Improving bee health through genomics.

Declines in bee populations across the world threaten food security and ecosystem function. It is currently not possible to routinely predict which specific stressors lead to declines in different populations or contexts, hindering efforts to improve bee health. Genomics has the potential to dramatically improve our ability to identify, monitor and predict the effects of stressors, as well as to mitigate their impacts through the use of marker-assisted selection, RNA interference and potentially gene editing. Here we discuss the most compelling recent applications of genomics to investigate the mechanisms underpinning bee population declines and to improve the health of both wild and managed bee populations.

Evaluating the role of social context and environmental factors in mediating overwintering physiology in honey bees (Apis mellifera).

In temperate climates, honey bees show strong phenotypic plasticity associated with seasonal changes. In summer, worker bees typically only survive for about a month and can be further classified as young nurse bees (which feed the developing brood) and older forager bees. In winter, brood production and foraging halt and the worker bees live for several months. These differences in task and longevity are reflected in their physiology, with summer nurses and long-lived winter bees typically having large fat bodies, high expression levels of vitellogenin (a longevity-, nutrition- and immune-related gene), and large provisioning glands in their head. The environmental factors (both within the colony and within the surrounding environment) that trigger this transition to long-lived winter bees are poorly understood. One theory is that winter bees are an extended nurse bee state, brought on by a reduction in nursing duties in autumn (i.e. lower brood area). We examined that theory here by assessing nurse bee physiology in both the summer and autumn, in colonies with varying levels of brood. We found that season is a better predictor of nurse bee physiology than brood area. This suggests that seasonal factors beyond brood area, such as pollen availability and colony demography, may be necessary for inducing the winter bee phenotype. This finding furthers our understanding of winter bee biology, which could have important implications for colony management for winter, a critical period for colony survival.

Effective pest management approaches can mitigate honey bee (Apis mellifera) colony winter loss across a range of weather conditions in small-scale, stationary apiaries.

Managed honey bee (Apis mellifera L.) colonies in North America and Europe have experienced high losses in recent years, which have been linked to weather conditions, lack of quality forage, and high parasite loads, particularly the obligate brood parasite, Varroa destructor. These factors may interact at various scales to have compounding effects on honey bee health, but few studies have been able to simultaneously investigate the effects of weather conditions, landscape factors, and management of parasites. We analyzed a dataset of 3,210 survey responses from beekeepers in Pennsylvania from 2017 to 2022 and combined these with remotely sensed weather variables and novel datasets about seasonal forage availability into a Random Forest model to investigate drivers of winter loss. We found that beekeepers who used treatment against Varroa had higher colony survival than those who did not treat. Moreover, beekeepers who used multiple types of Varroa treatment had higher colony survival rates than those who used 1 type of treatment. Our models found weather conditions are strongly associated with survival, but multiple-treatment type colonies had higher survival across a broader range of climate conditions. These findings suggest that the integrated pest management approach of combining treatment types can potentially buffer managed honey bee colonies from adverse weather conditions.

Examining parent-of-origin effects on transcription and RNA methylation in mediating aggressive behavior in honey bees (Apis mellifera).

Conflict between genes inherited from the mother (matrigenes) and the father (patrigenes) is predicted to arise during social interactions among offspring if these genes are not evenly distributed among offspring genotypes. This intragenomic conflict drives parent-specific transcription patterns in offspring resulting from parent-specific epigenetic modifications. Previous tests of the kinship theory of intragenomic conflict in honey bees (Apis mellifera) provided evidence in support of theoretical predictions for variation in worker reproduction, which is associated with extreme variation in morphology and behavior. However, more subtle behaviors - such as aggression - have not been extensively studied. Additionally, the canonical epigenetic mark (DNA methylation) associated with parent-specific transcription in plant and mammalian model species does not appear to play the same role as in honey bees, and thus the molecular mechanisms underlying intragenomic conflict in this species is an open area of investigation. Here, we examined the role of intragenomic conflict in shaping aggression in honey bee workers through a reciprocal cross design and Oxford Nanopore direct RNA sequencing. We attempted to probe the underlying regulatory basis of this conflict through analyses of parent-specific RNA m6A and alternative splicing patterns. We report evidence that intragenomic conflict occurs in the context of honey bee aggression, with increased paternal and maternal allele-biased transcription in aggressive compared to non-aggressive bees, and higher paternal allele-biased transcription overall. However, we found no evidence to suggest that RNA m6A or alternative splicing mediate intragenomic conflict in this species.

