American Bee Research Conference Abstracts

Judy Wu-Smart1, Shelby Kittle1, Margaret J. Couvillon2, Margarita López-Uribe3 1:Department of Entomology, Univ of NE-Lincoln 2:Department of Entomology, Virginia Tech Univ 3:Department of Entomology, Center for Pollinator Research, Penn State University

Introduction and Overview
The American Association of Professional Apiculturists (AAPA) is an organization consisting of professors, state apiarists, scientists and students who study and work with honey bees. The goals of this organization are to (1) promote communication within and between industry, academia and the beekeeping community, (2) develop and foster research on fundamental and applied questions to gain a greater understanding of honey bee biology that can assist and improve the beekeeping industry; and (3) create a venue to rapidly share new techniques and current research to advance the field. The AAPA held its annual American Bee Research Conference (ABRC) on January 7th and 8th 2021 virtually over Zoom conferencing due to COVID-19 pandemic travel restrictions. The two-day conference had over 130 participants and showcased 46 oral presentations including research talks from 25 students. Keynote addresses were provided by Drs. Thomas Seeley (Cornell University) and Madeleine Beekman (University of Sydney). Research presented covered current projects and training efforts to address the following four research topics: (1) role of abiotic stressors on honey bee colony health; (2) role of biotic stressors on honey bee colony health; (3) interactions between abiotic and biotic stressors on colony health and survival; and (4) fundamental investigations on honey bee ecology and behavior. The AAPA is pleased to present the submitted abstracts of many of the presentations given over the course of the two-day 2021 conference and 10 unpublished abstracts from ABRC 2020.

Abstracts of Presentations
Session I – Abiotic Stressors
Colony-level pesticide exposure affects honey bee (Apis mellifera L.) royal jelly production and nutritional composition
Priyadarshini Chakrabarti1, Joseph P. Milone2, Ramesh R. Sagili1 and David R. Tarpy2
1:Department of Horticulture, Oregon State University
2:Department of Entomology and Plant Pathology, NC State Univ
Honey bees provision glandular secretions in the form of royal jelly as larval nourishment to developing queens. Exposure to chemicals and nutritional conditions can influence queen development and thus impact colony fitness. Previous research reports that royal jelly remains pesticide-free during colony-level exposure and that chemical residues are buffered by the nurse bees. However, the impacts of pesticides can also manifest in quality and quantity of royal jelly produced by nurse bees. Here, we tested how colony exposure to a multi-pesticide pollen treatment influences the amount of royal jelly provisioned per queen and the additional impacts on royal jelly nutritional quality. We observed differences in the metabolome, proteome, and phytosterol compositions of royal jelly synthesized by nurse bees from multi-pesticide exposed colonies, including significant reductions of key nutrients such as 24-methylenecholesterol, major royal jelly proteins, and 10-hydroxy-2-decenoic acid. Additionally, the quantity of royal jelly provisioned per queen was lower in colonies exposed to pesticides, but this effect was colony-dependent. Pesticide treatment had a greater impact on royal jelly nutritional composition than the weight of royal jelly provisioned per queen cell. These novel findings highlight the indirect effects of pesticide exposure on queen developmental nutrition and allude to social consequences of nurse bee glandular degeneration. More information about the study can be found at Chemosphere (2021) Volume 263, pp. 128183.

Superorganism toxicology: Modeling the exposure and effects of metals in the honey bee colony
Dylan Ricke1, Reed Johnson1
1:Department of Entomology, Ohio State University
The honey bee (Apis mellifera) is a textbook example of a superorganism. As such, their responses to environmental chemicals are best understood at the colony level, but due to issues of scale and replicability, are more often assessed from laboratory assays with small groups of caged bees. Consequently, there’s high demand for modeling approaches that can extrapolate observations from the lab to the colony context. To do so, models must account for key differences between lab and field conditions: Whereas laboratory assays are typically brief (<4 days) and utilize acute dosages, colonies in the field are exposed to lower levels of anthropogenic chemicals over prolonged periods of time. Besides being important environmental contaminants in their own right, metals offer a variety of promising “model toxicants” that can be used to better understand the movement of food-borne chemicals within colonies and the gradual effects of chronic levels of exposure. Notably, certain metals (Li and Zn) are currently under development as the active ingredients of systemic pesticides to which honey bees are liable to be exposed. Utilizing data from a combination of lab and semi-field assays, I compare two approaches to modeling the time-cumulative effects of metals (Li, Zn, Cd) exposure in honey bee colonies. I show that these approaches can result in contrasting population predictions, depending on the exposure scenario and chemical in question.

The sublethal effects of IGRs on queens, workers, and colony reproduction
Julia Fine1
1:Invasive Species and Pollinator Health Research Unit, USDA-ARS, Davis, CA
As part of the USDA-ARS Invasive Species and Pollinator Health Research Unit, the new Pollinator Health Lab in Davis, CA is dedicated to performing longitudinal studies related to the health and productivity of managed honey bee colonies in an effort to help inform and develop improved management practices for beekeepers. As a part of this mission, current research efforts are focused on understanding how agrochemicals like insect growth regulators affect honey bee reproduction, behavior and the long term consequences of exposure scenarios. Recently, we have shown that IGR formulations commonly used in almond orchards during bloom can negatively influence queen productivity. Initial findings suggest that IGRs act transovarially, resulting in impaired hatching rates in eggs laid by exposed queens. This phenomenon could have potential consequences on metrics such as colony population stability, the development of surviving embryos, and their performance as adults.

Acute exposure to sublethal doses of neonicotinoid insecticides increases heat tolerance in honey bees
Victor H Gonzalez1, John M. Hranitz2, Mercedes B. McGonigle1, Rachel E. Manweiler1, Deborah R. Smith1, John F. Barthell3
1:Undergraduate Biology Program and Department of Ecology and Evolutionary Biology, University of Kansas
2:Biological and Allied Health Sciences, Bloomsburg University
3:Department of Biology, University of Central Oklahoma
Climate change is expected to accentuate the effects of abiotic stressors affecting honey bees. We tested the hypothesis that exposure to acute, sublethal doses of neonicotinoid insecticides reduce thermal tolerance in honey bees. We administered to bees oral doses of imidacloprid and acetamiprid at 1/5, 1/20, and 1/100 of LD50 and measured their heat tolerance 4 h post-feeding, using both dynamic and static protocols. Contrary to our expectations, bees fed with insecticides exhibited higher thermal tolerance and greater survival rates. Our study suggests a resilience of honey bees to high temperatures when other stressors are present, which is consistent with studies in other insects. We hypothesize that this compensatory effect is likely due to induction of heat shock proteins by the insecticides, which provides temporary protection from elevated temperatures.

Acute toxic effects of insecticide-fungicide-adjuvant combination on honey bees
Emily Walker1, Reed Johnson1, Guy Brock2
1:Department of Entomology, Ohio State University
2:Department of Biomedical Informatics, Ohio State University
Significant decreases in honey bee (Apis mellifera) populations have been reported by beekeepers and farmers over the last couple decades without a clear explanation. This decrease in the honey bee population poses a major problem for the California almond industry because of its dependence on honey bees as pollinators. This research aimed to determine if combinations of “bee-safe” pesticides applied during almond bloom were a possible explanation for this decrease in the honey bee population. In this study, we aimed to mimic the spray application route of exposure by using a Potter Tower to spray adult honey bees with the various treatments. This research determined that the combination of the fungicide Tilt (a.i. propiconazole) and the insecticide Altacor (a.i. chlorantraniliprole) displayed synergistic toxicity that was not observed when the treatments were applied individually. This study also looked at the toxic effects of adding adjuvants to pesticide mixtures. Adjuvants are exempt from bee testing that is required for other pesticides, so this study aimed to determine how these compounds may affect honey bee health. We showed that the adjuvant Dyne-Amic was toxic to honey bees at concentrations slightly above the recommended field applications. Dyne-Amic also showed synergistic toxicity when combined with the fungicide Pristine (a.i. pyraclostrobin and boscalid) and the addition of Dyne-Amic increased toxicity in the Tilt and Altacor combination treatment. These results suggest that the application of Tilt and Altacor in combination with an adjuvant at the recommended field application rates could cause significant mortality in adult honey bees. These findings highlight a potential explanation for honey bee losses around almond bloom and emphasize that adjuvants should receive the same testing as other pesticides.

