Compliance with recommended Varroa destructor treatment regimens improves the survival of honey bee colonies over winter.

The ectoparasitic mite Varroa destructor affects honey bee colony health and survival negatively, thus compelling beekeepers to treat their colonies every year. A broadly used mite control regimen is based on two organic molecules: formic and oxalic acids. To ensure optimal efficiency, several applications of these acids at pre-defined time points are recommended. These recommendations are mainly based on experiments conducted under controlled conditions. Studies evaluating the effectiveness under natural field conditions are lacking. We enrolled 30 beekeepers in a longitudinal study in three cantons in Switzerland and monitored the management and health of their colonies for two years. We assessed compliance with mite control recommendations and measured V. destructor infestation rates, indexes of colony productivity (brood size and honey harvest), and colony mortality in 300 colonies. We observed a 10-fold increased risk of colony death when beekeepers deviated slightly from the recommended treatment regimen compared to compliant beekeepers (odds ratio: 11.9, 95% CI: 2.6-55.2, p = 0.002). The risk of colony death increased 25-fold in apiaries with substantial deviations from the recommendations (odds ratio: 50.4, 95% CI: 9.7-262.5, p < 0.0001). The deviations led to increased levels of V. destructor infestation ahead of wintering, which was likely responsible for colony mortality. After communicating the apparent link between low compliance and poor colony survival at the end of the first year to the beekeepers, we observed better compliance and colony survival in the second year. Our results highlight the positive impact of compliance with the recommended V. destructor treatment regimen on the health of honeybee colonies and the need to better communicate the consequences of deviating from the recommendations to improve compliance. Compliance also occasionally decreased, which hints at concept implementation constraints that could be identified and possibly addressed in detail with the help of social sciences to further promote honey bee health.

α-Costic acid, a plant sesquiterpene with acaricidal activity against Varroa destructor parasitizing the honey bee.

The organic extract of the aerial parts of Dittrichia viscosa, a perennial native plant of the Mediterranean basin, showed a significant acaricidal activity against Varroa destructor, the parasite mite of Apis mellifera, commonly called honey bee. Among the metabolites isolated from the organic extract of this Asteraceae, α-costic acid showed to be one of the compounds responsible for the toxic activity exhibited by the crude plant extract on this parasite mite species. In addition to the toxic effect a clear acaricidal response has been recorded when the parasitic mite was exposed to 1 mg/mL concentration of α-costic acid while no effects have been showed on honey bees using the same compound at the same concentration. This finding suggests a potential use of α-costic acid to control Varroa mites. The possibility to reliably achieve absolute configuration of α-costic acid by DFT computational analysis of chiroptical spectra has been also demonstrated.†.

Comparative transcriptomics indicates endogenous differences in detoxification capacity after formic acid treatment between honey bees and varroa mites.

Formic acid (FA) has been used for decades to control Varroa destructor, one of the most important parasites of the western honey bee, Apis mellifera. The rather unselective molecular mode of action of FA and its possible effects on honeybees have long been a concern of beekeepers, as it has undesirable side effects that affect the health of bee colonies. This study focuses on short-term transcriptomic changes as analysed by RNAseq in both larval and adult honey bees and in mites after FA treatment under applied conditions. Our study aims to identify those genes in honey bees and varroa mites differentially expressed upon a typical FA hive exposure scenario. Five detoxification-related genes were identified with significantly enhanced and one gene with significantly decreased expression under FA exposure. Regulated genes in our test setting included members of various cytochrome P450 subfamilies, a flavin-dependent monooxygenase and a cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH), known to be involved in formate metabolism in mammals. We were able to detect differences in the regulation of detoxification-associated genes between mites and honey bees as well as between the two different developmental stages of the honey bee. Additionally, we detected repressed regulation of Varroa genes involved in cellular respiration, suggesting mitochondrial dysfunction and supporting the current view on the mode of action of FA-inhibition of oxidative phosphorylation. This study shows distinct cellular effects induced by FA on the global transcriptome of both host and parasite in comparison. Our expression data might help to identify possible differences in the affected metabolic pathways and thus make a first contribution to elucidate the mode of detoxification of FA.