Signatures of adaptive decreased virulence of deformed wing virus in an isolated population of wild honeybees (Apis mellifera).

Understanding the ecological and evolutionary processes that drive host-pathogen interactions is critical for combating epidemics and conserving species. The Varroa destructor mite and deformed wing virus (DWV) are two synergistic threats to Western honeybee (Apis mellifera) populations across the globe. Distinct honeybee populations have been found to self-sustain despite Varroa infestations, including colonies within the Arnot Forest outside Ithaca, NY, USA. We hypothesized that in these bee populations, DWV has been selected to produce an avirulent infection phenotype, allowing for the persistence of both host and disease-causing agents. To investigate this, we assessed the titre of viruses in bees from the Arnot Forest and managed apiaries, and assessed genomic variation and virulence differences between DWV isolates. Across groups, we found viral abundance was similar, but DWV genotypes were distinct. We also found that infections with isolates from the Arnot Forest resulted in higher survival and lower rates of symptomatic deformed wings, compared to analogous isolates from managed colonies, providing preliminary evidence to support the hypothesis of adaptive decreased viral virulence. Overall, this multi-level investigation of virus genotype and phenotype indicates that host ecological context can be a significant driver of viral evolution and host-pathogen interactions in honeybees.

Beyond conflict: Kinship theory of intragenomic conflict predicts individual variation in altruistic behaviour.

Behavioural variation is essential for animals to adapt to different social and environmental conditions. The Kinship Theory of Intragenomic Conflict (KTIC) predicts that parent-specific alleles can support different behavioural strategies to maximize allele fitness. Previous studies, including in honey bees (Apis mellifera), supported predictions of the KTIC for parent-specific alleles to promote selfish behaviour. Here, we test the KTIC prediction that for altruism-promoting genes (i.e. those that promote behaviours that support the reproductive fitness of kin), the allele with the higher altruism optimum should be selected to be expressed while the other is silenced. In honey bee colonies, workers act altruistically when tending to the queen by performing a 'retinue' behaviour, distributing the queen's mandibular pheromone (QMP) throughout the hive. Workers exposed to QMP do not activate their ovaries, ensuring they care for the queen's brood instead of competing to lay unfertilized eggs. Due to the haplodiploid genetics of honey bees, the KTIC predicts that response to QMP is favoured by the maternal genome. We report evidence for parent-of-origin effects on the retinue response behaviour, ovarian development and gene expression in brains of worker honey bees exposed to QMP, consistent with the KTIC. Additionally, we show enrichment for genes with parent-of-origin expression bias within gene regulatory networks associated with variation in bees' response to QMP. Our study demonstrates that intragenomic conflict can shape diverse social behaviours and influence expression patterns of single genes as well as gene networks.

Beat the heat: thermal respites and access to food associated with increased bumble bee heat tolerance.

Climate change poses a threat to organisms across the world, with cold-adapted species such as bumble bees (Bombus spp.) at particularly high risk. Understanding how organisms respond to extreme heat events associated with climate change as well as the factors that increase resilience or prime organisms for future stress can inform conservation actions. We investigated the effects of heat stress within different contexts (duration, periodicity, with and without access to food, and in the laboratory versus field) on bumble bee (Bombus impatiens) survival and heat tolerance. We found that both prolonged (5 h) heat stress and nutrition limitation were negatively correlated with worker bee survival and thermal tolerance. However, the effects of these acute stressors were not long lasting (no difference in thermal tolerance among treatment groups after 24 h). Additionally, intermittent heat stress, which more closely simulates the forager behavior of leaving and returning to the nest, was not negatively correlated with worker thermal tolerance. Thus, short respites may allow foragers to recover from thermal stress. Moreover, these results suggest there is no priming effect resulting from short- or long-duration exposure to heat - bees remained equally sensitive to heat in subsequent exposures. In field-caught bumble bees, foragers collected during warmer versus cooler conditions exhibited similar thermal tolerance after being allowed to recover in the lab for 16 h. These studies offer insight into the impacts of a key bumble bee stressor and highlight the importance of recovery duration, stressor periodicity and context on bumble bee thermal tolerance outcomes.