Integrated Pesticide Management for Beekeepers and Why We Need Another Approach
Judy Wu-Smart1, Surabhi Vakil, Jennifer Wiesbrod1, Rogan Tokach1
1:Department of Entomology, University of Nebraska-Lincoln
The use of pesticides is a complex multi-faceted problem impacting honey bee (Apis mellifera L.) health. Research show alarming levels of pesticides from the surrounding environment as well as from beekeeper-applied treatments in bees and hive products such as brood comb and food stores that cause direct and indirect effects from the compound or formulation as well as potential interaction effects with other stressors (malnutrition, mites, and pathogens). Pesticide “kills” leading to acute mortality of bees are noticed immediately, however, chronic abnormal losses often go unnoticed by beekeepers and can lead to stagnant growth or weakening of hives. These pesticide “incidents” where only a small proportion of bees are affected may have cascading effects throughout the colony affecting age-based division of hive tasks (brood care, food processing, and foraging). Currently, there are no monitoring tools for beekeepers to identify abnormal losses due to pesticide incidents. And while recommendations for reducing pesticide exposure to foraging bees exist, management guides to reduce in-hive contamination of nestmates, comb, and food stores is limited. This effort seeks to better understand the sources of hive contamination and to develop integrated pesticide management for beekeepers which includes monitoring using dead bee traps and mitigation steps that will prevent further health decline in affected hives.

Session II – Biotic Stressors: Pest and Pathogens
Brood hygiene-eliciting signal as a tool for assaying honey bee colony pest and disease-resistance
Kaira Wagoner1, Marla Spivak2, Jocelyn Millar3, Coby Schal4, Olav Rueppell5
1:University of North Carolina Greensboro, Department of Biology
2:University of Minnesota, Department of Entomology
3:University of California Riverside, Department of Entomology
4:North Carolina State University, Department of Entomology
5:University of Alberta, Department of Biology
Despite numerous management and breeding interventions, the ectoparasitic mite Varroa destructor and the pathogens it vectors remain the primary biological threat to honey bee (Apis mellifera) health. Hygienic behavior, the ability to detect, uncap, and remove unhealthy brood from the colony, has been selectively bred for over two decades, and continues to be a promising avenue for improved Varroa management. Although the hygienic trait is elevated in many Varroa-resistant colonies, hygiene does not always confer Varroa-resistance, as some hygienic colonies still require miticides to limit mite infestations. Additionally, existing Varroa-resistance selection methods tend to trade efficacy for efficiency, as those achieving the highest levels of Varroa resistance can be time-consuming, and thus expensive and impractical for commercial use. Here we demonstrate that a mixture of synthetic honey bee brood compounds associated with Varroa infestation and/or Deformed-Wing Virus infection can be used to trigger hygienic behavior in a two-hour assay. High-performing colonies (colonies exhibiting hygienic response to >= 60% of treated cells within two hours) have significantly fewer phoretic mites, remove significantly more introduced mites, and are significantly more likely to overwinter successfully compared to low-performing colonies (colonies exhibiting hygienic response to <60% of treated cells within two hours). We discuss the efficacy and efficiency of this Varroa-specific assay as a tool for facilitating apiary management decisions and for selection of honey bee colonies more resistant to Varroa.

The Understudied Honey Bee: A case study exploring the prevalence of viral disease in feral honey bee colonies in San Diego County
Amy Geffre1, Dillon Travis1, Joshua Kohn1, James Nieh1
1:Department of Ecology, Behavior and Evolution, University of California San Diego
Bees provide important pollination services, but are threatened by many increasingly disruptive stressors, including pathogens. Currently, the majority of pollinator health studies consider the well-known agricultural species such as Apis mellifera. Multiple honey bee-associated viruses (HBAV) are implicated in managed honey bee (MHB) colony losses. However, we know little about how these HBAV affect the broader pollinator community. Importantly, we rarely consider the interactions between feral honey bees (FHB) and MHB. Although the ways in which viral diseases manifest in MHB are reasonably understood, the prevalence and effects of viruses in FHB remain a relative black box. These FHB may prove important in considerations of pollinator health, as FHB colonies appear to coexist successfully alongside pathogen-stressed MHB, although FHB are not treated for diseases. This suggests that FHB have some hitherto unexplored strategy that allows them to mitigate pathogen pressure. Such strategies may prove useful in disease mitigation among MHB. In this case study, we show evidence for the notion that FHB and MHB colonies maintain similar viral species, at similar levels. As such, we propose that FHB play a hitherto poorly described, but important role in pathogen dynamics of honey bees and the pollinator community as a whole.

The role of Varroa mite (Acari: Varroidae) host selection on forager-mediated mite migration
Emily Watkins de Jong1, Gloria DeGrandi-Hoffman1
1:Carl Hayden Bee Research Center, USDA-ARS, Tucson, AZ
Varroa mites represent a serious threat to honey bee (Apis mellifera) populations globally through the costs of parasitism and the transmission of virus from mite to host. Although the relatively low reproductive rates of Varroa should not allow for rapid population growth, populations are often observed to increase quickly, predominantly in the late Fall. This discrepancy between reproduction and larger than expected mite populations suggests that other factors may be contributing to Varroa populations within colonies. We found that forager-mediated migration of mites and a shift in mite host selection behavior were possible mechanisms for rapid Varroa population growth in the Fall. GC-MS analysis of cuticular hydrocarbon profiles in nurse and forager age bees revealed differences in cuticular hydrocarbon composition early in the study period, these differences in nurse and forager profiles disappear in the late fall at the same time when populations of foragers with mites increase. In a host selection assay, Varroa showed a significant preference for nurse odors over forager odors, in the late Fall there was no preference for either odor. The proportion of time that Varroa chose a forager scent in a choice assay correlated with the proportion of foragers with mites captured. These data shed light on the mechanisms underlying destructive seasonal mite titers in honey bee colonies.

The utility of cultured honey bee primary cells to investigate host-virus interactions
Alex McMenamin1,2, Fenali Parekh1,2, Katie Daughenbaugh1,3, Michelle Flenniken1,2,3
1:Pollinator Health Center, Montana State University, Bozeman, MT
2:Microbiology and Immunology Department, Montana State University, Bozeman, MT
3:Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT
Virus infections contribute to honey bee colony losses worldwide, therefore ongoing research aims to understand the impact of viruses on honey bee health from the colony to the cellular level. However, honey bee virology is limited by a dearth of immortalized cell lines. To address this need, we further developed the use of primary honey bee cells, model viruses, and semi-purified honey bee virus stocks to investigate host-virus interactions at the cellular level. Hemocytes, which are macrophage-like immune cells were isolated from larvae and exposed to a purified Lake Sinai virus 2 (LSV2) stock in either WH2 or Schneider’s insect medium. While healthy primary cells could be maintained in either medium for 4 weeks, LSV2 replicated (i.e., 2.5x at 72 hours post-infection) only in cells maintained in WH2 medium. Therefore, WH2 medium was utilized for subsequent viral time course experiments with a model virus (i.e., Flock House virus) which replicated 23x over 96 hours. In addition, we determined that hemocytes isolated from larvae obtained from sacbrood virus (SBV) infected colonies supported ex vivo virus propagation (i.e., 1000x over 96 hours). Together, these data show that honey bee hemocytes support virus infection and are a tractable system for investigating honey bee host-virus interactions. Similarly, cells derived from purple eyed pupae were cultured in WH2 media and used as a more complex cell culture model to study virus infection. We determined that pupa cell cultures supported replication of semi-purified SBV and deformed wing virus (DWV) and thus demonstrate that tissue-obtained primary cell culture is an additional tool to study honey bee host-virus interactions.

Immune response of different developmental stages of honey bee queens to Israeli acute paralysis virus infection
Esmaeil Amiri1,2*, Bin Han1,3*, Micheline K. Strand4, David R. Tarpy2, Olav Rueppell1,5
1:Department of Biology, University of North Carolina at Greensboro, Greensboro, NC 27402, USA
2:Department of Entomology & Plant Pathology, North Carolina State University, Raleigh, NC 27695, USA
3:Institute of Apicultural Research/Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China
4:Life Sciences Division, U.S. Army Research Office, CCDC-ARL, Research Triangle Park, Durham, NC 27709, USA
5:Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada
*Contribute equality to this work
The honey bee queen’s health is crucial to colony survival since reproduction and colony growth rely solely on the queen. Among many environmental stresses, viruses are a major concern since they can infect queens at different developmental stages. Although queens are protected by worker bees and other mechanisms of social immunity, they rely on evolved individual antiviral defense mechanisms to cope with viral infections. To understand the maturation of the queen immune system from before emergence to the onset of reproduction, we investigated how virus infection influences 18 immune genes from different conserved immune pathways including Toll, Imd, JAK/STAT and RNA interference at different developmental stages. We used Israeli acute paralysis virus (IAPV) as an experimental pathogen because it is relevant to bee health, but rarely has been detected in honey bee queens. Our results are discussed in the context of the ontogeny of immunity and regulation of queen immune genes in response to virus infection.

