Virus Prevalence in Egg Samples Collected from Naturally Selected and Traditionally Managed Honey Bee Colonies across Europe.

Monitoring virus infections can be an important selection tool in honey bee breeding. A recent study pointed towards an association between the virus-free status of eggs and an increased virus resistance to deformed wing virus (DWV) at the colony level. In this study, eggs from both naturally surviving and traditionally managed colonies from across Europe were screened for the prevalence of different viruses. Screenings were performed using the phenotyping protocol of the 'suppressed in ovo virus infection' trait but with qPCR instead of end-point PCR and a primer set that covers all DWV genotypes. Of the 213 screened samples, 109 were infected with DWV, 54 were infected with black queen cell virus (BQCV), 3 were infected with the sacbrood virus, and 2 were infected with the acute bee paralyses virus. It was demonstrated that incidences of the vertical transmission of DWV were more frequent in naturally surviving than in traditionally managed colonies, although the virus loads in the eggs remained the same. When comparing virus infections with queen age, older queens showed significantly lower infection loads of DWV in both traditionally managed and naturally surviving colonies, as well as reduced DWV infection frequencies in traditionally managed colonies. We determined that the detection frequencies of DWV and BQCV in honey bee eggs were lower in samples obtained in the spring than in those collected in the summer, indicating that vertical transmission may be lower in spring. Together, these patterns in vertical transmission show that honey bee queens have the potential to reduce the degree of vertical transmission over time.

Using Citizen Science to Scout Honey Bee Colonies That Naturally Survive Varroa destructor Infestations.

Citizen Science contributes significantly to the conservation of biodiversity, but its application to honey bee research has remained minimal. Even though certain European honey bee (Apis mellifera) populations are known to naturally survive Varroa destructor infestations, it is unclear how widespread or common such populations are. Such colonies are highly valuable for investigating the mechanisms enabling colony survival, as well as for tracking the conservation status of free-living honey bees. Here, we use targeted Citizen Science to identify potentially new cases of managed or free-living A. mellifera populations that survive V. destructor without mite control strategies. In 2018, a survey containing 20 questions was developed, translated into 13 languages, and promoted at beekeeping conferences and online. After three years, 305 reports were collected from 28 countries: 241 from managed colonies and 64 from free-living colonies. The collected data suggest that there could be twice as many naturally surviving colonies worldwide than are currently known. Further, online and personal promotion seem to be key for successful recruitment of participants. Although the survivor status of these colonies still needs to be confirmed, the volume of reports and responses already illustrate how effectively Citizen Science can contribute to bee research by massively increasing generated data, broadening opportunities for comparative research, and fostering collaboration between scientists, beekeepers, and citizens. The success of this survey spurred the development of a more advanced Citizen Science platform, Honey Bee Watch, that will enable a more accurate reporting, confirmation, and monitoring of surviving colonies, and strengthen the ties between science, stakeholders, and citizens to foster the protection of both free-living and managed honey bees.

Adaptive population structure shifts in invasive parasitic mites, Varroa destructor.

Comparative studies of genetic diversity and population structure can shed light on the ecological and evolutionary factors governing host-parasite interactions. Even though invasive parasites are considered of major biological importance, little is known about their adaptative potential when infesting the new hosts. Here, the genetic diversification of Varroa destructor, a novel parasite of Apis mellifera originating from Asia, was investigated using population genetics to determine how the genetic structure of the parasite changed in distinct European populations of its new host. To do so, mites infesting two categories of hosts in four European regions were compared: (a) adapted hosts surviving through means of natural selection, thereby expected to impose strong selective pressure on the mites, and (b) treated host populations, surviving mite infestations because acaricides are applied, therefore characterized by a relaxed selection imposed by the host on the mites. Significant genetic divergence was found across regions, partially reflecting the invasion pattern of V. destructor throughout Europe and indicating local adaptation of the mite to the host populations. Additionally, varying degrees of genotypic changes were found between mites from adapted and treated colonies. Altogether, these results indicate that V. destructor managed to overcome the genetic bottlenecks following its introduction in Europe and that host-mediated selection fostered changes in the genetic structure of this mite at diverse geographic scales. These findings highlight the potential of parasites to adapt to their local host populations and confirm that adaptations developed within coevolutionary dynamics are a major determinant of population genetic changes.

Adapted tolerance to virus infections in four geographically distinct Varroa destructor-resistant honeybee populations.

