Heightened sensitivity in high-grooming honey bees (Hymenoptera: Apidae).

Honey bees use grooming to defend against the devastating parasite Varroa destructor Anderson and Trueman. We observed the grooming responses of individual bees from colonies previously chosen for high- and low-grooming behavior using a combination of mite mortality and mite damage. Our aim was to gain insight into specific aspects of grooming behavior to compare if high-grooming bees could discriminate between a standardized stimulus (chalk dust) and a stimulus of live Varroa mites and if bees from high-grooming colonies had greater sensitivity across different body regions than bees from low-grooming colonies. We hypothesized that individuals from high-grooming colonies would be more sensitive to both stimuli than bees from low-grooming colonies across different body regions and that bees would have a greater response to Varroa than a standardized irritant (chalk dust). Individuals from high-grooming colonies responded with longer bouts of intense grooming when either stimulus was applied to the head or thorax, compared to sham-stimulated controls, while bees from low-grooming colonies showed no differences between stimulated and sham-stimulated bees. Further, high-grooming bees from colonies with high mite damage exhibited greater grooming to Varroa than high-grooming colonies with only moderate mite damage rates. This study provides new insights into Varroa-specific aspects of grooming, showing that although a standardized stimulus (chalk dust) may be used to assess general grooming ability in individual bee grooming assays, it does not capture the same range of responses as a stimulus of Varroa. Thus, continuing to use Varroa mites in grooming assays should help select colonies with more precise sensitivity to Varroa.

The influence of Nosema (Microspora: Nosematidae) infection on honey bee (Hymenoptera: Apidae) defense against Varroa destructor (Mesostigmata: Varroidae).

The objectives of this study were to quantify the costs and benefits of co-parasitism with Varroa (Varroa destructor Anderson and Trueman) and Nosema (Nosema ceranae Fries and Nosema apis Zander) on honey bees (Apis mellifera L.) with different defense levels. Newly-emerged worker bees from either high-mite-mortality-rate (high-MMR) bees or low-mite-mortality-rate (low-MMR) bees were confined in forty bioassay cages which were either inoculated with Nosema spores [Nosema (+) group] or were left un-inoculated [Nosema (-) group]. Caged-bees were then inoculated with Varroa mites [Varroa (+) group] or were left untreated [Varroa (-) group]. This established four treatment combinations within each Nosema treatment group: (1) low-MMR Varroa (-), (2) high-MMR Varroa (-), (3) low-MMR Varroa (+) and (4) high-MMR Varroa (+), each with five replicates. Overall mite mortality in high-MMR bees (0.12±0.02 mites per day) was significantly greater than in the low-MMR bees (0.06±0.02 mites per day). In the Nosema (-) groups bee mortality was greater in high-MMR bees than low-MMR bees but only when bees had a higher mite burden. Overall, high-MMR bees in the Nosema (-) group showed greater reductions in mean abundance of mites over time compared with low-MMR bees, when inoculated with additional mites. However, high-MMR bees could not reduce mite load as well as in the Nosema (-) group when fed with Nosema spores. Mean abundance of Nosema spores in live bees and dead bees of both strains of bees was significantly greater in the Nosema (+) group. Molecular analyses confirmed the presence of both Nosema species in inoculated bees but N. ceranae was more abundant than N. apis and unlike N. apis increased over the course of the experiment. Collectively, this study showed differential mite mortality rates among different genotypes of bees, however, Nosema infection restrained Varroa removal success in high-MMR bees.

The Potential of Bee-Generated Carbon Dioxide for Control of Varroa Mite (Mesostigmata: Varroidae) in Indoor Overwintering Honey bee (Hymenoptera: Apidae) Colonies.

