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.

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.

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.

Native habitat mitigates feast-famine conditions faced by honey bees in an agricultural landscape.

Intensive agriculture can contribute to pollinator decline, exemplified by alarmingly high annual losses of honey bee colonies in regions dominated by annual crops (e.g., midwestern United States). As more natural or seminatural landscapes are transformed into monocultures, there is growing concern over current and future impacts on pollinators. To forecast how landscape simplification can affect bees, we conducted a replicated, longitudinal assessment of honey bee colony growth and nutritional health in an intensively farmed region where much of the landscape is devoted to production of corn and soybeans. Surprisingly, colonies adjacent to soybean fields surrounded by more cultivated land grew more during midseason than those in areas of lower cultivation. Regardless of the landscape surrounding the colonies, all experienced a precipitous decline in colony weight beginning in August and ended the season with reduced fat stores in individual bees, both predictors of colony overwintering failure. Patterns of forage availability and colony nutritional state suggest that late-season declines were caused by food scarcity during a period of extremely limited forage. To test if habitat enhancements could ameliorate this response, we performed a separate experiment in which colonies provided access to native perennials (i.e., prairie) were rescued from both weight loss and reduced fat stores, suggesting the rapid decline observed in these agricultural landscapes is not inevitable. Overall, these results show that intensively farmed areas can provide a short-term feast that cannot sustain the long-term nutritional health of colonies; reintegration of biodiversity into such landscapes may provide relief from nutritional stress.

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.

Transcriptomic responses to diet quality and viral infection in Apis mellifera.

BACKGROUND: Parts of Europe and the United States have witnessed dramatic losses in commercially managed honey bees over the past decade to what is considered an unsustainable extent. The large-scale loss of bees has considerable implications for the agricultural economy because bees are one of the leading pollinators of numerous crops. Bee declines have been associated with several interactive factors. Recent studies suggest nutritional and pathogen stress can interactively contribute to bee physiological declines, but the molecular mechanisms underlying interactive effects remain unknown. In this study, we provide insight into this question by using RNA-sequencing to examine how monofloral diets and Israeli acute paralysis virus inoculation influence gene expression patterns in bees. RESULTS: We found a considerable nutritional response, with almost 2000 transcripts changing with diet quality. The majority of these genes were over-represented for nutrient signaling (insulin resistance) and immune response (Notch signaling and JaK-STAT pathways). In our experimental conditions, the transcriptomic response to viral infection was fairly limited. We only found 43 transcripts to be differentially expressed, some with known immune functions (argonaute-2), transcriptional regulation, and muscle contraction. We created contrasts to explore whether protective mechanisms of good diet were due to direct effects on immune function (resistance) or indirect effects on energy availability (tolerance). A similar number of resistance and tolerance candidate differentially expressed genes were found, suggesting both processes may play significant roles in dietary buffering from pathogen infection. CONCLUSIONS: Through transcriptional contrasts and functional enrichment analysis, we contribute to our understanding of the mechanisms underlying feedbacks between nutrition and disease in bees. We also show that comparing results derived from combined analyses across multiple RNA-seq studies may allow researchers to identify transcriptomic patterns in bees that are concurrently less artificial and less noisy. This work underlines the merits of using data visualization techniques and multiple datasets to interpret RNA-sequencing studies.

Pollen Contaminated With Field-Relevant Levels of Cyhalothrin Affects Honey Bee Survival, Nutritional Physiology, and Pollen Consumption Behavior.

Honey bees are exposed to a variety of environmental factors that impact their health, including nutritional stress, pathogens, and pesticides. In particular, there has been increasing evidence that sublethal exposure to pesticides can cause subtle, yet important effects on honey bee health and behavior. Here, we add to this body of knowledge by presenting data on bee-collected pollen containing sublethal levels of cyhalothrin, a pyrethroid insecticide, which, when fed to young honey bees, resulted in significant changes in lifespan, nutritional physiology,and behavior. For the first time, we show that when young, nest-aged bees are presented with pollen containing field-relevant levels of cyhalothrin, they reduce their consumption of contaminated pollen. This indicates that, at least for some chemicals, young bees are able to detect contamination in pollen and change their behavioral response, even if the contamination levels do not prevent foraging honey bees from collecting the contaminated pollen.

Juvenile hormone regulation of Drosophila aging.

