qPCR. The antibody therapy caused up to seven-fold and statistically significant viral load reductions in DWV-infected bees. Our findings demonstrate the potential for antibody therapies to help mitigate the damaging effects of DWV. This treatment modality could be used to target other crippling pathogens and parasites of A. mellifera.

Do hygienic honey bees detect and remove virus-inoculated brood?
Levin-Nikulin, S1; Hesketh-Best, PJ2; Erez, T3; Osabutey, AF4; Soroker, V4; Spivak, M1; Schroeder, DC2
1Department of Entomology, University of Minnesota, Saint Paul, MN, USA
2Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN, USA
3Department of Entomology, Agricultural Research Organization, The Volcani Institute, Israel; The Department of Environmental Economics and Management, The Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, Israel.
4Department of Entomology, Agricultural Research Organization, The Volcani Institute, Israel
Hygienic handling of virus-infested brood could either increase or decrease transmission of the pathogen through the colony, depending on the timing of hygienic removal, the viral load in the pupae and the immune response of the hygienic bee. Using three hygienic and three non-hygienic colonies, as determined using a freeze-killed brood assay, we inoculated pupae with Deformed wing virus (DWV) in the white to pale-eye stage by injecting DWV at the dose of 55 viral copies directly into the pupae (after uncapping the cell). Pupae that were mock-injected with 1X PBS buffer and un-injected pupae were used as controls. Only pupae that were not infested with Varroa were injected. The challenged pupae were recapped and replaced in their respective colonies. We quantified the number of experimentally-inoculated and control pupae that were uncapped and/or removed by bees within the hygienic and non-hygienic colonies at several time points and up to 52 hours post-injection. Our preliminary results show that bees from hygienic colonies uncapped and removed the virus-injected brood more quickly than bees from non-hygienic colonies.

Discriminating active and background infections in honey bee (Apis mellifera) viruses through RT-qPCR and metatranscriptomic sequencing
Hesketh-Best, PJ; Levin-Nikulin, S; Erez, T; Osabutey, AF; Soroker, V; Spivak, M; Schroeder, DC
Honey bees (Apis mellifera) are vital pollinators, and are infected by viral pathogens that can contribute to colony loss. Here we explore the challenge of distinguishing between genuine active viral infections and incidental background signals in honey bees using quantitative RT-qPCR and metatranscriptomic sequencing. Bee pupae were injected with a low virus copy number inoculum of a recombinant Deformed wing virus (DWV), and sampled over 96 hours. The inoculum DWV genome was successfully assembled from infected pupae by de novo genome assembly. Notably, the presence of other viruses could be observed through a semi-automated metagenomic binning analysis. In addition, real-time PCR data were used as a comparative dataset against various parameters such as genome frequency, and unique k-mer frequency. These data were used to better understand the interpretation of virus detection based on RT-qPCR outcomes alone, particularly in studies involving environmentally acquired viral titers, a common practice in honey bee viral research. Environmentally acquired viral titers used in experimentation exhibit a diverse viral population due to co-infections, but may not be readily discernible through qPCR alone. A potential overlook of background signals could influence the interpretation of infection study results. RT-PCR results confirmation by unbiased meta-trascriptomics provides a crucial cross-validation.

Engineered microalgae feed additives to bolster honey bee disease resistance
Martin Ewert, A; McMenamin, A; Adjaye, D; Rainey, V; Ricigliano, V
Pathogenic diseases threaten the global beekeeping industry by weakening honey bee (Apis mellifera) health and productivity. To combat record colony losses, beekeepers need effective, practical and sustainable treatments against pathogens. Addressing this problem, we have genetically engineered microalgae to express double-stranded RNA (dsRNA) specific to honey bee pathogens. When consumed, these engineered strains provide dietary amino acids that support honey bee nutrition and stimulate the RNAi immune pathway against target pathogens. In previous work, we determined that adult bees fed DWV-targeting microalgae strains show reduced viral load after injection with DWV compared to controls. Here, we test whether this microalgae-induced disease resistance can also be conferred to younger life stages. In this study, larvae were reared on royal jelly diets containing either control engineered microalgae (Vector), non-specific dsRNA-producing engineered microalgae (YFP) or DWV-targeting engineered microalgae (DWV). The pupae reared on DWV-targeting diet as larvae had lower viral load after injection with DWV-A than controls. Our results show that engineered microalgae feed additives have potential to benefit whole colonies by dually supporting proper nutrition and improving disease resistance across all life stages of bees. Engineered microalgae feed additives represent a scalable, sustainable and effective opportunity to bolster honey bee health.

