Knock‐on community impacts of a novel vector: spillover of emerging DWV‐B from Varroa‐infested honeybees to wild bumblebees

Novel transmission routes can directly impact the evolutionary ecology of infectious diseases, with potentially dramatic effect on host populations and knock‐on effects on the wider host community. The invasion of Varroa destructor, an ectoparasitic viral vector in Western honeybees, provides a unique opportunity to examine how a novel vector affects disease epidemiology in a host community. This specialist honeybee mite vectors deformed wing virus (DWV), an important re‐emerging honeybee pathogen that also infects wild bumblebees. Comparing island honeybee and wild bumblebee populations with and without V. destructor, we show that V. destructor drives DWV prevalence and titre in honeybees and sympatric bumblebees. Viral genotypes are shared across hosts, with the potentially more virulent DWV‐B overtaking DWV‐A in prevalence in a current epidemic. This demonstrates disease emergence across a host community driven by the acquisition of a specialist novel transmission route in one host, with dramatic community level knock‐on effects.     

Relationships among leaf functional traits,litter traits,and mass loss during early phases of leaf litter decomposition in 12 woody plant species

Slow bee paralysis virus (SBPV)—previously considered an obligate honeybee disease—is now known to be prevalent in bumblebee species. SBPV is highly virulent in honeybees in association with Varroa mites, but has been considered relatively benign otherwise. However, condition-dependent pathogens can appear asymptomatic under good, resource abundant conditions, and negative impacts on host fitness may only become apparent when under stressful or resource-limited conditions. We tested whether SBPV expresses condition-dependent virulence in its bumblebee host, Bombus terrestris, by orally inoculating bees with SBPV and recording longevity under satiated and starvation conditions. SBPV infection resulted in significant virulence under starvation conditions, with infected bees 1.6 times more likely to die at any given time point (a median of 2.3 h earlier than uninfected bees), whereas there was no effect under satiated conditions. This demonstrates clear condition-dependent virulence for SBPV in B. terrestris. Infections that appear asymptomatic in non-stressful laboratory assays may nevertheless have significant impacts under natural conditions in the wild. For multi-host pathogens such as SBPV, the use of sentinel host species in laboratory assays may further lead to the underestimation of pathogen impacts on other species in nature. In this case the impact of ‘honeybee viruses’ on wild pollinators may be underestimated, with detrimental effects on conservation and food security. Our results highlight the importance of multiple assays and multiple host species when testing for virulence, in order for laboratory studies to accurately inform conservation policy and mitigate disease impacts in wild pollinators.     

On the Front Line: Quantitative Virus Dynamics in Honeybee (Apis mellifera L.) Colonies along a New Expansion Front of the Parasite Varroa destructor

Over the past fifty years, annual honeybee (Apis mellifera) colony losses have been steadily increasing worldwide. These losses have occurred in parallel with the global spread of the honeybee parasite Varroa destructor. Indeed, Varroa mite infestations are considered to be a key explanatory factor for the widespread increase in annual honeybee colony mortality. The host-parasite relationship between honeybees and Varroa is complicated by the mite''s close association with a range of honeybee viral pathogens. The 10-year history of the expanding front of Varroa infestation in New Zealand offered a rare opportunity to assess the dynamic quantitative and qualitative changes in honeybee viral landscapes in response to the arrival, spread and level of Varroa infestation. We studied the impact of de novo infestation of bee colonies by Varroa on the prevalence and titres of seven well-characterised honeybee viruses in both bees and mites, using a large-scale molecular ecology approach. We also examined the effect of the number of years since Varroa arrival on honeybee and mite viral titres. The dynamic shifts in the viral titres of black queen cell virus and Kashmir bee virus mirrored the patterns of change in Varroa infestation rates along the Varroa expansion front. The deformed wing virus (DWV) titres in bees continued to increase with Varroa infestation history, despite dropping infestation rates, which could be linked to increasing DWV titres in the mites. This suggests that the DWV titres in mites, perhaps boosted by virus replication, may be a major factor in maintaining the DWV epidemic after initial establishment. Both positive and negative associations were identified for several pairs of viruses, in response to the arrival of Varroa. These findings provide important new insights into the role of the parasitic mite Varroa destructor in influencing the viral landscape that affects honeybee colonies.     

