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.

The effects of protein supplementation, fumagillin treatment, and colony management on the productivity and long-term survival of honey bee (Apis mellifera) colonies.

In this study, we intensively measured the longitudinal productivity and survival of 362 commercially managed honey bee colonies in Canada, over a two-year period. A full factorial experimental design was used, whereby two treatments were repeated across apiaries situated in three distinct geographic regions: Northern Alberta, Southern Alberta and Prince Edward Island, each having unique bee management strategies. In the protein supplemented treatment, colonies were continuously provided a commercial protein supplement containing 25% w/w pollen, in addition to any feed normally provided by beekeepers in that region. In the fumagillin treatment, colonies were treated with the label dose of Fumagilin-B® each year during the fall. Neither treatment provided consistent benefits across all sites and dates. Fumagillin was associated with a large increase in honey production only at the Northern Alberta site, while protein supplementation produced an early season increase in brood production only at the Southern Alberta site. The protein supplement provided no long-lasting benefit at any site and was also associated with an increased risk of death and decreased colony size later in the study. Differences in colony survival and productivity among regions, and among colonies within beekeeping operations, were far larger than the effects of either treatment, suggesting that returns from extra feed supplements and fumagillin were highly contextually dependent. We conclude that use of fumagillin is safe and sometimes beneficial, but that beekeepers should only consider excess protein supplementation when natural forage is limiting.

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.

Requeening queenright honey bee colonies with queen cells in honey supers.

Many Canadian beekeepers replace a subset of their honey bee queens annually. However, introducing a new queen to a honey bee colony is a management practice with a high degree of uncertainty. Despite the consensus that it is most effective to introduce queens to queenless colonies, some commercial beekeepers claim success with introducing queen cells into the honey super of queenright colonies. We tested the success rate of this practice by introducing queen cells to 100 queenright colonies in southern Alberta during a honey flow. The genotypes of the resultant offspring drones were determined using the microsatellite marker A76 to identify their laying queen mothers. Our results show that new queens successfully supersede original queens in 6% of queenright colonies, suggesting that the practice does not result in the new queen taking over leadership in most colonies. Additionally, supersedure by daughter queens is more common (13%) than new queen supersedure when introducing queen cells to queenright colonies during a honey flow. However, there could be a benefit to the practice of requeening queenright colonies with queen cells in honey supers if the colonies that accepted a new queen (whether a daughter of or unrelated to the old queen) were colonies with a failing queen.

Carbohydrate flow through agricultural ecosystems: Implications for synthesis and microbial conversion of carbohydrates.

Carbohydrates are chemically and structurally diverse biomolecules, serving numerous and varied roles in agricultural ecosystems. Crops and horticulture products are inherent sources of carbohydrates that are consumed by humans and non-human animals alike; however carbohydrates are also present in other agricultural materials, such as soil and compost, human and animal tissues, milk and dairy products, and honey. The biosynthesis, modification, and flow of carbohydrates within and between agricultural ecosystems is intimately related with microbial communities that colonize and thrive within these environments. Recent advances in -omics techniques have ushered in a new era for microbial ecology by illuminating the functional potential for carbohydrate metabolism encoded within microbial genomes, while agricultural glycomics is providing fresh perspective on carbohydrate-microbe interactions and how they influence the flow of functionalized carbon. Indeed, carbohydrates and carbohydrate-active enzymes are interventions with unrealized potential for improving carbon sequestration, soil fertility and stability, developing alternatives to antimicrobials, and circular production systems. In this manner, glycomics represents a new frontier for carbohydrate-based biotechnological solutions for agricultural systems facing escalating challenges, such as the changing climate.

Winter mortality, diversification, and self-sufficiency affect honey bee (Hymenoptera: Apidae) colony profit in Canada: a model of commercial Alberta beekeepers.