Varroa destructor mite decision-making process regarding honey bee worker cell invasion and size implications for developing bee brood
Taylor Reams1, Juliana Rangel1
1:Department of Entomology, TX A&M Univ, College Station, TX
Parasitization of honey bees (Apis mellifera) by the mite Varroa destructor is one of the main causes for the decline of honey bee health. To begin its reproductive cycle, a female mite enters the comb cell of a bee larva just before it is capped, undergoes development and reproduction within the cell, and exits the cell as the adult bee emerges. The main difference between Varroa infestations in their original host, Apis cerana, and Apis mellifera is that in the latter the mites are able to invade and successfully reproduce in worker larval cells. This study examines if worker brood is differently at risk for Varroa invasion with proximity to drone brood. This study also measures developmental brood size with Varroa invasion and feeding. Understanding distribution of Varroa in worker brood and how mite feeding impacts brood development will give us a better picture of Varroa impact in our honey bee colonies.

Longitudinal DWV and immune gene expression dynamics in colonies managed under conventional, organic, and treatment-free systems
Margarita M. López-Uribe1, Brooke Lawrence1, Robyn M. Underwood1
1:Department of Entomology, Center for Pollinator Research, Penn State University
Varroa mites and associated viruses are among the most serious stressors to honey bee colonies and are the focus of beekeeping management. Despite efforts to control mites, these parasites are widespread across all colonies in the United States and are a major threat to the industry. Here, we investigated the levels of DWV and immune gene expression in colonies managed under three management systems (conventional, organic, and treatment-free) over two years to determine the role of beekeeping in colony overwintering survival. Our results suggest that high levels of DWV and expression of Defensin-1 are important predictors of colony survival for first-year colonies across all management systems. However, we did not find evidence of an association between DWV and expression of Defensin-1 for established two-year-old colonies. In contrast, higher expression of vitellogenin was consistently associated with higher overwintering survival over the two years. This study highlights the importance of longitudinal studies of disease dynamics and immune gene expression for honey bee colonies to better understand the role of pathogens on pollinator health.

Beekeeping economics: A comparison of the profitability of conventional, organic and treatment-free management systems
Robyn M Underwood1, Timothy W Kelsey2 and Margarita M López-Uribe1
1:Department of Entomology, Center for Pollinator Research, Penn State University
2:Department of Agricultural Economics, Sociology, and Education, Penn State University
Honey bee colony management is an important factor in bee health, colony productivity, and profits for beekeepers. Parasitic mites are a major focus of management, with most beekeepers applying in-hive chemicals to control them. We followed 144 honey bee colonies for 2.5 years, from colony installation in April 2018 through late Summer 2020. Colonies were managed, with sister queens for genetic consistency, using a conventional, organic, or treatment-free management system (N = 48). Inputs and outputs for each colony were carefully measured to determine the profitability of each system. We found that mites were successfully controlled using chemical inputs and Winter survival was high (over 80%) using both the conventional and organic system, while colonies that were treatment-free (avoiding chemical inputs) had high mite levels and low Winter survival. Honey production was significantly higher in the colonies managed organically in both 2019 and 2020. In addition, both the organic and conventional systems allowed for the sale of excess colonies during Spring splitting, while treatment-free splits had to be kept as replacements for Winter losses. Overall, the organic system was the most profitable, while the treatment-free system was not profitable, due to high colony mortality.

Session III – Interacting Biotic and Abiotic Stressors
Investigating the impact of nutrition and organic compounds on honey bee virus infections
Fenali Parekh1,2, Katie F. Daughenbaugh2,4, Priyadarshini Chakrabarti3, Ramesh Sagili3, Michelle L. Flenniken1,2,4
1:Department of Microbiology and Immunology, Montana State University, Bozemam, MT, USA
2:Pollinator Health Center, MT State Univ, Bozemam, MT, USA
3:Department of Horticulture, Oregon State University, Corvallis, OR, USA
4:Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, USA.
Honey bees are exposed to diverse environments and multiple stressors. These include close interactions with numerous plant species while foraging for nectar and pollen of varying nutritional quality and collecting oils/resins, and in-hive compounds introduced by beekeepers and agrochemicals used in cropping systems. Bees are also exposed to viruses which are horizontally transmitted via shared floral resources, trophallaxis, and close contact. Recent studies indicate that dietary supplements and plant extracts may reduce the impact of viruses on honey bees, but the mechanisms of mitigation have not been characterized. To further investigate the impact of orally administered organic compounds on the outcome of honey bee virus infections we performed laboratory-based infection assays using deformed wing virus and two model viruses. Virus-infected bees were fed diets of varying composition, including protein and sterol augmented (24-methylenecholesterol) diets, or sucrose containing thyme oil (160 ppb), the antifungal Fumagilin-B (25 or 75 ppm), or the insecticide clothianidin (1 or 10 ppb), and virus abundance was assessed up to four days post-infection. Sindbis and Flock House virus abundance was reduced in bees fed UltraBee®, OSU diet supplemented with 24-mc, or multifloral pollen, and surprisingly clothianidin (10 ppb). SINV, DWV, and FHV abundance was also reduced in bees fed 160 ppb thyme oil, and greater in bees fed Fumagillin-B compared to sucrose-syrup fed bees. The expression of key antiviral genes, including dicer and ago-2 was upregulated in virus infected bees fed 0.16 ppm thyme oil compared to control. Better understanding the outcome of virus infections in the context of diet and chemical exposure may lead to the development of strategies to reduce virus-associated colony losses.

Varroa mites and neonicotinoid insecticides: Effects on drone survival and reproductive health
Selina Bruckner1, Lars Straub2, Geoffrey R. Williams1
1:Department of Entomology and Plant Pathology, Auburn University, Auburn, AL
2:Institute of Bee Health, University of Bern, Bern, Switzerland
The parasitic mite Varroa destructor and neonicotinoid insecticides represent important biotic and abiotic stressors, respectively, to Apis mellifera honey bees. Despite this, their potential interaction effects, especially concerning male drones, are severely understudied. We employed a fully crossed experimental design to assess potential interaction effects of neonicotinoids and V. destructor on drone survival and sperm quality traits. Known age cohorts were obtained from colonies that either received pollen patties containing field-relevant concentrations of two neonicotinoids (4.5ppb thiamethoxam and 1.5ppb clothianidin), or not. Upon emergence, drones were assessed for V. destructor infestation, and kept in laboratory cages based on treatment group allocation:

  1. No neonicotinoid/No V. destructor,
  2. No neonicotinoid/Yes V. destructor,
  3. Yes neonicotinoid/No V. destructor, and
  4. Yes neonicotinoid/Yes V. destructor.

Once drones reached sexual maturity, sperm quality traits were measured. Our findings confirm that neonicotinoids and V. destructor individually can significantly reduce drone survival, but also provide novel evidence for a synergistic interaction between the two stressors. Contrary to our expectations, sperm quality traits were not affected by neonicotinoids and V. destructor, when pressured alone or in combination. Nonetheless, reduced drone survival until sexual maturity could severely affect honey bee colony and population health given the importance of drones to mating.

Honey bee (Apis mellifera) workers prematurely remove themselves from the colony due to developmental stressors
Jordan Twombly Ellis1, Juliana Rangel1
1 Department of Entomology, Texas A&M University, College Station, Texas
Honey bees (Apis mellifera) provide a tractable system for studying the behavioral consequences of eusociality. As eusocial insects, honey bees live in colonies composed of thousands of sterile female workers with only one reproductively active queen. Therefore, a sterile worker’s own genetic fitness is best served by acting in the interest of her colony, even if her behavior curtails her own lifespan. In this study, we tested the hypothesis that developmentally stressed worker bees remove themselves from the hive to protect their colony from the negative costs of an inefficient workforce. To confirm that this self-removal behavior is a reaction to severe stress, and not parasite-driven or a social immune response, we developmentally stressed bees with either cold shock or parasitization by Varroa destructor mites. Stressed bees, as well as their control counterparts, were tagged upon emergence and introduced to a common observation hive. We took daily attendance of the focal bees and checked a trap engineered to capture self-removing bees every hour. For both treatments, we found that bees stressed by either mites or cold temperatures lived for significantly less time and self-removed from the hive in significantly higher numbers than their control counterparts. This indicates that self-removal behavior is probably stress driven. Going forward, we plan to measure the hypopharyngeal glands and juvenile hormone titers of the bees that self-removed to further confirm the drivers of this behavior. This will ultimately enable us to model the effects of this behavior on the entire colony.