The objective of this study was to manipulate ventilation rate to characterize interactions between stocks of honey bees (Apis mellifera L.) and ventilation setting on varroa mite (Varroa destructor Anderson and Trueman) mortality in honey bee colonies kept indoors over winter. The first experiment used colonies established from stock selected locally for wintering performance under exposure to varroa (n = 6) and unselected bees (n = 6) to assess mite and bee mortality and levels of carbon dioxide (CO2) and oxygen (O2) in the bee cluster when kept under a simulated winter condition at 5°C. The second experiment, used colonies from selected bees (n = 10) and unselected bees (n = 12) that were exposed to either standard ventilation (14.4 liter/min per hive) or restricted ventilation (0.24 liter/min per hive, in a Plexiglas ventilation chamber) during a 16-d treatment period to assess the influence of restricted air flow on winter mortality rates of varroa mites and honey bees. Experiment 2 was repeated in early, mid-, and late winter. The first experiment showed that under unrestricted ventilation with CO2 concentrations averaging <2% there was no correlation between CO2 and varroa mite mortality when colonies were placed under low temperature. CO2 was negatively correlated with O2 in the bee cluster in both experiments. When ventilation was restricted, mean CO2 level (3.82 ± 0.31%, range 0.43-8.44%) increased by 200% relative to standard ventilation (1.29 ± 0.31%; range 0.09-5.26%) within the 16-d treatment period. The overall mite mortality rates and the reduction in mean abundance of varroa mite over time was greater under restricted ventilation (37 ± 4.2%) than under standard ventilation (23 ± 4.2%) but not affected by stock of bees during the treatment period. Selected bees showed overall greater mite mortality relative to unselected bees in both experiments. Restricting ventilation increased mite mortality, but did not affect worker bee mortality relative to that for colonies under standard ventilation. Restricted ventilation did not affect the overall level of Nosema compared with the control. However, there was an interaction between stock, season, and time of the trial. Unselected stock showed an increase in Nosema over time in the late winter trial that did not occur in the selected stock. In conclusion, these findings suggested that restricted ventilation has potential to suppress varroa mite in overwintering honey bee colonies via a low-cost and environmentally friendly measure.

Influence of Honey Bee Genotype and Wintering Method on Wintering Performance of Varroa destructor (Parasitiformes: Varroidae)-Infected Honey Bee (Hymenoptera: Apidae) Colonies in a Northern Climate.

The objective of this study was to assess the effectiveness of a cooperative breeding program designed to enhance winter survival of honey bees (Apis mellifera L.) when exposed to high levels of varroa (Varroa destructor Anderson and Trueman) in outdoor-wintered and indoor-wintered colonies. Half of the colonies from selected and unselected stocks were randomly assigned to be treated with late autumn oxalic acid treatment or to be left untreated. Colonies were then randomly assigned to be wintered either indoors (n = 37) or outdoors (n = 40). Late autumn treatment with oxalic acid did not improve wintering performance. However, genotype of bees affected colony survival and the proportion of commercially viable colonies in spring, as indicated by greater rates of colony survival and commercially viable colonies for selected stock (43% survived and 33% were viable) in comparison to unselected stock (19% survived and 9% were viable) across all treatment groups. Indoor wintering improved spring bee population score, proportion of colonies surviving, and proportion of commercially viable colonies relative to outdoor wintering (73% of selected stock and 41% of unselected stock survived during indoor wintering). Selected stock showed better "tolerance" to varroa as the selected stock also maintained higher bee populations relative to unselected stock. However, there was no evidence of "resistance" in selected colonies (reduced mite densities). Collectively, this experiment showed that breeding can improve tolerance to varroa and this can help minimize colony loss through winter and improve colony wintering performance. Overall, colony wintering success of both genotypes of bees was better when colonies were wintered indoors than when colonies were wintered outdoors.

The effect of queen pheromone status on Varroa mite removal from honey bee colonies with different grooming ability.

