Representative honey bee viruses do not replicate in the small hive beetle, Aethina tumida Murray.

The small hive beetle (SHB), Aethina tumida Murray, is an invasive pest of the honey bee and causes significant damage through the consumption of colony resources and brood. Two assumptions related to honey bee virus transmission have been made about SHB: first, that SHB vectors honey bee viruses and second, that these viruses replicate in SHB based on the detection of both positive and negative strand viral genomic RNA within SHB. To clarify the role of SHB in virus transmission, we sought to address whether selected honey bee viruses replicate in SHB. Sequences derived from five honey bee viruses were identified in the transcriptomes of field-caught SHB from the U.S., but not in those of lab-reared SHB, suggesting that these viruses do not replicate in SHB. To elucidate whether the representative viruses, Israeli acute paralysis virus (IAPV; Dicistroviridae) and Deformed wing virus (DWV; Iflaviridae) replicate in SHB, we tested for replication in vitro in an SHB-derived cell line (BCIRL-AtumEN-1129-D6). Following treatment of the cell line with viral particles or viral RNA, the number of virus genomes was monitored by reverse transcription quantitative PCR (RT-qPCR). In contrast to the positive control, IAPV and DWV RNA levels steadily decreased over a period of 8 days. Collectively, these results from bioinformatic observations and in vitro experiments indicate that IAPV and DWV do not replicate in SHB. These results are consistent with the host specificity of most insect viruses within a single insect order and indicate that while SHB may serve as a mechanical vector of honey bee viruses within and between hives, this insect does not serve as a biological vector for these honey bee viruses.

Nutrition, pesticide exposure, and virus infection interact to produce context-dependent effects in honey bees (Apis mellifera).

Declines in pollinator health are frequently hypothesized to be the combined result of multiple interacting biotic and abiotic stressors; namely, nutritional limitations, pesticide exposure, and infection with pathogens and parasites. Despite this hypothesis, most studies examining stressor interactions have been constrained to two concurrent factors, limiting our understanding of multi-stressor dynamics. Using honey bees as a model, we addressed this gap by studying how variable diet, field-realistic levels of multiple pesticides, and virus infection interact to affect survival, infection intensity, and immune and detoxification gene expression. Although we found evidence that agrochemical exposure (a field-derived mixture of chlorpyrifos and two fungicides) can exacerbate infection and increase virus-induced mortality, this result was nutritionally-dependent, only occurring when bees were provided artificial pollen. Provisioning with naturally-collected polyfloral pollen inverted the effect, reducing virus-induced mortality and suggesting a hormetic response. To test if the response was pesticide specific, we repeated our experiment with a pyrethroid (lambda-cyhalothrin) and a neonicotinoid (thiamethoxam), finding variable results. Finally, to understand the underpinnings of these effects, we measured viral load and expression of important immune and detoxification genes. Together, our results show that multi-stressor interactions are complex and highly context-dependent, but have great potential to affect bee health and physiology.

Indoor tent management for extending honey bee research season: benefits and caveats.

Honey bees are important organisms for research in many fields, including physiology, behavior, and ecology. Honey bee colonies are relatively easy and affordable to procure, manage, and replace. However, some difficulties still exist in honey bee research, specifically that honey bee colonies have a distinct seasonality, especially in temperate regions. Honey bee colonies transition from a large society in which workers have a strict temporal division of labor in the summer, to a group of behaviorally flexible workers who manage the colony over winter. Furthermore, opening colonies or collecting bees when they are outside has the potential to harm the colony because of the disruption in thermoregulation. Here, we present a simple and affordable indoor management method utilizing a mylar tent and controlled environmental conditions that allows bees to freely fly without access to outdoor space. This technique permits research labs to successfully keep several colonies persistently active during winter at higher latitudes. Having an extended research period is particularly important for training students, allowing preliminary experiments to be performed, and developing methods. However, we find distinct behavioral differences in honey bees managed in this situation. Specifically learning and thermoregulatory behaviors were diminished in the bees managed in the tent. Therefore, we recommend caution in utilizing these winter bees for full experiments until more is known. Overall, this method expands the research potential on honey bees, and calls attention to the additional research that is needed to understand how indoor management might affect honey bees.