The vectoring competence of Varroa destructor for Deformed wing-like viruses is affected by methods for controlling the mite in honey bee hives
Cook, SC; Ryabov, EV; Johnson, JD; Nearman, AJ; Rogers, CW; Powell, NS; Evans, JD; Chen, YP
Varroa destructor are harmful ectoparasitic mites of Apis mellifera honey bees. Deformed wing-like viruses (DWV-A and B (VDV1)) are ubiquitous honey bee viruses that are vectored by Varroa; mite vectoring can cause an increase in virus infectivity and diversity of genetic variants. Beekeepers use both chemical (e.g., amitraz) and non-chemical (e.g., ‘brood break’) means to control mite populations in honey bee colonies, and these methods may be combined into an integrated pest management strategy. Here we explore how these control methods affected the DWV/VDV1 population in honey bee colonies, the diversity of viral genetic variants, and the competence of Varroa to transmit an overt DWV infection. We found that ‘brood break’ treatment significantly elevated Varroa populations in colonies with a concomitant increase in DWV-A and VDV1 levels, but not when combined with amitraz application. Our results demonstrate negative implications to honey bee colony health from chemical treatment failures on the levels of DWV-like viruses in adult bees and mites, and the ability of mites to transmit overt infections.

Using AmE-711 honey bee cells in basic virological methods
Goblirsch, M; McMenamin, A
Honey bee viruses impose a significant burden on the beekeeping industry. Viral infections can cause individual bee death, and depending on the prevalence and severity of disease, can contribute to the decline and death of colonies. Single-stranded RNA (+ssRNA) viruses are common infections of colonies. High viral loads, in conjunction with exposure to other common stressors, such as pesticides, Varroa mites and poor nutrition, can accelerate the progression of viral disease. Understanding the pathology associated with honey bee viral infections requires studies conducted at not only the organismal and colony levels, but also at the cellular and molecular levels. To help fill gaps in our knowledge regarding honey bee-virus interactions at the cellular level, we utilized the continuous honey bee cell line, AmE-711, in experimental-infections with select +ssRNA viruses, i.e., Black queen cell virus, Acute bee paralysis virus and Chronic bee paralysis virus. We demonstrate that AmE-711 permits characterization of the cytopathology and immune response of honey bee cells to different virus infections. We also provide a comparative approach for quantifying viral genome copies using RT-qPCR to infectious dose using plaque assays. Our goal is to show the utility of AmE-711 honey bee cells for virological studies.

A method for quantifying Vairimorpha spore maturity
Gehefer, K; Webster, T
Vairimorpha (Nosema) ceranae, is a microsporidian parasite that reproduces within the midgut cells of the honey bee. This fungal infection causes high mortality rates within honey bee populations globally. Worker bees are being assessed to quantify the maturity of Vairimorpha spores in the midgut. Midguts were extracted from honey bees 15 days post-inoculation. Spores collected from the infected midguts were stained with calcofluor and viewed under a fluorescent microscope. The software cellSens was used in combination with an Olympus DP74 color camera to view and save images of the Vairimorpha spores. The line profile tool within the software was then extended to assess the intensity of light, via image, and its range of penetration throughout the spore. This provided visual representation, as well as a data set, useful for spore identification. For example, when comparing mature spores to immature (primary) spores, contrast is visually represented with immature spores having a darker end, but quantitatively by the intensity of light able to penetrate the chitinous layers or lack thereof. Utilizing this method allows spores at various stages of disease development to be identified and quantified. This method applies to research on treatments for and evaluation of V. ceranae infections.

Life-history stage determines within-host dietary alternation of ectoparasitic mites
Han, B1; Wu, J1; Wei, Q1; Liu, F1; Cui, L2; Rueppell, O3; Xu, S1
1State Key Laboratory of Resource Insects, Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, China
2Cell Biology Facility, Center of Biomedical Analysis, Tsinghua University, China
3Department of Biological Sciences, University of Alberta, Canada
Parasitic mites of the genera Varroa and Tropilaelaps are serious threats to honey bee health. Both have specialized in exploiting their honey bee hosts as food and habitat. While Varroa shows a clear alternation between dispersal and reproductive life history stages, Tropilaelaps has a drastically shortened dispersal stage and exclusively feeds on brood. In a series of experiments, we show that Varroa destructor varies its main food source: While feeding on adults, Varroa feed primarily on fat body, as reported previously by S. Ramsey and colleagues. In contrast, Varroa that feed on honey bee pupae inside brood cells select different feeding sites and primarily consume hemolymph. This conclusion is supported by wound analysis, differential biostaining of nymphal and adult mites, and a proteomic comparison between parasite and host tissues. These results were paralleled by findings in Tropilaelaps, which also showed an enrichment of hemolymph and bee proteins associated with the hemolymph. In addition, the metabolism of dispersing and reproducing Varroa is fundamentally different and confirms that reproductive mites have a very active protein metabolism. Thus, the complex life cycle of Varroa is accompanied by dietary specialization on the same host. The differences in food acquisition are important to consider when determining integrative pest control of Varroa and Tropilaelaps.