The objective of this study was to assess the effects of honey bees (Apis mellifera L.) with different grooming ability and queen pheromone status on mortality rates of Varroa mites (Varroa destructor Anderson and Trueman), mite damage, and mortality rates of honey bees. Twenty-four small queenless colonies containing either stock selected for high rates of mite removal (n = 12) or unselected stock (n = 12) were maintained under constant darkness at 5 °C. Colonies were randomly assigned to be treated with one of three queen pheromone status treatments: (1) caged, mated queen, (2) a synthetic queen mandibular pheromone lure (QMP), or (3) queenless with no queen substitute. The results showed overall mite mortality rate was greater in stock selected for grooming than in unselected stock. There was a short term transitory increase in bee mortality rates in selected stock when compared to unselected stock. The presence of queen pheromone from either caged, mated queens or QMP enhanced mite removal from clusters of bees relative to queenless colonies over short periods of time and increased the variation in mite mortality over time relative to colonies without queen pheromone, but did not affect the proportion of damaged mites. The effects of source of bees on mite damage varied with time but damage to mites was not reliably related to mite mortality. In conclusion, this study showed differential mite removal of different stocks was possible under low temperature. Queen status should be considered when designing experiments using bioassays for grooming response.

Treatment of waxborne honey bee (Hymenoptera: Apidae) viruses using time, temperature, and electron-beam irradiation.

Viruses are one of many serious threats to honey bee (Apis mellifera L.) health. There are many transmission routes for honey bee viruses, and there is potential for wax comb to act as a reservoir for transmission of viruses. Some work has been done on treating viruses on wax, focusing on irradiation as a potential treatment. However, irradiation is not universally available or economically viable for beekeepers in many regions. With increased colony deaths over winter beekeepers potentially risk further loss from reusing contaminated equipment from dead colonies. Here we explored the use of storage time and temperature on the reduction of waxborne virus levels from winter loss colony wax over 30 days and at -20, 5, and 20 °C. Furthermore, because irradiation has previously worked against waxborne viruses, we performed a dosage experiment with electron-beam irradiation. Winter loss wax was again used, and exposed to 10, 25, 35, and 45 kGy irradiation, including a nonirradiated transport control. Storage time decreased abundance of black queen cell virus and deformed wing virus at times equal or greater than 30 days but temperatures had no significant effect on virus levels. All irradiation doses decreased virus abundance and prevalence, yet only 35 and 45 kGy did so at a greater rate than the effect of transport alone.

Honey bee stressor networks are complex and dependent on crop and region.

Honey bees play a major role in crop pollination but have experienced declining health throughout most of the globe. Despite decades of research on key honey bee stressors (e.g., parasitic Varroa destructor mites and viruses), researchers cannot fully explain or predict colony mortality, potentially because it is caused by exposure to multiple interacting stressors in the field. Understanding which honey bee stressors co-occur and have the potential to interact is therefore of profound importance. Here, we used the emerging field of systems theory to characterize the stressor networks found in honey bee colonies after they were placed in fields containing economically valuable crops across Canada. Honey bee stressor networks were often highly complex, with hundreds of potential interactions between stressors. Their placement in crops for the pollination season generally exposed colonies to more complex stressor networks, with an average of 23 stressors and 307 interactions. We discovered that the most influential stressors in a network-those that substantively impacted network architecture-are not currently addressed by beekeepers. Finally, the stressor networks showed substantial divergence among crop systems from different regions, which is consistent with the knowledge that some crops (e.g., highbush blueberry) are traditionally riskier to honey bees than others. Our approach sheds light on the stressor networks that honey bees encounter in the field and underscores the importance of considering interactions among stressors. Clearly, addressing and managing these issues will require solutions that are tailored to specific crops and regions and their associated stressor networks.

Higher prevalence of sacbrood virus in Apis mellifera (Hymenoptera: Apidae) colonies after pollinating highbush blueberries.

