Antagonistic interactions between honey bee bacterial symbionts and implications for disease

Background  

Honey bees, Apis mellifera, face many parasites and pathogens and consequently rely on a diverse set of individual and group-level defenses to prevent disease. One route by which honey bees and other insects might combat disease is through the shielding effects of their microbial symbionts. Bees carry a diverse assemblage of bacteria, very few of which appear to be pathogenic. Here we explore the inhibitory effects of these resident bacteria against the primary bacterial pathogen of honey bees, Paenibacillus larvae.     

Social Apoptosis in Varroa Mite Resistant Western Honey Bees (Apis mellifera)

Honey bees are eusocial animals that exhibit both individual and social immune responses, which influence colony health. This is especially well-studied regarding the mite Varroa destructor Anderson and Trueman (Parasitiformes: Varroidae), a parasite of honey bee brood and disease vector. Varroa was introduced relatively recently to Apis mellifera L. (Hymenoptera: Apidae) and is a major driver of the catastrophic die-off of honey bee colonies in the last decade. In contrast, the original host species, Apis cerana Fabricius (Hymenoptera: Apidae) is able to survive mite infestations with little effect on colony health and survival. This resilience is due in part to a newly identified social immune response expressed by developing worker brood. Varroa infested female A. cerana brood experience delayed development and eventually die in a process called ‘social apoptosis’. Here, an individual’s susceptibility to Varroa results in colony level resistance. We tested for the presence of the social apoptosis trait in two Varroa resistant stocks of A. mellifera (Pol-line and Russian) with different selection histories and compared them to a known Varroa-susceptible stock (Italian). We assessed the survival and development of worker brood reared in either highly or lightly infested host colonies, then receiving one of three treatments: uninfested, experimentally inoculated with a Varroa mite, or wounded to simulate Varroa damage. We found that response to treatment was only differentiated in brood reared in lightly infested host colonies, where experimentally infested Russian honey bees had decreased survival relative to the mite-susceptible Italian stock. This is the first evidence that social apoptosis can exist in Western honey bee populations.     

Honey Bees (Apis mellifera Hymenoptera: Apidae) Preferentially Avoid Sugar Solutions Supplemented with Field-Relevant Concentrations of Hydrogen Peroxide Despite High Tolerance Limits

Honey bees (Apis mellifera L. Hymeoptera: Apidae) use hydrogen peroxide (synthesized by excreted glucose oxidase) as an important component of social immunity. However, both tolerance of hydrogen peroxide and the production of glucose oxidase in honey is costly. Hydrogen peroxide may also be encountered by honey bees at high concentrations in nectar while foraging, however despite its presence both in their foraged and stored foods, it is unclear if and how bees monitor concentrations of, and their behavioral responses to, hydrogen peroxide. The costs of glucose oxidase production and the presence of hydrogen peroxide in both nectar and honey suggest hypotheses that honey bees preferentially forage on hydrogen peroxide supplemented feed syrups at certain concentrations, and avoid feed syrups supplemented with hydrogen peroxide at concentrations above some tolerance threshold. We test these hypotheses and find that, counter to expectation, honey bees avoid glucose solutions supplemented with field-relevant hydrogen peroxide concentrations and either avoid or don’t differentiate supplemented sucrose solutions when given choice assays. This is despite honey bees showing high tolerance for hydrogen peroxide in feed solutions, with no elevated mortality until concentrations of hydrogen peroxide exceed 1% (v/v) in solution, with survival apparent even at concentrations up to 10%. The behavioral interaction of honey bees with hydrogen peroxide during both within-colony synthesis in honey and when foraging on nectar therefore likely relies on interactions with other indicator molecules, and maybe constrained evolutionarily in its plasticity, representing a constitutive immune mechanism.     

Context-Dependent Effect of Dietary Phytochemicals on Honey Bees Exposed to a Pesticide,Thiamethoxam