BACKGROUND: Juvenile hormone (JH) has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone's source. In contrast, little is known about how juvenile hormone affects adult Drosophila melanogaster. Previous work suggests that insulin signaling may modulate Drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control Drosophila life span. Here we adapt a genetic approach to knock out the corpora allata in adult Drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. With this system we test potential explanations for how juvenile hormone modulates aging. RESULTS: A tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. Corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. Treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. Knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. Dietary restriction further extended the lifespan of knockout females. In an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about 100 genes changed in response to loss of juvenile hormone independent of reproductive state. CONCLUSIONS: Reduced juvenile hormone alone is sufficient to extend the lifespan of Drosophila melanogaster. Reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. Our data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. Nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. We identify transcripts that change in response to juvenile hormone independent of reproductive state and suggest these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity.

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.

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.

Down-regulation of honey bee IRS gene biases behavior toward food rich in protein.

Food choice and eating behavior affect health and longevity. Large-scale research efforts aim to understand the molecular and social/behavioral mechanisms of energy homeostasis, body weight, and food intake. Honey bees (Apis mellifera) could provide a model for these studies since individuals vary in food-related behavior and social factors can be controlled. Here, we examine a potential role of peripheral insulin receptor substrate (IRS) expression in honey bee foraging behavior. IRS is central to cellular nutrient sensing through transduction of insulin/insulin-like signals (IIS). By reducing peripheral IRS gene expression and IRS protein amount with the use of RNA interference (RNAi), we demonstrate that IRS influences foraging choice in two standard strains selected for different food-hoarding behavior. Compared with controls, IRS knockdowns bias their foraging effort toward protein (pollen) rather than toward carbohydrate (nectar) sources. Through control experiments, we establish that IRS does not influence the bees' sucrose sensory response, a modality that is generally associated with food-related behavior and specifically correlated with the foraging preference of honey bees. These results reveal a new affector pathway of honey bee social foraging, and suggest that IRS expressed in peripheral tissue can modulate an insect's foraging choice between protein and carbohydrate sources.

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.

Worker division of labor and endocrine physiology are associated in the harvester ant, Pogonomyrmex californicus.

In Pogonomyrmex californicus harvester ants, an age-associated division of labor occurs in the worker caste, in which young workers perform in-nest tasks and older workers forage for food. Here, we tested whether this behavioral division is age based or age flexible, and whether it coincides with differential expression of systemic hormones with known roles in behavioral regulation. Whole-body content of juvenile hormone (JH) and ecdysteroids was determined in workers from (1) age-typical colonies, in which a typical age structure is maintained and workers transition across behaviors naturally, and (2) single-cohort colonies, which are entirely composed of same-aged workers, facilitating the establishment of age-independent division of labor. Foragers from both colony types had higher JH and lower ecdysteroid content than workers performing in-nest tasks, suggesting that age is not the sole determinant of worker behavior. This association between hormone content and behavior of P. californicus workers is similar to that previously observed in founding queens of this species. Because these hormones are key regulators of development and reproductive behavior, our data are consistent with the reproductive ground plan hypothesis (RGPH), which posits that the reproductive regulatory mechanisms of solitary ancestors were co-opted to regulate worker behavior.

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.

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.

IRS and TOR nutrient-signaling pathways act via juvenile hormone to influence honey bee caste fate.

Regardless of genetic makeup, a female honey bee becomes a queen or worker depending on the food she receives as a larva. For decades, it has been known that nutrition and juvenile hormone (JH) signaling determine the caste fate of the individual bee. However, it is still largely unclear how these factors are connected. To address this question, we suppressed nutrient sensing by RNA interference (RNAi)-mediated gene knockdown of IRS (insulin receptor substrate) and TOR (target of rapamycin) in larvae reared on queen diet. The treatments affected several layers of organismal organization that could play a role in the response to differential nutrition between castes. These include transcript profiles, proteomic patterns, lipid levels, DNA methylation response and morphological features. Most importantly, gene knockdown abolished a JH peak that signals queen development and resulted in a worker phenotype. Application of JH rescued the queen phenotype in either knockdown, which demonstrates that the larval response to JH remains intact and can drive normal developmental plasticity even when IRS or TOR transcript levels are reduced. We discuss our results in the context of other recent findings on honey bee caste and development and propose that IRS is an alternative substrate for the Egfr (epidermal growth factor receptor) in honey bees. Overall, our study describes how the interplay of nutritional and hormonal signals affects many levels of organismal organization to build different phenotypes from identical genotypes.

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.