The ectoparasitic mite, Varroa destructor, is unarguably the leading cause of honeybee (Apis mellifera) mortality worldwide through its role as a vector for lethal viruses, in particular, strains of the Deformed wing virus (DWV) and Acute bee paralysis virus (ABPV) complexes. Several honeybee populations across Europe have well-documented adaptations of mite-resistant traits but little is known about host adaptations towards the virus infections vectored by the mite. The aim of this study was to assess and compare the possible contribution of adapted virus tolerance and/or resistance to the enhanced survival of four well-documented mite-resistant honeybee populations from Norway, Sweden, The Netherlands and France, in relation to unselected mite-susceptible honeybees. Caged adult bees and laboratory reared larvae, from colonies of these four populations, were inoculated with DWV and ABPV in a series of feeding infection experiments, while control groups received virus-free food. Virus infections were monitored using RT-qPCR assays in individuals sampled over a time course. In both adults and larvae the DWV and ABPV infection dynamics were nearly identical in all groups, but all mite-resistant honeybee populations had significantly higher survival rates compared to the mite-susceptible honeybees. These results suggest that adapted virus tolerance is an important component of survival mechanisms.

Adaptation to vector-based transmission in a honeybee virus.

Global pollinator declines as a result of emerging infectious diseases are of major concern. Managed honeybees Apis mellifera are susceptible to numerous parasites and pathogens, many of which appear to be transmissible to sympatric non-Apis taxa. The ectoparasitic mite Varroa destructor is considered to be the most significant threat to honeybees due to its role in vectoring RNA viruses, particularly Deformed wing virus (DWV). Vector transmission of DWV has resulted in the accumulation of high viral loads in honeybees and is often associated with colony death. DWV has two main genotypes, A and B. DWV-A was more prevalent during the initial phase of V. destructor establishment. In recent years, the global prevalence of DWV-B has increased, suggesting that DWV-B is better adapted to vector transmission than DWV-A. We aimed to determine the role vector transmission plays in DWV genotype prevalence at a colony level. We experimentally increased or decreased the number of V. destructor mites in honeybee colonies, and tracked DWV-A and DWV-B loads over a period of 10 months. Our results show that the two DWV genotypes differ in their response to mite numbers. DWV-A accumulation in honeybees was positively correlated with mite numbers yet DWV-A was largely undetected in the absence of the mite. In contrast, colonies had high loads of DWV-B even when mite numbers were low. DWV-B loads persisted in miticide-treated colonies, indicating that this genotype has a competitive advantage over DWV-A irrespective of mite numbers. Our findings suggest that the global increase in DWV-B prevalence is not driven by selective pressure by the vector. Rather, DWV-B is able to persist in colonies at higher viral loads relative to DWV-A in the presence and absence of V. destructor. The interplay between V. destructor and DWV genotypes within honeybee colonies may have broad consequences upon viral diversity in sympatric taxa as a result of spillover.

Host-Parasite Co-Evolution in Real-Time: Changes in Honey Bee Resistance Mechanisms and Mite Reproductive Strategies.

Co-evolution is a major driving force shaping the outcome of host-parasite interactions over time. After host shifts, the lack of co-evolution can have a drastic impact on novel host populations. Nevertheless, it is known that Western honey bee (Apismellifera) populations can cope with host-shifted ectoparasitic mites (Varroa destructor) by means of natural selection. However, adaptive phenotypic traits of the parasites and temporal variations in host resistance behavior are poorly understood. Here, we show that mites made adaptive shifts in reproductive strategy when associated with resistant hosts and that host resistance traits can change over time. In a fully-crossed field experiment, worker brood cells of local adapted and non-adapted (control) A.mellifera host colonies were infested with mites originating from both types of host colonies. Then, mite reproduction as well as recapping of cells and removal of infested brood (i.e., Varroa Sensitive Hygiene, VSH) by host workers were investigated and compared to data from the same groups of host colonies three years earlier. The data suggest adaptive shifts in mite reproductive strategies, because mites from adapted hosts have higher probabilities of reproduction, but lower fecundity, when infesting their associated hosts than mites in treated colonies. The results confirm that adapted hosts can reduce mite reproductive success. However, neither recapping of cells nor VSH were significantly expressed, even though the latter was significantly expressed in this adapted population three years earlier. This suggests temporal variation in the expression of adaptive host traits. It also appears as if mechanisms not investigated here were responsible for the reduced mite reproduction in the adapted hosts. In conclusion, a holistic view including mite adaptations and studies of the same parasite/host populations over time appears overdue to finally understand the mechanisms enabling survival of V.destructor-infested honey bee host colonies.

Honey bee predisposition of resistance to ubiquitous mite infestations.

