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.

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.

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.

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.

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.

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.

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.

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.

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.