The expansion of agriculture has shaped the recent evolutionary history of a specialized squash pollinator.

The expansion of agriculture is responsible for the mass conversion of biologically diverse natural environments into managed agroecosystems dominated by a handful of genetically homogeneous crop species. Agricultural ecosystems typically have very different abiotic and ecological conditions from those they replaced and create potential niches for those species that are able to exploit the abundant resources offered by crop plants. While there are well-studied examples of crop pests that have adapted into novel agricultural niches, the impact of agricultural intensification on the evolution of crop mutualists such as pollinators is poorly understood. We combined genealogical inference from genomic data with archaeological records to demonstrate that the Holocene demographic history of a wild specialist pollinator of Cucurbita (pumpkins, squashes, and gourds) has been profoundly impacted by the history of agricultural expansion in North America. Populations of the squash bee Eucera pruinosa experienced rapid growth in areas where agriculture intensified within the past 1,000 y, suggesting that the cultivation of Cucurbita in North America has increased the amount of floral resources available to these bees. In addition, we found that roughly 20% of this bee species' genome shows signatures of recent selective sweeps. These signatures are overwhelmingly concentrated in populations from eastern North America where squash bees were historically able to colonize novel environments due to human cultivation of Cucurbita pepo and now exclusively inhabit agricultural niches. These results suggest that the widespread cultivation of crops can prompt adaptation in wild pollinators through the distinct ecological conditions imposed by agricultural environments.

Evolutionarily diverse origins of deformed wing viruses in western honey bees.

Novel transmission routes can allow infectious diseases to spread, often with devastating consequences. Ectoparasitic varroa mites vector a diversity of RNA viruses, having switched hosts from the eastern to western honey bees (Apis cerana to Apis mellifera). They provide an opportunity to explore how novel transmission routes shape disease epidemiology. As the principal driver of the spread of deformed wing viruses (mainly DWV-A and DWV-B), varroa infestation has also driven global honey bee health declines. The more virulent DWV-B strain has been replacing the original DWV-A strain in many regions over the past two decades. Yet, how these viruses originated and spread remains poorly understood. Here, we use a phylogeographic analysis based on whole-genome data to reconstruct the origins and demography of DWV spread. We found that, rather than reemerging in western honey bees after varroa switched hosts, as suggested by previous work, DWV-A most likely originated in East Asia and spread in the mid-20th century. It also showed a massive population size expansion following the varroa host switch. By contrast, DWV-B was most likely acquired more recently from a source outside East Asia and appears absent from the original varroa host. These results highlight the dynamic nature of viral adaptation, whereby a vector's host switch can give rise to competing and increasingly virulent disease pandemics. The evolutionary novelty and rapid global spread of these host-virus interactions, together with observed spillover into other species, illustrate how increasing globalization poses urgent threats to biodiversity and food security.

Repeated shifts in sociality are associated with fine-tuning of highly conserved and lineage-specific enhancers in a socially flexible bee.

Comparative genomic studies of social insects suggest that changes in gene regulation are associated with evolutionary transitions in social behavior, but the activity of predicted regulatory regions has not been tested empirically. We used STARR-seq, a high-throughput enhancer discovery tool, to identify and measure the activity of enhancers in the socially variable sweat bee, Lasioglossum albipes. We identified over 36,000 enhancers in the L. albipes genome from three social and three solitary populations. Many enhancers were identified in only a subset of L. albipes populations, revealing rapid divergence in regulatory regions within this species. Population-specific enhancers were often proximal to the same genes across populations, suggesting compensatory gains and losses of regulatory regions may preserve gene activity. We also identified 1182 enhancers with significant differences in activity between social and solitary populations, some of which are conserved regulatory regions across species of bees. These results indicate that social trait variation in L. albipes is associated with the fine-tuning of ancient enhancers as well as lineage-specific regulatory changes. Combining enhancer activity with population genetic data revealed variants associated with differences in enhancer activity and identified a subset of differential enhancers with signatures of selection associated with social behavior. Together, these results provide the first empirical map of enhancers in a socially flexible bee and highlight links between cis-regulatory variation and the evolution of social behavior.