Host species and geography impact bee-associated RNA virus communities with evidence for isolation by distance in viral populations.

Virus symbionts are important mediators of ecosystem function, yet we know little of their diversity and ecology in natural populations. The alarming decline of pollinating insects in many regions of the globe, especially the European honey bee, Apis mellifera, has been driven in part by worldwide transmission of virus pathogens. Previous work has examined the transmission of known honey bee virus pathogens to wild bee populations, but only a handful of studies have investigated the native viromes associated with wild bees, limiting epidemiological predictors associated with viral pathogenesis. Further, variation among different bee species might have important consequences in the acquisition and maintenance of bee-associated virome diversity. We utilized comparative metatranscriptomics to develop a baseline description of the RNA viromes associated with wild bee pollinators and to document viral diversity, community composition, and structure. Our sampling includes five wild-caught, native bee species that vary in social behavior as well as managed honey bees. We describe 26 putatively new RNA virus species based on RNA-dependent RNA polymerase phylogeny and show that each sampled bee species was associated with a specific virus community composition, even among sympatric populations of distinct host species. From 17 samples of a single host species, we recovered a single virus species despite over 600 km of distance between host populations and found strong evidence for isolation by distance in associated viral populations. Our work adds to the small number of studies examining viral prevalence and community composition in wild bees.

The effect of Israeli acute paralysis virus infection on honey bee brood care behavior.

To protect themselves from communicable diseases, social insects utilize social immunity-behavioral, physiological, and organizational means to combat disease transmission and severity. Within a honey bee colony, larvae are visited thousands of times by nurse bees, representing a prime environment for pathogen transmission. We investigated a potential social immune response to Israeli acute paralysis virus (IAPV) infection in brood care, testing the hypotheses that bees will respond with behaviors that result in reduced brood care, or that infection results in elevated brood care as a virus-driven mechanism to increase transmission. We tested for group-level effects by comparing three different social environments in which 0%, 50%, or 100% of nurse bees were experimentally infected with IAPV. We investigated individual-level effects by comparing exposed bees to unexposed bees within the mixed-exposure treatment group. We found no evidence for a social immune response at the group level; however, individually, exposed bees interacted with the larva more frequently than their unexposed nestmates. While this could increase virus transmission from adults to larvae, it could also represent a hygienic response to increase grooming when an infection is detected. Together, our findings underline the complexity of disease dynamics in complex social animal systems.

Consequences of microsporidian prior exposure for virus infection outcomes and bumble bee host health.

Host-parasite interactions do not occur in a vacuum, but in connected multi-parasite networks that can result in co-exposures and coinfections of individual hosts. These can affect host health and disease ecology, including disease outbreaks. However, many host-parasite studies examine pairwise interactions, meaning we still lack a general understanding of the influence of co-exposures and coinfections. Using the bumble bee Bombus impatiens, we study the effects of larval exposure to a microsporidian Nosema bombi, implicated in bumble bee declines, and adult exposure to Israeli Acute Paralysis Virus (IAPV), an emerging infectious disease from honey bee parasite spillover. We hypothesize that infection outcomes will be modified by co-exposure or coinfection. Nosema bombi is a potentially severe, larval-infecting parasite, and we predict that prior exposure will result in decreased host resistance to adult IAPV infection. We predict double parasite exposure will also reduce host tolerance of infection, as measured by host survival. Although our larval Nosema exposure mostly did not result in viable infections, it partially reduced resistance to adult IAPV infection. Nosema exposure also negatively affected survival, potentially due to a cost of immunity in resisting the exposure. There was a significant negative effect of IAPV exposure on survivorship, but prior Nosema exposure did not alter this survival outcome, suggesting increased tolerance given the higher IAPV infections in the bees previously exposed to Nosema. These results again demonstrate that infection outcomes can be non-independent when multiple parasites are present, even when exposure to one parasite does not result in a substantial infection.