Herbivory and Time Since Flowering Shape Floral Rewards and Pollinator-Pathogen Interactions.

Herbivory can induce chemical changes throughout plant tissues including flowers, which could affect pollinator-pathogen interactions. Pollen is highly defended compared to nectar, but no study has examined whether herbivory affects pollen chemistry. We assessed the effects of leaf herbivory on nectar and pollen alkaloids in Nicotiana tabacum, and how herbivory-induced changes in nectar and pollen affect pollinator-pathogen interactions. We damaged leaves of Nicotiana tabacum using the specialist herbivore Manduca sexta and compared nicotine and anabasine concentrations in nectar and pollen. We then pooled nectar and pollen by collection periods (within and after one month of flowering), fed them in separate experiments to bumble bees (Bombus impatiens) infected with the gut pathogen Crithidia bombi, and assessed infections after seven days. We did not detect alkaloids in nectar, and leaf damage did not alter the effect of nectar on Crithidia counts. In pollen, herbivory induced higher concentrations of anabasine but not nicotine, and alkaloid concentrations rose and then fell as a function of days since flowering. Bees fed pollen from damaged plants had Crithidia counts 15 times higher than bees fed pollen from undamaged plants, but only when pollen was collected after one month of flowering, indicating that both damage and time since flowering affected interaction outcomes. Within undamaged treatments, bees fed late-collected pollen had Crithidia counts 10 times lower than bees fed early-collected pollen, also indicating the importance of time since flowering. Our results emphasize the role of herbivores in shaping pollen chemistry, with consequences for interactions between pollinators and their pathogens.

Defining Pollinator Health: A Holistic Approach Based on Ecological, Genetic, and Physiological Factors.

Evidence for global bee population declines has catalyzed a rapidly evolving area of research that aims to identify the causal factors and to effectively assess the status of pollinator populations. The term pollinator health emerged through efforts to understand causes of bee decline and colony losses, but it lacks a formal definition. In this review, we propose a definition for pollinator health and synthesize the available literature on the application of standardized biomarkers to assess health at the individual, colony, and population levels. We focus on biomarkers in honey bees, a model species, but extrapolate the potential application of these approaches to monitor the health status of wild bee populations. Biomarker-guided health measures can inform beekeeper management decisions, wild bee conservation efforts, and environmental policies. We conclude by addressing challenges to pollinator health from a One Health perspective that emphasizes the interplay between environmental quality and human, animal, and bee health.

The associations among the breeding performance of Osmia bicornis L. (Hymenoptera: Megachilidae), burden of pathogens and nest parasites along urbanisation gradient.

Urban environments may negatively affect the development of organisms. In host-pathogen/parasite systems, this impact may lead to increased manifestations of pathogens that decrease the success of their hosts in urban environments compared to rural ones. We tested this hypothesis in the solitary bee Osmia bicornis L. We estimated the development of bees, their reproductive success and the manifestation of different pathogens and nest parasites along an urbanisation gradient. We conducted the experiment in an urbanisation gradient in sites representing three environments: urban, suburban and rural. First, we analysed the manifestation of bacterial and fungal microorganisms in pollen loads, within dried/mummified individuals, on the surface of cocoons and on the surface of diapausing adult individuals by using the MALDI-TOF MS technique. We also verified genetic samples from diapausing individuals for the presence of the parasitic Nosema apis (Zandler, 1909) and N. ceranae (Fries et al., 1996) species. Finally, we assessed the level of reproductive success and manifestations of brood parasites. Not any biological material from the nests was infected by pathogenic microorganisms. This result indicates that the nests are not a reservoir of the pathogenic bacteria and that O. bicornis offspring are not a source or vectors of these pathogens to the surrounding environment and indirectly to other bee species. In urban sites, there was a lower number of parasites than in suburban and rural environments. The presence of parasites was negatively correlated with the reproductive success and may be a limiting factor for O. bicornis populations. We also found that urban sites had the highest indices of reproductive success and the lowest number of breeding failures compared to suburban and rural sites. Moreover, bacterial and fungal transmission is not a serious threat in the studied region.

Flagellum Removal by a Nectar Metabolite Inhibits Infectivity of a Bumblebee Parasite.