Highbush blueberry pollination depends on managed honey bees (Apis mellifera) L. for adequate fruit sets; however, beekeepers have raised concerns about the poor health of colonies after pollinating this crop. Postulated causes include agrochemical exposure, nutritional deficits, and interactions with parasites and pathogens, particularly Melisococcus plutonius [(ex. White) Bailey and Collins, Lactobacillales: Enterococcaceae], the causal agent of European foulbrood disease, but other pathogens could be involved. To broadly investigate common honey bee pathogens in relation to blueberry pollination, we sampled adult honey bees from colonies at time points corresponding to before (t1), during (t2), at the end (t3), and after (t4) highbush blueberry pollination in British Columbia, Canada, across 2 years (2020 and 2021). Nine viruses, as well as M. plutonius, Vairimorpha ceranae, and V. apis [Tokarev et al., Microsporidia: Nosematidae; formerly Nosema ceranae (Fries et al.) and N. apis (Zander)], were detected by PCR and compared among colonies located near and far from blueberry fields. We found a significant interactive effect of time and blueberry proximity on the multivariate pathogen community, mainly due to differences at t4 (corresponding to ~6 wk after the beginning of the pollination period). Post hoc comparisons of pathogens in near and far groups at t4 showed that detections of sacbrood virus (SBV), which was significantly higher in the near group, not M. plutonius, was the primary driver. Further research is needed to determine if the association of SBV with highbush blueberry pollination is contributing to the health decline that beekeepers observe after pollinating this crop.

Pollen foraging mediates exposure to dichotomous stressor syndromes in honey bees.

Recent declines in the health of honey bee colonies used for crop pollination pose a considerable threat to global food security. Foraging by honey bee workers represents the primary route of exposure to a plethora of toxins and pathogens known to affect bee health, but it remains unclear how foraging preferences impact colony-level patterns of stressor exposure. Resolving this knowledge gap is crucial for enhancing the health of honey bees and the agricultural systems that rely on them for pollination. To address this, we carried out a national-scale experiment encompassing 456 Canadian honey bee colonies to first characterize pollen foraging preferences in relation to major crops and then explore how foraging behavior influences patterns of stressor exposure. We used a metagenetic approach to quantify honey bee dietary breadth and found that bees display distinct foraging preferences that vary substantially relative to crop type and proximity, and the breadth of foraging interactions can be used to predict the abundance and diversity of stressors a colony is exposed to. Foraging on diverse plant communities was associated with increased exposure to pathogens, while the opposite was associated with increased exposure to xenobiotics. Our work provides the first large-scale empirical evidence that pollen foraging behavior plays an influential role in determining exposure to dichotomous stressor syndromes in honey bees.

Effect of Fumagilin-B treatment timing on nosema (Vairimorpha spp.; Microspora: Nosematidae) abundance and honey bee (Hymenoptera: Apidae) colonies under winter management in the Canadian Prairies.

Fumagilin-B is used to treat nosema infection in honey bee colonies; however, it is unclear whether treatment consistently reduces Vairimorpha ceranae (Fries et al.) abundance and improves colony strength and survival in the Canadian Prairies. This study assessed spring and fall fumagillin treatments on nosema abundance, colony strength, and mortality in 2 different beekeeping regions within Alberta, using both indoor and outdoor wintering management at each site. We compared 4 fumagillin treatments: Spring-only, Fall-only, Spring-and-Fall, and Control (no treatment). The spring treatment dose was ~68 mg/colony, whereas the fall treatment dose was 120 or 48 mg/colony, depending on the year. We found that the colonies were infected predominately with V. ceranae, with V. apis (Zander) present only in mixed infections in a subset of colonies. Although treatment in either the spring or fall did reduce nosema abundance in the short term, it did not eliminate the infection, making continued monitoring necessary. Colony strength was improved by spring treatment in some locations but not consistently, possibly due to the treatment timing or low dose. The combined spring and fall treatment increased colony survival over winter in one of 2 yr. Wintering method did not interact with treatment to affect nosema abundance in the spring. There does not appear to be a significant residual benefit of fall treatment as it did not reduce spring nosema abundance or increase colony population. Therefore, spring treatment should be applied to reduce spring V. ceranae abundance rather than relying on residual efficacy from previous fall treatments.