Promiscuous feeding on multiple adult honey bee hosts amplifies the vectorial capacity of Varroa destructor.

Varroa destructor is a cosmopolitan pest and leading cause of colony loss of the European honey bee. Historically described as a competent vector of honey bee viruses, this arthropod vector is the cause of a global pandemic of Deformed wing virus, now endemic in honeybee populations in all Varroa-infested regions. Our work shows that viral spread is driven by Varroa actively switching from one adult bee to another as they feed. Assays using fluorescent microspheres were used to indicate the movement of fluids in both directions between host and vector when Varroa feed. Therefore, Varroa could be in either an infectious or naïve state dependent upon the disease status of their host. We tested this and confirmed that the relative risk of a Varroa feeding depended on their previous host's infectiousness. Varroa exhibit remarkable heterogeneity in their host-switching behavior, with some Varroa infrequently switching while others switch at least daily. As a result, relatively few of the most active Varroa parasitize the majority of bees. This multiple-feeding behavior has analogs in vectorial capacity models of other systems, where promiscuous feeding by individual vectors is a leading driver of vectorial capacity. We propose that the honeybee-Varroa relationship offers a unique opportunity to apply principles of vectorial capacity to a social organism, as virus transmission is both vectored and occurs through multiple host-to-host routes common to a crowded society.

HBeeID: a molecular tool that identifies honey bee subspecies from different geographic populations.

BACKGROUND: Honey bees are the principal commercial pollinators. Along with other arthropods, they are increasingly under threat from anthropogenic factors such as the incursion of invasive honey bee subspecies, pathogens and parasites. Better tools are needed to identify bee subspecies. Genomic data for economic and ecologically important organisms is increasing, but in its basic form its practical application to address ecological problems is limited. RESULTS: We introduce HBeeID a means to identify honey bees. The tool utilizes a knowledge-based network and diagnostic SNPs identified by discriminant analysis of principle components and hierarchical agglomerative clustering. Tests of HBeeID showed that it identifies African, Americas-Africanized, Asian, and European honey bees with a high degree of certainty even when samples lack the full 272 SNPs of HBeeID. Its prediction capacity decreases with highly admixed samples. CONCLUSION: HBeeID is a high-resolution genomic, SNP based tool, that can be used to identify honey bees and screen species that are invasive. Its flexible design allows for future improvements via sample data additions from other localities.

Susceptible and infectious states for both vector and host in a dynamic pathogen-vector-host system.

Deformed wing virus (DWV) is a resurgent insect pathogen of honeybees that is efficiently transmitted by vectors and through host social contact. Continual transmission of DWV between hosts and vectors is required to maintain the pathogen within the population, and this vector-host-pathogen system offers unique disease transmission dynamics for pathogen maintenance between vectors and a social host. In a series of experiments, we measured vector-vector, host-host and host-vector transmission routes and show how these maintain DWV in honeybee populations. We found co-infestations on shared hosts allowed for movement of DWV from mite to mite. Additionally, two social behaviours of the honeybee, trophallaxis and cannibalization of pupae, provide routes for horizontal transmission from bee to bee. Circulation of the virus solely among hosts through communicable modes provides a reservoir of DWV for naïve Varroa to acquire and subsequently vector the pathogen. Our findings illustrate the importance of community transmission between hosts and vector transmission. We use these results to highlight the key avenues used by DWV during maintenance and infection and point to similarities with a handful of other infectious diseases of zoonotic and medical importance.

Host switch by honey bee parasitic mites leads to symbiont diversification.

