Apis mellifera Solinvivirus-1, a Novel Honey Bee Virus That Remained Undetected for over a Decade, Is Widespread in the USA.

A metagenomic analysis of the virome of honey bees (Apis mellifera) from an apiary with high rates of unexplained colony losses identified a novel RNA virus. The virus, which was named Apis mellifera solinvivirus 1 (AmSV1), contains a 10.6 kb positive-strand genomic RNA with a single ORF coding for a polyprotein with the protease, helicase, and RNA-dependent RNA polymerase domains, as well as a single jelly-roll structural protein domain, showing highest similarity with viruses in the family Solinviviridae. The injection of honey bee pupae with AmSV1 preparation showed an increase in virus titer and the accumulation of the negative-strand of AmSV1 RNA 3 days after injection, indicating the replication of AmSV1. In the infected worker bees, AmSV1 was present in heads, thoraxes, and abdomens, indicating that this virus causes systemic infection. An analysis of the geographic and historic distribution of AmSV1, using over 900 apiary samples collected across the United States, showed AmSV1 presence since at least 2010. In the year 2021, AmSV1 was detected in 10.45% of apiaries (95%CI: 8.41-12.79%), mostly sampled in June and July in Northwestern and Northeastern United States. The diagnostic methods and information on the AmSV1 distribution will be used to investigate the connection of AmSV1 to honey bee colony losses.

Best Management Practices Increase Profitability of Small-Scale US Beekeeping Operations.

Little is known about the economics of small-scale beekeeping, due in part because many of these beekeepers are motivated by personal enjoyment and not profit. These beekeepers, however, represent more than 90% of US beekeeping population, so economic analysis of this majority group is warranted. Understanding how management practices impact colony profitability in small apiaries can inform beekeeper management decisions. Best management practices (BMPs) can increase colony productivity and survival, but often require additional labor and materials compared to less intensive beekeeping practices. Here, we investigate the impact of BMPs on the profitability of small-scale beekeeping. We found that BMPs required higher costs in labor and materials, but that they also produced higher revenue from honey and nucleus colony production. As a result, after three years, BMP apiaries were 8 times more profitable than less intensively managed apiaries. The increased profitability in BMP apiaries is largely attributed to improved colony health and survival due to more active Varroa management and reduced Varroa and viral loads. These results can inform small scale beekeeper budgeting and management, and also support extension efforts to increase BMP adoption.

Water provisioning increases caged worker bee lifespan and caged worker bees are living half as long as observed 50 years ago.

The high loss rates of honey bee colonies drive research for solutions aimed to mitigate these losses. While honey bee colonies are superorganisms, experiments that measure the response to stressors often use caged individuals to allow for inference in a controlled setting. In an initial experiment, we showed that caged honey bees provisioned with various types of water (deionized, 1%NaCl in deionized, or tap) have greater median lifespans than those that did not. While researching the history of water provisioning in cage studies, we observed that the median lifespan of caged honey bees has been declining in the US since the 1970's, from an average of 34.3 days to 17.7 days. In response to this, we again turned to historical record and found a relationship between this trend and a decline in the average amount of honey produced per colony per year in the US over the last 5 decades. To understand the relationship between individual bee lifespan and colony success we used an established honey bee population model (BEEHAVE) to simulate the predicted effects of decreased worker lifespans. Declines in downstream measures of colony population, overall honey production, and colony lifespan resulted from reduced worker bee lifespans. Modeled colony lifespans allowed us to estimate colony loss rates in a beekeeping operation where lost colonies are replaced annually. Resulting loss rates were reflective of what beekeepers' experience today, which suggests the average lifespan of individual bees plays an important role in colony success.

Lethal, sublethal, and combined effects of pesticides on bees: A meta-analysis and new risk assessment tools.