Validating a multi-locus metabarcoding approach for characterizing mixed-pollen samples.

BACKGROUND: The mutualistic interaction between entomophilous plants and pollinators is fundamental to the structure of most terrestrial ecosystems. The sensitive nature of this relationship has been disrupted by anthropogenic modifications to natural landscapes, warranting development of new methods for exploring this trophic interaction. Characterizing the composition of pollen collected by pollinators, e.g. Apis mellifera, is a common means of exploring this relationship, but traditional methods of microscopic pollen assessment are laborious and limited in their scope. The development of pollen metabarcoding as a method of rapidly characterizing the abundance and diversity of pollen within mixed samples presents a new frontier for this type of work, but metabarcoding may have limitations, and validation is warranted before any suite of primers can be confidently used in a research program. We set out to evaluate the utility of an integrative approach, using a set of established primers (ITS2 and rbcL) versus melissopalynological analysis for characterizing 27 mixed-pollen samples from agricultural sites across Canada. RESULTS: Both individual markers performed well relative to melissopalynology at the family level with decreases in the strength of correlation and linear model fits at the genus level. Integrating data from both markers together via a multi-locus approach provided the best rank-based correlation between metagenetic and melissopalynological data at both the genus (ρ = 0.659; p < 0.001) and family level (ρ = 0.830; p < 0.001). Species accumulation curves indicated that, after controlling for sampling effort, melissopalynological characterization provides similar or higher species richness estimates than either marker. The higher number of plant species discovered via the metabarcoding approach simply reflects the vastly greater sampling effort in comparison to melissopalynology. CONCLUSIONS: Pollen metabarcoding performed well at characterizing the composition of mixed pollen samples relative to a traditional melissopalynological approach. Limitations to the quantitative application of this method can be addressed by adopting a multi-locus approach that integrates information from multiple markers.

Phenomic analysis of the honey bee pathogen-web and its dynamics on colony productivity, health and social immunity behaviors.

Many pathogens and parasites have evolved to overwhelm and suppress their host's immune system. Nevertheless, the interactive effects of these agents on colony productivity and wintering success have been relatively unexplored, particularly in large-scale phenomic studies. As a defense mechanism, honey bees have evolved remarkable social behaviors to defend against pathogen and parasite challenges, which reduce the impact of disease and improve colony health. To investigate the complex role of pathogens, parasites and social immunity behaviors in relation to colony productivity and outcomes, we extensively studied colonies at several locations across Canada for two years. In 2016 and 2017, colonies founded with 1-year-old queens of diverse genetic origin were evaluated, which represented a generalized subset of the Canadian bee population. During each experimental year (May through April), we collected phenotypic data and sampled colonies for pathogen analysis in a standardized manner. Measures included: colony size and productivity (colony weight, cluster size, honey production, and sealed brood population), social immunity traits (hygienic behavior, instantaneous mite population growth rate, and grooming behavior), as well as quantification of gut parasites (Nosema spp., and Lotmaria passim), viruses (DWV-A, DWV-B, BQCV and SBV) and external parasites (Varroa destructor). Our goal was to examine: 1) correlations between pathogens and colony phenotypes; 2) the dynamics of pathogens and parasites on colony phenotypes and productivity traits; and 3) the effects of social immunity behaviors on colony pathogen load. Our results show that colonies expressing high levels of some social immunity behaviors were associated with low levels of pathogens/parasites, including viruses, Nosema spp., and V. destructor. In addition, we determined that elevated viral and Nosema spp. levels were associated with low levels of colony productivity, and that five out of six pathogenic factors measured were negatively associated with colony size and weight in both fall and spring periods. Finally, this study also provides information about the incidence and abundance of pathogens, colony phenotypes, and further disentangles their inter-correlation, so as to better understand drivers of honey bee colony health and productivity.