Host-parasite co-evolution is a reciprocal genetic change; however, the parasite may switch to a novel host, deviating from conventional co-evolution. Varroa destructor is a native parasite of the honey bee Apis cerana, and the mite has established infestation in another honey bee, Apis mellifera, causing colony failure. When mites switched to the novel host, they formed a distinct population from mites that remained on the native host. Consequently, this led to divergence in the microbiota associated with mites in two host populations. The microbes were conserved at the species level reflected by alpha diversity, with substantial relative abundance variance. Microbes found in mites were distinct from the bee microbiota. They mainly were pathogenic with antibiotic resistance, while a few bacterial taxa were previously found in honey bees, including Klebsiella pneumoniae and Pseudomanas aeruginosa. These symbionts may transfer between the mites and honey bees.

The effects of queen mandibular pheromone on Nosema (Vairimorpha) ceranae infections in caged honey bees.

Honey bees utilize queen mandibular pheromone (QMP) for maintaining social hierarchy and colony development. In controlled cage studies, synthetic QMP is often introduced to mimic natural conditions. However, questions have arisen about the effects of QMP on nosema disease studies. This short report identifies significant early-stage suppression effects of QMP on Nosema (Vairimorpha) ceranae infections. QMP was found to significantly lower infection rates below the reported infectious dose for 50 % infectivity (ID50) and to slow disease development in a dose-independent manner. These effects diminished at doses exceeding ID100. We recommend that studies investigating treatment effects using caged bees avoid QMP to ensure unambiguous results. Additionally, employing multiple infectious doses with shorter incubation times would be useful for evaluating other treatments that may have subtle effects. Furthermore, our findings support previous field studies suggesting that queen replacement reduces nosema disease at levels similar to treatment with fumagillin.

The recent revision of the genera Nosema and Vairimorpha (Microsporidia: Nosematidae) was flawed and misleads the bee scientific community.

The genus Vairimorpha was proposed for several species of Nosema in 1976 (Pilley, 1976), almost 70 years after Nosema apis Zander (Zander, 1909). Tokarev and colleagues proposed the redefinition of 17 microsporidian species in four genera, Nosema, Vairimorpha, Rugispora, and Oligosporidium, based on phylogenetic trees of two genetic markers (SSU rRNA and RPB1) (Tokarev et al., 2020). Several issues should invalidate this new classification, leading to the synonymization of Vairimorpha within Nosema.

Bacterial diversity using metagenomics of 16s rDNA in water kefir, an innovative source of probiotics for bee nutrition.

Water kefir is a sparkling, slightly acidic fermented beverage made from sugar, water, and water kefir grains, which are a mixture of yeast and bacteria. These grains produce a variety of fermentation compounds such as lactic acid, acetaldehyde, acetoin, ethanol and carbon dioxide. In this study, a high-throughput sequencing technique was used to characterize the bacterial composition of the original water kefir from which potential probiotics were obtained. We studied the bacterial diversity of both water kefir grains and beverages. DNA was extracted from three replicate samples of both grains and beverages using the Powerlyzer Microbial Kit. The hypervariable V1-V2 region of the bacterial 16S ribosomal RNA gene was amplified to prepare six DNA libraries. Between 1.4M and 2.4M base-pairs were sequenced for the library. In total, 28721971 raw reads were obtained from all the samples. Estimated species richness was higher in kefir beverage samples compared to grain samples. Moreover, a higher level of microbial alpha diversity was observed in the beverage samples. Particularly, the predominant bacteria in beverages were Anaerocolumna and Ralstonia, while in grains Liquorilactobacillus dominated, with lower levels of Leuconostoc and Oenococcus. Although the bacterial diversity in kefir grains was low because only three genera were the most represented, all of them are LAB bacteria with the potential to serve as probiotics in the artificial feeding of bees.

Transferrin-mediated iron sequestration suggests a novel therapeutic strategy for controlling Nosema disease in the honey bee, Apis mellifera.

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee's ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees.

High temperatures augment inhibition of parasites by a honey bee gut symbiont.