Multiple stressors threaten bee health, a major one being pesticides. Bees are simultaneously exposed to multiple pesticides that can cause both lethal and sublethal effects. Risk assessment and most research on bee health, however, focus on lethal individual effects. Here, we performed a systematic literature review and meta-analysis that summarizes and re-interprets the available qualitative and quantitative information on the lethal, sublethal, and combined toxicity of a comprehensive range of pesticides on bees. We provide results (1970-2019) for multiple bee species (Bombus, Osmia, Megachile, Melipona, Partamona, Scaptotrigona), although most works focused on Apis mellifera L. (78 %). Our harmonised results document the lethal toxicity of pesticides in bees (n = 377 pesticides) and the types of sublethal testing methods and related effects that cause a sublethal effect (n = 375 sublethal experiments). We identified the most common combinations of pesticides and mode of actions tested, and summarize the experimental methods, magnitude of the interactions, and robustness of available data (n = 361 experiments). We provide open access searchable, comprehensive, and integrated list of pesticides and their levels causing lethal, sublethal, and combined effects. We report major data gaps related to pesticide's sublethal (71 %) and combined (e.g., ~99 %) toxicity. We identified pesticides and mode of actions of greatest concern in terms of sublethal (chlorothalonil, pymetrozine, glyphosate; neonicotinoids) and combined (tau-fluvalinate combinations; acetylcholinesterase inhibitors and neonicotinoids) effects. Although certain pesticides have faced regulatory restrictions in specific countries (chlorothalonil, pymetrozine, neonicotinoids), most are still widely used worldwide (e.g., glyphosate). This work aims at facilitating the implementation of more comprehensive and harmonised research and risk assessments, considering sublethal and combined effects. To ensure safeguarding pollinators and the environment, we advocate for a more refined and holistic assessment that do not only focus on lethality but uses harmonised methods to test sublethal and relevant combinations.

Honey bee populations of the USA display restrictions in their mtDNA haplotype diversity.

The genetic diversity of the USA honey bee (Apis mellifera L.) populations was examined through a molecular approach using two mitochondrial DNA (mtDNA) markers. A total of 1,063 samples were analyzed for the mtDNA intergenic region located between the cytochrome c oxidase I and II (COI-COII) and 401 samples were investigated for the NADH dehydrogenase 2 (ND2) coding gene. The samples represented 45 states, the District of Colombia and two territories of the USA. Nationwide, three maternal evolutionary lineages were identified: the North Mediterranean lineage C (93.79%), the West Mediterranean lineage M (3.2%) and the African lineage A (3.01%). A total of 27 haplotypes were identified, 13 of them (95.11%) were already reported and 14 others (4.87%) were found to be novel haplotypes exclusive to the USA. The number of haplotypes per state/territory ranged between two and eight and the haplotype diversity H ranged between 0.236-0.763, with a nationwide haplotype diversity of 0.597. Furthermore, the honey bee populations of the USA were shown to rely heavily (76.64%) on two single haplotypes (C1 = 38.76%, C2j = 37.62%) of the same lineage characterizing A. m. ligustica and A. m. carnica subspecies, respectively. Molecular-variance parsimony in COI-COII and ND2 confirmed this finding and underlined the central and ancestral position of C2d within the C lineage. Moreover, major haplotypes of A. m. mellifera (M3a, M7b, M7c) were recorded in six states (AL, AR, HI, MO, NM and WA). Four classic African haplotypes (A1e, A1v, A4, A4p) were also identified in nine states and Puerto Rico, with higher frequencies in southern states like LA, FL and TX. This data suggests the need to evaluate if a restricted mtDNA haplotype diversity in the US honey bee populations could have negative impacts on the beekeeping sustainability of this country.

Accelerated Varroa destructor population growth in honey bee (Apis mellifera) colonies is associated with visitation from non-natal bees.

