Nutritional stress exacerbates impact of a novel insecticide on solitary bees' behaviour, reproduction and survival.

Pesticide exposure and food stress are major threats to bees, but their potential synergistic impacts under field-realistic conditions remain poorly understood and are not considered in current pesticide risk assessments. We conducted a semi-field experiment to examine the single and interactive effects of the novel insecticide flupyradifurone (FPF) and nutritional stress on fitness proxies in the solitary bee Osmia bicornis. Individually marked bees were released into flight cages with monocultures of buckwheat, wild mustard or purple tansy, which were assigned to an insecticide treatment (FPF or control) in a crossed design. Nutritional stress, which was high in bees foraging on buckwheat, intermediate on wild mustard and low on purple tansy, modulated the impact of insecticide exposure. Within the first day after application of FPF, mortality of bees feeding on buckwheat was 29 times higher compared with control treatments, while mortality of FPF exposed and control bees was similar in the other two plant species. Moreover, we found negative synergistic impacts of FPF and nutritional stress on offspring production, flight activity, flight duration and flower visitation frequency. These results reveal that environmental policies and risk assessment schemes that ignore interactions among anthropogenic stressors will fail to adequately protect bees and the pollination services they provide.

MALDI-MS Profiling to Address Honey Bee Health Status under Bacterial Challenge through Computational Modeling.

Honey bees play a critical role in the maintenance of plant biodiversity and sustainability of food webs. In the past few decades, bees have been subjected to biotic and abiotic threats causing various colony disorders. Therefore, monitoring solutions to help beekeepers to improve bee health are necessary. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) profiling has emerged within this decade as a powerful tool to identify in routine micro-organisms and is currently used in real-time clinical diagnosis. MALDI BeeTyping is developed to monitor significant hemolymph molecular changes in honey bees upon infection with a series of entomopathogenic Gram-positive and -negative bacteria. A Serratia marcescens strain isolated from one naturally infected honey bee collected from the field is also considered. A series of hemolymph molecular mass fingerprints is individually recorded and to the authors' knowledge, the first computational model harboring a predictive score of 97.92% and made of nine molecular signatures that discriminate and classify the honey bees' systemic response to the bacteria is built. Hence, the model is challenged by classifying a training set of hemolymphs and an overall recognition of 91.93% is obtained. Through this work, a novel, time and cost saving high-throughput strategy that addresses honey bee health on an individual scale is introduced.

Temperature-driven changes in viral loads in the honey bee Apis mellifera.

Many of the physiological traits in insects are shaped by environmental temperatures, which can influence their interactions with pathogens. Therefore, quantifying the thermal responses of the host-pathogen system is crucial for better understanding and predicting their dynamics due to environmental changes. This is particularly important in honey bees, which are experiencing severe colony losses around the world, notably due to infection with the Deformed wing virus (DWV). To investigate the influence of temperature on the honey bee/DWV relationship we exposed adult bees to low or high temperatures and determined the effects on viral titers and bee survival. Emerging bees naturally infected with DWV were reared in vitro at different temperatures ranging from 15 °C to 37 °C. In addition, some bees reared at 37 °C were exposed daily to acute heat treatments (40 and 43 °C). High temperatures significantly decreased DWV titers close to the initial viral load at emergence but increased bee mortality. The lowest temperature resulted in higher mortality, but virus load was not significantly impacted. In conclusion, our results indicate that temperature could contribute to seasonal variations in viral loads but do not suggest temperature to be used as a tool to eliminate viruses, even given that high temperatures limit viral multiplication.

Transcriptome profiling of the honeybee parasite Varroa destructor provides new biological insights into the mite adult life cycle.