Molecular disease diagnosis of Brazilian Africanized honey bees
Kadri, SM; Orsi, RO; Ellis, JD; Epperson, KJ; Fulton, JC
The purpose of this research was to evaluate Brazilian honey bee colonies selected for high and low honey production (HHP and LHP respectively) from commercial operations in Polo Cuesta, São Paulo for bacterial (Melissococcus plutonius, Paenibacillus larvae), fungal (Ascosphaera apis, Nosema ceranae) and viral (Acute Bee Paralysis Virus, Black Queen Cell Virus, Chronic Bee Paralysis Virus, Deformed Wing Virus – A, Deformed Wing Virus – B, Israeli Acute Paralysis Virus, Kashmir Bee Virus, Lake Sinai Virus, Moku Virus, Sacbrood Virus and Slow Bee Paralysis Virus) infections. Thirty-eight honey bee colonies standardized for queen age, population and honey production management were sampled, with N=20 HHP (89±23.9 kg/colony) and N=18 LHP(25±9.3 kg/colony) from 16 apiaries. DNA and RNA were extracted from 12 worker bee pupae from each colony and screened for pathogens using standard PCR and qPCR techniques. Analysis of variance was used to evaluate honey production between selected colonies and odds ratios between honey production and incidence calculated for each tested pathogen. LHP colonies were 15.9 times more likely to have N. ceranae infections than were HHP colonies (χ2 1.746468x10E-07). No other pathogens were present at significantly different quantities. Kadri was supported by FAPESP (2022/14754-6). Fulton and Epperson were supported by FDACS-DPI.

The potential for indoor storage to improve control of Varroa mites in honey bee colonies
Reed, R¹; Hopkins, BK¹
¹Department of Entomology, Washington State University
It has become increasingly common for beekeepers to overwinter their hives inside cold storage facilities. However, indoor storage may also be a useful tool at other times of the year. Here we tested the use of Spring indoor storage as a method of forcing a break in brood production to improve control of Varroa destructor. Immediately following almond pollination, 72 colonies were placed in a cold storage facility and 39 hives were left outside. After 18 days, the colonies were removed from cold storage and transferred to another outdoor location near the 39 colonies. At that time the stored colonies had an average of 4.78 mites per 100 bees. At the same time, the colonies left outdoors had significantly lower infestations at 2.98 mites per 100 bees. All colonies were treated for varroa mites at that time. Approximately one month later, the opposite was true. The colonies previously placed in cold storage had an average of 1.83 mites per 100 bees, significantly fewer than the 3.85 mites per 100 bees found in the colonies that were not placed in storage. The colonies placed in cold storage started out with one frame of bees less than the outdoor colonies on average, but by the end of the study there was no longer a significant difference between groups. These results demonstrate the strong potential of Spring cold storage of honey bee colonies as a valuable tool in the fight against varroa mites.

Varroa destructor economic injury levels and pathogens associated with colony losses in British Columbia
Morfin, N; Foster, LJ; Guzman-Novoa, E; vanWestendorp, P; Currie, RW; Higo, H
An Integrated Pest Management strategy to control Varroa destructor is based on monitoring mite levels to treat the colonies before they reach damaging levels (economic injury level; EIL). Possible interactions between V. destructor and other pathogens may lead to high colony mortality. The aim of this study was to record varroa mite levels in colonies from five regions of British Columbia, Canada, and analyze them for associations with health parameters as well as with other pathogens identified using total RNAseq. Significant differences in varroa levels, colony strength, and colony mortality between regions were found. Also, varroa levels in the Fall significantly predicted the odds of colony mortality in the Spring. Colonies with ≥3% mite infestation in the Fall had a significantly higher mortality rate compared to colonies with <3% mite levels. Additionally, deformed wing virus-B (DWV-B) levels were higher than those of the DWV-A variant in all the regions. Malpighamoeba mellificae and Nosema (Vairimorpha) apis transcripts were identified, along with other viruses like Apis filamentous virus and Lake Sinai virus. The dynamic nature of host-pathogen interactions urges constant pathogen surveillance and revising EIL for V. destructor.

Variation in susceptibility to Deformed wing virus in honey bee drones in relation to quality metrics
Simone-Finstrom M; Walsh, E; Slater, GP; Evans J; Weaver, D
A principle threat to honey bee health continues to be viral infections, which often compound with other abiotic and biotic stressors to impact individual bee health and colony productivity and survival. Despite the significant impacts of viruses, there are no antiviral treatments available to bees. However, bees vary in their susceptibility to viral infection, and this variance can be used as a target for selection of virus resistant bees. To do this, we have screened the viral resistance of (haploid) male bees in two different honey bee populations, one from a commercial beekeeping operation that has exhibited some viral resistance and one from a research derived Varroa mite resistant population. Resistance in this case is defined as reduced replication and maintenance of low levels of Deformed wing virus (DWV) for 48 hours post-injection of adult drone honey bees. Proportion of bees considered resistant or susceptible to DWV differed across populations. Additional measures of drone quality, including thorax width and gene expression of targets associated with sperm quality and flight performance, will be discussed in relation to susceptibility to viral infection. Overall, this initial work shows promise in drone selection as a target for breeding for virus resistance in honey bees.

