Population genetic diversity and dynamics of the honey bee brood pathogen Melissococcus plutonius in a region with high prevalence.

European foulbrood (EFB) is a honey bee brood disease caused by the bacterium Melissococcus plutonius. Large-scale EFB outbreaks have been reported in several countries in recent decades, which entail costly sanitation measures of affected apiaries to restrict the spread of this contagious pathogen. To mitigate its impact, a better understanding of the population dynamics of the etiological agent is required. We here used multi-locus sequence typing (MLST) to infer the genetic diversity and geographical distribution of 160 M. plutonius isolates collected from EFB symptomatic honey bee colonies seven years apart. Isolates belonged to three clonal complexes (CCs) known worldwide and to 12 sequence types (STs), of which five were novel. Phylogenetic and clustering analyses showed that some of these novel sequence types have likely evolved locally during a period of outbreak, but most disappeared again. We further screened the isolates for melissotoxin A (mtxA), a putative virulence gene. The prevalence of STs in which mtxA was frequent increased over time, suggesting that this gene promotes spread. Despite the increased frequency of this gene in the population, the total number of cases decreased, which could be due to stricter control measures implemented before the second sampling period. Our results provide a better understanding of M. plutonius population dynamics and help identify knowledge gaps that limit efficient control of this emerging disease.

Identifying bacterial predictors of honey bee health.

Non-targeted approaches are useful tools to identify new or emerging issues in bee health. Here, we utilise next generation sequencing to highlight bacteria associated with healthy and unhealthy honey bee colonies, and then use targeted methods to screen a wider pool of colonies with known health status. Our results provide the first evidence that bacteria from the genus Arsenophonus are associated with poor health in honey bee colonies. We also discovered Lactobacillus and Leuconostoc spp. were associated with healthier honey bee colonies. Our results highlight the importance of understanding how the wider microbial population relates to honey bee colony health.

Biogeography of Paenibacillus larvae, the causative agent of American foulbrood, using a new multilocus sequence typing scheme.

American foulbrood is the most destructive brood disease of honeybees (Apis mellifera) globally. The absence of a repeatable, universal typing scheme for the causative bacterium Paenibacillus larvae has restricted our understanding of disease epidemiology. We have created the first multilocus sequence typing scheme (MLST) for P. larvae, which largely confirms the previous enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR)-based typing scheme's divisions while providing added resolution and improved repeatability. We have used the new scheme to determine the distribution and biogeography of 294 samples of P. larvae from across six continents. We found that of the two most epidemiologically important ERIC types, ERIC I was more diverse than ERIC II. Analysis of the fixation index (FST ) by distance suggested a significant relationship between genetic and geographic distance, suggesting that population structure exists in populations of P. larvae. Interestingly, this effect was only observed within the native range of the host and was absent in areas where international trade has moved honeybees and their disease. Correspondence analysis demonstrated similar sequence type (ST) distributions between native and non-native countries and that ERIC I and II STs mainly have differing distributions. The new typing scheme facilitates epidemiological study of this costly disease of a key pollinator.

Clustering, persistence and control of a pollinator brood disease: epidemiology of American foulbrood.

American foulbrood (AFB), caused by Paenibacillus larvae, is the most damaging bacterial brood disease of the honeybee (Apis mellifera), causing colony deaths on all continents where honeybees are managed. AFB has been a persistent problem in the UK for over 70 years, with a fluctuating number of cases discovered annually. Once diseased colonies are identified, they are destroyed to reduce pathogen spread. We investigated the pattern of AFB cases recorded over the period 1994 to 2012 using spatial-statistical approaches, with a view to identifying the nature of spread across England and Wales. Our results indicated that AFB exhibits significant spatial aggregation at distances from 10 to 30 km, with aggregations lasting between 1 and 5 years. Kernel smoothing indicated areas of elevated relative risk in different years, and these were further detailed by spatial-scan statistics. We identified disease clusters and successfully estimated their size, location and duration. The majority of clusters did not persist in all years, indicating that management measures may lead to localized extinction of the disease. Whilst less common, persistent clusters likely indicate potential endemic or exotic risk points. The application of robust epidemiological approaches to improve the control of AFB is discussed.