BACKGROUND: The parasite Varroa destructor represents a significant threat to honeybee colonies. Indeed, development of Varroa infestation within colonies, if left untreated, often leads to the death of the colony. Although its impact on bees has been extensively studied, less is known about its biology and the functional processes governing its adult life cycle and adaptation to its host. We therefore developed a full life cycle transcriptomic catalogue in adult Varroa females and included pairwise comparisons with males, artificially-reared and non-reproducing females (10 life cycle stages and conditions in total). RESULTS: Extensive remodeling of the Varroa transcriptome was observed, with an upregulation of energetic and chitin metabolic processes during the initial and final phases of the life cycle (e.g. phoretic and post-oviposition stages), whereas during reproductive stages in brood cells genes showing functions related to transcriptional regulation were overexpressed. Several neurotransmitter and neuropeptide receptors involved in behavioural regulation, as well as active compounds of salivary glands, were also expressed at a higher level outside the reproductive stages. No difference was detected between artificially-reared phoretic females and their counterparts in colonies, or between females who failed to reproduce and females who successfully reproduced, indicating that phoretic individuals can be reared outside host colonies without impacting their physiology and that mechanisms underlying reproductive failure occur before oogenesis. CONCLUSIONS: We discuss how these new findings reveal the remarkable adaptation of Varroa to its host biology and notably to the switch from living on adults to reproducing in sealed brood cells. By spanning the entire adult life cycle, our work captures the dynamic changes in the parasite gene expression and serves as a unique resource for deciphering Varroa biology and identifying new targets for mite control.

Stress decreases pollen foraging performance in honeybees.

Foraging in honeybees is energetically demanding. Here, we examined whether stressors, which generally increase metabolic demands, can impair foraging performance. A controlled non-pathogenic stressor (immune challenge) resulted in a change in the foraging preferences of bees. It reduced pollen foraging and increased the duration of trips in pollen foragers. Stress also reduced the amount of octopamine in the brain of pollen foragers (a biogenic amine involved in the regulation of foraging and flight behaviour in insects). According to the literature, flight metabolic rate is higher during pollen foraging than during nectar foraging, and nectar gives a higher energetic return relative to the foraging effort when compared with pollen. We thus propose that stress might be particularly detrimental to the performance of pollen foragers, and stressed bees prefer the energy-rich resource of nectar. In conclusion, stress, even at low levels, could have consequences for bee foraging behaviour and thereby the nutritional balance of the colony.

Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens.

BACKGROUND: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses. RESULTS: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses. CONCLUSIONS: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.

New meta-analysis tools reveal common transcriptional regulatory basis for multiple determinants of behavior.

A fundamental problem in meta-analysis is how to systematically combine information from multiple statistical tests to rigorously evaluate a single overarching hypothesis. This problem occurs in systems biology when attempting to map genomic attributes to complex phenotypes such as behavior. Behavior and other complex phenotypes are influenced by intrinsic and environmental determinants that act on the transcriptome, but little is known about how these determinants interact at the molecular level. We developed an informatic technique that identifies statistically significant meta-associations between gene expression patterns and transcription factor combinations. Deploying this technique for brain transcriptome profiles from ca. 400 individual bees, we show that diverse determinants of behavior rely on shared combinations of transcription factors. These relationships were revealed only when we considered complex and variable regulatory rules, suggesting that these shared transcription factors are used in distinct ways by different determinants. This regulatory code would have been missed by traditional gene coexpression or cis-regulatory analytic methods. We expect that our meta-analysis tools will be useful for a broad array of problems in systems biology and other fields.

Parasitic and immune modulation of flight activity in honey bees tracked with optical counters.

Host-parasite interactions are often characterized by changes in the host behaviour, which are beneficial to either the parasite or the host, or are a non-adaptive byproduct of parasitism. These interactions are further complicated in animal society because individual fitness is associated with group performance. However, a better understanding of host-parasite interaction in animal society first requires the identification of individual host behavioural modification. Therefore, we challenged honey bee (Apis mellifera) workers with the parasite Nosema ceranae or an immune stimulation and tracked their flight activity over their lifetime with an optic counter. We found that bees responded differently to each stress: both Nosema-infected and immune-challenged bees performed a lower number of daily flights compared with control bees, but the duration of their flights increased and decreased over time, respectively. Overall, parasitized bees spent more time in the field each day than control bees, and the inverse was true for immune-challenged bees. Despite the stress of immune challenge, bees had a survival similar to that of control bees likely because of their restricted activity. We discuss how those different behavioural modifications could be adaptive phenotypes. This study provides new insights into how biological stress can affect the behaviour of individuals living in society and how host responses have evolved.