CRISPR-induced DNA reorganization for multiplexed nucleic acid detection.

Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence. Furthermore, we link the activation of CRISPR-Cas12a to the expression of proteins through a Reprogrammable PAIRing system (RePAIR). We then combine FACT and RePAIR to create FACTOR (FACT on RePAIR), a CRISPR-based diagnostic, that we use to detect infectious disease in an agricultural use case: honey bee viral infection. With high specificity and accuracy, we demonstrate the potential of FACTOR to be applied to the sensing of any nucleic acid of interest.

Honey bee hive covers reduce food consumption and colony mortality during overwintering.

Beekeepers regularly employ management practices to mitigate losses during the winter, often considered the most difficult time during a colony life cycle. Management recommendations involving covering or wrapping hives in insulation during winter have a long history; over 100 years ago, most recommendations for overwintering in cold climates involved heavy insulation wraps or moving hives indoors. These recommendations began to change in the mid-20th century, but hive covers are still considered useful and are described in contemporary beekeeping manuals and cooperative extension materials. However, most of the data supporting their use is published primarily in non-peer reviewed trade journals and was collected >40 years ago. In this time, the beekeeping environment has changed substantially, with new pressures from pathogens, agrochemicals, and land use changes. Here, we provide an update to the historical literature, reporting a randomized experiment testing the effectiveness of a common honey bee hive cover system across eight apiaries in central Illinois, USA, a temperate region dominated by conventional annual agriculture. We found that, when other recommended overwintering preparations are performed, covered colonies consumed less food stores and survived better than uncovered controls (22.5% higher survival). This study highlights the value of hive covers, even in an area not subject to extremely cold winter conditions, and these data can aid the production of evidence-based extension recommendations for beekeepers.

Can Native Plants Mitigate Climate-related Forage Dearth for Honey Bees (Hymenoptera: Apidae)?

Extreme weather events, like high temperatures and droughts, are predicted to become common with climate change, and may negatively impact plant growth. How honey bees (Apis mellifera L. [Hymenoptera: Apidae]) will respond to this challenge is unclear, especially when collecting pollen, their primary source of protein, lipids, and micro-nutrients. We explored this response with a data set from multiple research projects that measured pollen collected by honey bees during 2015-2017 in which above-average temperatures and a drought occurred in 2017. We summarized the abundance and diversity of pollen collected from July to September in replicated apiaries kept at commercial soybean and corn farms in Iowa, in the Midwestern USA. The most commonly collected pollen was from clover (Trifolium spp. [Fabales: Fabaceae]), which dramatically declined in absolute and relative abundance in July 2017 during a period of high temperatures and drought. Due to an apparent lack of clover, honey bees switched to the more drought-tolerant native species (e.g., Chamaecrista fasciculata [Michx.] Greene [Fabales: Fabaceae], Dalea purpurea Vent. [Fabales: Fabaceae], Solidago spp. [Asterales: Asteraceae]), and several species of Asteraceae. This was especially noticeable in August 2017 when C. fasciculata dominated (87%) and clover disappeared from bee-collected pollen. We discuss the potential implications of climate-induced forage dearth on honey bee nutritional health. We also compare these results to a growing body of literature on the use of native, perennial flowering plants found in Midwestern prairies for the conservation of beneficial insects. We discuss the potential for drought resistant-native plants to potentially promote resilience to climate change for the non-native, managed honey bee colonies in the United States.

Butenolide Insecticide Flupyradifurone Affects Honey Bee Worker Antiviral Immunity and Survival.