Quantitative and Qualitative Changes in the Deformed Wing Virus Population in Honey Bees Associated with the Introduction or Removal of Varroa destructor.

Varroa destructor is an ectoparasitic mite associated with significant losses of honeybee colonies globally. The mite vectors a range of pathogenic viruses, the most important of which is the Deformed wing virus (DWV). In the absence of Varroa, DWV exists as a low-level, highly diverse virus population. However, when transmitted by Varroa, certain variants become highly elevated, and may become near-clonal and cause symptomatic infections. Mite transmission between colonies can occur when parasitised workers drift from or rob adjacent hives. These activities can result in elevated mite levels, but the resulting change in the DWV population, the primary determinant of winter colony losses, has not been determined. In reciprocal studies, we investigated the influence of the removal of mites, or their acquisition, on the DWV population. When mites were removed from heavily infested colonies, there was a striking and rapid reduction in virus load. Conversely, siting Varroa-naïve colonies in a mite-infested apiary resulted in the acquisition of mites and concomitant changes in the virus population. We observed both near-clonal and highly divergent virus populations regardless of titre, suggesting changes were stochastic and colony-specific. Our findings have implications for the outcome of strategies in areas with total or patchy implementation of Varroa control plans.

Landscape-scale drivers of pollinator communities may depend on land-use configuration.

Research into pollinators in managed landscapes has recently combined approaches of pollination ecology and landscape ecology, because key stressors are likely to interact across wide areas. While laboratory and field experiments are valuable for furthering understanding, studies are required to investigate the interacting drivers of pollinator health and diversity across a broader range of landscapes and a wider array of taxa. Here, we use a network of 96 study landscapes in six topographically diverse regions of Britain, to test the combined importance of honeybee density, insecticide loadings, floral resource availability and habitat diversity to pollinator communities. We also explore the interactions between these drivers and the cover and proximity of semi-natural habitat. We found that among our four drivers, only honeybee density was positively related to wild pollinator abundance and diversity, and the positive association between abundance and floral resources depended on insecticide loadings and habitat diversity. By contrast, our exploratory models including habitat composition metrics revealed a complex suite of interactive effects. These results demonstrate that improving pollinator community composition and health is unlikely to be achieved with general resource enhancements only. Rather, local land-use context should be considered in fine-tuning pollinator management and conservation. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Detection of honey bee (Apis mellifera) viruses with an oligonucleotide microarray.

In recent years, declines in honey bee (Apis mellifera L.) colonies have been observed to varying degrees worldwide with the worst losses in the USA being termed Colony Collapse Disorder (CCD). Pathogen load and the prevalence of honey bee viruses have been implicated in these losses and many diseased hives have multiple viruses present. We have designed and tested an oligonucleotide microarray which enables the simultaneous detection of nine honey bee viruses: Acute bee paralysis virus, Black queen cell virus, Chronic bee paralysis virus, Deformed wing virus, Kashmir bee virus, Sacbrood virus, Israel acute paralysis virus, Varroa destructor virus 1 and Slow paralysis virus. The microarray can be used to robustly diagnose nine viruses in one test.

Identifying the climatic drivers of honey bee disease in England and Wales.

Honey bee colony health has received considerable attention in recent years, with many studies highlighting multifactorial issues contributing to colony losses. Disease and weather are consistently highlighted as primary drivers of colony loss, yet little is understood about how they interact. Here, we combined disease records from government honey bee health inspections with meteorological data from the CEDA to identify how weather impacts EFB, AFB, CBP, varroosis, chalkbrood and sacbrood. Using R-INLA, we determined how different meteorological variables influenced disease prevalence and disease risk. Temperature caused an increase in the risk of both varroosis and sacbrood, but overall, the weather had a varying effect on the six honey bee diseases. The risk of disease was also spatially varied and was impacted by the meteorological variables. These results are an important step in identifying the impacts of climate change on honey bees and honey bee diseases.

Transitions in symbiosis: evidence for environmental acquisition and social transmission within a clade of heritable symbionts.