Breeding, Genetics and Evolution

The Influence of Queens on Varroa Infestation Levels and Virus Profiles in Honey Bee Colonies
Waldbieser, S1; Hardy, J1; Wagoner, K2; Amiri, E1
1Delta Research and Extension Center, Mississippi State University, MS, USA
2Biology Department, University of North Carolina at Greensboro, NC, USA
The genetic profile of a honey bee colony plays a crucial role in its resistance/tolerance to pests and pathogens. With the increase of Varroa mite problem, bee breeding programs were developed to control Varroa mite populations in honey bee colonies. Breeding activities improved the hygienic behavior of the colonies to decrease mite populations. However, Varroa mites could potentially co-evolve with hygienic bees. The impact of such co-evolution on virus diversity and levels in honey bees and Varroa mite are not well understood. To address this, we established two apiaries with colonies headed by queens selected for hygienic and non-hygienic behavior in Mississippi and North Carolina. Over a beekeeping season, we measured Varroa infestation levels between hygienic and non-hygienic colonies in both locations. We collected honey bees and Varroa mite samples from all experimental colonies once every month to measure the virus diversity and infection level. Varroa infestation results diverged between hygienic and non-hygienic colonies over time in both locations. Laboratory analysis of single mites found lower virus levels in hygienic colonies compared to non-hygienic colonies. This suggests that hygienic queen genetics allow workers to control both infestation and virus levels.

Honey Bee Germplasm Importation, Cryopreservation, Distribution and Propagation
Cobey, S; Hopkins, B; Sheppard, W
Germplasm of four honey bee subspecies, A.m. carnica, A.m. caucasica, A.m. ligustica and A.m. pomonella, has been imported from original source populations in the Old World. U.S.-reared virgin queens were inseminated with fresh and cryopreserved semen from each subspecies and those genetics subsequently distributed to U.S. beekeepers. A cryogenic storage repository for honey bee germplasm was established at WSU for future use in honey bee breeding and conservation. The U.S. commercial beekeeping industry largely manages bees descended from Old World sources that were collected and introduced before 1922. The recent introduction and distribution of additional germplasm of A.m. carnica and A.m. ligustica has enhanced the genetic diversity of honey bee populations selected by queen breeders and producers. A new “Caucasian” strain of honey bees is also available to U.S. beekeepers, derived from backcrossing A. m. caucasica into a maternal Carniolan line. A.m. pomonella, a cold hardy bee endemic to the wild apple forests of Central Asia, may offer an advantage for early season pollination. We are introducing the genetics of this subspecies from cryopreserved semen and collaborating with colleagues from Cornell University to establish and propagate a honey bee strain well-suited as a pollinator of tree fruit.

Do Honey Bees Selected for Resistance to Varroa Parasites also Resist Pathogens?
Dyrbye-Wright, I; Spivak, M
To improve the health and vitality of honey bees, Apis mellifera, beekeepers can propagate stocks that demonstrate resistance to parasites and pathogens. One mechanism of resistance in honey bees is hygienic behavior, in which adult bees detect and remove mite-infested and/or diseased brood from the colony. A line of bees bred specifically for Varroa resistance shows effective hygienic removal of mite-infested brood but has not been tested for its ability to remove and resist diseased brood. I began by comparing disease resistance among three lines of bees: 1) POL line bred specifically for Varroa resistance; 2) MN Hygienic line bred for disease resistance but with limited Varroa resistance and 3) an unselected, commercially available line as a control. I challenged 12 colonies within each line with chalkbrood, Ascosphaera apis, and quantified signs of disease, mites and viral loads. I predicted that POL and MN Hygienic colonies would have similar levels of pathogen resistance, but the POL line would have lower mite and viral loads compared to MN Hygienic and control colonies. If confirmed, my findings would indicate POL line bees show both parasite and pathogen resistance, and use of this line by beekeepers would improve colony health and reduce economic costs for treatments.

Maternal effect in honey bees: Selection for egg size can improve the weight of new honey bee queens
Martinez Caranton, O1; Amiri, E1
1Delta Research and Extension Center, Mississippi State University, MS, USA
Queen is the sole reproductive individual in a honey bee colony. She invests resources into her eggs, which this investment reflects in the size of produced eggs. Queens have been observed to actively adjust the size of their eggs in response to variable environments including food scarcity and colony size. Egg size also varies among queens, with some naturally laying large eggs. In our investigation, we found a significant variation in the size of eggs produced by sister queens, even when they are kept in the same apiary and heading colonies with almost equal number of honey bee workers in each colony. Further analysis also confirmed that larger eggs confer a body size advantage in grafted larvae, which persist into adulthood. Queens reared from young larvae that were hatched from small and large eggs show that increase in maternal investment can enhance the weight of queens across all developmental stages, including larvae at 72 hours, larvae at six days, pupae at nine days after grafting as well as newly emerged queens. While the molecular mechanisms underlying these differences require further elucidation, our findings demonstrate the advantage of selecting queens that produce larger eggs to improve the weight of offspring queens.