Plant phytochemicals can act as natural "medicines" for animals against parasites [1-3]. Some nectar metabolites, for example, reduce parasite infections in bees [4-7]. Declining plant diversity through anthropogenic landscape change [8-11] could reduce the availability of medicinal nectar plants for pollinators, exacerbating their decline [12]. Existing studies are, however, limited by (1) a lack of mechanistic insights into how phytochemicals affect pollinator diseases and (2) the restriction to few, commercially available chemicals, thereby potentially neglecting plants with the biggest antiparasitic effects. To rapidly identify plants with the greatest potential as natural bee medicines, we developed a bioactivity-directed fractionation assay for nectar metabolites. We evaluated 17 important nectar plants against the bumblebee pathogen Crithidia bombi (Trypanosomatidae) [13-17]. The most bioactive species was heather (Calluna vulgaris), the second most productive UK nectar plant [10]. We identified 4-(3-oxobut-1-enylidene)-3,5,5-trimethylcyclohex-2-en-1-one (callunene) from heather nectar as a potent inhibitor of C. bombi. Wild bumblebees (Bombus terrestris) foraging on heather ingest callunene at concentrations causing complete C. bombi inhibition. Feeding on callunene was prophylactic against infections. We show that C. bombi establishes infections by flagellar anchoring to the ileum epithelium. Short-term callunene exposure induced flagellum loss in C. bombi choanomastigotes, resulting in a loss of infectivity. We conclude that plant secondary metabolites can disrupt parasite flagellum attachment, revealing a mechanism behind their prophylactic effects. The decline of heathlands [18-21] reduces the availability of natural bee "medicine" and could exacerbate the contribution of diseases to pollinator declines. VIDEO ABSTRACT.

Extracts of Polypore Mushroom Mycelia Reduce Viruses in Honey Bees.

Waves of highly infectious viruses sweeping through global honey bee populations have contributed to recent declines in honey bee health. Bees have been observed foraging on mushroom mycelium, suggesting that they may be deriving medicinal or nutritional value from fungi. Fungi are known to produce a wide array of chemicals with antimicrobial activity, including compounds active against bacteria, other fungi, or viruses. We tested extracts from the mycelium of multiple polypore fungal species known to have antiviral properties. Extracts from amadou (Fomes) and reishi (Ganoderma) fungi reduced the levels of honey bee deformed wing virus (DWV) and Lake Sinai virus (LSV) in a dose-dependent manner. In field trials, colonies fed Ganoderma resinaceum extract exhibited a 79-fold reduction in DWV and a 45,000-fold reduction in LSV compared to control colonies. These findings indicate honey bees may gain health benefits from fungi and their antimicrobial compounds.

Medicinal value of sunflower pollen against bee pathogens.

Global declines in pollinators, including bees, can have major consequences for ecosystem services. Bees are dominant pollinators, making it imperative to mitigate declines. Pathogens are strongly implicated in the decline of native and honey bees. Diet affects bee immune responses, suggesting the potential for floral resources to provide natural resistance to pathogens. We discovered that sunflower (Helianthus annuus) pollen dramatically and consistently reduced a protozoan pathogen (Crithidia bombi) infection in bumble bees (Bombus impatiens) and also reduced a microsporidian pathogen (Nosema ceranae) of the European honey bee (Apis mellifera), indicating the potential for broad anti-parasitic effects. In a field survey, bumble bees from farms with more sunflower area had lower Crithidia infection rates. Given consistent effects of sunflower in reducing pathogens, planting sunflower in agroecosystems and native habitat may provide a simple solution to reduce disease and improve the health of economically and ecologically important pollinators.

Seasonal Effects and the Impact of In-Hive Pesticide Treatments on Parasite, Pathogens, and Health of Honey Bees.