Viral transmission in honey bees and native bees,supported by a global black queen cell virus phylogeny

In recent decades, we have realized that honey bee viruses are not, in fact, exclusive to honey bees. The potential impact of Apis-affiliated viruses on native pollinators is prompting concern. Our research addresses the issue of virus crossover between honey bees and native bees foraging in the same localities. We measured the presence of black queen cell virus (BQCV), deformed wing virus (DWV) and sacbrood virus (SBV) in managed Apis mellifera (honey bees) and native Andrena spp. (subgenus Melandrena) bee populations in five commercial orchards. We identified viral presence across sites and bees and related these data to measures of bee community diversity. All viruses were found in both managed and native bees, and BQCV was the most common virus in each. To establish evidence for viral crossover between taxa, we undertook an additional examination of BQCV where 74 samples were sequenced and placed in a global phylogenic framework of hundreds of BQCV strains. We demonstrate pathogen sharing across managed honey bees and distantly related wild bees. This phylogenetic analysis contributes to growing evidence for host switching and places local incidence patterns in a worldwide context, revealing multispecies viral transmission.     

A gene for resistance to the Varroa mite (Acari) in honey bee (Apis mellifera) pupae

Social insect colonies possess a range of defences which protect them against highly virulent parasites and colony collapse. The host–parasite interaction between honey bees (Apis mellifera) and the mite Varroa destructor is unusual, as honey bee colonies are relatively poorly defended against this parasite. The interaction has existed since the mid‐20th Century, when Varroa switched host to parasitize A. mellifera. The combination of a virulent parasite and relatively naïve host means that, without acaricides, honey bee colonies typically die within 3 years of Varroa infestation. A consequence of acaricide use has been a reduced selective pressure for the evolution of Varroa resistance in honey bee colonies. However, in the past 20 years, several natural‐selection‐based breeding programmes have resulted in the evolution of Varroa‐resistant populations. In these populations, the inhibition of Varroa's reproduction is a common trait. Using a high‐density genome‐wide association analysis in a Varroa‐resistant honey bee population, we identify an ecdysone‐induced gene significantly linked to resistance. Ecdysone both initiates metamorphosis in insects and reproduction in Varroa. Previously, using a less dense genetic map and a quantitative trait loci analysis, we have identified Ecdysone‐related genes at resistance loci in an independently evolved resistant population. Varroa cannot biosynthesize ecdysone but can acquire it from its diet. Using qPCR, we are able to link the expression of ecdysone‐linked resistance genes to Varroa's meals and reproduction. If Varroa co‐opts pupal compounds to initiate and time its own reproduction, mutations in the host's ecdysone pathway may represent a key selection tool for honey bee resistance and breeding.     

New evidence that deformed wing virus and black queen cell virus are multi-host pathogens

The host-range breadth of pathogens can have important consequences for pathogens' long term evolution and virulence, and play critical roles in the emergence and spread of the new diseases. Black queen cell virus (BQCV) and Deformed wing virus (DWV) are the two most common and prevalent viruses in European honey bees, Apis mellifera. Here we provide the evidence that BQCV and DWV infect wild species of honey bees, Apis florea and Apis dorsata. Phylogenetic analyses suggest that these viruses might have moved from A. mellifera to wild bee species and that genetic relatedness as well as the geographical proximity of host species likely play an important role in host range of the viruses. The information obtained from this present study can have important implication for understanding the population structure of bee virus as well as host-virus interactions.     

Bidirectional Transfer of RNAi between Honey Bee and Varroa destructor: Varroa Gene Silencing Reduces Varroa Population

The mite Varroa destructor is an obligatory ectoparasite of the honey bee (Apis mellifera) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the Varroa mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and Varroa engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to Varroa control.     

Deformed wing virus

Deformed wing virus (DWV; Iflaviridae) is one of many viruses infecting honeybees and one of the most heavily investigated due to its close association with honeybee colony collapse induced by Varroa destructor. In the absence of V. destructor DWV infection does not result in visible symptoms or any apparent negative impact on host fitness. However, for reasons that are still not fully understood, the transmission of DWV by V. destructor to the developing pupae causes clinical symptoms, including pupal death and adult bees emerging with deformed wings, a bloated, shortened abdomen and discolouration. These bees are not viable and die soon after emergence. In this review we will summarize the historical and recent data on DWV and its relatives, covering the genetics, pathobiology, and transmission of this important viral honeybee pathogen, and discuss these within the wider theoretical concepts relating to the genetic variability and population structure of RNA viruses, the evolution of virulence and the development of disease symptoms.     