Female cryptic choice for sperm storage in honey bee (Apis mellifera) queens as a reproductive mechanism for strain differentiation
Ritchie, K1; Sheppard, W1
1Department of Entomology, Washington State University, WA, USA
Honey bee queens store and use sperm from mating with multiple drones early in life. Queens can mate with drones from multiple strains in the U.S. to produce viable offspring. There is evidence to suggest that queens differentially use sperm during fertilization, but it is unclear when and where sperm selection is taking place in the queen reproductive tract. To evaluate differential sperm storage in honey bee queens, queens from different U.S. strains representing Old World subspecies were instrumentally inseminated with pooled semen from individual strains. Queens inseminated with semen from one strain were sacrificed and dissected 40 hours after insemination and sperm counts from the dissected spermatheca were performed. Data suggests there is an asymmetric effect of sperm storage by queens from different evolutionary lineages. Caucasian queens stored more sperm when they were inseminated with semen from drones of the same strain; Italian queens stored semen in concentrations that were not significantly different. Future work will analyze sperm use via microsatellite paternity analysis of worker progeny from queens inseminated with multiple strains of semen. Findings from this research will improve the knowledge of honey bee mating biology and lead to more informed breeding practices.

Honey Bee Pangenome Highlights Structural Variation and Diversity of Commercial Stocks
Slater, GP; Avalos, A
The Apis mellifera genome is the most widely used resource in honey bee genetics and is due for a major update. Its current structure is a linear composite, with a single individual comprising most of the sequence. It contains biases and errors within a framework that does not represent commercial honey bee genomic variation. A high-quality reference with commercial representation of common variants, including single-nucleotide variants, structural variants and functional elements, is needed. We aim to create a more sophisticated and complete honey bee reference genome with a graph-based, telomere-to-telomere representation of genomic diversity. Here we leverage innovations in technology, study design and global partnerships with the goal of constructing the highest-possible quality human pangenome reference. Our goal is to improve data representation and streamline analyses to enable routine assembly of complete honey bee genomes. This honey bee pangenome reference will contain a more accurate and diverse representation of commercial genomic variation, improve gene–disease association studies across populations, expand the scope of genomics research to the most repetitive and polymorphic regions of the genome and serve as the ultimate genetic resource for future research.

Genetic diversity and Varroa destructor survey of commercial beekeeping operations across Southwest Saudi Arabia
Nichols, K; Kahn, K; Ghramh, H; Shepherd, T; Rangel, J
The honey bee subspecies native to Saudi Arabia, Apis mellifera jemenitica Rutter, is currently being threatened by genetic pressure from exotic subspecies imported by commercial beekeeping operations. Uncontrolled interbreeding between native and exotic subspecies could dilute advantageous adaptations and give rise to new haplotypes that are not well suited for the harsh climate of Saudi Arabia. In this study, we analyzed the mitochondrial genetic lineage, as well as the levels of parasitization by the mite Varroa destructor, in honey bee colonies managed by commercial beekeepers across the Southwest region of Saudi Arabia. We found that all of the 55 colonies analyzed belonged to the Apis mellifera jemenitica subspecies, which is part of the Z subgroup of the A lineage. Analysis of the mitochondrial genome revealed ten new haplotypes at four of the eleven locations, suggesting the possibility of interbreeding between various lineages at those sites. Furthermore, the average (±SEM) number of Varroa mites per 100 workers across all sites was 1.95 ± 0.96, and was below the 3% infestation threshold at nine of the eleven locations, indicating a pattern of low Varroa levels in commercial beekeeping operations in the sampled region. This preliminary look at the genetic diversity and Varroa mite levels in this population is the first step in a selection process for the future implementation of a breeding program aimed at improving disease resistance and honey production of A. m. jemenitica colonies in southwest Saudi Arabia.

Comparing the performance of commonly available honey bee stocks for Midwestern and Northeastern beekeepers
López-Uribe, MM1; Underwood, RM1,2; Dean, CAE1; Cambron-Kopco, L1; Given, JK3; Harpur, BA3
1Department of Entomology, Penn State University, PA, USA
2Penn State Extension, PA, USA
3Department of Entomology, Purdue University, IN, USA
The availability of honey bee stocks that are resistant to pests and pathogens is of critical importance for the implementation of integrated pest management practices in beekeeping that rely less on chemical controls. Currently, dozens of stocks are available to beekeepers in the United States, which are commercialized for different traits including resistance to varroa mites. However, there is a lack of reliable data demonstrating differences in their performance in field-relevant settings. Here, we quantified mite numbers, virus levels, expression of grooming-related genes and honey production in 400 colonies from five commonly commercialized stocks in the northeast and midwest of the U.S. The colonies were managed by beekeepers in Pennsylvania and Indiana after the introduction of the queens from the different lines. We found significant differences among stocks in DWV levels, expression of grooming-related genes and honey production demonstrating that the performance of the selected stocks is different. Our results highlight the importance of generating regional data on the performance of different honey bee stocks to facilitate data-driven decisions by beekeepers on which queens to purchase.