Characterizing user needs for Beescape: A spatial decision support tool focused on pollinator health.

Interactive maps can serve as powerful environmental decision-support tools. However, designing an interactive map that meets the needs of diverse constituencies is a challenge. In this article, we evaluate and characterize user needs for an interactive map and spatial decision-support tool called Beescape. Beescape is designed to visualize resources and environmental risks to bees and other pollinators (such as availability of nutritional resources from flowering plants and exposure to pesticides) in order to help users make informed decisions about managing bee populations and associated landscapes. We conducted a needs assessment workshop with twenty stakeholders from four user groups including beekeepers, growers, conservationists, and pollinator scientists to elicit their knowledge to guide future Beescape development. The results of the workshop identify current analytical gaps with the existing Beescape prototype, including the need for predictive and historical tools, more actionable data layers, finer-grain spatial data, and better explanations on what data represent and how they were created. Our findings on user's analytical, informational, and interface needs can be utilized to guide the future development of spatial decision support tools like Beescape, and our methodological approach may apply to other environmental informatics tools where it is important to design for multiple constituent user groups.

Tissue-specific transcriptional patterns underlie seasonal phenotypes in honey bees (Apis mellifera).

Faced with adverse conditions, such as winter in temperate regions or hot and dry conditions in tropical regions, many insect species enter a state of diapause, a period of dormancy associated with a reduction or arrest of physical activity, development and reproduction. Changes in common physiological pathways underlie diapause phenotypes in different insect species. However, most transcriptomic studies of diapause have not simultaneously evaluated and compared expression patterns in different tissues. Honey bees (Apis mellifera) represent a unique model system to study the mechanisms underpinning diapause-related phenotypes. In winter, honey bees exhibit a classic diapause phenotype, with reduced metabolic activity, increased physiological nutritional resources and altered hormonal profiles. However, winter bees actively heat their colony by vibrating their wing muscles; thus, this tissue is not quiescent. Here, we evaluated the transcriptional profiles of flight muscle tissue and fat body tissue (involved in nutrient storage, metabolism and immune function) of winter bees. We also evaluated two behavioural phenotypes of summer bees: nurses, which exhibit high nutritional stores and low flight activity, and foragers, which exhibit low nutritional stores and high flight activity. We found winter bees and nurses have similar fat body transcriptional profiles, whereas winter bees and foragers have similar flight muscle transcriptional profiles. Additionally, differentially expressed genes were enriched in diapause-related gene ontology terms. Thus, honey bees exhibit tissue-specific transcriptional profiles associated with seasonal phenotypes, laying the groundwork for future studies evaluating the mechanisms, evolution and consequences of this tissue-specific regulation.

Simulated vector transmission differentially influences dynamics of two viral variants of deformed wing virus in honey bees (Apis mellifera).

Understanding how vectors alter the interactions between viruses and their hosts is a fundamental question in virology and disease ecology. In honey bees, transmission of deformed wing virus (DWV) by parasitic Varroa mites has been associated with elevated disease and host mortality, and Varroa transmission has been hypothesized to lead to increased viral titres or select for more virulent variants. Here, we mimicked Varroa transmission by serially passaging a mixed population of two DWV variants, A and B, by injection through in vitro reared honey bee pupae and tracking these viral populations through five passages. The DWV-A and DWV-B variant proportions shifted dynamically through passaging, with DWV-B outcompeting DWV-A after one passage, but levels of both variants becoming equivalent by Passage 5. Sequencing analysis revealed a dominant, recombinant DWV-B strain (DWV-A derived 5' IRES region with the rest of the genome DWV-B), with low nucleotide diversity that decreased through passaging. DWV-A populations had higher nucleotide diversity compared to DWV-B, but this also decreased through passaging. Selection signatures were found across functional regions of the DWV-A and DWV-B genomes, including amino acid mutations in the putative capsid protein region. Simulated vector transmission differentially impacted two closely related viral variants which could influence viral interactions with the host, demonstrating surprising plasticity in vector-host-viral dynamics.