Laboratory study on the effects of temperature and three ventilation rates on infestations of Varroa destructor in clusters of honey bees (Hymenoptera: Apidae).

In this study, reduced levels of ventilation were applied to small clusters of bees under controlled conditions to determine whether lowered ventilation rates and the resulting increased levels of CO2 could increase the mortality rates of varroa. Two experiments were performed at two different temperatures (10 degrees C and 25 degrees C). Both experiments compared varroa mortality among high (360 liters/h), medium (42.5 liters/h), and low (14 liters/h) rates of ventilation. The clusters of bees (approximately 300 worker bees) in bioassay cages with 40 introduced varroa mites were placed into self-contained glass chambers and were randomly assigned to one of the three ventilation treatments within incubators set at either of the two temperatures. Bee and varroa mortality and the levels of CO2 concentration were measured in each of the experimental chambers. In both experiments, CO2 levels within the chamber increased, with a decrease in ventilation with CO2 reaching a maximum of 1.2 +/- 0.45% at 10 degrees C and 2.13 +/- 0.2% at 25 degrees C under low ventilation. At high ventilation rates, CO2 concentration in chamber air was similar at 10 degrees C (1.1 +/- 1.5%) and 25 degrees C (1.9 +/- 1.1%). Both humidity and CO2 concentration were higher at 25 degrees C than at 10 degrees C. Bee mortality was similar within all ventilation rate treatments at either 10 degrees C (11.5 +/- 2.7-19.3 +/- 3.8%) or 25 degrees C (15.2 +/- 1.9-20.7 +/- 3.5%). At 10 degrees C, varroa mortality (percentage dead) was greatest in the high ventilation treatment (12.2 +/- 2.1%), but only slightly higher than under low (3.7 +/- 1.7%) and medium ventilation (4.9 +/- 1.6%). At 25 degrees C, varroa mortality was greatest under low ventilation at 46.12 +/- 7.7% and significantly greater than at either medium (29.7 +/- 7.4%) or low ventilation (9.5 +/- 1.6.1%). This study demonstrates that at 25 degrees C, restricted ventilation, resulting in high levels of CO2 in the surrounding environment of small clusters of honey bees, has the potential to substantially increase varroa mortality.

Epidemiology of Nosema spp. and the effect of indoor and outdoor wintering on honey bee colony population and survival in the Canadian Prairies.

The epidemiology of Nosema spp. in honey bees, Apis mellifera, may be affected by winter conditions as cold temperatures and differing wintering methods (indoor and outdoor) provide varying levels of temperature stress and defecation flight opportunities. Across the Canadian Prairies, including Alberta, the length and severity of winter vary among geographic locations. This study investigates the seasonal pattern of Nosema abundance in two Alberta locations using indoor and outdoor wintering methods and its impact on bee population, survival, and commercial viability. This study found that N. ceranae had a distinct seasonal pattern in Alberta, with high spore abundance in spring, declining to low levels in the summer and fall. The results showed that fall Nosema monitoring might not be the best indicator of treatment needs or future colony health outcomes. There was no clear pattern for differences in N. ceranae abundance by location or wintering method. However, wintering method affected survival with colonies wintered indoors having lower mortality and more rapid spring population build-up than outdoor-wintered colonies. The results suggest that the existing Nosema threshold should be reinvestigated with wintering method in mind to provide more favorable outcomes for beekeepers. Average Nosema abundance in the spring was a significant predictor of end-of-study winter colony mortality, highlighting the importance of spring Nosema monitoring and treatments.

Indoor winter fumigation with formic acid for control of Acarapis woodi (Acari: Tarsonemidae) and nosema disease, Nosema sp.