Grow in petrochemical environment: Effects of commercial plastic queen cell cups on the rearing success and development of honey bee queens
Amiri, E1; Abou-Shaara, H1,2; Chen, J3
1Delta Research and Extension Center, Mississippi State University, MS, USA
2Department of Plant Protection, Damanhour University, Egypt
3USDA-ARS, Biological Control of Pests Research Unit, Stoneville, MS, USA
Plastic queen cell cups have become an integral part of queen rearing practice and gained widespread popularity among beekeepers. However, these petrochemical-based products have raised concerns regarding potential adverse effects on the success of queen rearing and the development of queens. To address this concern, we conducted a comprehensive survey to compare the success of queen rearing, birth characteristics of newly emerged queens and the expression of detoxification and immune genes in queens and larvae raised in beeswax cups with 10 different commercial plastic queen cups. The results indicate significant differences in larval acceptance rate, sealing and emergence rate among different types of queen cups. Although a negative correlation was observed between the increasing size of queen cups and the three components of queen rearing success (acceptance rate, sealing and queen emergence), the strength of this correlation was relatively modest. Potentially, the chemical components that queen cups are made of have also a role in queen rearing success and physiological changes of queen larvae. These chemical components showed to elevate some of the detoxification genes in both queen larvae and newly emerged queens, which highlights the impact of petrochemical composition of cups during development.

Using the Unhealthy Brood Odor (UBO) assay, a novel hygiene-eliciting assay, as a selection tool in bee breeding programs
Alger, SA1; Miller, MS1; Burnham, PA2; Wagoner, K 3; Palmer, M 4; Collins, A 5; Munkres, A6; Rath, J 7; Braman, B 5; Peck, D 8
1Plant and Soil Science Department, University of Vermont, VT, USA
2Biology Department, University of Vermont, VT, USA
3Biology Department, University of North Carolina Greensboro, NC, USA
4French Hill Apiaries
5Vermont Bees, LLC
6Lemon Fair Honeyworks
7Hundred Acre Wood Apiary
Vermont USA is home to several well-known bee producers whose bees are prized in the region due to their adaptiveness to the northeast climate. In 2021, the University of Vermont Bee Lab began leveraging diagnostic services for Vermont bee producers towards the development of more disease resistant stock. In 2022, we began to trial the Unhealthy Brood Odor (UBO) assay across three major bee producers with long-time selection programs. UBO is a novel tool to assess a colony’s performance of hygienic behavior. The assay measures a colony’s hygienic response to a blend of synthetic pheromones that simulate unhealthy brood. UBO is anticipated to be available to a broader market by Spring 2024. In preparation for its release, we present case study examples of three Vermont breeding operations that are using UBO to identify and select for stock that is more disease and pest resistant. We share results of this new assay in the context of both open mated and instrumentally inseminated programs. By pairing pest and pathogen sampling with UBO scores, we show how UBO performance correlates with lower pest and pathogen loads. In particular, we found that high UBO performers have reduced Varroa and Vairimorpha (Nosema) loads. We share lessons learned and how beekeepers are selecting for these traits in their operations.

Why diversity matters: non-additive fitness benefits of individual versus colony-level genetic variation in social insects
Ryals, DK; Given, KJ; Brito, LF; Harpur, BA
Numerous studies have shown queen polyandry increases the fitness of social insect nests. While increased genetic diversity is implicated, we lack sufficient evidence to show causality. In particular, previous experiments do not separate genetic diversity at the individual (worker) level from that at the social (colony) level. Fitness impacts of individual heterozygosity (i.e., heterosis) are already well-known in animal systems, while those of social diversity are comparatively novel. Through controlled, single-male breeding and brood mixing using two genetically distinct honey bee populations, we created an array of experimental colonies that range from high to low levels of social diversity and contain individuals ranging from high and low levels of heterozygosity. We quantified the fitness of each colony for two independent traits: survival against infection in a laboratory setting and variance of brood nest temperature in a field setting. We found both social and individual diversity increased colony fitness by similar effect sizes across both traits. This result highlights the importance of non-additive effects like directional dominance, indirect genetic effects and division of labor in social insect fitness, and holds implications for social evolution and bee breeding.

Beekeeping Management, Education and Outreach

The Pollinator Plexus Tapestry: Interlacing Indigenous perspectives with Western science for pollinator research & outreach
Kirby, M; Quintan, T; Quintana, P; Logan-Brayshaw, L
Based in Santa Fe, New Mexico – IAIA is a global leader of contemporary Indigenous artistry and diversity. Through the concept of art as a traditional path of creativity, IAIA excels at skill building, provoking thought and providing a place to embrace the past, enrich the present and create the future, moving ahead to establish paths where traditions are rediscovered, explored, deepened. And, where art and cultural identity are celebrated and revered. IAIA Land-Grant Programs is the agriculture extension department which focuses on the development of integrative approaches weaving the arts and earth sciences together through Traditional Ecological Knowledge (TEK) and Western sustainable ag science. This poster shares an introduction and overview of Indigenous Research Theory and practices as related to pollinator stewardship and conservation at IAIA. Recently, IAIA Land-Grant Programs signed a cooperative agreement with the USDA Office of Tribal Relations to establish Grassland and Pollinator Stewardship research of interest to tribal communities and also the development of resource guides for tribal college campuses developing pollinator conservation programs. Named one of the top art institutions by UNESCO and the International Association of Art, IAIA also recognizes that Agriculture and Apiculture are also Land Arts.