Diverse pollen nutrition can improve the development of solitary bees but does not mitigate negative pesticide impacts.

Floral resource loss and pesticide exposure are major threats to bees in intensively managed agroecosystems, but interactions among these drivers remain poorly understood. Altered composition and lowered diversity of pollen nutrition may reinforce negative pesticide impacts on bees. Here we investigated the development and survival of the solitary bee Osmia bicornis provisioned with three different pollen types, as well as a mixture of these types representing a higher pollen diversity. We exposed bees of each nutritional treatment to five pesticides at different concentrations in the laboratory. Two field-realistic concentrations of three nicotinic acetylcholine receptor (nAChR) modulating insecticides (thiacloprid, sulfoxaflor and flupyradifurone), as well as of two fungicides (azoxystrobin and tebuconazole) were examined. We further measured the expression of two detoxification genes (CYP9BU1, CYP9BU2) under exposure to thiacloprid across different nutrition treatments as a potential mechanistic pathway driving pesticide-nutrition interactions. We found that more diverse pollen nutrition reduced development time, enhanced pollen efficacy (cocoon weight divided by consumed pollen weight) and pollen consumption, and increased weight of O. bicornis after larval development (cocoon weight). Contrary to fungicides, high field-realistic concentrations of all three insecticides negatively affected O. bicornis by extending development times. Moreover, sulfoxaflor and flupyradifurone also reduced pollen efficacy and cocoon weight, and sulfoxaflor reduced pollen consumption and increased mortality. The expression of detoxification genes differed across pollen nutrition types, but was not enhanced after exposure to thiacloprid. Our findings highlight that lowered diversity of pollen nutrition and high field-realistic exposure to nAChR modulating insecticides negatively affected the development of O. bicornis, but we found no mitigation of negative pesticide impacts through increased pollen diversity. These results have important implications for risk assessment for bee pollinators, indicating that negative effects of nAChR modulating insecticides to developing solitary bees are currently underestimated.

Allele Frequencies of Genetic Variants Associated with Varroa Drone Brood Resistance (DBR) in Apis mellifera Subspecies across the European Continent.

Implementation of marker-assisted selection (MAS) in modern beekeeping would improve sustainability, especially in breeding programs aiming for resilience against the parasitic mite Varroa destructor. Selecting honey bee colonies for natural resistance traits, such as brood-intrinsic suppression of varroa mite reproduction, reduces the use of chemical acaricides while respecting local adaptation. In 2019, eight genomic variants associated with varroa non-reproduction in drone brood were discovered in a single colony from the Amsterdam Water Dune population in the Netherlands. Recently, a new study tested the applicability of these eight genetic variants for the same phenotype on a population-wide scale in Flanders, Belgium. As the properties of some variants varied between the two studies, one hypothesized that the difference in genetic ancestry of the sampled colonies may underly these contribution shifts. In order to frame this, we determined the allele frequencies of the eight genetic variants in more than 360 Apis mellifera colonies across the European continent and found that variant type allele frequencies of these variants are primarily related to the A. mellifera subspecies or phylogenetic honey bee lineage. Our results confirm that population-specific genetic markers should always be evaluated in a new population prior to using them in MAS programs.

Bridging the Gap between Field Experiments and Machine Learning: The EC H2020 B-GOOD Project as a Case Study towards Automated Predictive Health Monitoring of Honey Bee Colonies.