Honey bees continue to face challenges relating to the degradation of natural flowering habitats that limit their access to diverse floral resources. While it is known that nectar and pollen provide macronutrients, flowers also contain secondary metabolites (phytochemicals) that impart benefits including increased longevity, improved gut microbiome abundance, and pathogen tolerance. Our study aims to understand the role of phytochemicals in pesticide tolerance when worker bees were fed with sublethal doses (1 ppb and 10 ppb) of thiamethoxam (TMX), a neonicotinoid, in 20% (w/v) sugar solution supplemented with 25 ppm of phytochemicals—caffeine, kaempferol, gallic acid, or p-coumaric acid, previously shown to have beneficial impacts on bee health. The effect of phytochemical supplementation during pesticide exposure was context-dependent. With 1 ppb TMX, phytochemical supplementation increased longevity but at 10 ppb TMX, longevity was reduced suggesting a negative synergistic effect. Phytochemicals mixed with 1 ppb TMX increased mortality in bees of the forager-age group but with 10 ppb TMX, mortality of the inhive-age group increased, implying the possibility of accumulation effect in lower sublethal doses. Given that the phytochemical composition of pollen and nectar varies between plant species, we suggest that the negative impacts of agrochemicals on honey bees could vary based on the phytochemicals in pollen and nectar of that crop, and hence the effects may vary across crops. Analyzing the phytochemical composition for individual crops may be a necessary first step prior to determining the appropriate dosage of agrochemicals so that harm to bees Apis mellifera L. is minimized while crop pests are effectively controlled.     

Impact of Honey Bee Migratory Management on Pathogen Loads and Immune Gene Expression is Affected by Complex Interactions With Environment,Worker Life History,and Season

The effects of honey bee management, such as intensive migratory beekeeping, are part of the ongoing debate concerning causes of colony health problems. Even though comparisons of disease and pathogen loads among differently managed colonies indicate some effects, the direct impact of migratory practices on honey bee pathogens is poorly understood. To test long- and short-term impacts of managed migration on pathogen loads and immunity, experimental honey bee colonies were maintained with or without migratory movement. Individuals that experienced migration as juveniles (e.g., larval and pupal development), as adults, or both were compared to control colonies that remained stationary and therefore did not experience migratory relocation. Samples at different ages and life-history stages (hive bees or foragers), taken at the beginning and end of the active season, were analyzed for pathogen loads and physiological markers of health. Bees exposed to migratory management during adulthood had increased levels of the AKI virus complex (Acute bee paralysis, Kashmir bee, and Israeli acute bee paralysis viruses) and decreased levels of antiviral gene expression (dicer-like). However, those in stationary management as adults had elevated gut parasites (i.e. trypanosomes). Effects of environment during juvenile development were more complex and interacted with life-history stage and season. Age at collection, life-history stage, and season all influenced numerous factors from viral load to immune gene expression. Although the factors that we examined are not independent, the results illuminate potential factors in both migratory and nonmigratory beekeeping that are likely to contribute to colony stress, and also indicate potential mitigation measures.     

The Life Span and Levels of Oxidative Stress in Foragers Between Feral and Managed Honey Bee Colonies

Molecular damage caused by oxidative stress may lead to organismal aging and result in acute mortality to organisms. Thus, oxidative stress resistance and longevity are closely linked. Honey bees (Apis mellifera) are the most important managed pollinator in agriculture, but the long-term survival of honey bees is seriously threatened. Feral honey bee colonies can be used as natural resources to improve honey bee health. One question we ask here is whether feral honey bees are stress resistant or survive longer than managed bee populations. More work is needed to determine the impact of oxidative stress on honey bee health and survival. In this study, we used paired colony designs to compare the life span of worker bees (foragers) between feral and managed colonies and their levels of oxidative stress. Each pair of colonies shared similar foraging resources. The results indicated that foragers in feral colonies had longer survival times and life spans than those in managed colonies. The levels of oxidative stress from lipid damage content in feral colonies were higher than those in managed colonies, indicating that they used a tolerance mechanism rather than a repair mechanism to survive. Our study provides new insights into a colony difference in the physiology and oxidative stress resistance of feral honey bees compared with managed colony stocks.     

Effects of Dinotefuran on Brain miRNA Expression Profiles in Young Adult Honey Bees (Hymenopptera: Apidae)

Honey bees are important pollinators of wild plants and crops. MicroRNAs (miRNAs) are endogenous regulators of gene expression. In this study, we initially determined that the lethal concentration 50 (LC₅₀) of dinotefuran was 0.773 mg/l. Then, the expression profiles and differentially expressed miRNAs (DE miRNAs) in honey bee brains after 1, 5, and 10 d of treatment with the lethal concentration 10 (LC₁₀) of dinotefuran were explored via deep small-RNA sequencing and bioinformatics. In total, 2, 23, and 27 DE miRNAs were identified after persistent exposure to the LC₁₀ of dinotefuran for 1, 5, and 10 d, respectively. Some abundant miRNAs, such as ame-miR-375-3p, ame-miR-281-5p, ame-miR-3786-3p, ame-miR-10-5p, and ame-miR-6037-3p, were extremely significantly differentially expressed. Enrichment analysis suggested that the candidate target genes of the DE miRNAs are involved in the regulation of biological processes, cellular processes, and behaviors. These results expand our understanding of the regulatory roles of miRNAs in honey bee Apis mellifera (Hymenopptera: Apidae) responses to neonicotinoid insecticides and facilitate further studies on the functions of miRNAs in honey bees.     