The Adaptive Bee Breeders Alliance: Phase 1 – Building Comb Between the Field & the Lab
Kirby, M; Mahoney, M; Mahood, J
ABBA is a cross-country network of bee breeders and researchers collaborating to better nurture adaptive and regenerative approaches to queen honey bee reproduction for supporting healthy and productive hives. Based in northern New Mexico, the Adaptive Bee Breeders Alliance was founded by Melanie Kirby of Zia Queenbees Farm & Field Institute in 2021. The ABBA-Building Comb from Castle to Castle program will be sharing collaborative open-source findings and media guides helping to build bridges between the field and the lab that can help us all become better producers and stewards. Program launch will unfold over the next three years from 2022-2025 and includes: Genetic stock analysis for both Mitochondrial (maternal) assessment; Genomic techniques to understand drone fertility in each of the participating producers breeding areas; Instrumental Insemination (I.I.) training in advanced breeding techniques for sharing across distances and times; Cryopreservation of honey bee germplasm for inclusion in the USDA American Honey Bee Germplasm Repository Program; Determination of drone congregation area research with UAVs; Educational outreach opportunities via webinars; and in-service workshops for all interested beekeepers to support continued professional growth, development of climate smart practices and encourage resilience of stewards and of their bees.

Taking the sting out of honey bee medicine: training and tools for veterinarians
Milbrath, MO; Bammer, M; Chapman, H; Fowler, P; Harris, D; Heck, A; Lee, K; Rangel, J; Vu, A
In 2017, federal policy changes required that beekeepers work with a veterinarian to access needed honey bee medications – treatments that used to be directly available. Honey bees, like other animals, can be affected by viral, fungal and bacterial diseases, but finding good care can often be difficult, as honey bee medicine is not covered by most standard veterinary schools. Trained veterinarians are needed to provide access to needed antibiotics, but in time can also help to address other issues with the bee crisis, including optimizing nutrition and managing parasites. In 2022, four universities began working together on developing training materials for veterinary professionals. We first administered a survey to determine the level of interest, confidence, and ability of veterinarians to take on beekeeping clients. This survey also highlighted training gaps and needs to focus our materials. We have developed the first modules of an online training series, and we developed and disseminated over a hundred honey bee veterinary medicine kits at hands-on workshops. We have had positive results from our work and are developing materials that can be shared with other extension educators and specialists to help train veterinarians in each state. All results and resources are posted on www.beesneedvets.com.

BeeVision: Using Dynamic Vision Sensors to track Pollinators
Traynor, K; Wagner, A; Treder, M; Glück, M; Gebler, C; Pohle-Fröhlich, R
We are developing BeeVision, a radical, new, non-invasive insect monitoring method with significant potential for biodiversity research. BeeVision combines technical advances in dynamic vision sensor (DVS) image analysis with artificial intelligence methods and our expertise in pollinators. Unlike conventional cameras, DVS cameras record event streams, capturing only changes of brightness at each pixel position. These camera systems thus require less memory than conventional video recording, making them convenient for long-term monitoring. Our project partner has conducted successful initial tests with this sensor, automatically detecting insect flight from background noise. We intend to use DVS to monitor the flight patterns of pollinators in a landscape or agricultural field, detecting their presence and categorizing them into the main pollinator groups. Machine learning will be applied to tagged recordings with the goal of classifying patterns of flight activity. We seek to establish classifiers of six major insect groups which we currently use in our monitoring activities of flower-rich meadows: honey bees, bumblebees, other bees, hoverflies, butterflies and other insects. We have previously examined the impact of increased honey bee density on pollinator activity using trained entomologists, but this limits observations to a specific quadrant and time frame. BeeVision will permit continuous monitoring.

buzzdetect: a machine learning tool for automated acoustic detection of honey bee activity
Hearon, LH1; Johnson, RM1
1Department of Entomology, the Ohio State University, OH, USA
A common demand of bee research is the in situ detection of foraging honey bees and the quantification of their activity. Visual observation is a direct solution, but is labor intensive, requires expertise for accurate identification, and is not feasible when flowers are obstructed. Other methods, such as pan traps or sticky cards sample destructively and are often a loose proxy for desired endpoints such as pollination activity. To better detect bee activity we are developing a machine learning tool called “buzzdetect” that detects and quantifies the audible buzz produced by honey bees in flight. buzzdetect facilitates low-cost, non-destructive, accessible and scalable acoustic surveys of honey bee activity. Current performance of buzzdetect compares well with our previous semi-automated using human labeling. buzzdetect correctly identifies most of the human-made labels and finds previously undetected buzzes in the recordings. Performing these detections at a rate thousands of times faster than manual labeling, buzzdetect is a valuable tool for bioacoustic assays.