Honey bees face many environmental stressors, including exposure to pesticides and pathogens. A novel butenolide pesticide, flupyradifurone, was recently introduced to the US and shown to have a bee-friendly toxicity profile. Like the much-scrutinized neonicotinoids that preceded it, flupyradifurone targets the insect nervous system. Some neonicotinoids have been shown to interfere with antiviral immunity, which raised the concern that similar effects may be observed with flupyradifurone. In this study, we investigated how flupyradifurone and a neonicotinoid, clothianidin, affect the ability of honey bee workers to combat an infection of Israeli acute paralysis virus (IAPV). We exposed workers to field-realistic doses of the pesticides either with or without co-exposure with the virus, and then tracked survival and changes in viral titers. We repeated the experiment in the spring and fall to look for any seasonal effects. We found that flupyradifurone caused elevated mortality in the fall, but it did not lead to increased virus-induced mortality. Flupyradifurone also appeared to affect virus clearance, as bees co-exposed to the pesticide and virus tended to have higher viral titers after 48 hours than those exposed to the virus alone. Clothianidin had no effect on viral titers, and it actually appeared to increase resistance to viral infection in spring bees.

Developmental environment shapes honeybee worker response to virus infection.

The consequences of early-life experiences are far reaching. In particular, the social and nutritional environments that developing animals experience can shape their adult phenotypes. In honeybees, larval nutrition determines the eventual social roles of adults as reproductive queens or sterile workers. However, little is known about the effects of developmental nutrition on important adult worker phenotypes such as disease resilience. In this study, we manipulated worker developmental nutrition in two distinct ways under semi-natural field conditions. In the first experiment, we restricted access to nutrition via social isolation by temporarily preventing alloparental care. In the second experiment, we altered the diet quality experienced by the entire colony, leading to adult bees that had developed entirely in a nutritionally restricted environment. When bees from these two experiments reached the adult stage, we challenged them with a common bee virus, Israeli acute paralysis virus (IAPV) and compared mortality, body condition, and the expression of immune genes across diet and viral inoculation treatments. Our findings show that both forms of early life nutritional stress, whether induced by lack of alloparental care or diet quality restriction, significantly reduced bees' resilience to virus infection and affected the expression of several key genes related to immune function. These results extend our understanding of how early life nutritional environment can affect phenotypes relevant to health and highlight the importance of considering how nutritional stress can be profound even when filtered through a social group. These results also provide important insights into how nutritional stress can affect honeybee health on a longer time scale and its potential to interact with other forms of stress (i.e. disease).

Can Solar Energy Fuel Pollinator Conservation?

As the expansion of solar power spreads through much of the United States, members of the solar industry are working to change how solar energy facilities are designed and presented to the public. This includes the addition of habitat to conserve pollinators. We highlight and discuss ongoing efforts to couple solar energy production with pollinator conservation, noting recent legal definitions of these practices. We summarize key studies from the field of ecology, bee conservation, and our experience working with members of the solar industry (e.g., contribution to legislation defining solar pollinator habitat). Several recently published studies that employed similar practices to those proposed for solar developments reveal features that should be replicated and encouraged by the industry. These results suggest the addition of native, perennial flowering vegetation will promote wild bee conservation and more sustainable honey beekeeping. Going forward, there is a need for oversight and future research to avoid the misapplication of this promising but as of yet untested practice of coupling solar energy production with pollinator-friendly habitat. We conclude with best practices for the implementation of these additions to realize conservation and agricultural benefits.

North American Prairie Is a Source of Pollen for Managed Honey Bees (Hymenoptera: Apidae).

Prairie was a dominant habitat within large portions of North America before European settlement. Conversion of prairies to farmland resulted in the loss of a large proportion of native floral resources, contributing to the decline of native pollinator populations. Efforts to reconstruct prairie could provide honey bees (Apis mellifera) a source of much-needed forage, especially in regions dominated by crop production. To what extent honey bees, which were introduced to North America by European settlers, use plants native to prairies is unclear. We placed colonies with pollen traps within reconstructed prairies in central Iowa to determine which and how much pollen is collected from prairie plants. Honey bee colonies collected more pollen from nonnative than native plants during June and July. During August and September, honey bee colonies collected more pollen from plants native to prairies. Our results suggest that honey bees' use of native prairie plants may depend upon the seasonality of both native and nonnative plants present in the landscape. This finding may be useful for addressing the nutritional health of honey bees, as colonies in this region frequently suffer from a dearth of forage contributing to colony declines during August and September when crops and weedy plants cease blooming. These results suggest that prairie can be a significant source of forage for honey bees in the later part of the growing season in the Midwestern United States; we discuss this insight in the context of honey bee health and biodiversity conservation.