Honey bee colonies have great societal and economic importance. The main challenge that beekeepers face is keeping bee colonies healthy under ever-changing environmental conditions. In the past two decades, beekeepers that manage colonies of Western honey bees (Apis mellifera) have become increasingly concerned by the presence of parasites and pathogens affecting the bees, the reduction in pollen and nectar availability, and the colonies' exposure to pesticides, among others. Hence, beekeepers need to know the health condition of their colonies and how to keep them alive and thriving, which creates a need for a new holistic data collection method to harmonize the flow of information from various sources that can be linked at the colony level for different health determinants, such as bee colony, environmental, socioeconomic, and genetic statuses. For this purpose, we have developed and implemented the B-GOOD (Giving Beekeeping Guidance by computational-assisted Decision Making) project as a case study to categorize the colony's health condition and find a Health Status Index (HSI). Using a 3-tier setup guided by work plans and standardized protocols, we have collected data from inside the colonies (amount of brood, disease load, honey harvest, etc.) and from their environment (floral resource availability). Most of the project's data was automatically collected by the BEEP Base Sensor System. This continuous stream of data served as the basis to determine and validate an algorithm to calculate the HSI using machine learning. In this article, we share our insights on this holistic methodology and also highlight the importance of using a standardized data language to increase the compatibility between different current and future studies. We argue that the combined management of big data will be an essential building block in the development of targeted guidance for beekeepers and for the future of sustainable beekeeping.

Ecto- and endoparasite induce similar chemical and brain neurogenomic responses in the honey bee (Apis mellifera).

BACKGROUND: Exclusion from a social group is an effective way to avoid parasite transmission. This type of social removal has also been proposed as a form of collective defense, or social immunity, in eusocial insect groups. If parasitic modification of host behavior is widespread in social insects, the underlying physiological and neuronal mechanisms remain to be investigated. We studied this phenomenon in honey bees parasitized by the mite Varroa destructor or microsporidia Nosema ceranae, which make bees leave the hive precociously. We characterized the chemical, behavioral and neurogenomic changes in parasitized bees, and compared the effects of both parasites. RESULTS: Analysis of cuticular hydrocarbon (CHC) profiles by gas chromatography coupled with mass spectrophotometry (GC-MS) showed changes in honey bees parasitized by either Nosema ceranae or Varroa destructor after 5 days of infestation. Levels of 10-HDA, an antiseptic important for social immunity, did not change in response to parasitism. Behavioral analysis of N. ceranae- or V. destructor- parasitized bees revealed no significant differences in their behavioral acts or social interactions with nestmates. Digital gene expression (DGE) analysis of parasitized honey bee brains demonstrated that, despite the difference in developmental stage at which the bee is parasitized, Nosema and Varroa-infested bees shared more gene changes with each other than with honey bee brain expression gene sets for forager or nurse castes. CONCLUSIONS: Parasitism by Nosema or Varroa induces changes to both the CHC profiles on the surface of the bee and transcriptomic profiles in the brain, but within the social context of the hive, does not result in observable effects on her behavior or behavior towards her. While parasitized bees are reported to leave the hive as foragers, their brain transcription profiles suggest that their behavior is not driven by the same molecular pathways that induce foraging behavior.

Using physiology to better support wild bee conservation.

There is accumulating evidence that wild bees are experiencing a decline in terms of species diversity, abundance or distribution, which leads to major concerns about the sustainability of both pollination services and intrinsic biodiversity. There is therefore an urgent need to better understand the drivers of their decline, as well as design conservation strategies. In this context, the current approach consists of linking observed occurrence and distribution data of species to environmental features. While useful, a highly complementary approach would be the use of new biological metrics that can link individual bee responses to environmental alteration with population-level responses, which could communicate the actual bee sensitivity to environmental changes and act as early warning signals of bee population decline or sustainability. We discuss here through several examples how the measurement of bee physiological traits or performance can play this role not only in better assessing the impact of anthropogenic pressures on bees, but also in guiding conservation practices with the help of the documentation of species' physiological needs. Last but not least, because physiological changes generally occur well in advance of demographic changes, we argue that physiological traits can help in predicting and anticipating future population trends, which would represent a more proactive approach to conservation. In conclusion, we believe that future efforts to combine physiological, ecological and population-level knowledge will provide meaningful contributions to wild bee conservation-based research.