North American Prairie Is a Source of Pollen for Managed Honey Bees (Hymenoptera: Apidae)

Prairie was a dominant habitat within large portions of North America before European settlement. Conversion of prairies to farmland resulted in the loss of a large proportion of native floral resources, contributing to the decline of native pollinator populations. Efforts to reconstruct prairie could provide honey bees (Apis mellifera) a source of much-needed forage, especially in regions dominated by crop production. To what extent honey bees, which were introduced to North America by European settlers, use plants native to prairies is unclear. We placed colonies with pollen traps within reconstructed prairies in central Iowa to determine which and how much pollen is collected from prairie plants. Honey bee colonies collected more pollen from nonnative than native plants during June and July. During August and September, honey bee colonies collected more pollen from plants native to prairies. Our results suggest that honey bees’ use of native prairie plants may depend upon the seasonality of both native and nonnative plants present in the landscape. This finding may be useful for addressing the nutritional health of honey bees, as colonies in this region frequently suffer from a dearth of forage contributing to colony declines during August and September when crops and weedy plants cease blooming. These results suggest that prairie can be a significant source of forage for honey bees in the later part of the growing season in the Midwestern United States; we discuss this insight in the context of honey bee health and biodiversity conservation.     

Differential Expression of Three Dopamine Receptors in Varroa-Resistant Honey Bees

Various stocks of honey bees (Apis mellifera L. (Hymenoptera: Apidae)) employ multiple mechanisms to control varroa mite (Varroa destructor Anderson & Trueman (Mesostigmata: Varroidae)) infestations. Identification of trait-associated genes and markers can improve efficiency of selective breeding. Dopamine receptors show promise in this regard in their association with numerous traits in honey bees, high plasticity, and indicated association with varroa resistance through QTL analysis. We assessed the relationship between exposure to mite-infested brood and gene expression of the honey bee dopamine receptors, Amdop1, Amdop2, and Amdop3, in bees and stocks with known levels of varroa resistance, in Spring 2016 (VSH vs Italian) and Summer 2019 (Pol-line vs Italian). Relative mRNA expression levels varied both by honey bee stock and before/after exposure to varroa-infested brood, in 7-, 10-, and 14-day-old bees. However, the trials revealed contrasting patterns in expression of the three dopamine receptors. In 2016, downregulation was evident in VSH bees, but varied by days post-emergence and by gene. The 2019 trial showed upregulation post-exposure in both stocks, and at all ages, for Amdop1, Amdop2, and Amdop3, with the exception of 14 d Italian bees for Amdop2 and Amdop3. Stock comparison in 2019 showed upregulation of all three dopamine-like receptors in post-exposure bees of all ages. Season and associated differences in mite loads may have contributed to the differences observed across trials. Differential expression of all three dopamine receptors suggests a role for the dopaminergic system in varroa resistance and suggests that further characterization of these receptors for breeding potential is warranted.     

Pollen Treated with a Combination of Agrochemicals Commonly Applied During Almond Bloom Reduces the Emergence Rate and Longevity of Honey Bee (Hymenoptera: Apidae) Queens