Prevalence and Seasonal Variations of Six Bee Viruses in Apis mellifera L. and Varroa destructor Mite Populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Deformed wing virus associated with <Emphasis Type="Italic">Tropilaelaps mercedesae</Emphasis> infesting European honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>)

Mites in the genus Tropilaelaps (Acari: Laelapidae) are ectoparasites of the brood of honey bees (Apis spp.). Different Tropilaelaps subspecies were originally described from Apis dorsata, but a host switch occurred to the Western honey bee, Apis mellifera, for which infestations can rapidly lead to colony death. Tropilaelaps is hence considered more dangerous to A. mellifera than the parasitic mite Varroa destructor. Honey bees are also infected by many different viruses, some of them associated with and vectored by V. destructor. In recent years, deformed wing virus (DWV) has become the most prevalent virus infection in honey bees associated with V. destructor. DWV is distributed world-wide, and found wherever the Varroa mite is found, although low levels of the virus can also be found in Varroa free colonies. The Varroa mite transmits viral particles when feeding on the haemolymph of pupae or adult bees. Both the Tropilaelaps mite and the Varroa mite feed on honey bee brood, but no observations of DWV in Tropilaelaps have so far been reported. In this study, quantitative real-time RT-PCR was used to show the presence of DWV in infested brood and Tropilaelaps mercedesae mites collected in China, and to demonstrate a close quantitative association between mite-infested pupae of A. mellifera and DWV infections. Phylogenetic analysis of the DWV sequences recovered from matching pupae and mites revealed considerable DWV sequence heterogeneity and polymorphism. These polymorphisms appeared to be associated with the individual brood cell, rather than with a particular host.     

In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris

Recent studies suggest a potent role of the small interfering RNA (siRNA) pathway in the control of bee viruses and its usefulness to tackle these viral diseases. However, the involvement of the siRNA pathway in the defense against different bee viruses is still poorly understood. Therefore, in this report, we comprehensively analyzed the response of the siRNA pathway in bumblebees of Bombus terrestris to systemic infections of the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that IAPV and SBPV infections induced the expression of Dicer-2. IAPV infections also triggered the production of predominantly 22 nt-long virus-derived siRNAs (vsiRNAs). Intriguingly, these 22 nt-long vsiRNAs showed a high proportion of antigenomic IAPV sequences. Conversely, these predominantly 22 nt-long vsiRNAs of SBPV were not detected in SBPV infected bees. Furthermore, an “RNAi-of-RNAi” experiment on Dicer-2 did not result in altered genome copy numbers of IAPV (n = 17–18) and also not of SBPV (n = 11–12). Based on these results, we can speculate about the importance of the siRNA pathway in bumblebees for the antiviral response. During infection of IAPV, this pathway is probably recruited but it might be insufficient to control viral infection in our experimental setup. The host can control SBPV infection, but aside from the induction of Dicer-2 expression, no further evidence of the antiviral activity of the siRNA pathway was observed. This report may also enhance the current understanding of the siRNA pathway in the innate immunity of non-model insects upon different viral infections.     

Selection for outbreeding in Varroa parasitising resistant honey bee (Apis mellifera) colonies

Parasitism is expected to select for counter‐adaptations in the host: driving a coevolutionary arms race. However, human interference between honey bees (Apis mellifera) and Varroa mites removes the effect of natural selection and restricts the evolution of host counter‐adaptations. With full‐sibling mating common among Varroa, this can rapidly select for virulent, highly inbred, Varroa populations. We investigated how the evolution of host resistance could affect the infesting population of Varroa mites. We screened a Varroa‐resistant honey bee population near Toulouse, France, for a Varroa resistance trait: the inhibition of Varroa's reproduction in drone pupae. We then genotyped Varroa which had co‐infested a cell using microsatellites. Across all resistant honey bee colonies, Varroa's reproductive success was significantly higher in co‐infested cells but the distribution of Varroa between singly and multiply infested cells was not different from random. While there was a trend for increased reproductive success when Varroa of differing haplotypes co‐infested a cell, this was not significant. This suggests local mate competition, through the presence of another Varroa foundress in a pupal cell, may be enough to help Varroa overcome host resistance traits; with a critical mass of infesting Varroa overwhelming host resistance. However, the fitness trade‐offs associated with preferentially co‐infesting cells may be too high for Varroa to evolve a mechanism to identify already‐infested cells. The increased reproductive success of Varroa when co‐infesting resistant pupal cells may act as a release valve on the selective pressure for the evolution of counter resistance traits: helping to maintain a stable host–parasite relationship.     

Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees

The animal gut is a habitat for diverse communities of microorganisms (microbiota). Honeybees and bumblebees have recently been shown to harbour a distinct and species poor microbiota, which may confer protection against parasites. Here, we investigate diversity, host specificity and transmission mode of two of the most common, yet poorly known, gut bacteria of honeybees and bumblebees: Snodgrassella alvi (Betaproteobacteria) and Gilliamella apicola (Gammaproteobacteria). We analysed 16S rRNA gene sequences of these bacteria from diverse bee host species across most of the honeybee and bumblebee phylogenetic diversity from North America, Europe and Asia. These focal bacteria were present in 92% of bumblebee species and all honeybee species but were found to be absent in the two related corbiculate bee tribes, the stingless bees (Meliponini) and orchid bees (Euglossini). Both Snodgrassella alvi and Gilliamella apicola phylogenies show significant topological congruence with the phylogeny of their bee hosts, albeit with a considerable degree of putative host switches. Furthermore, we found that phylogenetic distances between Gilliamella apicola samples correlated with the geographical distance between sampling locations. This tentatively suggests that the environmental transmission rate, as set by geographical distance, affects the distribution of G. apicola infections. We show experimentally that both bacterial taxa can be vertically transmitted from the mother colony to daughter queens, and social contact with nest mates after emergence from the pupa greatly facilitates this transmission. Therefore, sociality may play an important role in vertical transmission and opens up the potential for co‐evolution or at least a close association of gut bacteria with their hosts.     

Resin foraging dynamics in Varroa destructor-infested hives: a case of medication of kin?

t Social insects have evolved colony behavioral, physiological, and organiza. tional adaptations (social immunity) to reduce the risks of parasitization and/or disease transmission. The collection of resin from various plants and its use in the hive as propolis is a clear example of behavioral defense. For Apis mellifera, an increased propolis content in the hive may correspond to variations in the microbial load of the colony and to a downregulation of an individual bee's immune response. However, many aspects of such antimicrobial mechanism still need to be clarified. Assuming that bacterial and fungal infection mechanisms differ from the action of a parasite, we studied the resin collection dynamics in Varroa destructor-infested honeybee colonies. Comparative experiments involving hives with different mite infestation levels were conducted in order to assess the amount of resin collected and propolis quality within the hive, over a 2-year period (2014 and 2015). Our study demonstrates that when A. mellifera colonies are under stress because of Varroa infestation, an increase in the number of resin foragers is recorded, even if a general intensification of the foraging activity is not observed. A reduction in the total polyphenolic content in propolis produced in infested versus uninfested hives was also noticed. Considering that different propolis types show varying levels of inhibition against a variety of honey bee pathogens in vitro, it would be very important to study the effects against Varroa of two diverse types of propolis: from Varroa-free and from Varroa-infested hives.     

The effect of the ectoparasitic mite, Varroa destructor on adult worker honeybee (Apis mellifera) emergence weights, water, protein, carbohydrate, and lipid levels

Wet weight, dry weight and water contents of emerging honeybees (Apis mellifera L. [Hymenoptera: Apidae]) infested with the ectoparasitic mite Varroa destructor (Anderson) (Acari: Varroidae) were all negatively correlated with increasing numbers of mites. It was estimated that for every female mite present during the bees' development, the host would lose three percent of its body water. Parasitised bees also emerged with lower head and abdomen concentrations of protein and with lower abdominal carbohydrate concentrations. Lipid concentrations were not detectably affected by V. destructor infestation. The losses of metabolic reserves were not, however, judged to be serious enough to be directly responsible for the high bee mortality and ultimate colony collapse that are associated with the arrival of Varroa in a hive. Some 8.5% of the emerging bees exhibited morphological deformities and deformity was positively correlated with increasing numbers of mites in brood cells. Deformed bees were, however, found in all categories of parasitosis, suggesting that other factors, such as infectious agents, may be involved. Mites that fed on either live or dead U¹⁴C- labelled bees acquired the label within 24 h and it was calculated that an adult female mite consumes 0.67 l haemolymph 24 h^–1. It was also demonstrated that ¹⁴C was transmitted to a previously non-radio-labelled bee when a mite that had been feeding on a labelled bee changed hosts. The level of transfer was above that which could have arisen through contamination of the mites' mouthparts and supports the suggestion that Varroa is an important vector of pathogens such as viruses.     