Do Viruses From Managed Honey Bees (Hymenoptera: Apidae) Endanger Wild Bees in Native Prairies?

Populations of wild and managed pollinators are declining in North America, and causes include increases in disease pressure and decreases in flowering resources. Tallgrass prairies can provide floral resources for managed honey bees (Hymenoptera: Apidae, Apis mellifera Linnaeus) and wild bees. Honey bees kept near prairies may compete with wild bees for floral resources, and potentially transfer viral pathogens to wild bees. Measurements of these potential interactions are lacking, especially in the context of native habitat conservation. To address this, we assessed abundance and richness of wild bees in prairies with and without honey bee hives present, and the potential spillover of several honey bee viruses to bumble bees (Hymenoptera: Apidae, Bombus Latrielle). We found no indication that the presence of honey bee hives over 2 yr had a negative effect on population size of wild bee taxa, though a potential longer-term effect remains unknown. All levels of viruses quantified in bumble bees were lower than those observed in honey bees. Higher levels of deformed wing virus and Israeli acute paralysis virus were found in Bombus griseocollis DeGeer (Hymenoptera: Apidae) collected at sites with hives than those without hives. These data suggest that the presence of honey bees in tallgrass prairie could increase wild bee exposure to viruses. Additional studies on cross-species transmission of viruses are needed to inform decisions regarding the cohabitation of managed bees within habitat utilized by wild bees.

Ameliorative Effects of Phytochemical Ingestion on Viral Infection in Honey Bees.

Honey bee viruses are capable of causing a wide variety of devastating effects, but effective treatments have yet to be discovered. Phytochemicals represent a broad range of substances that honey bees frequently encounter and consume, many of which have been shown to improve honey bee health. However, their effect on bee viruses is largely unknown. Here, we tested the therapeutic effectiveness of carvacrol, thymol, p-coumaric acid, quercetin, and caffeine on viral infection by measuring their ability to improve survivorship in honey bees inoculated with Israeli acute paralysis virus (IAPV) using high-throughput cage bioassays. Among these candidates, caffeine was the only phytochemical capable of significantly improving survivorship, with initial screening showing that naturally occurring concentrations of caffeine (25 ppm) were sufficient to produce an ameliorative effect on IAPV infection. Consequently, we measured the scope of caffeine effectiveness in bees inoculated and uninoculated with IAPV by performing the same type of high-throughput bioassay across a wider range of caffeine concentrations. Our results indicate that caffeine may provide benefits that scale with concentration, though the exact mechanism by which caffeine ingestion improves survivorship remains uncertain. Caffeine therefore has the potential to act as an accessible and inexpensive method of treating viral infections, while also serving as a tool to further understanding of honey bee-virus interactions at a physiological and molecular level.

Preparation of Virus-Enriched Inoculum for Oral Infection of Honey Bees (Apis Mellifera).

Honey bees are of great ecological and agricultural importance around the world but are also subject to a variety of pressures that negatively affect bee health, including exposure to viral pathogens. Such viruses can cause a wide variety of devastating effects and can often be challenging to study due to multiple factors that make it difficult to separate the effects of experimental treatments from preexisting background infection. Here we present a method to mass produce large quantities of virus particles along with a high throughput bioassay to test viral infection and effects. Necessitated by the current lack of a continuous, virus-free honey bee cell line, viral particles are amplified in vivo using honey bee pupae, which are extracted from the hive in large volumes using minimally stressful methodology. These virus particles can then be used in honey bee cage bioassays to test inocula viability, as well as various other virus infection dynamics, including interactions with nutrition, pesticides, and other pathogens. A major advantage of using such particles is that it greatly reduces the chances of introducing unknown variables in subsequent experimentation when compared to current alternatives, such as infection via infected bee hemolymph or homogenate, though care should still be taken when sourcing the bees, to minimize background virus contamination. The cage assays are not a substitute for large-scale, field-realistic experiments testing virus infection effects at a colony level, but instead function as a method to establish baseline viral responses that, in combination with the semi-pure virus particles, can serve as important tools to examine various dimensions of honey bee-virus physiological interactions.