Temperature affects growth, metabolism, and interspecific interactions in microbial communities. Within animal hosts, gut bacterial symbionts can provide resistance to parasitic infections. Both infection and populations of symbionts can be shaped by the host body temperature. However, the effects of temperature on the antiparasitic activities of gut symbionts have seldom been explored. The Lactobacillus-rich gut microbiota of facultatively endothermic honey bees is subject to seasonal and ontogenetic changes in host temperature that could alter the effects of symbionts against parasites. We used cell cultures of a Lactobacillus symbiont and an important trypanosomatid gut parasite of honey bees to test the potential for temperature to shape parasite-symbiont interactions. We found that symbionts showed greater heat tolerance than parasites and chemically inhibited parasite growth via production of acids. Acceleration of symbiont growth and acid production at high temperatures resulted in progressively stronger antiparasitic effects across a temperature range typical of bee colonies. Consequently, the presence of symbionts reduced both the peak growth rate and heat tolerance of parasites. Substantial changes in parasite-symbiont interactions were evident over a temperature breadth that parallels changes in diverse animals exhibiting infection-related fevers and the amplitude of circadian temperature variation typical of endothermic birds and mammals, implying the frequent potential for temperature to alter symbiont-mediated resistance to parasites in endo- and ectothermic hosts. Results suggest that the endothermic behavior of honey bees could enhance the impacts of gut symbionts on parasites, implicating thermoregulation as a reinforcer of core symbioses and possibly microbiome-mediated antiparasitic defense. IMPORTANCE Two factors that shape the resistance of animals to infection are body temperature and gut microbiota. However, temperature can also alter interactions among microbes, raising the question of whether and how temperature changes the antiparasitic effects of gut microbiota. Honey bees are agriculturally important hosts of diverse parasites and infection-mitigating gut microbes. They can also socially regulate their body temperatures to an extent unusual for an insect. We show that high temperatures found in honey bee colonies augment the ability of a gut bacterial symbiont to inhibit the growth of a common bee parasite, reducing the parasite's ability to grow at high temperatures. This suggests that fluctuations in colony and body temperatures across life stages and seasons could alter the protective value of bees' gut microbiota against parasites, and that temperature-driven changes in gut microbiota could be an underappreciated mechanism by which temperature-including endothermy and fever-alters animal infection.

Widespread distribution of honey bee-associated pathogens in native bees and wasps: Trends in pathogen prevalence and co-occurrence.

Pollinators have experienced significant declines in the past decade, in part due to emerging infectious diseases. Historically, studies have primarily focused on pathogens in the Western honey bee, Apis mellifera. However, recent work has demonstrated that these pathogens are shared by other pollinators and can negatively affect their health. Here, we surveyed honey bees and 15 native bee and wasp species for 13 pathogens traditionally associated with honey bees. The native bee and wasp species included 11 species not previously screened for pathogens. We found at least one honey bee-associated pathogen in 53% of native bee and wasp samples. The most widely distributed and commonly detected pathogens were the microsporidian Nosema ceranae, the bacterium Melissococcus plutonius, and the viruses deformed wing virus and black queen cell virus. The prevalence of viruses was generally higher in honey bees than in native bees and wasps. However, the prevalence of M. plutonius and the brood fungus Ascosphaera apis was significantly higher in some native bee species than in honey bees. The data also reveal novel trends in the association between co-occurring pathogens in honey bees and native bees and wasps at the pathogen community level. These results can inform the assessment of risks that native pollinator species face from pathogen stress, and indicate that many non-viral pathogens, notably M. plutonius and N. ceranae, are far more widely distributed and commonly found in native bees and wasps than previously thought.

Punch in the gut: parasite tolerance of phytochemicals reflects host diet.