Canadian beekeepers faced widespread levels of high honey bee colony mortality over the winter of 2021/2022, with an average winter loss of 45%. To understand the economic impact of winter colony mortality in Canada and the beekeeping management strategies used to mitigate these losses, we develop a profit model of commercial beekeeping operations in Alberta, Canada. Our model shows that for operations engaging in commercial pollination as well as honey production (compared to honey production alone), per colony profit is higher and operations are better able to withstand fluctuations in exogenous variables such as prices and environmental factors affecting productivity including winter mortality rates. The results also suggest that beekeeping operations that replace winter colony losses with splits instead of package bees accrue higher per colony profit than those importing packages to replace losses. Further, operations that produce their own queens to use in their replacement splits, accrue even higher profit. Our results demonstrate that the profitability of beekeeping operations is dependent on several factors including winter mortality rates, colony replacement strategies, and the diversification of revenue sources. Beekeepers who are not as susceptible to price and risk fluctuations in international markets and imported bee risks accrue more consistently positive profits.

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.

Consumption of Supplemental Spring Protein Feeds by Western Honey Bee (Hymenoptera: Apidae) Colonies: Effects on Colony Growth and Pollination Potential.

Adequate nutrition is required to support productive honey bee colonies, therefore beekeepers supplement colonies with additional protein at targeted time points. We tested the effects of commercially available protein feeds in spring, in advance of colonies being used for hybrid canola pollination. The feed treatments across the three-year study included the following patty types: Global 15% pollen, Global 0% pollen, Bee Pollen-Ate, FeedBee, and Healthy Bees, as well as an unsupplemented control in year two of the study only. The amount of feed consumed varied among colonies, treatments, date, and year. Similarly, there were also differences in feed efficiency (bees reared per gram of feed consumed), likely due to the relative availability of external forage sources to supplement the feed provided. Unsupplemented colonies were able to rear less brood, and subsequently had fewer adult bees than supplemented colonies, in an apiary where pollen was not abundant. Differences in consumption among treatments often failed to translate in to differences in amount of brood reared or subsequent adult population. All the protein feed treatments contained all ten amino acids essential to honey bees, however lysine and arginine were below the optimal proportion required for growth in all patties except the FeedBee patty. The amount of protein and amount and types of sugars and fats in the products also varied among product type and batch. The results of this study demonstrate a benefit to supplementary spring protein feeding to increase honey bee colony populations in advance of a summer pollination market.

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.

Impacts of COVID-19 on Canadian Beekeeping: Survey Results and a Profitability Analysis.

To gauge the impact of COVID-19 on the Canadian beekeeping sector, we conducted a survey of over 200 beekeepers in the fall of 2020. Our survey results show Canadian beekeepers faced two major challenges: 1) disrupted importation of honey bees (Hymenoptera: Apidae) (queen and bulk bees) that maintain populations; and 2) disrupted arrival of temporary foreign workers (TFWs). Disruptions in the arrival of bees and labor resulted in fewer colonies and less colony management, culminating in higher costs and lower productivity. Using the survey data, we develop a profitability analysis to estimate the impact of these disruptions on colony profit. Our results suggest that a disruption in either foreign worker or bee arrival allows beekeepers to compensate and while colony profits are lower, they remain positive. When both honey bee and foreign workers arrivals are disrupted for a beekeeper, even when the beekeeper experiences less significant colony health and cost impacts, a colony with a single pollination contract is no longer profitable, and a colony with two pollination contracts has significantly reduced profitability. As COVID-19 disruptions from 2020 and into 2021 become more significant to long-term colony health and more costly to a beekeeping operation, economic losses could threaten the industry's viability as well as the sustainability of pollination-dependent crop sectors across the country. The economic and agricultural impacts from the COVID-19 pandemic have exposed a vulnerability within Canada's beekeeping industry stemming from its dependency on imported labor and bees. Travel disruptions and border closures pose an ongoing threat to Canadian agriculture and apiculture in 2021 and highlight the need for Canada's beekeeping industry to strengthen domestic supply chains to minimize future risks.

Honey Bee Queen Production: Canadian Costing Case Study and Profitability Analysis.