Tissue-specific transcription patterns support the kinship theory of intragenomic conflict in honey bees (Apis mellifera).

Kin selection may act differently on genes inherited from parents (matrigenes and patrigenes), resulting in intragenomic conflict. This conflict can be observed as differential expression of matrigenes and patrigenes, or parent-specific gene expression (PSGE). In honey bees (Apis mellifera), intragenomic conflict is hypothesized to occur in multiple social contexts. Previously, we found that patrigene-biased expression in reproductive tissues was associated with increased reproductive potential in worker honey bees, consistent with the prediction that patrigenes are selected to promote selfish behaviour in this context. Here, we examined brain gene expression patterns to determine if PSGE is also found in other tissues. As before, the number of transcripts showing patrigene expression bias was significantly greater in the brains of reproductive vs. sterile workers, while the number of matrigene-biased transcripts was not significantly different. Twelve transcripts out of the 374 showing PSGE in either tissue showed PSGE in both brain and reproductive tissues; this overlap was significantly greater than expected by chance. However, the majority of transcripts show PSGE only in one tissue, suggesting the epigenetic mechanisms mediating PSGE exhibit plasticity between tissues. There was no significant overlap between transcripts that showed PSGE and transcripts that were significantly differentially expressed. Weighted gene correlation network analysis identified modules which were significantly enriched in both types of transcripts, suggesting that these genes may influence each other through gene networks. Our results provide further support for the kin selection theory of intragenomic conflict, and provide valuable insights into the mechanisms which may mediate this process.

Wild bees as winners and losers: Relative impacts of landscape composition, quality, and climate.

Wild bees, like many other taxa, are threatened by land-use and climate change, which, in turn, jeopardizes pollination of crops and wild plants. Understanding how land-use and climate factors interact is critical to predicting and managing pollinator populations and ensuring adequate pollination services, but most studies have evaluated either land-use or climate effects, not both. Furthermore, bee species are incredibly variable, spanning an array of behavioral, physiological, and life-history traits that can increase or decrease resilience to land-use or climate change. Thus, there are likely bee species that benefit, while others suffer, from changing climate and land use, but few studies have documented taxon-specific trends. To address these critical knowledge gaps, we analyzed a long-term dataset of wild bee occurrences from Maryland, Delaware, and Washington DC, USA, examining how different bee genera and functional groups respond to landscape composition, quality, and climate factors. Despite a large body of literature documenting land-use effects on wild bees, in this study, climate factors emerged as the main drivers of wild-bee abundance and richness. For wild-bee communities in spring and summer/fall, temperature and precipitation were more important predictors than landscape composition, landscape quality, or topography. However, relationships varied substantially between wild-bee genera and functional groups. In the Northeast USA, past trends and future predictions show a changing climate with warmer winters, more intense precipitation in winter and spring, and longer growing seasons with higher maximum temperatures. In almost all of our analyses, these conditions were associated with lower abundance of wild bees. Wild-bee richness results were more mixed, including neutral and positive relationships with predicted temperature and precipitation patterns. Thus, in this region and undoubtedly more broadly, changing climate poses a significant threat to wild-bee communities.

Pollen protein and lipid content influence resilience to insecticides in honey bees (Apis mellifera).