Dancing honey bees communicate monthly fluctuations in forage availability in a mixed-use landscape in Virginia
Bradley D. Ohlinger1, Roger Schürch1, Margaret J. Couvillon1
1:Department of Entomology, Virginia Tech, Blacksburg, Virginia
Bees and other flower-visiting insects, which provide critical ecosystem services, face declines in both diversity and abundance that threaten their ability to provide adequate pollination for both wild plant communities and agricultural crops. In response, management efforts have attempted to address pollinator loss through programs that provide supplemental forage or foraging habitat for the hungry pollinators; however, such efforts must consider the temporal and spatial dynamics of foraging and the phenology of flowering species against the nutritional needs of the pollinators. In this study, we monitored the waggle dances of honey bees foraging in a mixed-use landscape in Blacksburg, VA over two foraging seasons (2018-2019) to 1) identify seasonal fluctuations in the availability of honey bee forage, as determined by communicated foraging distance and to 2) determine the types of foraging habitat that honey bees prefer to visit. We decoded and analyzed waggle dances (n=3614) to identify monthly fluctuations in communicated foraging distance and to map the association between land cover type and honey bee foraging dynamics. Our results consistently show an increase in foraging distances in June and October during both years, suggesting that these months likely pose a challenge for locally feeding pollinators and should be the time when aid should be directed in our area. Additionally, our results demonstrate that the honey bee foragers advertised pasture lands with 40.7% of their waggle dances, more than the other available land cover types, despite the pasture land cover category comprising only 19% of the study area. However, foraging rates to the different land cover categories varied across months, with honey bees showing increased foraging to pasture lands and decreased foraging to forests during months with low median foraging distances and decreased foraging to pasture lands and increased foraging to forests during months with higher median foraging distances. These data suggest that local pastures provide attractive foraging resources throughout most of the honey bee foraging season at our field site, while forests provide transitory foraging resources during the early Summer.

Honey bee tolerance to Deformed wing virus infection when fed diets with varying macronutrient ratios
Alexandria N Payne1, Pierre Lau1, Jordan Gomez1, Cora Garcia1, Humberto Boncristiani2, Juliana Rangel1
1:Department of Entomology, Texas A&M University, College Station, Texas
2:Entomology and Nematology Department, University of Florida, Gainesville, Florida
It has been shown that the health of honey bees infected with pathogens can be improved by ensuring proper nutrition. The purpose of this study was to determine what protein (P) to lipid (L) ratio within artificial diets have a positive impact on the survivorship and overall health of honey bees infected with Deformed wing virus (DWV). We conducted a cage assay where newly emerged bees were assigned to one of three treatments: a DWV injected group, a PBS injected negative control, or a non-injected negative control group. Cages in the three infection groups were further divided into four treatment groups based on whether they were fed a high P: low L diet (40P:10L), a low P: high L diet (20P:30L), an intermediate diet ratio at which non-infected honey bee colonies self-select for in the field (30P:20L), or no diet whatsoever for a total of n=6 cages treatment group. Survivorship and the amount of diet consumed was measured for each cage over a 16 day period. It was found that bees across all three infection groups consumed the intermediate 30:20 the most. It was also determined that bees infected with DWV and fed this 30:20 diet or no diet at all had a higher rate of survival than bees fed one of the more protein/lipid extreme diets. Pollen and commercially available pollen substitutes vary widely in their macronutrient P:L ratios, and this work will help us better determine what target ratio can help bees better deal with DWV infection.

In vitro effects of fungicides on the susceptibility of honey bee (Apis mellifera) larvae to European foulbrood
Jenna Thebeau1, Dana Liebe1, Sarah Wood1, Larhonda Sobchishin1, Ivanna V. Kozii1, Colby D. Klein1, Igor Medici de Mattos1, Michael W. Zabrodski1, Melanie Roulin1, Jessica E. DeBruyne1, Igor Moshynskyy1, Mohsen Sharafi1, Meghan O. Milbrath2, Robyn McCallum3, M. Marta Guarna4, Patricia Wolf Veiga5, Eric M. Gerbrandt6, Elemir Simko1
1:Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan
2:Michigan Pollinator Initiative, Michigan State University
3:Atlantic Tech Transfer Team for Apiculture (ATTTA), Perennia Food and Agriculture Inc.
4:Beaverlodge Research Farm, Agriculture and Agri-Food Canada
5:National Bee Diagnostic Centre, Grand Prairie Regional College
6:British Columbia Blueberry Council
Pesticide exposure has been implicated in the immunosuppression of honey bees (Apis mellifera) and suspected to increase susceptibility to European foulbrood (EFB). EFB, caused by the bacterium Melissococcus plutonius, produces increased mortality in honey bee larvae in colonies under environmental and nutritional stress, particularly in association with commercial blueberry pollination. The effects of exposure to formulated fungicide products commonly used in blueberry production on susceptibility of honey bee larvae to EFB during blueberry pollination is currently unknown. Using an in vitro larval infection model of EFB, we tested the effects of chronic larval exposure to field-relevant concentrations of the formulated blueberry fungicides Captan® and Kenja® on larval mortality from M. plutonius infection. Surprisingly, we found that chronic exposure to Captan® or Kenja® during development significantly (P<0.0001) increased larval survival from EFB by 33% compared to infected control larvae which were unexposed to fungicides. One explanation for this finding could be an inhibitory effect of fungicides on the growth of M. plutonius. However, larvae chronically exposed to a combination of Captan® and Kenja® did not experience a significant difference in survival relative to infected controls. These in vitro results suggest that chronic exposure of honey bee colonies to formulated fungicide products during blueberry pollination does not predispose these colonies to EFB. Additional colony-level studies are necessary to verify the field-relevance of these in vitro results.

Evaluation of Active Ingredients for Potential Miticidal Activities Against Varroa destructor and Toxicity to Honey Bees
Rassol Bahreini1, Medhat Nasr1, Cassandra Docherty1, Olivia de Herdt1, Samantha Muirhead1, David Feindel1
1:Plant and Bee Health Surveillance Section, Alberta Agriculture and Forestry, 17507 Fort Road NW, Edmonton, Alberta, T5Y 6H3, Canada
The Varroa mite, Varroa destructor Anderson and Trueman, infestation has threatened honey bee survivorship. Low efficacy and development of Varroa mite resistance to currently used Varroacides has increased the demand for alternative effective treatment tool options that exhibit high efficacy, while minimizing adverse effects on honey bee fitness. In this investigation, the toxicity of 16 active ingredients and nine formulated products from 12 registered chemical families were evaluated on Varroa mites and honey bees. In the laboratory test, we used the vial test for contact surface and topical exposures for 4h and 24h. We found that compounds belong to Pyrazoles (93% mortality) and Tetronic acids (70-84% mortality) had greater toxicity to Varroa mites, but high dose rates caused high bee mortality (>60%). The results showed that high toxicity of active ingredients from Quinazolines and Oxazolines against Varroa mites caused 92% and 69% mortality, respectively; and were found to be safe on honey bees. These identified products will be further investigated for development of potential Varroacides. To simultaneously test bees infested with mites, a plastic cage was designed to expose a group of 100 – 120 bees infested with mite to three different concentrations of each tested active ingredient for 24 h. Then, the mite mortality and bee mortality were determined in comparison to Amitraz. Out of these tested active ingredients, four ingredients belong to Tetronic acid, Quinazoline, and Pyrazole, showed higher mite mortality rate and variable mortality rates to bees. These vetted products were then subjected to a semi field trial using modified beehives. Each hive had three compartments that had three frames of bees infested with Varroa mites in each compartment. Results of field assay indicated that candidates from Quinazoline and Pyrazole miticide classes had greater efficiency and reduced mite abundance by up to 80.12% and 67.71%, respectively, in comparison to 95.88% in positive control (Apivar®) treatment. Our research will continue to further develop an application method that is effective against mites, safe for bees, without resides, and safe for applicators for these products.