The decline in managed honey bee (Hymenoptera: Apidae) colony health worldwide has had a significant impact on the beekeeping industry. To mitigate colony losses, beekeepers in Canada and around the world introduce queens into replacement colonies; however, Canada's short queen rearing season has historically limited the production of early season queens. As a result, Canadian beekeepers rely on the importation of foreign bees, particularly queens from warmer climates. Importing a large proportion of (often mal-adapted) queens each year creates a dependency on foreign bee sources, putting beekeeping, and pollination sectors at risk in the event of border closures, transportation issues, and other restrictions as is currently happening due to the 2020 Covid-19 pandemic. Although traditional Canadian queen production is unable to fully meet early season demand, increasing domestic queen production to meet mid- and later season demand would reduce Canada's dependency. As well, on-going studies exploring the potential for overwintering queens in Canada may offer a strategy to have early season domestic queens available. Increasing the local supply of queens could provide Canadian beekeepers, farmers, and consumers with a greater level of agricultural stability and food security. Our study is the first rigorous analysis of the economic feasibility of queen production. We present the costs of queen production for three Canadian operations over two years. Our results show that it can be profitable for a beekeeping operation in Canada to produce queen cells and mated queens and could be one viable strategy to increase the sustainability of the beekeeping industry.

Pollinators enhance crop yield and shorten the growing season by modulating plant functional characteristics: A comparison of 23 canola varieties.

Insect pollination of flowers should change the within-season allocation of resources in plants. But the nature of this life-history response, particularly regarding allocation to roots, photosynthetic structures, and flowers, is empirically unresolved. This study uses a greenhouse experiment to investigate the effect of insect pollination on the reproductive output of 23 varieties of a globally important crop-canola (Brassica napus). Overall, insect pollination modified the functional characteristics (flower timing & effort, plant size & shape, seed packaging, root biomass) of canola, increasing seed production and quality, and pollinator dependence. Reproductive output and pollinator dependence were defined by strong trait trade-offs, which ranged from more pollinator-dependent plants favouring early reproductive effort, to less pollinator-dependent plants favouring a prolonged phenology with smaller plant size and lower seed quality. Seed production decreased with pollinator dependence in the absence of pollinators. The agricultural preference for hybrid varieties will increase seed production compared to open-pollinated varieties, but, even so, pollinators typically enhance seed production of both types. Our study elucidates how insect pollination alters the character and function of a globally important crop, supporting optimization of yield via intensification of insect pollination, and highlights the beneficial effects of insect pollination early in the season.

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.

Comparison of Honey Bee (Hymenoptera: Apidae) Colony Units of Different Sizes as Pollinators of Hybrid Seed Canola.

We compare two different sizes of honey bee colony units: singles (one brood chamber) and doubles (two brood chambers) in hybrid seed canola pollination in southern Alberta in 2014 and 2015. Currently, canola seed production companies only contract double-brood chamber units to pollinate canola in southern Alberta, but it may be advantageous to the industry if singles could also be contracted for pollination, as they are in many other crops. To evaluate the differences between the colony units, we measured population size, nectar and pollen foraging, nectar and pollen load weights, pollen collection, and honey production. The colony populations of both the single- and double-brood chamber hives in this study were highly variable. In 2015, there was no difference between the single- and the double-brood chamber colonies in adult bee populations, and the singles had more sealed brood than did the double-brood chamber colonies. Our findings indicate that in comparison to doubles, on a per-frame basis, singles yield more pollen, more nectar foragers, similar or more pollen foragers, and similar amounts of honey. Therefore, we conclude that singles could be used to provide the same level of pollination services as doubles currently do in hybrid seed canola pollination, and growers should focus on receiving healthy populous colonies, regardless of the number of brood boxes.

Pollen Collection, Honey Production, and Pollination Services: Managing Honey Bees in an Agricultural Setting.

Hybrid canola seed production is an important pollination market in Canada; typically both honey bees (Apis mellifera L. (Hymenoptera: Apidae)) and Alfalfa Leafcutting bees (Megachile rotundata Fab. (Hymenoptera: Megachilidae)) are concurrently managed to ensure pollination in this high-value crop. Beekeepers are paid to provide pollination services, and the colonies also produce a honey crop from the canola. Pollen availability from male-fertile plants is carefully managed in this crop to provide an abundance of pollen to fertilize male-sterile ('female') plants. This abundance of pollen represents an underutilized resource for beekeepers, and an opportunity to diversify the hive-products produced for market in this management system. We used a commercial-style pollen trap to collect pollen from colonies twice weekly for the duration of canola pollination, and compared the honey production and amount of sealed brood in colonies with pollen traps to those without pollen traps. We found that while pollen trapping reduced honey production, there was no negative impact on brood production, and at current market prices, the per-hive revenue was higher in colonies from which pollen was trapped. Pollen trapping honey bee colonies in the context of hybrid canola pollination, therefore, offers beekeepers an opportunity to diversify their products and increase their revenue.