In honey bees (Apis mellifera), there is growing evidence that the impacts of multiple stressors can be mitigated by quality nutrition. Pollen, which is the primary source of protein and lipids in bees diets, is particularly critical for generating more resilient phenotypes. Here, we evaluate the relationship between pollen protein-to-lipid ratios (P:Ls) and honey bee insecticide resilience. We hypothesized that pollen diets richer in lipids would lead to increased survival in bees exposed to insecticides, as pollen-derived lipids have previously been shown to improve bee resilience to pathogens and parasites. Furthermore, lipid metabolic processes are altered in bees exposed to insecticides.We fed age-matched bees pollen diets of different P:Ls by altering a base pollen by either adding protein (casein powder) or lipids (canola oil) and simulating chronic insecticide exposure by feeding bees an organophosphate (Chlorpyrifos). We also tested pollen diets of naturally different P:Ls to determine if results are consistent. Linear regression analysis revealed that mean survival time for altered diets was best explained by protein concentration (p =0.04 , adjusted R2 =0.92), and that mean survival time for natural diets was best explained by P:L ratio (p =0.008 , adjusted R2 =0.93). Our results indicate that higher ratios of dietary protein to lipid has a negative effect on bee physiology when combined with insecticide exposure, while lower ratios have a positive effect. These results suggest that protein and lipid intake differentially influence insecticide response in bees, laying the groundwork for future studies of metabolic processes and development of improved diets.

Economic Dependence and Vulnerability of United States Agricultural Sector on Insect-Mediated Pollination Service.

Deficits in insect-mediated pollination service undermine ecosystem biodiversity and function, human nutrition, and economic welfare. Global pollinator supply continues to decline, while production of pollination-dependent crops increases. Using publicly available price and production data and existing pollination field studies, we quantify economic dependence of United States crops on insect-mediated pollination service at the county level and update existing coefficients of insect dependence of sample crops when possible. Economic value dependent on pollination service totals 34.0 billion USD in 2012. Twenty percent of US counties produce 80% of total economic value attributable to insect pollinators. We compile county-level data and consider the spatial relationship between economic value dependent on insect-mediated pollination, region-specific forage suitability, and crop-specific agricultural areas within US landscapes. We identify vulnerable, highly dependent areas where habitat for wild pollinators has been reduced. These results can help inform future efforts to conserve and bolster managed and wild pollinator populations to ensure sustainable production of key agricultural crops.

Bee Viruses: Ecology, Pathogenicity, and Impacts.

Bees-including solitary, social, wild, and managed species-are key pollinators of flowering plant species, including nearly three-quarters of global food crops. Their ecological importance, coupled with increased annual losses of managed honey bees and declines in populations of key wild species, has focused attention on the factors that adversely affect bee health, including viral pathogens. Genomic approaches have dramatically expanded understanding of the diversity of viruses that infect bees, the complexity of their transmission routes-including intergenus transmission-and the diversity of strategies bees have evolved to combat virus infections, with RNA-mediated responses playing a prominent role. Moreover, the impacts of viruses on their hosts are exacerbated by the other major stressors bee populations face, including parasites, poor nutrition, and exposure to chemicals. Unraveling the complex relationships between viruses and their bee hosts will lead to improved understanding of viral ecology and management strategies that support better bee health.

Honey bee (Apis mellifera) larval pheromones may regulate gene expression related to foraging task specialization.

BACKGROUND: Foraging behavior in honey bees (Apis mellifera) is a complex phenotype that is regulated by physiological state and social signals. How these factors are integrated at the molecular level to modulate foraging behavior has not been well characterized. The transition of worker bees from nursing to foraging behaviors is mediated by large-scale changes in brain gene expression, which are influenced by pheromones produced by the queen and larvae. Larval pheromones can also stimulate foragers to leave the colony to collect pollen. However, the mechanisms underpinning this rapid behavioral plasticity in foragers that specialize in collecting pollen over nectar, and how larval pheromones impact these different behavioral states, remains to be determined. Here, we investigated the patterns of gene expression related to rapid behavioral plasticity and task allocation among honey bee foragers exposed to two larval pheromones, brood pheromone (BP) and (E)-beta-ocimene (EBO). We hypothesized that both pheromones would alter expression of genes in the brain related to foraging and would differentially impact brain gene expression depending on foraging specialization. RESULTS: Combining data reduction, clustering, and network analysis methods, we found that foraging preference (nectar vs. pollen) and pheromone exposure are each associated with specific brain gene expression profiles. Furthermore, pheromone exposure has a strong transcriptional effect on genes that are preferentially expressed in nectar foragers. Representation factor analysis between our study and previous landmark honey bee transcriptome studies revealed significant overlaps for both pheromone communication and foraging task specialization. CONCLUSIONS: Our results suggest that, as social signals, pheromones alter expression patterns of foraging-related genes in the bee's brain to increase pollen foraging at both long and short time scales. These results provide new insights into how social signals and task specialization are potentially integrated at the molecular level, and highlights the possible role that brain gene expression may play in honey bee behavioral plasticity across time scales.