A leading cause of managed honey bee colony mortality in the US, Varroa destructor populations typically exceed damaging levels in the fall. One explanation for rapid population increases is migration of mite carrying bees between colonies. Here, the degree to which bees from high and low mite donor colonies move between apiaries, and the effect visitation has on Varroa populations was monitored. More bees from low mite colonies (n = 37) were detected in receiver apiaries than bees from high mite colonies (n = 10, p < 0.001). Receiver colony Varroa population growth was associated with visitation by non-natal bees (p = 0.03), but not high mite bees alone (p = 0.19). Finally, colonies lacking robbing screens experienced faster Varroa population growth than screened neighbors (p = 0.01). Results indicate visiting non-natal bees may vector mites to receiver colonies. These results do not support the current two leading theories regarding mite immigration - the "mite bomb" theory (bees from high mite colonies emigrating to collapsing colonies), or the "robbing" theory (natal robbing bees return home with mites from collapsing colonies). Potential host-parasite effects to bee behavior, as well as important management implications both for Varroa treatment regimens and breeding Varroa resistant bees are discussed.

Social disruption: Sublethal pesticides in pollen lead to Apis mellifera queen events and brood loss.

Eusocial Apis mellifera colonies depend on queen longevity and brood viability to survive, as the queen is the sole reproductive individual and the maturing brood replenishes the shorter-lived worker bees. Production of many crops rely on both pesticides and bee pollination to improve crop quantity and quality, yet sublethal impacts of this pesticide exposure is often poorly understood. We investigated the resiliency of queens and their brood after one month of sublethal exposure to field relevant doses of pesticides that mimic exposure during commercial pollination contracts. We exposed full size colonies to pollen contaminated with field-relevant doses of the fungicides (chlorothalonil and propicanizole), insecticides (chlorypyrifos and fenpropathrin) or both, noting a significant reduction in pollen consumption in colonies exposed to fungicides compared to control. While we found no difference in the total amount of pollen collected per colony, a higher proportion of pollen to non-pollen foragers was detected in all pesticide exposed colonies. After ceasing treatments, we measured brood development, discovering a significant increase in brood loss and/or cannibalism across all pesticide exposed groups. Sublethal pesticide exposure in general was linked to reduced production of replacement workers and a change in protein acquisition (pollen vs. non-pollen foraging). Fungicide exposure also resulted in increased loss of the reproductive queen.

Mutations associated with pyrethroid resistance in Varroa mite, a parasite of honey bees, are widespread across the United States.

BACKGROUND: Managed honey bees are key pollinators of many crops and play an essential role in the United States food production. For more than ten years, beekeepers in the United States have been reporting high rates of colony losses. One of the drivers of these losses is the parasitic mite Varroa destructor. Maintaining healthy honey bee colonies in the United States is dependent on a successful control of this mite. The pyrethroid tau-fluvalinate (Apistan®) was among the first synthetic varroacides registered in the United States. With over 20 years of use, mites resistant to Apistan® have emerged, and so it is unsurprising that treatment failures have been reported. Resistance to tau-fluvalinate in US mite populations is associated with point mutations at position 925 of the voltage-gated sodium channel. RESULTS: Here, we have generated a distribution map of pyrethroid resistance alleles in Varroa samples collected from US apiaries in 2016 and 2017, using a high throughput allelic discrimination assay based on TaqMan®. Our results evidence that knockdown resistance (kdr)-type mutations are widely distributed in Varroa populations across the country showing high variability among apiaries. We used these data to predict the phenotype of the mites in the case of treatments with pyrethroids. CONCLUSION: We highlight the relevance of monitoring the resistance in mite populations to achieve an efficient control of this pest. We also put forward the benefits of implementing this methodology to provide data for designing pest management programs aiming to control Varroa. © 2021 Society of Chemical Industry.

Survey-derived best management practices for backyard beekeepers improve colony health and reduce mortality.

Honey bee colony losses in the US have exceeded acceptable levels for at least a decade, leaving beekeepers in need of management practices to improve colony health and survival. Here, an empirical Best Management Practice (BMP) regimen was tested, comprised of the top four management practices associated with reduced colony mortality in backyard beekeeping operations according to Bee Informed Partnership Loss and Management survey results. Seven study locations were established across the US, and each location consisted of ten colonies treated according to empirical BMPs and ten according to average beekeeping practice. After 3 years, colonies treated according to empirical BMPs experienced reduced Varroa infestation, viral infection, and mortality compared to colonies managed with Average practices. In addition, BMP colonies produced more new colonies via splits. The colonies under Average practices were given chemical Varroa treatments only once per year, and thus spent more months above economic threshold of 3.0 mites/100 bees. Increased time spent above the economic threshold was significantly correlated to both increased viral infection and colony mortality. This study demonstrates the cumulative effects of management and colony health stressors over months and years, especially the dire importance of regular Varroa monitoring and management.