A new look at honey bees foraging in the greenhouse: Does early experience matter?
Ashley Welchert1,2, Vanessa Corby-Harris1, Jack Welchert3
1:Carl Hayden Bee Research Center, USDA-ARS, Tucson, AZ
2:Department of Entomology, University of Arizona
3:Department of Biosystems Engineering, University of Arizona
As the climate continues to change, greenhouse agriculture will be increasingly important to our global food system. Greenhouse crops requiring pollination are mostly limited to bumble bee or hand pollination, but bumble bees are not ideal pollinators for all plants. Using honey bees for pollination would increase the number of viable greenhouse crops. Although there is some evidence that honey bees can be used for pollination in greenhouses, other research suggests that colonies decline in these environments and foraging behavior is altered. This may be because honey bees navigate via environmental cues that are altered in the greenhouse. Other studies show that bees learn to navigate their environments using experience gained during their first orientation flights. Here, we asked how environmental experience affects honeybee foraging activity and learning acquisition in greenhouses. We raised single-cohort hives outdoors or in the greenhouse until bees were several days past foraging age. The colonies were then moved into separate greenhouse or outdoor testing arenas, yielding four treatment combinations: outdoor-reared/outdoor-foraging, outdoor-reared/greenhouse-foraging, greenhouse-reared/greenhouse-foraging, and greenhouse-reared/outdoor-foraging. We observed bee activity at hive entrances and at feeder arrays containing high- to low-value pollen and sucrose resources. In a preliminary analysis, we found that greenhouse-raised bees foraging in the greenhouse collected more pollen and had more foragers visiting feeder arrays. Regardless of their experience, outdoor foraging bees collected more pollen per forager. Further analysis of our data is planned to help us better understand the effects of experience and foraging environment on honey bee foraging behavior.

Session IV – Behavior and Ecology
The grooming and biting behavior of Indiana mite biters and commercial honey bee colonies
Jada Smith1, Krispn Given2, Hongmei Li-Byarlay1,3
1:Department of Agricultural and Life Science, Central State University
2:Department of Entomology, Purdue University
3:Agricultural Research and Development Program, Central State University
The honey bees (Apis mellifera) are the most important managed pollinator for sustainable agriculture and our ecosystem. However, the managed honey bee colonies in the United States experience 30-40% of losses annually. Among all the biotic stressors, the parasitic mite Varroa destructor is considered as one of the main pests for colony losses. The mite biting behavior as a Varroa tolerant or resistant trait has been selected in Indiana for a decade. A survey of damaged mites from the bottom of a colony can be used as an extended phenotype of the mite biting behavior to evaluate a colony. Our results showed the mite biting rates from both Indiana mite biters and open-mated colonies are significantly higher than commercial colonies from Georgia. Even though we did not detect a significant difference in the number of missing legs in mites between Indiana mite biter open mated colonies and commercial colonies, we noticed a trend of more mite legs are missing in Indiana mite biters open-mated colonies. In addition, the morphology of pollen forager worker mandibles were compared between Indiana mite biters and commercial colonies via X-ray micro-computed tomography. A significant difference was detected in the long edge of the mandible. Our results showed novel scientific evidence to explain the potential defensive mechanism against Varroa mites via mandibles providing the significant knowledge of a defensive behavioral trait for mite resistance and efforts in honeybee breeding.

Fluctuating Forage: Honey bee hives located in fruit orchard systems experience boom and bust periods across the foraging season
Taylor N. Steele, Roger Schürch, Margaret J. Couvillon1
1:Department of Entomology, Virginia Tech, Blacksburg, Virginia, USA
Honey bees (Apis mellifera) are important pollinators of many foods, such as apples, and their presence in these landscapes is therefore beneficial to crops. However, less is known about the reciprocal of this relationship: are fruit orchards beneficial to honey bees? In particular, how are bees in an orchard feeding themselves, especially when they are located there across the entire foraging season from early Spring to late Autumn? To understand this relationship, we use the honey bee waggle dance, a unique behavior that communicates the location of quality forage, and pollen collection to investigate the foraging dynamics of honey bees in Northern Virginia, a landscape dominated by apple orchards. For two years’ foraging seasons, we video recorded for one hour/day (three or four times/week) the dances of returning foragers from three observation hives. Concurrently, we collected pollen two times/week from returning foragers for plant identification and pesticide residue purposes. We extracted the dance vectors (n = 3540 dances) and analyzed them spatially and temporally, which we also correlated with the pollen data. We found that honey bees are predominantly foraging (85% foraging) outside of apple orchards during the bloom in late April – early May. Pollen analysis demonstrates that bees are collecting pollen from apples very early in the season, but not as a primary protein source.

Honey bee workers fed fatty acid diets exhibit differences in learning and discrimination of brood odors
Meghan M. Bennett1, Ashley Gander1, Mark Carroll1, Sharoni Shafir2, Brian Smith3, Vanessa Corby-Harris1
1:Carl Hayden Bee Research Center USDA-ARS, Tucson, AZ
2:Hebrew University of Jerusalem, Rehovot, Israel
3:Arizona State University, Tempe, AZ
Honey bee colonies consume pollen to satisfy their dietary requirements for proteins and lipids. Young worker bees consume almost all of the pollen coming into the hive, while foragers consume mostly nectar. Young bees consume pollen because it provides the nutrients required for the production of food for developing brood. Young bees also play an integral role in hive health by performing hygienic behaviors, such as removing diseased and dead brood. Past research suggests that lipids, specifically fatty acids, impact olfactory learning in honey bees. Thus, we wanted to know how fatty acids affect olfactory learning to colony-relevant odors. We fed young workers diets that were balanced or unbalanced in their ratio of essential omega fatty acids. We then measured their ability to learn healthy and damaged brood odors as well as their ability to discriminate between the two. Workers fed balanced diets exhibited higher learning acquisition to brood odors compared to those fed unbalanced diets. In addition, workers fed balanced diets can discriminate between damaged and healthy brood odors better than workers fed unbalanced diets. These results reveal crucial insight about how diet affects young worker olfactory learning, which could have downstream effects on the hygienic ability of the hive.

Row crops provide mid-summer forage for honey bees
Mary Silliman, Roger Schürch, Sally Taylor, Margaret Couvillon1
1:Department of Entomology, Virginia Tech, Blacksburg, VA, USA
Insect pollination is necessary for many major food crops and is a vital ecological service. Hymenoptera and Lepidoptera species have faced declines in abundance and diversity due to external stressors including pests, pathogens, pesticides, and poor nutrition. One method of investigating underlying causes of these issues is assessing a landscape’s nutritional content. Honey bees communicate their foraging location through the waggle dance, a recruitment behavior foragers use to convey the location of a good resource (i.e., distance and direction relative to the hive). Researchers can observe and interpret these signals to estimate food availability. We placed three hives in a row crop system (e.g., corn, cotton, peanuts, soybeans, wetlands, forest ) and filmed bees throughout the foraging season (April – October) of 2018 and 2019. In all, we decoded and analyzed 3459 honey bee waggle dances. We found a difference in foraging distance, as communicated by dance duration, by month in both years (n = 3459, p< 0.0001). The shortest median foraging distances were observed in July of both years, suggesting that is when forage is more available. Percent foraging in cotton and soybean fields was between 19-30% and 10-18% respectively during full bloom in Summer. Our findings illustrate food availability within an agricultural landscape and show honey bee visitation to row crops throughout the foraging season.

Factors affecting attractiveness of soybeans to honey bees
Reed M. Johnson, Harper McMinn-Sauder, Nicole Sammons and Chia-Hua Lin1
1:Department of Entomology, The Ohio State University, Columbus, OH, USA
Soybean flowers have the potential to provide a substantial honey flow for beekeepers maintaining apiaries in agricultural regions. However, soybean flowers can be notoriously unreliable sources of nectar. Different soybean varieties display different floral characteristics including nectar production. Weather conditions can also influence nectar production, although some varieties are more sensitive to changes in temperature regimes than others. We monitored nectar production and floral visitation by honey bees in a range of soybean varieties grown in Ohio. A subset of the most and least bee-attractive varieties were grown under pollinator exclusion tents with or without nucleus colonies to assess the foraging preference of honey bees and soybean yield response to honey bee pollination. Data on soybean nectar production could help guide planting choices to maximize benefits to both farmers and beekeepers in areas hosting apiaries and may help inform the timing of pesticide applications to reduce impacts on bees.

Using colony weight monitoring to identify flowers important for honey production in the Ohio agroecosystem
Harper McMinn-Sauder, Chia-Hua Lin, Reed Johnson1
1:Department of Entomology, The Ohio State University, Columbus, Ohio, USA
The Ohio agricultural landscape provides high potential to support honey bee foraging with resources such as crops, weeds, and pollinator enhancement plantings embedded in the landscape. Though many Ohio honey bee colonies are located in this region, there is little definitive knowledge about the flowers that are supporting honey bees in this habitat. This study aims to identify the nectar resources most important for honey production in colonies located in the Ohio agricultural landscape. Colony weight was continuously monitored for 36 colonies at 12 apiaries in central Ohio located on a gradient of agricultural intensity in 2019 and 2020. These weight data not only help us better understand within-colony dynamics, but also indicate periods of nectar flow and dearth on the surrounding landscape. Colony weight data will be analyzed in tandem with pollen metabarcoding analysis of honey samples collected periodically from study colonies over the foraging season. This will allow us to identify the floral resources contributing to periods of colony weight gain and honey production. Preliminary results indicate a period of colony weight gain in many colonies in mid to late July, when soybeans are blooming in Ohio. Metabarcoding analysis of honey samples collected during this bloom period will be analyzed to identify the resources contributing to this weight gain. This study will expand our understanding of honey bee foraging in the Ohio agroecosystem, identifying the flowers most important for honey production in this region.