Indoor fumigation of honey bees, Apis mellifera L., with formic acid to control varroa mites, Varroa destructor Anderson & Trueman, allows simultaneous fumigation of multiple colonies with little labor input and good efficacy. Several experiments were designed to test the efficacy of formic acid as a treatment for honey bee mites, Acarapis woodi (Rennie) (Acari: Tarsonemidae), and nosema disease, Nosema sp., indoors in winter. The objectives of this study were (1) to determine the efficacy of formic acid fumigation for honey bee mite control by using both the thoracic slice and live dissection methods and (2) to determine whether indoor fumigation can reliably prevent the buildup of nosema disease in overwintering honey bee colonies. Indoor winter fumigation of honey bee colonies with formic acid was effective in killing a high percentage of honey bee mites but did not significantly reduce the proportion of bees with infested tracheae over the duration of the experiments. Thus, the method used to determine the efficacy of the treatment affected the results. Under conditions of relatively low or decreasing levels of nosema, fumigation tended to suppress the mean abundance of nosema spores relative to the controls. In three separate fumigation experiments using a range of formic acid concentrations, there was no statistical difference between the buildup or maintenance of nosema spore mean abundance over the winter in bees from formic acid fumigated colonies compared with untreated controls. However, fumigation with formic acid during winter at a low concentration for extended periods significantly suppressed spore buildup of mixed populations of nosema (Nosema apis and Nosema ceranae) in 1 yr.

Integrative Genomics Reveals the Genetics and Evolution of the Honey Bee's Social Immune System.

Social organisms combat pathogens through individual innate immune responses or through social immunity-behaviors among individuals that limit pathogen transmission within groups. Although we have a relatively detailed understanding of the genetics and evolution of the innate immune system of animals, we know little about social immunity. Addressing this knowledge gap is crucial for understanding how life-history traits influence immunity, and identifying if trade-offs exist between innate and social immunity. Hygienic behavior in the Western honey bee, Apis mellifera, provides an excellent model for investigating the genetics and evolution of social immunity in animals. This heritable, colony-level behavior is performed by nurse bees when they detect and remove infected or dead brood from the colony. We sequenced 125 haploid genomes from two artificially selected highly hygienic populations and a baseline unselected population. Genomic contrasts allowed us to identify a minimum of 73 genes tentatively associated with hygienic behavior. Many genes were within previously discovered QTLs associated with hygienic behavior and were predictive of hygienic behavior within the unselected population. These genes were often involved in neuronal development and sensory perception in solitary insects. We found that genes associated with hygienic behavior have evidence of positive selection within honey bees (Apis), supporting the hypothesis that social immunity contributes to fitness. Our results indicate that genes influencing developmental neurobiology and behavior in solitary insects may have been co-opted to give rise to a novel and adaptive social immune phenotype in honey bees.

Peptide biomarkers used for the selective breeding of a complex polygenic trait in honey bees.

We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype-based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

Effects of Wintering Environment and Parasite-Pathogen Interactions on Honey Bee Colony Loss in North Temperate Regions.

Extreme winter losses of honey bee colonies are a major threat to beekeeping but the combinations of factors underlying colony loss remain debatable. We monitored colonies in two environments (colonies wintered indoors or outdoors) and characterized the effects of two parasitic mites, seven viruses, and Nosema on honey bee colony mortality and population loss over winter. Samples were collected from two locations within hives in fall, mid-winter and spring of 2009/2010. Although fall parasite and pathogen loads were similar in outdoor and indoor-wintered colonies, the outdoor-wintered colonies had greater relative reductions in bee population score over winter. Seasonal patterns in deformed wing virus (DWV), black queen cell virus (BQCV), and Nosema level also differed with the wintering environment. DWV and Nosema levels decreased over winter for indoor-wintered colonies but BQCV did not. Both BQCV and Nosema concentration increased over winter in outdoor-wintered colonies. The mean abundance of Varroa decreased and concentration of Sacbrood virus (SBV), Kashmir bee virus (KBV), and Chronic bee paralysis virus (CBPV) increased over winter but seasonal patterns were not affected by wintering method. For most viruses, either entrance or brood area samples were reasonable predictors of colony virus load but there were significant season*sample location interactions for Nosema and BQCV, indicating that care must be taken when selecting samples from a single location. For Nosema spp., the fall entrance samples were better predictors of future infestation levels than were fall brood area samples. For indoor-wintered colonies, Israeli acute paralysis virus IAPV concentration was negatively correlated with spring population size. For outdoor-wintered hives, spring Varroa abundance and DWV concentration were positively correlated with bee loss and negatively correlated with spring population size. Multivariate analyses for fall collected samples indicated higher DWV was associated with colony death as did high SBV for spring-collected samples.