Prioritizing changes in management practices associated with reduced winter honey bee colony losses for US beekeepers.

Beekeepers attempt to manage their honey bee colonies in ways that optimize colony health. Disentangling the impact of management from other variables affecting colony health is complicated by the diversity of practices used and difficulties handling typically complex and incomplete observational datasets. We propose a method to 1) compress multi-factored management data into a single index, to holistically investigate the real world impact of management on colony mortality, and 2) simplify said index to identify the core practices for which a change in behavior is associated with the greatest improvement in survivorship. Experts scored the practices of US beekeepers (n = 18,971) documented using four years of retrospective surveys (2012-2015). Management Index scores significantly correlated with loss rates, with beekeepers most in line with recommendations suffering lower losses. The highest ranked practices varied by operation type, as recommendations accounted for the current prevalence of practices. These results validate experts' opinion using empirical data, and can help prioritize extension messages. Improving management will not prevent all losses; however, we show that few behavioral changes (in particular related to comb management, sources of new colonies and Varroa management) can lead to a non-negligible reduction in risk.

Are native and non-native pollinator friendly plants equally valuable for native wild bee communities?

Bees rely on floral pollen and nectar for food. Therefore, pollinator friendly plantings are often used to enrich habitats in bee conservation efforts. As part of these plantings, non-native plants may provide valuable floral resources, but their effects on native bee communities have not been assessed in direct comparison with native pollinator friendly plantings. In this study, we performed a common garden experiment by seeding mixes of 20 native and 20 non-native pollinator friendly plant species at separate neighboring plots at three sites in Maryland, USA, and recorded flower visitors for 2 years. A total of 3,744 bees (120 species) were collected. Bee abundance and species richness were either similar across plant types (midseason and for abundance also late season) or lower at native than at non-native plots (early season and for richness also late season). The overall bee community composition differed significantly between native and non-native plots, with 11 and 23 bee species being found exclusively at one plot type or the other, respectively. Additionally, some species were more abundant at native plant plots, while others were more abundant at non-natives. Native plants hosted more specialized plant-bee visitation networks than non-native plants. Three species out of the five most abundant bee species were more specialized when foraging on native plants than on non-native plants. Overall, visitation networks were more specialized in the early season than in late seasons. Our findings suggest that non-native plants can benefit native pollinators, but may alter foraging patterns, bee community assemblage, and bee-plant network structures.

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

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

Beeswax cleaning by solvent extraction of pesticides.

We set out to test if the methodology used to clean sheep wool wax (Lanolin) from pesticides could be used to clean beeswax as well. We first made an aggregate sample of brood comb wax from three different US beekeepers. Sub-samples of these aggregate wax samples were analyzed for pesticide contamination. The remaining wax, was then dissolved into hexane solution and run through four N, N-Dimethylformamide (DMF) washes. During these extractions, the pesticides partitioned into the DMF, and so were removed from the beeswax. Following the solvent extractions, the beeswax was tested again for pesticides. An average of 95% of the pesticide contamination was removed by the chemical wash procedure. •Beeswax is the beekeeping matrix with the highest pesticide content.•This study developed methodology for solvent-based removal of pesticides from beeswax (>95%).•Of 24 pesticides detected in beeswax samples before to the solvent extraction, only 3 pesticides were detected after the extraction with DMF.

Use of Chemical and Nonchemical Methods for the Control of Varroa destructor (Acari: Varroidae) and Associated Winter Colony Losses in U.S. Beekeeping Operations.