A dynamic continuum exists from free-living environmental microbes to strict host-associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in symbiotic lifestyle and transmission mode is lacking. Arsenophonus is a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genus Arsenophonus and the Western honey bee. The symbiont shares common genomic and predicted metabolic properties with the male-killing symbiont Arsenophonus nasoniae, however we present multiple lines of evidence that the bee Arsenophonus deviates from a heritable model of transmission. Field sampling uncovered spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and laboratory individuals. Fluorescent in situ hybridisation showed Arsenophonus localised in the gut, and detection was rare in screens of early honey bee life stages. We directly show horizontal transmission of Arsenophonus between bees under varying social conditions. We conclude that honey bees acquire Arsenophonus through a combination of environmental exposure and social contacts. These findings uncover a key link in the Arsenophonus clades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle.

Genetic characterization of slow bee paralysis virus of the honeybee (Apis mellifera L.).

Complete genome sequences were determined for two distinct strains of slow bee paralysis virus (SBPV) of honeybees (Apis mellifera). The SBPV genome is approximately 9.5 kb long and contains a single ORF flanked by 5'- and 3'-UTRs and a naturally polyadenylated 3' tail, with a genome organization typical of members of the family Iflaviridae. The two strains, labelled 'Rothamsted' and 'Harpenden', are 83% identical at the nucleotide level (94% identical at the amino acid level), although this variation is distributed unevenly over the genome. The two strains were found to co-exist at different proportions in two independently propagated SBPV preparations. The natural prevalence of SBPV for 847 colonies in 162 apiaries across five European countries was <2%, with positive samples found only in England and Switzerland, in colonies with variable degrees of Varroa infestation.

The Acute bee paralysis virus-Kashmir bee virus-Israeli acute paralysis virus complex.

Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV) are part of a complex of closely related viruses from the Family Dicistroviridae. These viruses have a widespread prevalence in honey bee (Apis mellifera) colonies and a predominantly sub-clinical etiology that contrasts sharply with the extremely virulent pathology encountered at elevated titres, either artificially induced or encountered naturally. These viruses are frequently implicated in honey bee colony losses, especially when the colonies are infested with the parasitic mite Varroa destructor. Here we review the historical and recent literature of this virus complex, covering history and origins; the geographic, host and tissue distribution; pathology and transmission; genetics and variation; diagnostics, and discuss these within the context of the molecular and biological similarities and differences between the viruses. We also briefly discuss three recent developments relating specifically to IAPV, concerning its association with Colony Collapse Disorder, treatment of IAPV infection with siRNA and possible honey bee resistance to IAPV.

First molecular detection of a viral pathogen in Ugandan honey bees.

Ugandan honey bees (Apis mellifera L.) produce honey, and are key pollinators within commercial crops and natural ecosystems. Real-time RT-PCR was used to screen immature and adult bees collected from 63 beekeeping sites across Uganda for seven viral pathogens. No samples tested positive for Chronic bee paralysis virus, Sacbrood virus, Deformed wing virus, Acute bee paralysis virus, Apis iridescent virus or Israeli acute paralysis virus. However, Black queen cell virus (BQCV) was found in 35.6% of samples. It occurred in adults and larvae, and was most prevalent in the Western highlands, accounting for over 40% of positive results nationally.

The occurrence of Melissococcus plutonius in healthy colonies of Apis mellifera and the efficacy of European foulbrood control measures.

European foulbrood (EFB) persists in England and Wales despite current treatment methods, all of which include feeding honey bee colonies with the antibiotic oxytetracycline (OTC). A large-scale field experiment was conducted to monitor a husbandry-based method, using comb replacement (known as Shook swarm), as a drug free EFB control option. The understanding of EFB epidemiology is limited, with little information on the presence of Melissococcus plutonius in disease free colonies. Additional samples were collected from diseased and disease free apiaries to identify symptomless infection. EFB reoccurrence was not significantly different between OTC and husbandry methods and real-time PCR data demonstrated that fewer Shook swarm treated colonies contained M. plutonius carryover to the Spring following treatment. Asymptomatic colonies from diseased apiaries showed an increased risk of testing positive for M. plutonius compared to asymptomatic colonies from disease free apiaries. The probability of a sample being symptomatic increased when a greater quantity of M. plutonius was detected in adult bees and larvae. The possibility of treating EFB as an apiary disease rather than a colony disease and the implications of a control strategy without antibiotics are discussed.