Honey bee, Apis mellifera (L.; Hymenoptera: Apidae), populations are in decline and their losses pose a serious threat for crop pollination and food production. The specific causes of these losses are believed to be multifactorial. Pesticides, parasites and pathogens, and nutritional deficiencies have been implicated in the losses due to their ability to exert energetic stress on bees. While our understanding of the role of these factors in honey bee colony losses has improved, there is still a lack of knowledge of how they impact the immune system of the honey bee. In this study, honey bee colonies were exposed to Fumagilin-B, Apistan (tau-fluvalinate), and chlorothalonil at field realistic levels. No significant effects of the antibiotic and two pesticides were observed on the levels of varroa mite, Nosema ceranae (Fries; Microsporidia: Nosematidae), black queen cell virus, deformed wing virus, or immunity as measured by phenoloxidase and glucose oxidase activity. Any effects on the parasites, pathogens, and immunity we observed appear to be due mainly to seasonal changes within the honey bee colonies. The results suggest that Fumagilin-B, Apistan, and chlorothalonil do not significantly impact the health of honey bee colonies, based on the factors analyzed and the concentration of chemicals tested.

Effect of pollen extract supplementation on the varroatosis tolerance of honey bee (Apis mellifera) larvae reared in vitro.

As the main source of lipids and proteins in honey bees, pollen is a major nutrient provider involved in development and health and has been studied for tolerance stimulation against pathogens and parasites. In the case of Varroa destructor Anderson & Trueman (Acari, Mesostigmata: Varroidae) parasitization, the lack of a complete laboratory system to rear both the bee larva and the acarian parasite limited the studies concerning larval nutrition effects on the bee tolerance and resistance against varroatosis. Due to the development of this complete rearing protocol, we managed to feed young honey bee larvae with pollen supplemented solutions and to study the effect on their later development under parasitism conditions. In our experimental conditions, pollen influences neither the deformity rate, nor the survival of bees both parasitized and unparasitized. However, pollen extract supplementation seems to significantly impact the weight of the spinning bee larvae without having an effect on the physiological weight loss during pupation, so the differences found at the larval stage remain the same as at emergence. Varroa has a deleterious effect on bee pupae and led to a steady increase of the physiological weight loss experienced during metamorphosis. Interestingly, this ponderal loss associated with Varroa parasitization seems to be reduced in the polyfloral pollen supplementation condition. Altogether, this work is to our knowledge the first to study in laboratory conditions the impact of larval nutrition on the tolerance to parasitism. A diverse pollen diet may be beneficial to the bees' tolerance against V. destructor parasitism.

Elucidating the mechanisms underlying the beneficial health effects of dietary pollen on honey bees (Apis mellifera) infested by Varroa mite ectoparasites.

Parasites and pathogens of the honey bee (Apis mellifera) are key factors underlying colony losses, which are threatening the beekeeping industry and agriculture as a whole. To control the spread and development of pathogen infections within the colony, honey bees use plant resins with antibiotic activity, but little is known about the properties of other substances, that are mainly used as a foodstuff, for controlling possible diseases both at the individual and colony level. In this study, we tested the hypothesis that pollen is beneficial for honey bees challenged with the parasitic mite Varroa destructor associated to the Deformed Wing Virus. First, we studied the effects of pollen on the survival of infested bees, under laboratory and field conditions, and observed that a pollen rich diet can compensate the deleterious effects of mite parasitization. Subsequently, we characterized the pollen compounds responsible for the observed positive effects. Finally, based on the results of a transcriptomic analysis of parasitized bees fed with pollen or not, we developed a comprehensive framework for interpreting the observed effects of pollen on honey bee health, which incorporates the possible effects on cuticle integrity, energetic metabolism and immune response.

Continuous release of oregano oil effectively and safely controls Varroa destructor infestations in honey bee colonies in a northern climate.

The ectoparasitic mite Varroa destructor is responsible for the death of millions of honey bee (Apis mellifera) colonies worldwide. Testing potential miticide compounds with different delivery methods that effectively control V. destructor and have low toxicity for honey bees is crucial to manage this parasite in hives. We determined the varroacide efficacy of three natural compounds delivered to hives with three application methods over a 4-week period. Oxalic acid in a sucrose solution was applied impregnated in cardboard (T1). A mixture of oregano and clove oils in an ethanol-gelatin solution was applied impregnated in absorbent pads (T2). Oregano oil alone was delivered using electric vaporizers (T3) to test the hypothesis that continuous release of miticides increases the varroacidal efficacy of essential oils. The varroa mite control rates for treatments T1-T3 were 76.5 ± 7.11, 57.8 ± 12.79 and 97.4 ± 0.68%, respectively, and there were no differences for bee mortality between control and treatments 1 and 3. Additionally, most mites were killed in the first 2 weeks in T3 colonies compared to the last 2 weeks in colonies of the other treatments. These results demonstrate the importance of continuously releasing natural miticides to achieve safe and high rates of mite control in hives. They also show that oregano oil may be an effective miticide against V. destructor infestations in colonies.