Lightning Talks
Assessing alternative tools for performing hygienic behavior testing for beekeepers
Sheldon Brummel1, Judy Wu-Smart1
1:Department of Entomology, University of Nebraska-Lincoln
Hygienic behavior testing in honey bees is used to assess mite or disease tolerance in colonies. Liquid nitrogen is applied to a portion of brood inside capped comb cells to freeze-killed pupating brood. The rate of removal of freeze-killed brood reflects how quickly workers may detect mite-infested or disease-infected brood and remove the infected brood from the hive before transmission to nestmates may occur. Currently, there are few options for hygienic testing of colonies for non-commercial apiaries due to the challenges associated with access, storage and use of liquid nitrogen. Many hobbyist and small scale beekeepers would like to learn the tools needed to test and select for hygienic behavior in local stock. This research seeks to develop and test novel tools for beekeepers to use in management of their hives for hygienic testing. Comparisons of the current hygienic test using liquid nitrogen will be compared with commercially available alternatives and measured for effectiveness and duration of treatment. Results will be used to inform beekeepers of cheap, fast, and easily replicable alternatives for hygienic testing in the field.

Comparing sample types, and sequencing approaches for the characterization of the honey bee (Apis mellifera) gut microbiome
Tara Newman1,2, Lance Lansing2,4, Lan Tran1,3, Amanda Gregoris1, Rodrigo Ortega Polo1,4, M. Marta Guarna1,3
1:Agriculture and Agri-Food Canada, Beaverlodge Research Farm, Beaverlodge, AB
2:University of Victoria, Victoria, BC, Canada
3:Agriculture and Agri-Food Canada, CFIA Centre for Plant Health, Sidney, BC.
4:Agriculture and Agri-Food Canada, Lethbridge Research and Development Centre, Lethbridge, AB.
The diverse members of the honey bee gut microbiome have a symbiotic and/or pathogenic relationship with honey bees, and they directly impact bee health and immunity. 16S rRNA amplicon and shotgun metagenomics sequencing are used to study microbiome composition of the honeybee gut microbial communities. In this study, we compared the resolution of shotgun metagenomic sequencing and 16S rRNA microbial community profiling for the characterization of bacterial members within the honey bee microbiome. We also investigated the relative representation of gut microbiome reads compared with all classified reads in different sample types. Our sample types included the whole bee and bee abdomen, as well as dissected whole gut and hindgut. We evaluated the impact of sample type and sequencing depth on the taxonomic profiling of the honeybee gut microbiome and compared different databases for bioinformatic analysis. We determined that, at the genus level, one pooled sample can represent a group of 10 honey bees, and all sequencing approaches are able to give an accurate representation of the core microbiota. However, metagenomics sequencing is advantageous at the species level and lower taxonomic ranks. Species rarefaction analysis showed that deep-sequenced samples discover species at a greater rate than shallow-sequenced samples, indicating that taxa abundance information is gained through deeper sequencing. The work presented here will help inform decisions on methodological approaches for bee microbiome studies tailored to specific research questions in bee health and nutrition.

Developing Integrated Pollination Conservation and Research Efforts at Kimmel Orchard
Courtney Brummel1, Louise Lynch-O’Brien1, Susan Weller1,2, Judy Wu-Smart1
1:University of Nebraska-Lincoln, Department of Entomology
2:University of Nebraska State Museum of Natural History
Beekeepers are facing increased challenges, from honey bee population loss to the spread of misinformation in management guidance. As the diverse audience of hobbyist beekeepers grows, innovations are essential, particularly as the reasons for keeping bees have also diversified. Beekeepers today are not just interested in honey production but also providing pollination services, producing value-added products, and offering educational opportunities to new beekeepers. This research examines how beekeeping management differs with alternative hive structures or the hive boxes that are used to house each honey bee colony. Data on various functional measures, such as honey production, brood rearing, thermoregulation and overwintering success will help elucidate advantages and disadvantages of each alternative hive structure compared to the traditional Langstroth hive. Results from this project may facilitate successful management of bees for educational purposes which will enhance external partnerships, increase educational opportunities, and further promote engagement with local communities regarding the importance of pollinator-friendly landscapes and practices that support healthy bee communities.

Effects of amitraz and amitraz+thymol acaricide treatments on Varroa destructor in Apis mellifera honey bee colonies
Dan Aurell, Selina Bruckner, Clint Wall, Geoffrey Williams1
1:Dept of Entomology and Plant Pathology, Auburn University
More than 30 years after the arrival of the ectoparasitic mite Varroa destructor, American beekeepers still struggle to manage this damaging pest of honey bees (Apis mellifera). While it is preferable to maintain low V. destructor levels throughout the year, it is also important to bring mite levels down rapidly if they reach a damaging level prior to the critical Winter season. During the Fall in Alabama, 32 colonies with high levels of V. destructor (mean infestation of adult bees 11.3%) were used to test if concurrent treatment with two registered active ingredients (amitraz and thymol) could improve the management of V. destructor compared to an amitraz only based treatment. This was achieved by allocating colonies in late September to two treatment groups that received: 1) Four Apivar® strips (amitraz 3.3%) per colony; and 2) Four Apivar® strips and 51 mL of Apiguard® (thymol 25%), all applied at one visit. Full colony assessments were performed before treatment application, mid-treatment (21 days post-application), and upon completion of the 42-day treatment period. This included visual estimation of the number of adult bees and the area of sealed worker brood per colony. At the same time, V. destructor abundance on adult bees was also assessed by an alcohol wash method, as was percent brood infestation, which allowed an estimate of the total V. destructor population per colony. Sealed worker brood area was also estimated on Day 10. Preliminary analyses indicate that both treatments reduced the V. destructor population in the colonies by Day 42, and that the regimens were similarly effective. There was no treatment effect on the number of adult bees, but estimation of sealed brood area on Day 10 was suggestive of a reduced brood area in the Apivar®+Apiguard® treatment. This and the pieces of pupae observed on the bottom boards in the combination treatment point to brood removal in this group in the first days of the treatment. Of the 27 colonies that remained at the end of the trial, 10 still had V. destructor abundances on adult bees that exceeded 3.0%; 7/14 for Apivar® and 3/13 for Apivar®+Apiguard® treatments. The colonies will be monitored through the winter to detect any delayed treatment effects.

Reusing equipment from dead outs and the potential impacts on queen rearing capacity
Rogan Tokach1, Autumn Smart1, Judy Wu-Smart1
1:Department of Entomology, University of Nebraska
This study evaluates the potential impact of reusing combs from colonies that have previously died. Consistently high losses of colonies within apiaries may be the result of environmental or abiotic stressors (such as pesticide exposure or malnutrition) or biotic pressure from high mite or disease loads. Reusing combs from previous dead colonies to restock new hives is a common beekeeping practice that may adversely affect survival of subsequent colonies. Exposure to pesticides through contaminated comb and food may impact queen development, mating, and egg viability, thus impairing a colony’s ability to replace their queen if necessary. This research examines the role old “deadout” comb plays on requeening success and the viability of replacement queens reared by queenless microcolonies. We established two treatment groups that used, 1) combs from good performing apiaries, or 2) combs from “deadout” colonies originating from an apiary suspected to have chronic pesticide exposure and contaminated food stores. The number of queen cells formed, adult emergence success rate, and measures of virgin queens (weight, wing length, head length, and head width) will be compared among treatments. A separate subset of reared queens will be observed for timing of egging laying, and egg laying behavior, to determine if colonies are able to successfully rear viable, functional replacement queens.