Host-parasite co-evolution history is lacking when parasites switch to novel hosts. This was the case for Western honey bees (Apis mellifera) when the ectoparasitic mite, Varroa destructor, switched hosts from Eastern honey bees (Apis cerana). This mite has since become the most severe biological threat to A. mellifera worldwide. However, some A. mellifera populations are known to survive infestations, largely by suppressing mite population growth. One known mechanism is suppressed mite reproduction (SMR), but the underlying genetics are poorly understood. Here, we take advantage of haploid drones, originating from one queen from the Netherlands that developed Varroa-resistance, whole exome sequencing and elastic-net regression to identify genetic variants associated with SMR in resistant honeybees. An eight variants model predicted 88% of the phenotypes correctly and identified six risk and two protective variants. Reproducing and non-reproducing mites could not be distinguished using DNA microsatellites, which is in agreement with the hypothesis that it is not the parasite but the host that adapted itself. Our results suggest that the brood pheromone-dependent mite oogenesis is disrupted in resistant hosts. The identified genetic markers have a considerable potential to contribute to a sustainable global apiculture.

A Diverse Range of Novel RNA Viruses in Geographically Distinct Honey Bee Populations.

Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees (Apis mellifera) has changed dramatically since the emergence of the parasitic mite Varroa destructor, which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management.IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales Collapsing Varroa-infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa-resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator.

Three years of banning neonicotinoid insecticides based on sub-lethal effects: can we expect to see effects on bees?

The 2013 EU ban of three neonicotinoids used in seed coating of pollinator attractive crops was put in place because of concern about declining wild pollinator populations and numbers of honeybee colonies. It was also concluded that there is an urgent need for good field data to fill knowledge gaps. In the meantime such data have been generated. Based on recent literature we question the existence of recent pollinator declines and their possible link with the use of neonicotinoids. Because of temporal non-coincidence we conclude that declines of wild pollinators and of honeybees are not likely caused by neonicotinoids. Even if bee decline does occur and if there is a causal relationship with the use of neonicotinoids, we argue that it is not possible on such short term to evaluate the effects of the 2013 ban. In order to supply future debate with realistic (field) data and to discourage extrapolating the effects of studies using overdoses that are not of environmental relevance, we propose - in addition to field studies performed by the chemical industry - to use the 'semi-field worst case' treated artificial diet studies approach to free flying colonies in the field. This kind of study may provide realistic estimates for risk and be useful to study realistic interactions with non-pesticide stressors. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Darwin cure for apiculture? Natural selection and managed honeybee health.

Recent major losses of managed honeybee, Apis mellifera, colonies at a global scale have resulted in a multitude of research efforts to identify the underlying mechanisms. Numerous factors acting singly and/or in combination have been identified, ranging from pathogens, over nutrition to pesticides. However, the role of apiculture in limiting natural selection has largely been ignored. This is unfortunate, because honeybees are more exposed to environmental stressors compared to other livestock and management can severely compromise bee health. Here, we briefly review apicultural factors that influence bee health and focus on those most likely interfering with natural selection, which offers a broad range of evolutionary applications for field practice. Despite intense breeding over centuries, natural selection appears to be much more relevant for the health of managed A. mellifera colonies than previously thought. We conclude that sustainable solutions for the apicultural sector can only be achieved by taking advantage of natural selection and not by attempting to limit it.

A restatement of recent advances in the natural science evidence base concerning neonicotinoid insecticides and insect pollinators.

A summary is provided of recent advances in the natural science evidence base concerning the effects of neonicotinoid insecticides on insect pollinators in a format (a 'restatement') intended to be accessible to informed but not expert policymakers and stakeholders. Important new studies have been published since our recent review of this field (Godfray et al. 2014 Proc. R. Soc. B 281, 20140558. (doi:10.1098/rspb.2014.0558)) and the subject continues to be an area of very active research and high policy relevance.

A restatement of the natural science evidence base concerning neonicotinoid insecticides and insect pollinators.

There is evidence that in Europe and North America many species of pollinators are in decline, both in abundance and distribution. Although there is a long list of potential causes of this decline, there is concern that neonicotinoid insecticides, in particular through their use as seed treatments are, at least in part, responsible. This paper describes a project that set out to summarize the natural science evidence base relevant to neonicotinoid insecticides and insect pollinators in as policy-neutral terms as possible. A series of evidence statements are listed and categorized according to the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

Winter survival of individual honey bees and honey bee colonies depends on level of Varroa destructor infestation.