Gut parasites of plant-eating insects are exposed to antimicrobial phytochemicals that can reduce infection. Trypanosomatid gut parasites infect insects of diverse nutritional ecologies as well as mammals and plants, raising the question of how host diet-associated phytochemicals shape parasite evolution and host specificity. To test the hypothesis that phytochemical tolerance of trypanosomatids reflects the chemical ecology of their hosts, we compared related parasites from honey bees and mosquitoes - hosts that differ in phytochemical consumption - and contrasted our results with previous studies on phylogenetically related, human-parasitic Leishmania. We identified one bacterial and 10 plant-derived substances with known antileishmanial activity that also inhibited honey bee parasites associated with colony collapse. Bee parasites exhibited greater tolerance of chrysin - a flavonoid found in nectar, pollen and plant resin-derived propolis. In contrast, mosquito parasites were more tolerant of cinnamic acid - a product of lignin decomposition present in woody debris-rich larval habitats. Parasites from both hosts tolerated many compounds that inhibit Leishmania, hinting at possible trade-offs between phytochemical tolerance and mammalian infection. Our results implicate the phytochemistry of host diets as a potential driver of insect-trypanosomatid associations and identify compounds that could be incorporated into colony diets or floral landscapes to ameliorate infection in bees.

Honeybee intestines retain low yeast titers, but no bacterial mutualists, at emergence.

Honeybee symbionts, predominantly bacteria, play important roles in honeybee health, nutrition, and pathogen protection, thereby supporting colony health. On the other hand, fungi are often considered indicators of poor bee health, and honeybee microbiome studies generally exclude fungi and yeasts. We hypothesized that yeasts may be an important aspect of early honeybee biology, and if yeasts provide a mutual benefit to their hosts, then honeybees could provide a refuge during metamorphosis to ensure the presence of yeasts at emergence. We surveyed for yeast and fungi during pupal development and metamorphosis in worker bees using fungal-specific quantitative polymerase chain reaction (qPCR), next-generation sequencing, and standard microbiological culturing. On the basis of yeast presence in three distinct apiaries and multiple developmental stages, we conclude that yeasts can survive through metamorphosis and in naïve worker bees, albeit at relatively low levels. In comparison, known bacterial mutualists, like Gilliamella and Snodgrassella, were generally not found in pre-eclosed adult bees. Whether yeasts are actively retained as an important part of the bee microbiota or are passively propagating in the colony remains unknown. Our demonstration of the constancy of yeasts throughout development provides a framework to further understand the honeybee microbiota.

Dynamic evolution in the key honey bee pathogen deformed wing virus: Novel insights into virulence and competition using reverse genetics.

The impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health, which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to coexisting DWV genotypes, thereby devising a reverse-genetics system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone-derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversification. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.

Arsenophonus apicola sp. nov., isolated from the honeybee Apis mellifera.

The genus Arsenophonus has been traditionally considered to comprise heritable bacterial symbionts of arthropods. Recent work has reported a microbe related to the type species Arsenophonus nasoniae as infecting the honey bee, Apis mellifera. The association was unusual for members of the genus in that the microbe-host interaction arose through environmental and social exposure rather than vertical transmission. In this study, we describe the in vitro culture of ArsBeeUST, a strain of this microbe isolated from A. mellifera in the USA. The 16S rRNA sequence of the isolated strain indicates it falls within the genus Arsenophonus. Biolog analysis indicates the bacterium has a restricted range of nutrients that support growth. In vivo experiments demonstrate the strain proliferates rapidly on injection into A. mellifera hosts. We further report the closed genome sequence for the strain. The genome is 3.3 Mb and the G+C content is 37.6 mol%, which is smaller than A. nasoniae but larger than the genomes reported for non-culturable Arsenophonus symbionts. The genome is complex, with six extrachromosomal elements and 11 predicted intact phage elements, but notably less complex than A. nasoniae. Strain ArsBeeUST is clearly distinct from the type species A. nasoniae on the basis of genome sequence, with 92 % average nucleotide identity. Based on our results, we propose Arsenophonus apicola sp. nov., with the type strain ArsBeeUST (CECT 30499T=DSM113403T=LMG 32504T).

Honey Bee Habitat Sharing Enhances Gene Flow of the Parasite Nosema ceranae.