Real-time monitoring of honeybee colony daily activity and bee loss rates can highlight the risk posed by a pesticide.

Information on honeybee foraging performance and especially bee loss rates at the colony level are crucial for evaluating the magnitude of effects due to pesticide exposure, thereby ensuring that protection goals for honeybee colonies are met (i.e. threshold of acceptable effects). However, current methods for monitoring honeybee foraging activity and mortality are very approximate (visual records) or are time-limited and mostly based on single cohort analysis. We therefore assess the potential of bee counters, that enable a colony-level and continuous monitoring of bee flight activity and mortality, in pesticide risk assessment. After assessing the background activity and bee loss rates, we exposed colonies to two concentrations of sulfoxaflor (a neurotoxic insecticide) in sugar syrup: a concentration that was considered to be field realistic (0.59 µg/ml) and a higher concentration (2.36 µg/ml) representing a worst-case exposure scenario. We did not find any effect of the field-realistic concentration on flight activity and bee loss rates. However, a two-fold decrease in daily flight activity and a 10-fold increase in daily bee losses were detected in colonies exposed to the highest sulfoxaflor concentration as compared to before exposure. When compared to the theoretical trigger values associated with the specific protection goal of 7 % colony-size reduction, the observed fold changes in daily bee losses were often found to be at risk for colonies. In conclusion, the real-time and colony-level monitoring of bee loss rates, combined with threshold values indicating at which levels bee loss rates threaten the colony, have great potential for improving regulatory pesticide risk assessments for honeybees under field conditions.

Brain, physiological and behavioral modulation induced by immune stimulation in honeybees (Apis mellifera): a potential mediator of social immunity?

Social removal is often an adaptive response for preventing the entry and spread of parasitic infection between kin members of a group. Social isolation via removal or the switching of social tasks has also been observed in insect societies; however, the underlying mechanisms are unclear. We tested in honeybees the role of the immune system in physiological and behavioral modulation. Forager bees are often located in the outer area of the colony, and thus have reduced contacts with individuals of high importance, who are located in the inner area of the colony (e.g. queen and brood). We thus expected that an immune challenge would induce a forager profile. This was confirmed by measuring brain (foraging and malvolio gene expression), physiological (hypopharyngeal glands size) and behavioral (queen attendance) parameters of nurse/forager profiles after a treatment with an immune-activator (lipopolysaccharides). Our results support the idea that the interplay between the brain and immune system may be an important regulatory factor of social immunity in insects.

Honey bee aggression supports a link between gene regulation and behavioral evolution.

A prominent theory states that animal phenotypes arise by evolutionary changes in gene regulation, but the extent to which this theory holds true for behavioral evolution is not known. Because "nature and nurture" are now understood to involve hereditary and environmental influences on gene expression, we studied whether environmental influences on a behavioral phenotype, i.e., aggression, could have evolved into inherited differences via changes in gene expression. Here, with microarray analysis of honey bees, we show that aggression-related genes with inherited patterns of brain expression are also environmentally regulated. There were expression differences in the brain for hundreds of genes between the highly aggressive Africanized honey bee compared with European honey bee (EHB) subspecies. Similar results were obtained for EHB in response to exposure to alarm pheromone (which provokes aggression) and when comparing old and young bees (aggressive tendencies increase with age). There was significant overlap of the gene lists generated from these three microarray experiments. Moreover, there was statistical enrichment of several of the same cis regulatory motifs in promoters of genes on all three gene lists. Aggression shows a remarkably robust brain molecular signature regardless of whether it occurs because of inherited, age-related, or environmental (social) factors. It appears that one element in the evolution of different degrees of aggressive behavior in honey bees involved changes in regulation of genes that mediate the response to alarm pheromone.

Pesticide risk assessment: honeybee workers are not all equal regarding the risk posed by exposure to pesticides.