The parasitic mite Varroa destructor (Acari: Varroidae) is a major cause of overwintering honey bee (Apis mellifera) colony losses in the United States, suggesting that beekeepers must control Varroa populations to maintain viable colonies. Beekeepers have access to several chemical varroacides and nonchemical practices to control Varroa populations. However, no studies have examined large-scale patterns in Varroa control methods in the United States. Here we used responses from 4 yr of annual surveys of beekeepers representing all regions and operation sizes across the United States to investigate use of Varroa control methods and winter colony losses associated with use of different methods. We focused on seven varroacide products (amitraz, coumaphos, fluvalinate, hop oil, oxalic acid, formic acid, and thymol) and six nonchemical practices (drone brood removal, small-cell comb, screened bottom boards, powdered sugar, mite-resistant bees, and splitting colonies) suggested to aid in Varroa control. We found that nearly all large-scale beekeepers used at least one varroacide, whereas small-scale beekeepers were more likely to use only nonchemical practices or not use any Varroa control. Use of varroacides was consistently associated with the lowest winter losses, with amitraz being associated with lower losses than any other varroacide product. Among nonchemical practices, splitting colonies was associated with the lowest winter losses, although losses associated with sole use of nonchemical practices were high overall. Our results suggest potential control methods that are effective or preferred by beekeepers and should therefore inform experiments that directly test the efficacy of different control methods. This will allow beekeepers to incorporate Varroa control methods into management plans that improve the overwintering success of their colonies.

Varroa destructor feeds primarily on honey bee fat body tissue and not hemolymph.

The parasitic mite Varroa destructor is the greatest single driver of the global honey bee health decline. Better understanding of the association of this parasite and its host is critical to developing sustainable management practices. Our work shows that this parasite is not consuming hemolymph, as has been the accepted view, but damages host bees by consuming fat body, a tissue roughly analogous to the mammalian liver. Both hemolymph and fat body in honey bees were marked with fluorescent biostains. The fluorescence profile in the guts of mites allowed to feed on these bees was very different from that of the hemolymph of the host bee but consistently matched the fluorescence profile unique to the fat body. Via transmission electron microscopy, we observed externally digested fat body tissue in the wounds of parasitized bees. Mites in their reproductive phase were then fed a diet composed of one or both tissues. Mites fed hemolymph showed fitness metrics no different from the starved control. Mites fed fat body survived longer and produced more eggs than those fed hemolymph, suggesting that fat body is integral to their diet when feeding on brood as well. Collectively, these findings strongly suggest that Varroa are exploiting the fat body as their primary source of sustenance: a tissue integral to proper immune function, pesticide detoxification, overwinter survival, and several other essential processes in healthy bees. These findings underscore a need to revisit our understanding of this parasite and its impacts, both direct and indirect, on honey bee health.

Beekeeper stewardship, colony loss, and Varroa destructor management.

Varroa (Varroa destructor) is a leading cause of honey bee mortality worldwide. In a U.S. national survey of beekeepers, 3519 respondents noted what they believe are the advantages and disadvantages of managing for Varroa, what good stewardship means in beekeeping, and whether they treated for Varroa. Dominant attitudes were keeping bees healthy, minimizing disturbance, and monitoring hives. We found a bifurcation in Varroa management beliefs. Decision tree analyses show group distinctions. Treatment Skeptics tend to say that stewardship means bees should not be disturbed or subjected to chemicals, and should be given forage to do their 'normal business.' This group was less likely to treat for Varroa. Treatment Adherents identify themselves as bee stewards and say stewardship means active hive management and keeping bees healthy and alive. Illuminating beekeeper stewardship is essential for a socioecological understanding of how to address challenging Varroa management and complex human-environmental production systems that have landscape-level effects.

Drivers of colony losses.