Plausibility of novel honey bee semen cryopreservation methods
Connor A. Auth1, Brandon K. Auth1
1:Department of Entomology, Washington State University, Pullman, Washington, USA
Cryopreservation of germplasm is used in several livestock species to accelerate artificial selection of desirable traits. Recently, semen cryopreservation has been successfully introduced to honey bees, bolstering trait selection for bee breeders and aiding conservation efforts for threatened bee populations. The leading cryopreservation method uses slow-programmable freezing to achieve long-term storage of honey bee germplasm; however, this method is costly and time consuming, making it less accessible to bee breeders and researchers. Additionally, the success of this method is inconsistent, producing inseminated queens with variable fertilization success, prompting the need for an alternative freezing method. Here we test two freezing devices, the CryoLock and microdialysis tube, with varying levels of cryoprotectant (20%, 40%, or 60%). Each device was either plunged directly into liquid nitrogen (vitrification) or submerged in liquid nitrogen vapor (vapor immersion) before being stored. The post-thaw sperm viability and subjective motility of these techniques were compared to those of slow-frozen semen and non-frozen controls. In general, semen frozen in dialysis tubes produced higher motility and sperm viability than those frozen with the CryoLock. The same trend was observed between vapor immersion and vitrification, with vapor immersion proving superior. Compared to slow-programmable freezing, only dialyzed semen immersed in vapor with 20% cryoprotectant produced comparable sperm motility and viability. Future brood evaluation studies on queens inseminated with dialyzed semen immersed in vapor with 20% cryoprotectant should be performed to determine if this method can effectively replace slow-freezing and alleviate financial barriers imposed on bee breeders.

Introducing the Bee Health Collective
Grace Kunkel, Nathalie Steinhauer2
1:Project Apis m.
2:The Bee Informed Partnership
The Bee Health Collective is a collaboration of stakeholder organizations to gather and share current, credible information about honey bee health. Founded by Project Apis m. and the National Honey Board in 2019, the mission of this project is to provide a ‘one-stop shop’ resource with information about honey bee health, honey bee research, and the beekeeping industry. This information is presented as infographics, images, and narrative, and focuses on how these topics relate to important things like agriculture, resource management, and food. In addition to the general information, there are two other main features of the site: A comprehensive research database, and a bulletin board for bee-related opportunities. The database is searchable by topic, funding source, researcher, or institution, and contains funded honey bee projects in the United States since 2009. The bulletin board is a free resource where anyone can post or find honey bee related jobs, research funding, and scholarships. In addition to Project Apis m. and the National Honey Board, The Bee Health Collective is supported by the USDA, the Almond Board of California, and the Bee Informed Partnership.

Abstracts from ABRC 2020
The physiological effects of oxidative stress in feral honey bees
Kilea Ward1 and Hongmei Li-Byarlay1,2
1:Department of Agriculture and Life Science, Central State University
2:Agricultural Research and Development Program, Central State University
Honey bees are the most important managed pollinator in agriculture but the long-term survival of their colonies is seriously threatened. Oxidative stress is closely related to the lifespan and ageing of honey bees. Many synthetic pesticides and herbicides are thought to increase oxidative stress in honey bees. More work is needed to determine the impact of increased oxidative stress on honey bee health and survival. Feral colonies have displayed higher mite resistance comparing to commercial package bees. However, it is unknown whether there is any difference between the aging and levels of oxidative stress between these two populations. We sought to determine the impact of oxidative stress of forager workers by collecting them from paired colonies (a feral colony and a managed commercial colony) with similar foraging resources. Results exhibit a significant difference of survival time and oxidative stress via lipid damage between feral and commercial honey bees. Our study provides new evidence of the difference physiology and oxidative stress between feral and commercial stocks.

Neonicotinoid pesticides are more toxic to honey bees at lower temperature: implications for overwintering bees
Muhammad Shoaib Saleem1,2, Zachary Y. Huang2
1:Department of Agriculture and Agribusiness Management, University of Karachi, Karachi, Pakistan.
2:Department of Entomology, Michigan State University, East Lansing, Michigan
The honey bee (Apis mellifera L.) is an important pollinator and the best model for pesticide effects on insect pollinators. The effects of agricultural pesticides on honey bee health have consequently raised concern. Honey bees are suffering from eminent colony losses in the northern and eastern hemisphere possibly because of a variety of growing problems, with which pesticides may interact to exacerbate their impacts. Effects of various pesticides have been measured for multiple responses such as learning, memory performance, feeding activity, and thermoregulation. These studies were conducted at many different temperatures (11-35°C), however, few studies compared the toxicity of the same pesticide to bees at different temperatures. It is possible that the same pesticide might show different toxicity to honey bees at different temperatures. To reveal such potential interactions, we administered low doses of two neonicotinoid insecticides (imidacloprid @ 250 ppb and thiamethoxam @ 125 ppb) at three different temperature scenarios (35°C, 24°C and a varying temperature) and determined the effects on honey bee survivorship. We discovered that honey bees are much more sensitive to the neonicotinoid pesticides imidacloprid and thiamethoxam at a constant 24°C or at a varying temperature (night at 13°C and day at 24°C) compared to bees at 35°C. These results suggest that honey bee colonies during wintertime will be more sensitive to pesticides. Doses of neonicotinoids that are safe to colonies during Summer might kill them during the wintertime.

Extended-release Oxalic Acid in Virginia
Soren Roberts1, Aris Roberts1, Randy Oliver2
1:Aurora Apiary, LLC
2:Scientific Beekeeping
Extended-release oxalic acid Scott shop towels seem an effective method of treating Varroa mites in beehives in the Summer in California. These shop towels were soaked in a 1:1 mixture of glycerin and oxalic acid and applied to the hives. We repeated Randy Oliver’s study in our high humidity climate of Northern Virginia and found mixed results. Four hives were responsive, two hives were neutrally affected, and two hives had high increase in mite count. The response to extended-release OA might be to multiple factors. The bees could be hygienic and resistant to mites, the high humidity might affect the chemistry of the glycerin, the bees behavior might affect the rate of shop towel removal and thus their exposure to the oxalic acid. Further studies are needed to elucidate reason(s) for varied responses.

Honey bee queen flight ability negatively impacted by Nosema infection
Yuan Zhang1,2, Fang Liu1,3, Zachary Y. Huang1
1:Department of Entomology, MI State Univ, East Lansing, MI
2:Yunnan Academy of Biodiversity, Southwest Forestry University, Kunming, Yunnan, China.
3:College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui, China.
Nosema ceranae (Nc), a fungal pathogen that infects honey bee’s midgut epithelial cells, can affect honey bees in many aspects. However, whether infection with this pathogen can affect honey bee queen’s ability to fly has not been investigated. We fed each newly emerged queen with 100,000 Nc spores (via 5 ul sugar syrup) and let the queens age inside cages for 10 days. On the age of 11 days (day 1=emergence), we tested their flight performance using flight mills. We found significant negative effects on queen flight distance, flight duration, maximum flight speed, and both the flight duration and flight distance of the longest flight episode. No significant differences were found for other parameters, such as number of stops, stopping time, and the average flight speed. Our results suggest that Nosema ceranae infection can have significant negative impacts on queen flight ability and thus may affect their mating success. The fungal infection by Nosema ceranae thus can have more serious negative impacts on honey bee health, than previously considered.

Effect of various pollen substitute on honey bee worker longevity and size of hypopharyngeal glands
Jing Zhang1, Zachary Huang1
1:Department of Entomology, Michigan State University, Michigan
There are currently many types of pollen substitutes available in the market, which are used to feed honey bee colonies when there is pollen dearth. We tested several common pollen substitutes in the market, including Beepro, Megabee, Nutrabee, and Ultrabee, with sugar only as a negative control, and mixed bee collected pollen as a positive control, and also a yeast extract. All pollen substitutes were mixed with 50% syrup to yield the same soft pliable consistency and provided to caged bees. Longevity was tested at 34.5°C and 50% RH. We measured pollen consumption every five days and on day 10 sampled five bees per cage for the size of hypopharyngeal glands. We replicated the study with bees from five different colonies. In general, pollen yielded the longest survival bees while sugar gave the shortest longevity. Pollen substitutes yielded longevity between these two extremes and their rankings varied among different trials.