A 'Landscape physiology' approach for assessing bee health highlights the benefits of floral landscape enrichment and semi-natural habitats.

Understanding how anthropogenic landscape alteration affects populations of ecologically- and economically-important insect pollinators has never been more pressing. In this context, the assessment of landscape quality typically relies on spatial distribution studies, but, whether habitat-restoration techniques actually improve the health of targeted pollinator populations remains obscure. This gap could be filled by a comprehensive understanding of how gradients of landscape quality influence pollinator physiology. We therefore used this approach for honey bees (Apis mellifera) to test whether landscape patterns can shape bee health. We focused on the pre-wintering period since abnormally high winter colony losses have often been observed. By exposing colonies to different landscapes, enriched in melliferous catch crops and surrounded by semi-natural habitats, we found that bee physiology (i.e. fat body mass and level of vitellogenin) was significantly improved by the presence of flowering catch crops. Catch crop presence was associated with a significant increase in pollen diet diversity. The influence of semi-natural habitats on bee health was even stronger. Vitellogenin level was in turn significantly linked to higher overwintering survival. Therefore, our experimental study, combining landscape ecology and bee physiology, offers an exciting proof-of-concept for directly identifying stressful or suitable landscapes and promoting efficient pollinator conservation.

The glass is not yet half empty: agitation but not Varroa treatment causes cognitive bias in honey bees.

Honey bees (Apis mellifera) are prone to judge an ambiguous stimulus negatively if they had been agitated through shaking which simulates a predator attack. Such a cognitive bias has been suggested to reflect an internal emotional state analogous to humans who judge more pessimistically when they do not feel well. In order to test cognitive bias experimentally, an animal is conditioned to respond to two different stimuli, where one is punished while the other is rewarded. Subsequently a third, ambiguous stimulus is presented and it is measured whether the subject responds as if it expects a reward or a punishment. Generally, it is assumed that negative experiences lower future expectations, rendering the animals more pessimistic. Here we tested whether a most likely negatively experienced formic acid treatment against the parasitic mite Varroa destructor also affects future expectations of honey bees. We applied an olfactory learning paradigm (i.e., conditioned proboscis extension response) using two odorants and blends of these odorants as the ambiguous stimuli. Unlike agitating honey bees, exposure to formic acid did not significantly change the response to the ambiguous stimuli in comparison with untreated bees. Overall evidence suggests that the commonest treatment against one of the most harmful bee pests has no detrimental effects on cognitive bias in honey bees.

Chemical Composition and Acaricidal Activity of Thymus algeriensis Essential Oil against Varroa destructor.

The aim of the present study was to determine the chemical composition and evaluate the acaricidal activity of Thymus algeriensis essential oil (TAEO) against Varroa destructor. This ectoparasitic mite is a pest of the honey bee Apis mellifera. The essential oil from the, aerial parts of T. algeriensis, obtained by hydrodistillation, was obtained in a yield of 2.8± 0.2%, w/w. The TAEO was analyzed by GC and GC/MS. Thirty-four compounds were identified, representing 99.3% of the oil. The main constituents were carvacrol (48.4%), γ-terpinene (14.9%), p-cymene (14.7%), and thymol (5.6%). Four lots 'were constituted at the level of an apiary in order to study the dynamics of the Varroa destructor and its host, Apis mellifera. After diagnosis by the.biological method "install of diapers", the lots were treated at different doses of TAEO (0.1, 0.3 and 0.5%). TAEO was sprayed on top of the hives. The results show that TAEO at 0.5% resulted in a decrease in the rate of infestation of Varroa destructor, causing a mortality rate of 32.6% without negative effect on the nesting of the queen. The essential oil of T. algeriensis could be used as a bioacaricidal agent.