Honey bee (Apis mellifera L.) colonies that pollinate California’s almond orchards are often exposed to mixtures of agrochemicals. Although agrochemicals applied during almond bloom are typically considered bee-safe when applied alone, their combined effects to honey bees are largely untested. In recent years, beekeepers providing pollination services to California’s almond orchards have reported reductions in queen quality during and immediately after bloom, raising concerns that pesticide exposure may be involved. Previous research identified a synergistic effect between the insecticide active ingredient chlorantraniliprole and the fungicide active ingredient propiconazole to lab-reared worker brood, but their effects to developing queens are unknown. To test the individual and combined effects of these pesticides on the survival and emergence of developing queens, we fed worker honey bees in closed queen rearing boxes with pollen artificially contaminated with formulated pesticides containing these active ingredients as well as the spray adjuvant Dyne-Amic, which contains both organosilicone and alkyphenol ethoxylate. The translocation of pesticides from pesticide-treated pollen into the royal jelly secretions of nurse bees was also measured. Despite consistently low levels of all pesticide active ingredients in royal jelly, the survival of queens from pupation to 7 d post-emergence were reduced in queens reared by worker bees fed pollen containing a combination of formulated chlorantraniliprole (Altacor), propiconazole (Tilt), and Dyne-Amic, as well as the toxic standard, diflubenzuron (Dimilin 2L), applied in isolation. These results support recommendations to protect honey bee health by avoiding application of pesticide tank-mixes containing insecticides and adjuvants during almond bloom.     

Understanding the Enemy: A Review of the Genetics,Behavior and Chemical Ecology of Varroa destructor,the Parasitic Mite of Apis mellifera

Varroa destructor (Mesostigmata: Varroidae) is arguably the most damaging parasitic mite that attacks honey bees worldwide. Since its initial host switch from the Asian honey bee (Apis cerana) (Hymenoptera: Apidae) to the Western honey bee (Apis mellifera) (Hymenoptera: Apidae), Varroa has become a widely successful invasive species, attacking honey bees on almost every continent where apiculture is practiced. Two haplotypes of V. destructor (Japanese and Korean) parasitize A. mellifera, both of which vector various honey bee-associated viruses. As the population of Varroa grows within a colony in the spring and summer, so do the levels of viral infections. Not surprisingly, high Varroa parasitization impacts bees at the individual level, causing bees to exhibit lower weight, decreased learning capacity, and shorter lifespan. High levels of Varroa infestation can lead to colony-wide varroosis and eventually colony death, especially when no control measures are taken against the mites. Varroa has become a successful parasite of A. mellifera because of its ability to reproduce within both drone cells and worker cells, which allows populations to expand rapidly. Varroa uses several chemical cues to complete its life cycle, many of which remain understudied and should be further explored. Given the growing reports of pesticide resistance by Varroa in several countries, a better understanding of the mite’s basic biology is needed to find alternative pest management strategies. This review focuses on the genetics, behavior, and chemical ecology of V. destructor within A. mellifera colonies, and points to areas of research that should be exploited to better control this pervasive honey bee enemy.     

Honey Bees (Hymenoptera: Apidae) Decrease Foraging But Not Recruitment After Neonicotinoid Exposure

Honey bees (Linnaeus, Hymenoptera: Apidae) are widely used as commercial pollinators and commonly forage in agricultural and urban landscapes containing neonicotinoid-treated plants. Previous research has demonstrated that honey bees display adverse behavioral and cognitive effects after treatment with sublethal doses of neonicotinoids. In laboratory studies, honey bees simultaneously increase their proportional intake of neonicotinoid-treated solutions and decrease their total solution consumption to some concentrations of certain neonicotinoids. These findings suggest that neonicotinoids might elicit a suboptimal response in honey bees, in which they forage preferentially on foods containing pesticides, effectively increasing their exposure, while also decreasing their total food intake; however, behavioral responses in semifield and field conditions are less understood. Here we conducted a feeder experiment with freely flying bees to determine the effects of a sublethal, field-realistic concentration of imidacloprid (IMD) on the foraging and recruitment behaviors of honey bees visiting either a control feeder containing a sucrose solution or a treatment feeder containing the same sucrose solution with IMD. We report that IMD-treated honey bees foraged less frequently (–28%) and persistently (–66%) than control foragers. Recruitment behaviors (dance frequency and dance propensity) also decreased with IMD, but nonsignificantly. Our results suggest that neonicotinoids inhibit honey bee foraging, which could potentially decrease food intake and adversely affect colony health.     

Classic Hoarding Cages Increase Gut Bacterial Abundance and Reduce the Individual Immune Response of Honey Bee (Apis mellifera) Workers

Laboratory experiments have advanced our understanding of honey bee (Apis mellifera) responses to environmental factors, but removal from the hive environment may also impact physiology. To examine whether the laboratory environment alters the honey bee gut bacterial community and immune responses, we compared bacterial community structure (based on amplicon sequence variant relative abundance), total bacterial abundance, and immune enzyme (phenoloxidase and glucose oxidase) activity of cohort honey bee workers kept under laboratory and hive conditions. Workers housed in the laboratory showed differences in the relative abundance of their core gut taxa, an increase in total gut bacterial abundance, and reduced phenoloxidase activity, compared to bees housed in hives.     