Over the past decade, in some regions of the world, honey bee (Apis mellifera L.) colonies have experienced rates of colony loss that are difficult for beekeepers to sustain. The reasons for losses are complex and interacting, with major drivers including Varroaand related viruses, pesticides, nutrition and beekeeper practices. In these endeavors it has also become apparent that defining a dead colony, and singling out the effects of specific drivers of loss, is not so straightforward. Using the class of neonicotinoid pesticides as an example we explain why quantifying risk factor impact at the colony level is at times elusive and in some cases unpractical. In this review, we discuss the caveats of defining and quantifying dead colonies. We also summarize the current leading drivers of colony losses, their interactions and the most recent research on their effects on colony mortality.

Tropilaelaps mite: an emerging threat to European honey bee.

The risk of transmission of honey bee parasites has increased substantially as a result of trade globalization and technical developments in transportation efficacy. Great concern over honey bee decline has accelerated research on newly emerging bee pests and parasites. These organisms are likely to emerge from Asia as it is the only region where all 10 honey bee species co-occur. Varroa destructor, an ectoparasitic mite, is a classic example of a pest that has shifted from A. cerana, a cavity nesting Asian honey bee to A. mellifera, the European honey bee. In this review, we will describe the potential risks to global apiculture of the global expansion of Tropilaelaps mercedesae, originally a parasite of the open-air nesting Asian giant honey bee, compared to the impact of V. destructor.

Recent spread of Varroa destructor virus-1, a honey bee pathogen, in the United States.

RNA viruses impact honey bee health and contribute to elevated colony loss rates worldwide. Deformed wing virus (DWV) and the closely related Varroa destructor virus-1 (VDV1), are the most widespread honey bee viruses. VDV1 is known to cause high rates of overwintering colony losses in Europe, however it was unknown in the United States (US). Using next generation sequencing, we identified VDV1 in honey bee pupae in the US. We tested 603 apiaries the US in 2016 and found that VDV1 was present in 66.0% of them, making it the second most prevalent virus after DWV, which was present in 89.4% of the colonies. VDV1 had the highest load in infected bees (7.45*1012 ± 1.62*1012 average copy number ± standard error) compared to other tested viruses, with DWV second (1.04*1012 ± 0.53*1012). Analysis of 75 colonies sourced in 2010 revealed that VDV1 was present in only 2 colonies (2.7%), suggesting its recent spread. We also detected newly emerged recombinants between the US strains of VDV1 and DWV. The presence of these recombinants poses additional risk, because similar VDV1-DWV recombinants constitute the most virulent honeybee viruses in the UK.

Colony Collapse Disorder (CCD) and bee age impact honey bee pathophysiology.

Honey bee (Apis mellifera) colonies continue to experience high annual losses that remain poorly explained. Numerous interacting factors have been linked to colony declines. Understanding the pathways linking pathophysiology with symptoms is an important step in understanding the mechanisms of disease. In this study we examined the specific pathologies associated with honey bees collected from colonies suffering from Colony Collapse Disorder (CCD) and compared these with bees collected from apparently healthy colonies. We identified a set of pathological physical characteristics that occurred at different rates in CCD diagnosed colonies prior to their collapse: rectum distension, Malpighian tubule iridescence, fecal matter consistency, rectal enteroliths (hard concretions), and venom sac color. The multiple differences in rectum symptomology in bees from CCD apiaries and colonies suggest effected bees had trouble regulating water. To ensure that pathologies we found associated with CCD were indeed pathologies and not due to normal changes in physical appearances that occur as an adult bee ages (CCD colonies are assumed to be composed mostly of young bees), we documented the changes in bees of different ages taken from healthy colonies. We found that young bees had much greater incidences of white nodules than older cohorts. Prevalent in newly-emerged bees, these white nodules or cellular encapsulations indicate an active immune response. Comparing the two sets of characteristics, we determined a subset of pathologies that reliably predict CCD status rather than bee age (fecal matter consistency, rectal distension size, rectal enteroliths and Malpighian tubule iridescence) and that may serve as biomarkers for colony health. In addition, these pathologies suggest that CCD bees are experiencing disrupted excretory physiology. Our identification of these symptoms is an important first step in understanding the physiological pathways that underlie CCD and factors impacting bee health.