Probiotic Treatment with a Gut Symbiont Leads to Parasite Susceptibility in Honey Bees.

Gut symbionts are critical for host health and as such might be used as probiotics. In a recent study, Schwarz et al. showed that pretreatment of honey bees with a dominant gut bacterium causes dysbiosis and increases pathogen susceptibility, showing that probiotic applications for animal health can have unwanted effects.

Food Limitation Affects Parasite Load and Survival of Bombus impatiens (Hymenoptera: Apidae) Infected With Crithidia (Trypanosomatida: Trypanosomatidae).

Bumble bees (genus Bombus) are globally important insect pollinators, and several species have experienced marked declines in recent years. Both nutritional limitation and pathogens may have contributed to these declines. While each of these factors may be individually important, there may also be synergisms where nutritional stress could decrease pathogen resistance. Understanding interactions between bumble bees, their parasites, and food availability may provide new insight into the causes of declines. In this study, we examined the combined impacts of pollen and nectar limitation on Crithidia, a common gut parasite in Bombus impatiens Cresson. Individual worker bees were inoculated with Crithidia and then assigned in a factorial design to two levels of pollen availability (pollen or no pollen) and two nectar sugar concentrations (high [30%] or low [15%] sucrose). We found that lack of pollen and low nectar sugar both reduced Crithidia cell counts, with the most dramatic effect from lack of pollen. Both pollen availability and nectar sugar concentration were also important for bee survival. The proportion of bees that died after seven days of infection was ∼25% lower in bees with access to pollen and high nectar sugar concentration than any other treatment. Thus, nectar and pollen availability are both important for bee survival, but may come at a cost of higher parasite loads. Our results illustrate the importance of understanding environmental context, such as resource availability, when examining a host-parasite interaction.

Asteraceae Pollen Provisions Protect Osmia Mason Bees (Hymenoptera: Megachilidae) from Brood Parasitism.

Many specialist herbivores eat foods that are apparently low quality. The compensatory benefits of a poor diet may include protection from natural enemies. Several bee lineages specialize on pollen of the plant family Asteraceae, which is known to be a poor-quality food. Here we tested the hypothesis that specialization on Asteraceae pollen protects bees from parasitism. We compared rates of brood parasitism by Sapyga wasps on Asteraceae-specialist, Fabeae-specialist, and other species of Osmia bees in the field over several years and sites and found that Asteraceae-specialist species were parasitized significantly less frequently than other species. We then tested the effect of Asteraceae pollen on parasites by raising Sapyga larvae on three pollen mixtures: Asteraceae, Fabeae, and generalist (a mix of primarily non-Asteraceae pollens). Survival of parasite larvae was significantly reduced on Asteraceae provisions. Our results suggest that specialization on low-quality pollen may evolve because it helps protect bees from natural enemies.

Hygienic food to reduce pathogen risk to bumblebees.

Bumblebees are ecologically and economically important pollinators, and the value of bumblebees for crop pollination has led to the commercial production and exportation/importation of colonies on a global scale. Commercially produced bumblebee colonies can carry with them infectious parasites, which can both reduce the health of the colonies and spillover to wild bees, with potentially serious consequences. The presence of parasites in commercially produced bumblebee colonies is in part because colonies are reared on pollen collected from honey bees, which often contains a diversity of microbial parasites. In response to this threat, part of the industry has started to irradiate pollen used for bumblebee rearing. However, to date there is limited data published on the efficacy of this treatment. Here we examine the effect of gamma irradiation and an experimental ozone treatment on the presence and viability of parasites in honey bee pollen. While untreated pollen contained numerous viable parasites, we find that gamma irradiation reduced the viability of parasites in pollen, but did not eliminate parasites entirely. Ozone treatment appeared to be less effective than gamma irradiation, while an artificial pollen substitute was, as expected, entirely free of parasites. The results suggest that the irradiation of pollen before using it to rear bumblebee colonies is a sensible method which will help reduce the incidence of parasite infections in commercially produced bumblebee colonies, but that further optimisation, or the use of a nutritionally equivalent artificial pollen substitute, may be needed to fully eliminate this route of disease entry into factories.