Physicochemical Parameters and Antioxidant Activity of Bee Honey Enriched With Herbs

Three groups of products enriched with herbs were studied: (1) commercial herb honeys (n?=?5) produced by bees fed a syrup with an herbal extract, (2) natural herbal honey (n?=?3) produced by bees from the nectar of herbs, and (3) creamed multifloral honey with added dried herbs (n?=?5). As a control, multifloral honey (n?=?5) was used. The physicochemical parameters (i.e., sugar extract, water content, specific rotation, conductivity, hydroxymethylfurfural content, pH and acidity), sugar profiles (HPLC analysis), antioxidant activity and total phenolic compounds content of the studied samples were compared. Although great diversity in the basic properties of the studied products was observed, they were comparable to multifloral honey and complied with honey regulations. Significant differences in sugar composition were observed, and adversely positive rotation (excluding nettle herb honey) was detected in group 1, likely resulting from the change in bee feeding. The best antioxidant activity for creamed honeys with dried herbs (group 2) was investigated, whereas herb honeys (group 1) exhibited similar antioxidant properties as multifloral honey. The use of controlled feeding of bees appears to be an effective method of enriching honey with desirable plant bioactive components to create innovative bee products.     

Evaluating and Comparing the Natural Cell Structure and Dimensions of Honey Bee Comb Cells of Chinese Bee,Apis cerana cerana (Hymenoptera: Apidae) and Italian Bee,Apis mellifera ligustica (Hymenoptera: Apidae)

The hexagonal structure of the honey bee comb cell has been the source of many studies attempting to understand its structure and function. In the storage area of the comb, only honey is stored and no brood is reared. We predicted that honey bees may construct different hexagonal cells for brood rearing and honey storage. We used quantitative analyses to evaluate the structure and function of the natural comb cell in the Chinese bee, Apis cerana cerana and the Italian bee, A. mellifera ligustica. We made cell molds using a crystal glue solution and measured the structure and inclination of cells. We found that the comb cells of A. c. cerana had both upward-sloping and downward-sloping cells; while the A. m. ligustica cells all tilted upwards. Interestingly, the cells did not conform to the regular hexagonal prism structure and showed irregular diameter sizes. In both species, comb cells also were differentiated into worker, drone and honey cells, differing in their diameter and depth. This study revealed unique differences in the structure and function of comb cells and showed that honey bees design their cells with precise engineering to increase storage capacity, and to create adequate growing room for their brood.     

Antibiotics Alter the Expression of Genes Related to Behavioral Development in Honey Bees (Hymenoptera: Apidae)

Honey bees, as many species of social insects, display a division of labor among colony members based on behavioral specializations related to age. Adult worker honey bees perform a series of tasks in the hive when they are young (such as brood care or nursing) and at ca. 2–3 wk of age, shift to foraging for nectar and pollen outside the hive. The transition to foraging involves changes in metabolism and neuroendocrine activities. These changes are associated with a suite of developmental genes. It was recently demonstrated that antibiotics influence behavioral development by accelerating or delaying the onset of foraging depending on timing of antibiotic exposure. To understand the mechanisms of these changes, we conducted a study on the effects of antibiotics on expression of candidate genes known to regulate behavioral development. We demonstrate a delay in the typical changes in gene expression over the lifetime of the individuals that were exposed to antibiotics during immature stage and adulthood. Additionally, we show an acceleration in the typical changes in gene expression on individuals that were expose to antibiotics only during immature stage. These results show that timing of antibiotic exposure alter the typical regulation of behavioral development by metabolic and neuroendocrine processes.     

The use of bees for the purpose of inter-mating in potato

The pollination behavior of bumblebees and honey bees was studied on potato flowers in screened enclosures and in the field. In enclosures, the domestic honey bee (Apis mellifera L.) and the bumblebee species,Bombus fervidus Fabricius, seemed to lack any “cue” to initiate visitation of the flowers. When honey was placed on a few flowers, visitation was stimulated. The honey bee tore and chewed at the anther cone to collect pollen, whileB. fervidus probed for nectar which was not present. Shortly afterward, both species ceased visitation and could not be induced to visit further, regardless of the honey stimulus. Neither species was effective as a pollinator. It is concluded that neither species will be useful for large-scale crossing of potato populations. However, another bumblebee species,Bombus impatiens Cresson, is very effective in pollinating potatoes in the field. Manipulating the behavior of such indigenous populations of bumblebees is likely to be the most effective method of exploiting insect pollination in the potato.     