Patterns in Varroa destructor depend on bee host abundance,availability of natural resources,and climate in Mediterranean apiaries

1. Varroa destructor Linnaeus (Acari: Varroidae) is one of the greatest threats to apiculture. This study examines the role of host density, natural resource availability for bees, the management and climate in driving spatial and annual variability in the abundance of Varroa, and the occurrence of colony losses, recorded in apiaries across a Mediterranean island over a 2‐year period, using a hierarchical generalised linear model framework. 2. The seasonal abundance of Varroa showed a bimodal pattern with two peaks, the first one being in spring and larger than the second one located in summer/autumn. In contrast, bee colony losses were mainly concentrated in autumn/winter. 3. The abundance patterns of Varroa were best explained by models combining host, climate, and resource availability. A key novel finding was that low availability of natural flowering resources leads to high levels of infestation of Varroa, highlighting the importance of preserving natural resources around apiaries for the maintenance of pollination services in the landscape. Varroa abundance was also found to increase as bee density increased, probably as a result of the greater brood availability. Moreover, Varroa abundance increased as temperatures decrease and decreases as relative humidity increases, which is consistent with previous studies. Anti‐varroa treatments were only found to impact Varroa levels in the second 6 months of the year. Organic treatments outperformed synthetic treatments. 4. Empirical research on optimal seasonal timing and combinations of treatments, as well as impacts of climate and resource availability on natural dynamics of bees and Varroa in different climate zones, is urgently required.     

Viral gut metagenomics of sympatric wild and domestic canids,and monitoring of viruses: Insights from an endangered wolf population

Animal host–microbe interactions are a relevant concern for wildlife conservation, particularly regarding generalist pathogens, where domestic host species can play a role in the transmission of infectious agents, such as viruses, to wild animals. Knowledge on viral circulation in wild host species is still scarce and can be improved by the recent advent of modern molecular approaches. We aimed to characterize the fecal virome and identify viruses of potential conservation relevance of diarrheic free‐ranging wolves and sympatric domestic dogs from Central Portugal, where a small and threatened wolf population persists in a highly anthropogenically modified landscape. Using viral metagenomics, we screened diarrheic stools collected from wolves (n = 8), feral dogs (n = 4), and pet dogs (n = 6), all collected within wolf range. We detected novel highly divergent viruses as well as known viral pathogens with established effects on population dynamics, including canine distemper virus, a novel bocavirus, and canine minute virus. Furthermore, we performed a 4‐year survey for the six wolf packs comprising this endangered wolf population, screening 93 fecal samples from 36 genetically identified wolves for canine distemper virus and the novel bocavirus, previously identified using our metagenomics approach. Our novel approach using metagenomics for viral screening in noninvasive samples of wolves and dogs has profound implications on the knowledge of both virology and wildlife diseases, establishing a complementary tool to traditional screening methods for the conservation of threatened species.     

Honey bee and bumblebee trypanosomatids: specificity and potential for transmission

Abstract 1. Experimental studies of multihost parasite dynamics are scarce. Understanding the transmission dynamics of parasites in these systems is a key task in developing better models of parasite evolution and to make more accurate predictions of disease dynamics. 2. Bumblebee species (Bombus spp.) host the trypanosomatid parasite, Crithidia bombi. Its transmission in the field occurs through the shared use of flowers. Flowers are a perfect scenario for inter‐taxa transmission of diseases because they are used by a wide range of animals. 3. Honey bees host a poorly studied trypanosomatid, Crithidia mellificae. In this study, five questions have been experimentally addressed: (a) Can C. bombi infect honey bees? (b) Can C. mellificae infect bumblebees? (c) Can the honey bee act as a vector for C. bombi? (d) Are C. bombi cells present in honey‐bee faeces? (e) Does C. bombi have an effect on the mortality of honey bees after ingestion? 4. While both parasites were found to be specific to their hosts at the genus level, results suggest that honey bees may play a role in the epidemiology of C. bombi transmission.     

Pathogen effects on vegetative and floral odours mediate vector attraction and host exposure in a complex pathosystem

Pathogens can alter host phenotypes in ways that influence interactions between hosts and other organisms, including insect disease vectors. Such effects have implications for pathogen transmission, as well as host exposure to secondary pathogens, but are not well studied in natural systems, particularly for plant pathogens. Here, we report that the beetle‐transmitted bacterial pathogen Erwinia tracheiphila – which causes a fatal wilt disease – alters the foliar and floral volatile emissions of its host (wild gourd, Cucurbita pepo ssp. texana) in ways that enhance both vector recruitment to infected plants and subsequent dispersal to healthy plants. Moreover, infection by Zucchini yellow mosaic virus (ZYMV), which also occurs at our study sites, reduces floral volatile emissions in a manner that discourages beetle recruitment and therefore likely reduces the exposure of virus‐infected plants to the lethal bacterial pathogen – a finding consistent with our previous observation of dramatically reduced wilt disease incidence in ZYMV‐infected plants.