Chronic bee paralysis as a serious emerging threat to honey bees.

Chronic bee paralysis is a well-defined viral disease of honey bees with a global distribution that until recently caused rare but severe symptomatology including colony loss. Anecdotal evidence indicates a recent increase in virus incidence in several countries, but no mention of concomitant disease. We use government honey bee health inspection records from England and Wales to test whether chronic bee paralysis is an emerging infectious disease and investigate the spatiotemporal patterns of disease. The number of chronic bee paralysis cases increased exponentially between 2007 and 2017, demonstrating chronic bee paralysis as an emergent disease. Disease is highly clustered spatially within most years, suggesting local spread, but not between years, suggesting disease burnt out with periodic reintroduction. Apiary and county level risk factors are confirmed to include scale of beekeeping operation and the history of honey bee imports. Our findings offer epidemiological insight into this damaging emerging disease.

Characterisation of the British honey bee metagenome.

The European honey bee (Apis mellifera) plays a major role in pollination and food production. Honey bee health is a complex product of the environment, host genetics and associated microbes (commensal, opportunistic and pathogenic). Improved understanding of these factors will help manage modern challenges to bee health. Here we used DNA sequencing to characterise the genomes and metagenomes of 19 honey bee colonies from across Britain. Low heterozygosity was observed in many Scottish colonies which had high similarity to the native dark bee. Colonies exhibited high diversity in composition and relative abundance of individual microbiome taxa. Most non-bee sequences were derived from known honey bee commensal bacteria or pathogens. However, DNA was also detected from additional fungal, protozoan and metazoan species. To classify cobionts lacking genomic information, we developed a novel network analysis approach for clustering orphan DNA contigs. Our analyses shed light on microbial communities associated with honey bees and demonstrate the power of high-throughput, directed metagenomics for identifying novel biological threats in agroecosystems.

Variability of residue concentrations of ciprofloxacin in honey from treated hives.

Honey bees (Apis mellifera L.) were treated with a model veterinary drug compound (ciprofloxacin) in a 3-year study (2012-14) to investigate the variability of residue concentration in honey. Sucrose solution containing ciprofloxacin was administered to 45 hives (1 g of ciprofloxacin per hive) at the beginning of the honey flow in late May/mid-June 2012, 2013 and 2014. Buckfast honey bees (A. mellifera - hybrid) were used in years 2012 and 2013. Carniolan honey bees (A. mellifera carnica) were used instead of the Buckfast honey bees as a replacement due to unforeseen circumstances in the final year of the study (2014). Honey was collected over nine scheduled time points from May/June till late October each year. Up to five hives were removed and their honey analysed per time point. Honey samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine ciprofloxacin concentration. Statistical assessment of the data shows that the inter-hive variation of ciprofloxacin concentrations in 2012/13 is very different compared with that of 2014 with relative standard deviations (RSDs) of 138% and 61%, respectively. The average ciprofloxacin concentration for 2014 at the last time point was more than 10 times the concentration compared with samples from 2012/13 at the same time point. The difference between the 2012/13 data compared with the 2014 data is likely due to the different type of honey bees used in this study (2012/13 Buckfast versus 2014 Carniolan). Uncertainty estimates for honey with high ciprofloxacin concentration (upper 95th percentile) across all hives for 55-day withdrawal samples gave residual standard errors (RSEs) of 22%, 20% and 11% for 2012, 2013 and 2014, respectively. If the number of hives were to be reduced for future studies, RSEs were estimated to be 52% (2012), 54% (2013) and 26% (2014) for one hive per time point (nine total hives).

Predicting the spread of the Asian hornet (Vespa velutina) following its incursion into Great Britain.