Neonicotinoid pesticides are more toxic to honey bees at lower temperature: implications for overwintering bees
Muhammad Shoaib Saleem1,2, Zachary Y. Huang2
1:Department of Agriculture and Agribusiness Management, University of Karachi, Karachi, Pakistan
2:Department of Entomology, Michigan State University, East Lansing, Michigan
The honey bee (Apis mellifera L.) is an important pollinator and the best model for pesticide effects on insect pollinators. The effects of agricultural pesticides on honey bee health have consequently raised concern. Honey bees are suffering from eminent colony losses in the northern and eastern hemisphere possibly because of a variety of growing problems, with which pesticides may interact to exacerbate their impacts. Effects of various pesticides have been measured for multiple responses such as learning, memory performance, feeding activity, and thermoregulation. These studies were conducted at many different temperatures (11-35°C), however, few studies compared the toxicity of the same pesticide to bees at different temperatures. It is possible that the same pesticide might show different toxicity to honey bees at different temperatures. To reveal such potential interactions, we administered low doses of two neonicotinoid insecticides (imidacloprid @ 250 ppb and thiamethoxam @ 125 ppb) at three different temperature scenarios (35°C, 24°C and a varying temperature) and determined the effects on honey bee survivorship. We discovered that honey bees are much more sensitive to the neonicotinoid pesticides imidacloprid and thiamethoxam at a constant 24°C or at a varying temperature (night at 13°C and day at 24°C) compared to bees at 35°C. These results suggest that honey bee colonies during wintertime will be more sensitive to pesticides. Doses of neonicotinoids that are safe to colonies during Summer might kill them during the wintertime.

A Dataset of Varroa Survey
Megan MacDonald1, Awad Hassan1, Michael Rubinigg1,2, Ed Hassler1, Joseph Cazier1
1:Center for Analytics Research and Education, Appalachian State University
2:Biene Österreich (Austrian Beekeepers Association)
Varroa destructor is classified as an ectoparasitic mite and currently poses one of the most serious threats to the western honey bees (Apis mellifera L.). This mite has invaded many western honey bee populations throughout most of the world, leading to diminished colonies for hobbyist and commercial beekeepers. This research will utilize survey data on the varroa infestation rates of honey bee colonies in Austria, and examine the potential factors affecting these infestation rates. The Varroa survey data is collected from three sources of varying quality by both software and a mix of trained and untrained individuals. The data of factors that we believe is most likely to affect Varroa infestation levels was collected and consists of two categories. The first is beekeeping factors which includes the location and elevation of the beeyard (the spatial dimension), and time of the year (temporal dimension). The second is weather factors which include daily values of temperature, dew point, wind speed, and precipitation. The goal of this research is to build a prediction model for Varroa distribution, and to enhance the existing models by utilizing this data. Although this model will be limited to Austria, it will be able to be used in other parts of the world with similar climate and elevation zones. By predicting the spatial distribution of Varroa on these factors, there might be an opportunity to give beekeepers advanced warning of infestations nearby so they can take precautions and protect their hives from varroa outbreak in the future.

A data mining to validating good beekeeping practices
Andrew Scott1, Ed Hassler1, Awad Hassan1, Giovanni Formato2, Joseph Cazier1, James Wilkes1
1:Department of Computer Information Systems, Appalachian State University; North Carolina
2:Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “Mariano Aleandri”, Italy
As humanity continues to understand and battle the effects of climate change on the world today, pushing research forward in all segments of natural life is critical. The environment and habitat of almost all species around the world are being affected by these changes, and pollinators are definitely not an exception. As part of the United Nations 17 Sustainable Development Goals, the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana (Veterinary Beekeeping Laboratory Office) and affiliated with the Food and Agricultural Organizations took on this global best beekeeping practices project. The first part of this project culminated in a list of 234 beekeeping practices, that are proven to help with key hive metrics like survival and honey flow. In the next chapter of the BPractices project, we were searching to prove a handful of practices legitimacy using data from apiculture integrated software systems. During this iteration of the project we have proven five of the seven original hypothesized practices to be legitimate. Record keeping, treatments, Fall, Spring, and Winter inspections have all been proven as significant factors in the hives survival rates through this study. Further analysis of the queen acceptance and yard address variables would be needed to prove these. The final phase of this project will be transferring the information we have gleaned from the database analysis, to an economic metric for impact. This will be conducted by officials qualified for this analysis at the United Nations. Preparing the data and understanding what the differences are between the groups that survived and those that died is critical to the forward progress of this project in the coming months.

Prospective Advantages of GIS in apiculture
Awad Hassan1, Megan MacDonald1, Ed Hassler1, Andrew Scott1, Max Rünzel1, James Wilkes1, Joseph Cazier1
1:Center for Analytics Research and Education, Appalachian State University, North Carolina
Geographic Information Systems (GIS) are frameworks designed to capture, store, manipulate, analyse, manage, and present spatial data. GIS integrate combinations of hardware tools such as GPS and remote sensing, and software platforms such as Google maps, and ESRI packages. GIS are used in hundreds of different applications ranging from the daily used car and smartphone navigators to the complicated astronomy issues. GIS have appeared in apiculture almost 20 years ago. This review work has mined the online information about the efforts had done to employ GIS in apiculture; in both research and business. Scientific literature, R&D initiatives, and commercial start-ups that have used GIS in apiculture has been screened. Moreover, the future prospective services that GIS can provide to apiculture are brain-storming here. Finally, we are announcing our proposed “US Bee Map” initiative. The very brief conclusion is that the past uses of GIS in apiculture were extremely less than the potentials. However, the future is promising.

Pathogenic Associations with Winter Colony Loss in Canada
Stephen F. Pernal1, Renata Borba1, Shelly E. Hoover2, Robert W. Currie3, Marta M. Guarna1, Pierre Giovenazzo4, Amro Zayed5, Leonard J. Foster6
1:Agriculture & Agri-Food Canada, Beaverlodge Research Farm, Beaverlodge, AB, Canada
2:Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, Canada
3:Department of Entomology, University of Manitoba, Winnipeg, MB, Canada
4:Department of Biology, Université Laval, Quebec City, QC, Canada
5:Department of Biology, York University, Toronto, ON, Canada
6:Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
Canadian beekeepers report that high pathogen/parasite infestation levels, poor queen quality and severe weather conditions are the leading causes of elevated wintering losses. In order to replenish annual losses or maintain their operations, beekeepers in Canada face a unique and difficult situation for purchasing new queens or package bees. Scarce local supply drives local producers to import approximately 300,000 queens and packages each year, predominantly from foreign sources. This large-scale importation of stock may contribute to the introduction of undesirable pathogens or genetics, and supply bees that have not been selected to survive and prosper in northern temperate climates, thereby influencing wintering success. Honey bees act as a host for a multitude of pathogens and parasites. Nevertheless, the interactive effects that many of these pathogens, endoparasites and ectoparasites have on colony wintering success remains poorly understood. In order to better understand these interrelationships, we studied colony health and wintering success as a part of an ongoing national-scale study. In 2016 and 2017, we sampled 1025 and 520 colonies, respectively, across five Canadian provinces. During each experimental year (May through April), we collected pre-winter phenotypic data (Fall colony weight and cluster size), and samples for pathogen analysis (Nosema spp., Lotmaria passim, DWV-A, DWV-B, BQCV, SBV, and phoretic loads of Varroa destructor) from colonies in all locations to investigate the main drivers of colony Winter mortality. We also studied colonies wintered outdoors, as well as those wintered inside specialized wintering facilities. Although Winter mortality was statistically similar between 2016 and 2017, indoor-wintered colonies had greater survival than those wintered outdoors (92% vs 77%). Irrespective of wintering method, consistent influences were seen across both experimental years, based on logistic regression modelling. Elevated levels of DWV-A, DWV-B, BQCV and Fall phoretic mite loads increased the risk of colony death during Winter, whereas higher fall colony weights, larger cluster sizes and increased sealed brood areas exerted positive influences on survival outcomes.

Using 48-hour queen cells to study the queen quality of honey bees
Xaryn Cleare1, Hongmei Li-Byarlay1,2
1:Department of Agriculture and Life Science, Central State University
2:Agricultural Research and Development Program, Central State University
Apiculturists desire to maximize their investment in their apiaries to obtain healthy colonies but often struggle on which source of bees is the best: commercial package bees or feral bees stocks. The fitness of a colony often time depends on the quality of the queen who lays approximately 2,000 eggs per day in the Summer. In addition, virgin queens go out for mating once and store drone sperm inside the spermathecal. The quality of the queen is measured upon her ability to reproduce in the colony. To ensure that the queen is viable beekeepers have developed an inexpensive way to make virgin queens mobile using 48-hour queen cells. A queen cell of larvae after grafting was built by nurse bees for 48 hours in a queenless colony. Then the queen cell can be moved around into different apiaries for increasing the genetic diversity. It is unknown whether a difference occurs in the quality of 48-hour queen cells between feral and commercial honeybees. We monitored and test the survival rate and development of three queen cells from each population. Comparing to 100% of successful emerging and returning from mating flights in feral colonies, commercial package colonies only had 33% of survival rate. In the coming field season, we will increase the sample size and compare the queen quality in the numbers of eggs laid by each queen. Our research will provide unique information on the quality of queens between feral and commercial colonies.