BACKGROUND: Recent elevated winter loss of honey bee colonies is a major concern. The presence of the mite Varroa destructor in colonies places an important pressure on bee health. V. destructor shortens the lifespan of individual bees, while long lifespan during winter is a primary requirement to survive until the next spring. We investigated in two subsequent years the effects of different levels of V. destructor infestation during the transition from short-lived summer bees to long-lived winter bees on the lifespan of individual bees and the survival of bee colonies during winter. Colonies treated earlier in the season to reduce V. destructor infestation during the development of winter bees were expected to have longer bee lifespan and higher colony survival after winter. METHODOLOGY/PRINCIPAL FINDINGS: Mite infestation was reduced using acaricide treatments during different months (July, August, September, or not treated). We found that the number of capped brood cells decreased drastically between August and November, while at the same time, the lifespan of the bees (marked cohorts) increased indicating the transition to winter bees. Low V. destructor infestation levels before and during the transition to winter bees resulted in an increase in lifespan of bees and higher colony survival compared to colonies that were not treated and that had higher infestation levels. A variety of stress-related factors could have contributed to the variation in longevity and winter survival that we found between years. CONCLUSIONS/SIGNIFICANCE: This study contributes to theory about the multiple causes for the recent elevated colony losses in honey bees. Our study shows the correlation between long lifespan of winter bees and colony loss in spring. Moreover, we show that colonies treated earlier in the season had reduced V. destructor infestation during the development of winter bees resulting in longer bee lifespan and higher colony survival after winter.

Neonicotinoids in bees: a review on concentrations, side-effects and risk assessment.

Neonicotinoid insecticides are successfully applied to control pests in a variety of agricultural crops; however, they may not only affect pest insects but also non-target organisms such as pollinators. This review summarizes, for the first time, 15 years of research on the hazards of neonicotinoids to bees including honey bees, bumble bees and solitary bees. The focus of the paper is on three different key aspects determining the risks of neonicotinoid field concentrations for bee populations: (1) the environmental neonicotinoid residue levels in plants, bees and bee products in relation to pesticide application, (2) the reported side-effects with special attention for sublethal effects, and (3) the usefulness for the evaluation of neonicotinoids of an already existing risk assessment scheme for systemic compounds. Although environmental residue levels of neonicotinoids were found to be lower than acute/chronic toxicity levels, there is still a lack of reliable data as most analyses were conducted near the detection limit and for only few crops. Many laboratory studies described lethal and sublethal effects of neonicotinoids on the foraging behavior, and learning and memory abilities of bees, while no effects were observed in field studies at field-realistic dosages. The proposed risk assessment scheme for systemic compounds was shown to be applicable to assess the risk for side-effects of neonicotinoids as it considers the effect on different life stages and different levels of biological organization (organism versus colony). Future research studies should be conducted with field-realistic concentrations, relevant exposure and evaluation durations. Molecular markers may be used to improve risk assessment by a better understanding of the mode of action (interaction with receptors) of neonicotinoids in bees leading to the identification of environmentally safer compounds.

Honeybee interference as a novel aid to reduce pollen exposure and nasal symptoms among greenhouse workers allergic to sweet bell pepper (Capsicum annuum) pollen.

BACKGROUND: In 1999, an extensive study among bell pepper growers showed a prevalence of 53.8% work-related symptoms and 35.4% sensitisation to bell pepper pollen. Experiments with bees to remove pollen from bell pepper flowers have been done for a few years. OBJECTIVES: It was the aim of this study to investigate whether bees can reduce the pollen exposure in bell pepper greenhouses and whether this reduction results in a decrease in allergic complaints in the greenhouse workers. METHODS: Eighteen greenhouses were selected for the study. In each greenhouse, pollen exposure was estimated. In 6 and 3 of the greenhouses, high, respectively low, numbers of honeybees were placed throughout the pollen season of the sweet bell pepper plant. Nine greenhouses without honeybees were used as control. At baseline, as well as after 4 and 8 months, nasal symptoms assessed with a visual analogue score and lung function parameters were used as outcome measure. RESULTS: Forty-four of the 133 employees invited reported work-related symptoms. High numbers of bees reduced the pollen amount in a dose-dependent way to 18% of the baseline exposure. A significant trend relationship between the visual analogue scale in nasal symptoms and the number of colonies of bees was seen. CONCLUSIONS: The interference of bees in bell pepper greenhouses significantly reduces the pollen amount. This reduction is associated with less work-related rhinitis symptoms in allergic greenhouse workers. This intervention study supports the hypothesis that allergic work-related complaints of greenhouse workers, sensitised to bell pepper pollen, are caused by occupational exposure to this pollen in the greenhouse.