Host-parasite co-evolution is a process of reciprocal, adaptive genetic change. In natural conditions, parasites can shift to other host species, given both host and parasite genotypes allow this. Even though host-parasite co-evolution has been extensively studied both theoretically and empirically, few studies have focused on parasite gene flow between native and novel hosts. Nosema ceranae is a native parasite of the Asian honey bee Apis cerana, which infects epithelial cells of mid-guts. This parasite successfully switched to the European honey bee Apis mellifera, where high virulence has been reported. In this study, we used the parasite N. ceranae and both honey bee species as model organisms to study the impacts of two-host habitat sharing on parasite diversity and virulence. SNVs (Single Nucleotide Variants) were identified from parasites isolated from native and novel hosts from sympatric populations, as well as novel hosts from a parapatric population. Parasites isolated from native hosts showed the highest levels of polymorphism. By comparing the parasites isolated from novel hosts between sympatric and parapatric populations, habitat sharing with the native host significantly enhanced parasite diversity, suggesting there is continuing gene flow of parasites between the two host species in sympatric populations.

Antiparasitic effects of three floral volatiles on trypanosomatid infection in honey bees.

Trypanosomatid gut parasites are common in pollinators and costly for social bees. The recently described honey bee trypanosomatid Lotmaria passim is widespread, abundant, and correlated with colony losses in some studies. The potential for amelioration of infection by antimicrobial plant compounds has been thoroughly studied for closely related trypanosomatids of humans and is an area of active research in bumble bees, but remains relatively unexplored in honey bees. We recently identified several floral volatiles that inhibited growth of L. passim in vitro. Here, we tested the dose-dependent effects of four such compounds on infection, mortality, and food consumption in parasite-inoculated honey bees. We found that diets containing the monoterpenoid carvacrol and the phenylpropanoids cinnamaldehyde and eugenol at > 10-fold the inhibitory concentrations for cell cultures reduced infection, with parasite numbers decreased by > 90 % for carvacrol and cinnamaldehyde and > 99 % for eugenol; effects of the carvacrol isomer thymol were non-significant. However, both carvacrol and eugenol also reduced bee survival, whereas parasite inoculation did not, indicating costs of phytochemical exposure that could exceed those of infection itself. To our knowledge, this is the first controlled screening of phytochemicals for effects on honey bee trypanosomatid infection, identifying potential treatments for managed bees afflicted with a newly characterized, cosmopolitan intestinal parasite.

Multi-tiered analyses of honey bees that resist or succumb to parasitic mites and viruses.

BACKGROUND: Varroa destructor mites, and the numerous viruses they vector to their honey bee hosts, are among the most serious threats to honey bee populations, causing mortality and morbidity to both the individual honey bee and colony, the negative effects of which convey to the pollination services provided by honey bees worldwide. Here we use a combination of targeted assays and deep RNA sequencing to determine host and microbial changes in resistant and susceptible honey bee lineages. We focus on three study sets. The first involves field sampling of sympatric western bees, some derived from resistant stock and some from stock susceptible to mites. The second experiment contrasts three colonies more deeply, two from susceptible stock from the southeastern U.S. and one from mite-resistant bee stock from Eastern Texas. Finally, to decouple the effects of mites from those of the viruses they vector, we experimentally expose honey bees to DWV in the laboratory, measuring viral growth and host responses. RESULTS: We find strong differences between resistant and susceptible bees in terms of both viral loads and bee gene expression. Interestingly, lineages of bees with naturally low levels of the mite-vectored Deformed wing virus, also carried lower levels of viruses not vectored by mites. By mapping gene expression results against current ontologies and other studies, we describe the impacts of mite parasitism, as well as viruses on bee health against two genetic backgrounds. We identify numerous genes and processes seen in other studies of stress and disease in honey bee colonies, alongside novel genes and new patterns of expression. CONCLUSIONS: We provide evidence that honey bees surviving in the face of parasitic mites do so through their abilities to resist the presence of devastating viruses vectored by these mites. In all cases, the most divergence between stocks was seen when bees were exposed to live mites or viruses, suggesting that gene activation, rather than constitutive expression, is key for these interactions. By revealing responses to viral infection and mite parasitism in different lineages, our data identify candidate proteins for the evolution of mite tolerance and virus resistance.