Toxicological studies in honeybees have long shown that a single pesticide dose or concentration does not necessarily induce a single response. Inter-individual differences in pesticide sensitivity and/or the level of exposure (e.g., ingestion of pesticide-contaminated matrices) may explain this variability in risk posed by a pesticide. Therefore, to better inform pesticide risk assessment for honeybees, we studied the risk posed by pesticides to two behavioral castes, nurse, and forager bees, which are largely represented within colonies and which exhibit large differences in their physiological backgrounds. For that purpose, we determined the sensitivity of nurses and foragers to azoxystrobin (fungicide) and sulfoxaflor (insecticide) upon acute or chronic exposure. Azoxystrobin was found to be weakly toxic to both types of bees. However, foragers were more sensitive to sulfoxaflor than nurses upon acute and chronic exposure. This phenomenon was not explained by better sulfoxaflor metabolization in nurses, but rather by differences in body weight (nurses being 1.6 times heavier than foragers). Foragers consistently consumed more sugar syrup than nurses, and this increased consumption was even more pronounced with pesticide-contaminated syrup (at specific concentrations). Altogether, the stronger susceptibility and exposure of foragers to sulfoxaflor contributed to increases of 2 and tenfold for the acute and chronic risk quotients, respectively, compared to nurses. In conclusion, to increase the safety margin and avoid an under-estimation of the risk posed by insecticides to honeybees, we recommend systematically including forager bees in regulatory tests.

Delayed effects of a single dose of a neurotoxic pesticide (sulfoxaflor) on honeybee foraging activity.

Pesticide risk-assessment guidelines for honeybees (Apis mellifera) generally require determining the acute toxicity of a chemical over the short-term through fix-duration tests. However, potential long-lasting or delayed effects resulting from an acute exposure (e.g. a single dose) are often overlooked, although the modification of a developmental process may have life-long consequences. To investigate this question, we exposed young honeybee workers to a single sublethal field-realistic dose of a neurotoxic pesticide, sulfoxaflor, at one of two amounts (16 or 60 ng), at the moment when they initiated orientation flights (preceding foraging activity). We then tracked in the field their flight activity and lifespan with automated life-long monitoring devices. Both amounts of sulfoxaflor administered reduced the total number of flights but did not affect bee survival and flight duration. When looking at the time series of flight activity, effects were not immediate but delayed until foraging activity with a decrease in the daily number of foraging flights and consequently in their total number (24 and 33% less for the 16 and 60 ng doses, respectively). The results of our study therefore blur the general assumption in honeybee toxicology that acute exposure results in immediate and rapid effects and call for long-term recording and/or time-to-effect measurements, even upon exposure to a single dose of pesticide.

Warmer winters are associated with lower levels of the cryoprotectant glycerol, a slower decrease in vitellogenin expression and reduced virus infections in winter honeybees.

Within the context of climate change, winter temperatures at high latitudes are predicted to rise faster than summer temperatures. This phenomenon is expected to negatively affect the diapause performance and survival of insects, since they largely rely on low temperatures to lower their metabolism and preserve energy. However, some insects like honeybees, remain relatively active during the winter and elevate their metabolic rate to produce endothermic heat when temperatures drop. Warming winters are thus expected to improve overwintering performance of honeybees. In order to verify this hypothesis, for two consecutive years, we exposed honeybee colonies to either a mild or cold winter. We then monitored the influence of wintering conditions on several parameters of honeybee overwintering physiology, such as levels of the cryoprotectant glycerol, expression levels of immune and antioxidant genes, and genes encoding multifunctional proteins, including vitellogenin, which promotes bee longevity. Winter conditions had no effect on the expression of antioxidant genes, and genes related to immunity were not consistently affected. However, mild winters were consistently associated with a lower investment in glycerol synthesis and a higher expression of fat body genes, especially apidaecin and vitellogenin. Finally, while we found that viral loads generally decreased through the winter, this trend was more pronounced under mild winter conditions. In conclusion, and without considering how warming temperatures might affect other aspects of honeybee biology before overwintering, our data suggest that warming temperatures will likely benefit honeybee vitality by notably reducing their viral loads over the winter.