Bee Health Assessment in Kentucky: Stressors and Management Practices
Olden, F; Palmer, J; Holbrook, K
The substantial decline in the honey bee population has prompted research efforts to uncover the underlying causes, revealing a complex interplay of contributing factors. These findings underscore the need for addressing this issue through an integrated approach that examines various local stressors. To comprehensively assess seasonal diseases, pesticide residues and stored-pollen quality in Kentucky’s apiaries, we systematically collected hive matrices and bees from 60 apiaries throughout Kentucky during the Fall of 2022, Spring of 2023 and Summer of 2023, totaling 900 sampled hives. These hives were also assessed for colony strength, categorized as strong, moderate or weak. The analysis of pesticide residues in the Fall 2022 pollen has been completed, while disease diagnosis and discovery through RNA-seq in bee samples are currently underway. Furthermore, a metagenomics analysis to identify different species in the Fall 2022 pollen samples is in progress. An epidemiological pilot survey was also conducted to evaluate colony management and Winter colony losses among all participating beekeepers. The results of the pesticide analysis of the Fall samples indicate that out of 468 analytes, only 15 were identified, with some falling under the Method Detection Limit (MDL). The most prevalent pesticides were coumaphos, carbendazim and EPN. Aside from one sample containing imidacloprid at a dose lower than the MDL, no neonicotinoid was found. Regarding colony management, the pilot survey revealed that beekeepers, regardless of their operation size, consider varroa and Fall colony weakness as the most significant factors contributing to their Winter colony losses. Surprisingly, many beekeepers with operations of one to 10 hives and 10-30 hives do not monitor varroa, despite recognizing its importance.

Enhancement of a Pollen Substitute to Promote Honey bee Health
Ghimire, S; Palmer, J; Holbrook, K; Olden, F
The honey bee, essential for both pollination services and honey production, faces several environmental challenges, including pathogen exposure, chemical toxicity and malnutrition. Previous research has demonstrated that commercial honey bee diets often fall short in comparison to natural pollen, impacting overall bee health. This study aims to improve the impact of the pollen substitute MegaBee on bee health. Newly emerged bees were distributed into four diet groups: 1) Sugar-only (Control), 2) MegaBee (MB), 3) MegaBee with invert sugar, honey bee health supplement and vitamin C (MB+) and 4) MB+ with the probiotic superDMF (MB+/DMF). Daily records were maintained for mortality and data on diet consumption and body weight were collected at various time intervals, including at the conclusion of the 14-day experiment. Bee samples were also collected and frozen for subsequent analysis of physiological characteristics and gene expression. The initial findings suggest that, while there is no significant difference between the diets in terms of mortality and food consumption, there is a noticeable difference in the body weight of bees fed MB or MB+/DMF compared to the control. Further analysis of physiological and molecular aspects will provide insights into the nutritional status of bees fed MB, MB+/DMF or MB+.

The path to organic beekeeping in the U.S.: factors affecting pesticide loads in colonies on organic farms
DeMoras, BX; Baert, N; Zhao, C; Anderson, WB; Underwood, RM; López-Uribe, MM; McArt, SH
While organic agriculture is growing in popularity and prevalence, a lack of formal USDA Organic Apiculture standards prevents American beekeepers from reaping the economic benefits of Organic certification. While Underwood et al. 2023 found that organically managed colonies are just as healthy and productive as those managed conventionally, data on the foraging and pesticide exposure of colonies placed on organic farms are needed before Organic certification is possible. In this poster, I will describe preliminary pesticide exposure data from (A) colonies managed in Underwood et al. 2023 and (B) an ongoing study of another 72 colonies established on Organic farms in Pennsylvania and Upstate New York in the Spring of 2023. Analysis of these data is still in progress and will be connected to measurements of colony populations and estimates of habitat quality from the Beescape landscape model. Results will clarify factors that affect pesticide loads in colonies placed on organic farms and will be incorporated into discussions with the USDA and the National Organic Standards Board to redefine standards for organic apiculture.

Quantifying Texas honey bee health risks associated with dominant beekeeper business models to improve colony survival, honey production and crop pollination services
Rangel, J; Houston, B; Aurell, D; Steinhauer, NA; Fauvel, AM; Meredith, A
Texas commercial beekeeping operations can be categorized into four groups: 1) stationary honey producers; 2) stationary honey producers who are also pollination providers; 3) migratory honey producers who produce two honey crops per year – one in Texas and one in the upper Midwest (ND, SD, MN); and 4) migratory honey producers who only produce out-of-state honey. Each of these operational types differ in several factors that can affect colony health, disease susceptibility and management options that can be used. Such factors include: the timing and distance of colony movement, optimal timing for reaching maximum colony strength and pesticide exposure rates. The goal of this project was to identify whether there is variation in management styles adopted by the most prevalent Texas beekeeper operational classes, and if so, to determine the major risk factors (e.g., disease loads, pesticide exposure, mortality) that beekeepers in each operational class experience throughout the season. Across all beekeeper participants, 288 colonies were monitored each year. Colony mortality was 58.8% in 2020 and 20.8% in 2021. Five types of management actions were tracked monthly from September 2019 to January 2021: Pest/Disease Treatment, Requeening, honey harvest, feeding and equipment change. Migratory beekeeping operations executed a greater total number of management actions than the stationary operations, especially in treatment and feeding categories. There was wide variation among operations in all the colony health metrics examined, but there were no significant differences between beekeeper operational category in any of these metrics, including Varroa load, Nosema load and colony size.