Surfing the Sweet Wave: Migrating Giant Honey Bees (Hymenoptera: Apidae: Apis dorsata) Display Spatial and Temporal Fidelity to Annual Stopover Site in Thailand

Apis dorsata F. (Hymenoptera: Apidae), the giant honey bee of southern Asia, is an important pollinator of crops and non-cultivated angiosperms, and a producer of honey and beeswax. Its populations are in decline in many areas. Colonies migrate seasonally between highland and lowland nesting sites, taking advantage of available food sources. In 2009, a stopover site was discovered in Thailand where numerous migrating colonies bivouacked near one another. Bivouacs used the site again in 2010. I went to the site in 2016 to test the hypothesis that bees use the site regularly as part of an annual migration. I witnessed many bivouacs, spanning almost precisely the same time period and occupying the same area as in 2010. Here I describe their migratory dances in preparation for departure and their subsequent flights as well as periodic mass flight and defensive behavior. Analysis of photographs indicated that the bivouacking bees aged slowly and may thus live long enough to be capable of intergenerational transmission of migratory route knowledge. I describe attributes of the stopover site, e.g., abundant food and water availability, its location along a major river, and other possible navigational cues. Although the site is the only one of its kind so far known to researchers, such stopover sites probably exist wherever giant honey bees undertake long seasonal migrations. I recommend searching for bivouacking sites, particularly along rivers, wherever giant honey bees migrate. Stopover sites are undoubtedly essential to the life history and health of migratory bee populations, and thus warrant conservation policies.     

Honey Bees and Industrial Agriculture: What Researchers are Missing,and Why it’s a Problem

Industrial agriculture is the root cause of many health problems that honey bees (Apis mellifera Linneaus, 1758) face, but honey bee researchers seldom call attention to this fact. We often discuss the stressors that contribute to colony loss (e.g., pathogens, pesticides, poor nutrition), but we rarely talk about where those stressors come from. This is a problem because we cannot resolve honey bee health issues unless we confront the systems that cause them harm. In this forum article, I unpack the relationship between honey bee health and industrial agriculture. I propose steps we can take to reframe our research to account for the impacts of this destructive system, and I discuss the uncomfortable questions that surface when we engage in this process. The goal of this article is to encourage conversation within the honey bee research community around the impacts of industrial agriculture, so that we can fully engage in the transformative change needed to support honey bee health.     

Hygiene-Eliciting Brood Semiochemicals as a Tool for Assaying Honey Bee (Hymenoptera: Apidae) Colony Resistance to Varroa (Mesostigmata: Varroidae)

Despite numerous interventions, the ectoparasitic mite Varroa (Varroa destructor Anderson and Trueman [Mesostigmata: Varroidae]) and the pathogens it vectors remain a primary threat to honey bee (Apis mellifera Linnaeus [Hymenoptera: Apidae]) health. Hygienic behavior, the ability to detect, uncap, and remove unhealthy brood from the colony, has been bred for selectively for over two decades and continues to be a promising avenue for improved Varroa management. Although hygienic behavior is expressed more in Varroa-resistant colonies, hygiene does not always confer resistance to Varroa. Additionally, existing Varroa resistance selection methods trade efficacy for efficiency, because those achieving the highest levels of Varroa resistance can be time-consuming, and thus expensive and impractical for apicultural use. Here, we tested the hypothesis that hygienic response to a mixture of semiochemicals associated with Varroa-infested honey bee brood can serve as an improved tool for predicting colony-level Varroa resistance. In support of our hypothesis, we demonstrated that a mixture of the compounds (Z)-10-tritriacontene, (Z)-8-hentriacontene, (Z)-8-heptadecene, and (Z)-6-pentadecene triggers hygienic behavior in a two-hour assay, and that high-performing colonies (hygienic response to ≥60% of treated cells) have significantly lower Varroa infestations, remove significantly more introduced Varroa, and are significantly more likely to survive the winter compared to low-performing colonies (hygienic response to <60% of treated cells). We discuss the relative efficacy and efficiency of this assay for facilitating apiary management decisions and selection of Varroa-resistant honey bees, as well as the relevance of these findings to honey bee health, pollination services, and social insect communication.