Effect of concentration and exposure time on treatment efficacy against Varroa mites (Acari: Varroidae) during indoor winter fumigation of honey bees (Hymenoptera: Apidae) with formic acid.

The combination of the concentration of formic acid and the duration of fumigation (CT product) during indoor treatments of honey bee, Apis mellifera L., colonies to control the varroa mite, Varroa destructor Anderson & Trueman, determines the efficacy of the treatment. Because high concentrations can cause queen mortality, we hypothesized that a high CT product given as a low concentration over a long exposure time rather than as a high concentration over a short exposure time would allow effective control of varroa mites without the detrimental effects on queens. The objective of this study was to assess different combinations of formic acid concentration and exposure time with similar CT products in controlling varroa mites while minimizing the effect on worker and queen honey bees. Treated colonies were exposed to a low, medium, or high concentration of formic acid until a mean CT product of 471 ppm*d in room air was realized. The treatments consisted of a long-term low concentration of 19 ppm for 27 d, a medium-term medium concentration of 42 ppm for 10 d, a short-term high concentration of 53 ppm for 9 d, and an untreated control. Both short-term high-concentration and medium-term medium-concentration fumigation with formic acid killed varroa mites, with averages of 93 and 83% mortality, respectively, but both treatments also were associated with an increase in mortality of worker bees, queen bees, or both. Long-term low-concentration fumigation had lower efficacy (60% varroa mite mortality), but it did not increase worker or queen bee mortality. This trend differed slightly in colonies from two different beekeepers. Varroa mite mean abundance was significantly decreased in all three acid treatments relative to the control. Daily worker mortality was significantly increased by the short-term high concentration treatment, which was reflected by a decrease in the size of the worker population, but not an increase in colony mortality. Queen mortality was significantly greater under the medium-term medium concentration and the short-term high concentration treatments than in controls.

Effect of formic acid formulations on honey bee (Hymenoptera: Apidae) colonies and influence of colony and ambient conditions on formic acid concentration in the hive.

The interaction between the effects of varroa, Varroa destructor Anderson & Trueman, and formic acid treatments on colonies of honey bees, Apis mellifera L., were examined in two field experiments. In experiment 1, colonies with low varroa levels were exposed to two different slow-release formulations and compared with untreated colonies. In experiment 2, colonies inoculated with varroa and uninoculated colonies were exposed to a slow-release formulation, a pour-on formulation, or were left untreated. The effects of treatments, hive temperature, and hive relative humidity on formic acid concentration in hive air also were examined. Slow-release formic acid application improved colony development in colonies that had been inoculated with varroa. However, in uninoculated colonies where the mean abundance of varroa was low, slow-release formic acid application suppressed colony development. The pour-on application did not have a negative impact on worker population growth in uninoculated colonies, but also it was not as effective as the slow-release treatment in improving population growth in varroa-inoculated colonies. Equivalent volumes of acid applied in pour-on and slow-release formulations provided the same cumulative dose in hive air but differed in the daily pattern of formic acid release. Colonies that were not inoculated with varroa had higher concentrations of formic acid in hive air than colonies that were inoculated with varroa on three of the five pour-on application dates. The data suggest that reductions in worker population and/or activity caused by varroa can interact with ambient conditions to affect the volatilization or sorption of formic acid in the hive.