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.

A Bio-Economic Case Study of Canadian Honey Bee (Hymenoptera: Apidae) Colonies: Marker-Assisted Selection (MAS) in Queen Breeding Affects Beekeeper Profits.

Over the past decade in North America and Europe, winter losses of honey bee (Hymenoptera: Apidae) colonies have increased dramatically. Scientific consensus attributes these losses to multifactorial causes including altered parasite and pathogen profiles, lack of proper nutrition due to agricultural monocultures, exposure to pesticides, management, and weather. One method to reduce colony loss and increase productivity is through selective breeding of queens to produce disease-, pathogen-, and mite-resistant stock. Historically, the only method for identifying desirable traits in honey bees to improve breeding was through observation of bee behavior. A team of Canadian scientists have recently identified markers in bee antennae that correspond to behavioral traits in bees and can be tested for in a laboratory. These scientists have demonstrated that this marker-assisted selection (MAS) can be used to produce hygienic, pathogen-resistant honey bee colonies. Based on this research, we present a beekeeping case study where a beekeeper's profit function is used to evaluate the economic impact of adopting colonies selected for hygienic behavior using MAS into an apiary. Our results show a net profit gain from an MAS colony of between 2% and 5% when Varroa mites are effectively treated. In the case of ineffective treatment, MAS generates a net profit benefit of between 9% and 96% depending on the Varroa load. When a Varroa mite population has developed some treatment resistance, we show that MAS colonies generate a net profit gain of between 8% and 112% depending on the Varroa load and degree of treatment resistance.

Warming, CO2, and nitrogen deposition interactively affect a plant-pollinator mutualism.

Environmental changes threaten plant-pollinator mutualisms and their critical ecosystem service. Drivers such as land use, invasions and climate change can affect pollinator diversity or species encounter rates. However, nitrogen deposition, climate warming and CO(2) enrichment could interact to disrupt this crucial mutualism by altering plant chemistry in ways that alter floral attractiveness or even nutritional rewards for pollinators. Using a pumpkin model system, we show that these drivers non-additively affect flower morphology, phenology, flower sex ratios and nectar chemistry (sugar and amino acids), thereby altering the attractiveness of nectar to bumble bee pollinators and reducing worker longevity. Alarmingly, bees were attracted to, and consumed more, nectar from a treatment that reduced their survival by 22%. Thus, three of the five major drivers of global environmental change have previously unknown interactive effects on plant-pollinator mutualisms that could not be predicted from studies of individual drivers in isolation.

Genome-wide analysis reveals differences in brain gene expression patterns associated with caste and reproductive status in honey bees (Apis mellifera).

A key characteristic of eusocial species is reproductive division of labour. Honey bee colonies typically have a single reproductive queen and thousands of sterile workers. Adult queens differ dramatically from workers in anatomy, physiology, behaviour and lifespan. Young female workers can activate their ovaries and initiate egg laying; these 'reproductive' workers differ from sterile workers in anatomy, physiology, and behaviour. These differences, however, are on a much smaller scale than those observed between the queen and worker castes. Here, we use microarrays to monitor expression patterns of several thousand genes in the brains of same-aged virgin queens, sterile workers, and reproductive workers. We found large differences in expression between queens and both worker groups (~2000 genes), and much smaller differences between sterile and reproductive workers (221 genes). The expression patterns of these 221 genes in reproductive workers are more queen-like, and may represent a core group of genes associated with reproductive physiology. Furthermore, queens and reproductive workers preferentially up-regulate genes associated with the nurse bee behavioural state, which supports the hypothesis of an evolutionary link between worker division of labour and molecular pathways related to reproduction. Finally, several functional groups of genes associated with longevity in other species are significantly up-regulated in queens. Identifying the genes that underlie the differences between queens, sterile workers, and reproductive workers will allow us to begin to characterize the molecular mechanisms underlying the evolution of social behaviour and large-scale remodelling of gene networks associated with polyphenisms.