The yellow-legged or Asian hornet (Vespa velutina) is native to South-East Asia, and is a voracious predator of pollinating insects including honey bees. Since its accidental introduction into South-Western France in 2004, V. velutina has spread to much of western Europe. The presence of V. velutina in Great Britain was first confirmed in September 2016. The likely dynamics following an initial incursion are uncertain, especially the risk of continued spread, and the likely success of control measures. Here we extrapolate from the situation in France to quantify the potential invasion of V. velutina in Great Britain. We find that, without control, V. velutina could colonise the British mainland rapidly, depending upon how the Asian hornet responds to the colder climate in Britain compared to France. The announcement that a second hornet had been discovered in Somerset, increases the chance that the invasion first occurred before 2016. We therefore consider the likely site of first invasion and the probabilistic position of additional founding nests in late 2016 and early 2017. Given the potential dispersion of V. velutina, we conclude that vigilance is required over a large area to prevent the establishment of this threat to the pollinator population.

Invasion dynamics of Asian hornet, Vespa velutina (Hymenoptera: Vespidae): a case study of a commune in south-west France.

Asian hornet, Vespa velutina Lepeletier nests were discovered in 2007 in Andernos-les-Bains on the south-west coast of France, 3 years after the first reported sightings in France. The number of nests increased in the commune over the following 7 years, despite local authorities enacting a destruction policy. The nests existed in close proximity to one another leading to a high density of over 10 nests per square kilometre in urban areas. New information on the chosen habitat for nests is presented, and the differences between primary and secondary locations are evident, with primary nests mostly occupying buildings and man-made structures, while secondary nests were found on trees. Using Bayesian inference methods, we fit a basic model to the observational data, which allows us to estimate key demographic parameters. This model fit is highly informative for predicting V. velutina spread and colonisation of other at-risk regions, and suggests that local control has a limited impact on the spread of V. velutina once established within a region.

Efficient use of sentinel sites: detection of invasive honeybee pests and diseases in the UK.

Sentinel sites, where problems can be identified early or investigated in detail, form an important part of planning for exotic disease outbreaks in humans, livestock and plants. Key questions are: how many sentinels are required, where should they be positioned and how effective are they at rapidly identifying new invasions? The sentinel apiary system for invasive honeybee pests and diseases illustrates the costs and benefits of such approaches. Here, we address these issues with two mathematical modelling approaches. The first approach is generic and uses probabilistic arguments to calculate the average number of affected sites when an outbreak is first detected, providing rapid and general insights that we have applied to a range of infectious diseases. The second approach uses a computationally intensive, stochastic, spatial model to simulate multiple outbreaks and to determine appropriate sentinel locations for UK apiaries. Both models quantify the anticipated increase in success of sentinel sites as their number increases and as non-sentinel sites become worse at detection; however, unexpectedly sentinels perform relatively better for faster growing outbreaks. Additionally, the spatial model allows us to quantify the substantial role that carefully positioned sentinels can play in the rapid detection of exotic invasions.

The invasion, provenance and diversity of Vespa velutina Lepeletier (Hymenoptera: Vespidae) in Great Britain.

The yellow-legged or Asian hornet (Vespa velutina colour form nigrithorax) was introduced into France from China over a decade ago. Vespa velutina has since spread rapidly across Europe, facilitated by suitable climatic conditions and the ability of a single nest to disperse many mated queens over a large area. Yellow-legged hornets are a major concern because of the potential impact they have on populations of many beneficial pollinators, most notably the western honey bee (Apis mellifera), which shows no effective defensive behaviours against this exotic predator. Here, we present the first report of this species in Great Britain. Actively foraging hornets were detected at two locations, the first around a single nest in Gloucestershire, and the second a single hornet trapped 54 km away in Somerset. The foraging activity observed in Gloucestershire was largely restricted to within 700 m of a single nest, suggesting highly localised movements. Genetic analyses of individuals from the Gloucestershire nest and the single hornet from Somerset suggest that these incursions represent an expansion of the European population, rather than a second incursion from Asia. The founding queen of the Gloucestershire nest mated with a single male, suggesting that sexual reproduction may have occurred in an area of low nest density. Whilst the nest contained diploid adult males, haploid 'true' males were only present at the egg stage, indicating that the nest was detected and removed before the production of queens. Members of the public reported additional dead hornets associated with camping equipment recently returned from France and imported timber products, highlighting possible pathways of incursion. The utility of microsatellites to inform surveillance during an incursion and the challenge of achieving eradication of this damaging pest are discussed.