Pan Traps for Tracking Honey Bee Activity-Density: A Case Study in Soybeans.

To study how honey bees utilize forage resources and guide pollination management plans in crops, a multitude of methods have been developed, but most are time consuming, costly, and require specialized skills. Colored pan traps for monitoring activity-density are a simple, efficient, and cost-effective alternative; however, their usefulness for studying honey bees is not well described. We examined if trap color, location within a field, and the presence of managed colonies affected estimates of honey bee activity-density within soybean fields. Soybeans are visited by pollinators but do not require these visits for seed development. Pan traps, especially those colored blue, captured more honey bees when colonies were present. There were no differences in activity-density based on placement of traps within a field nor with increasing distance from colonies. Throughout the season, activity-density in soybeans was constant but tripled after soybean ceased blooming, suggesting spikes in pan trap captures may indicate periods of forage scarcity. Activity-density did not correlate with the population size of worker bees at a site, but did correlate with number of colonies present. We conclude that pan traps can be useful for assessing honey bee activity, particularly for estimating colony presence and identifying times of forage scarcity.

Pesticide-Virus Interactions in Honey Bees: Challenges and Opportunities for Understanding Drivers of Bee Declines.

Honey bees are key agricultural pollinators, but beekeepers continually suffer high annual colony losses owing to a number of environmental stressors, including inadequate nutrition, pressures from parasites and pathogens, and exposure to a wide variety of pesticides. In this review, we examine how two such stressors, pesticides and viruses, may interact in additive or synergistic ways to affect honey bee health. Despite what appears to be a straightforward comparison, there is a dearth of studies examining this issue likely owing to the complexity of such interactions. Such complexities include the wide array of pesticide chemical classes with different modes of actions, the coupling of many bee viruses with ectoparasitic Varroa mites, and the intricate social structure of honey bee colonies. Together, these issues pose a challenge to researchers examining the effects pesticide-virus interactions at both the individual and colony level.

Honey bee virus causes context-dependent changes in host social behavior.

Anthropogenic changes create evolutionarily novel environments that present opportunities for emerging diseases, potentially changing the balance between host and pathogen. Honey bees provide essential pollination services, but intensification and globalization of honey bee management has coincided with increased pathogen pressure, primarily due to a parasitic mite/virus complex. Here, we investigated how honey bee individual and group phenotypes are altered by a virus of concern, Israeli acute paralysis virus (IAPV). Using automated and manual behavioral monitoring of IAPV-inoculated individuals, we find evidence for pathogen manipulation of worker behavior by IAPV, and reveal that this effect depends on social context; that is, within versus between colony interactions. Experimental inoculation reduced social contacts between honey bee colony members, suggesting an adaptive host social immune response to diminish transmission. Parallel analyses with double-stranded RNA (dsRNA)-immunostimulated bees revealed these behaviors are part of a generalized social immune defensive response. Conversely, inoculated bees presented to groups of bees from other colonies experienced reduced aggression compared with dsRNA-immunostimulated bees, facilitating entry into susceptible colonies. This reduction was associated with a shift in cuticular hydrocarbons, the chemical signatures used by bees to discriminate colony members from intruders. These responses were specific to IAPV infection, suggestive of pathogen manipulation of the host. Emerging bee pathogens may thus shape host phenotypes to increase transmission, a strategy especially well-suited to the unnaturally high colony densities of modern apiculture. These findings demonstrate how anthropogenic changes could affect arms races between human-managed hosts and their pathogens to potentially affect global food security.