Haemolymph removal by Varroa mite destabilizes the dynamical interaction between immune effectors and virus in bees, as predicted by Volterra's model.

The association between the deformed wing virus and the parasitic mite Varroa destructor has been identified as a major cause of worldwide honeybee colony losses. The mite acts as a vector of the viral pathogen and can trigger its replication in infected bees. However, the mechanistic details underlying this tripartite interaction are still poorly defined, and, particularly, the causes of viral proliferation in mite-infested bees. Here, we develop and test a novel hypothesis that mite feeding destabilizes viral immune control through the removal of both virus and immune effectors, triggering uncontrolled viral replication. Our hypothesis is grounded on the predator-prey theory developed by Volterra, which predicts prey proliferation when both predators and preys are constantly removed from the system. Consistent with this hypothesis, we show that the experimental removal of increasing volumes of haemolymph from individual bees results in increasing viral densities. By contrast, we do not find consistent support for alternative proposed mechanisms of viral expansion via mite immune suppression or within-host viral evolution. Our results suggest that haemolymph removal plays an important role in the enhanced pathogen virulence observed in the presence of feeding Varroa mites. Overall, these results provide a new model for the mechanisms driving pathogen-parasite interactions in bees, which ultimately underpin honeybee health decline and colony losses.

Bee community preference for an invasive thistle associated with higher pollen protein content.

Non-native plant species reliant on insect pollination must attract novel pollinators in their introduced habitat to reproduce. Indeed, pollination services provided by resident floral visitors may contribute to the spread of non-native species, which may then affect the pollination services received by native plants. To determine the mechanisms by which an invasive thistle attracts pollinators in its introduced range, and whether its presence changes the pollinator visitation to native plant species, we compared bee visitation to native plants in the presence or absence of the invader. We experimentally tested the effect of a thistle invasion into a native plant community. We found that the non-native thistle was the most attractive of the plant species to visiting bee species. However, there was no effect of experimental treatment (presence of thistle) on bee abundance or visitation rate (bees per unit floral area per sample) to native plant species. Across 68 bee and 6 plant species, we found a significant correlation between pollen protein content and bee abundance and visitation rate. Thistle pollen also had a similar protein:lipid ratio to legumes, which correlated with bumble bee visitation. The high protein content of the thistle pollen, as compared to four native asters, may allow it to attract pollinators in novel ecosystems, and potentially contribute to its success as an invader. At the same time, this high protein pollen may act as a novel resource to pollinators in the thistle's invaded range.

Evaluating the Effect of Honey Bee (Apis mellifera) Queen Reproductive State on Pheromone-Mediated Interactions with Male Drone Bees.

Honey bee (Apis mellifera) queens produce pheromones responsible for mediating both male mating behavior and many critical facets of worker social organization within their colony. These pheromones are dynamic multi-component blends, allowing the communication of detailed information. Indeed, variation in the queen's mating and reproductive state is associated with significant changes in her pheromone profiles, and these different pheromone profiles elicit different behavioral and physiological responses in female workers. Here we evaluate behavioral responses of male drones to the chemical blends produced by two exocrine glands in queens, and determine if the blends and responses are altered by the queen's mating and reproductive state. We find that drone attraction to the chemical blends of mandibular glands produced by mated, laying queens versus virgin queens is reduced, suggesting that the queens produce a reliable signal of their mating receptivity. Interestingly, while the chemical blends of mating, laying queens and virgins queens largely overlap, mated, laying queens produce a greater number of chemicals and greater quantities of certain chemicals than virgin queens, suggesting that these chemicals may serve to inhibit behavioral responses of drones to mated, laying queens. Thus, our results highlight the importance of considering chemical cues and signals that serve to both stimulate and inhibit behavioral responses during social interactions in animals.