<figure><img src="image_1.png" /></figure>

HAL
open science

# The virulent, emerging genotype B of Deformed wing virus is closely linked to overwinter honeybee worker loss

Myrsini E Natsopoulou, Dino P Mcmahon, Vincent Doublet, Eva Frey, Peter Rosenkranz, Robert J Paxton

► To cite this version:

Myrsini E Natsopoulou, Dino P Mcmahon, Vincent Doublet, Eva Frey, Peter Rosenkranz, et al.. The virulent, emerging genotype B of Deformed wing virus is closely linked to overwinter honeybee worker loss. Scientific Reports, 2017, 7 (1), pp.5242. 10.1038/s41598-017-05596-3. hal-05361538

HAL Id: hal-05361538

https://hal.inrae.fr/hal-05361538v1

Submitted on 12 Nov 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
SCIENTIFIC REPORTS

<figure><img src="image_3.png" /></figure>

OPEN

# The virulent, emerging genotype B of *Deformed wing virus* is closely linked to overwinter honeybee worker loss

Myrsini E. Natsopoulou<fnref n="1" />,⁷, Dino P. McMahon<fnref n="2" />,³,⁴, Vincent Doublet<fnref n="1" />,⁵,⁸, Eva Frey⁶, Peter Rosenkranz⁶ & Robert J. Paxton<fnref n="1" />,<fnref n="2" />,⁵

Received: 21 February 2017

Accepted: 31 May 2017

Published online: 12 July 2017

Bees are considered to be threatened globally, with severe overwinter losses of the most important commercial pollinator, the Western honeybee, a major concern in the Northern Hemisphere. Emerging infectious diseases have risen to prominence due to their temporal correlation with colony losses. Among these is *Deformed wing virus* (DWV), which has been frequently linked to colony mortality. We now provide evidence of a strong statistical association between overwintering colony decline in the field and the presence of DWV genotype-B (DWV-B), a genetic variant of DWV that has recently been shown to be more virulent than the original DWV genotype-A. We link the prevalence of DWV-B directly to a quantitative measure of overwinter decline (workforce mortality) of honeybee colonies in the field. We demonstrate that increased prevalence of virus infection in individual bees is associated with higher overwinter mortality. We also observed a substantial reduction of infected colonies in the spring, suggesting that virus-infected individuals had died during the winter. Our findings demonstrate that DWV-B, plus possible A/B recombinants exhibiting DWV-B at PCR primer binding sites, may be a major cause of elevated overwinter honeybee loss. Its potential emergence in naïve populations of bees may have far-reaching ecological and economic impacts.

Insect pollination, which is carried out mostly by bees, is required for 75% of all food crops<fnref n="1" />; the global value of insect pollination in 2005 has been estimated at €153 billion<fnref n="2" />, although this is likely to be an underestimate³. In addition, 85% of wild plants are pollinated by animals⁴, chief among these being bees. Bees are therefore of considerable economic and ecological importance. The honeybee *Apis mellifera* is by far the most important commercial pollinator and is relied upon heavily for the successful pollination of many food crops⁵. However, over the last decade severe yearly losses of honeybees in the Northern Hemisphere⁶⁻⁹ have raised concerns about food security¹⁰.

The emergence of several infectious diseases has coincided with elevated honeybee colony losses¹¹⁻¹³. Notable among these are positive single stranded RNA (positive ssRNA) viruses that have risen to prominence since the arrival of a novel biological vector from Asia, the ectoparasitic mite *Varroa destructor*. The mite feeds on the hemolymph of honey bee pupae and adults¹⁴. Its ability to act as a viral vector and potential incubator of several honeybee RNA viruses has given rise to a new viral transmission route, thereby aiding the spread and re-emergence of several bee viruses¹⁵⁻¹⁷.

<footnote n="1">¹Institute for Biology, Martin-Luther-University Halle-Wittenberg, Hoher Weg 8, 06120, Halle (Saale), Germany.</footnote>

<footnote n="2">²School of Biological Sciences, MBC, Queen's University Belfast, Belfast, BT9 7BL, UK. ³Institute of Biology, Free University Berlin, Schwendenerstr. 1, 14195, Berlin, Germany. ⁴Department for Materials and Environment, BAM Federal Institute for Materials Research and Testing, Unter den Eichen 87, 12205, Berlin, Germany. ⁵German Centre for Integrative Biodiversity Research Halle-Jena-Leipzig (iDiv), Deutscher Platz 5e, 04103, Leipzig, Germany. ⁶Apicultural State Institute, University of Hohenheim, 70599, Stuttgart, Germany. ⁷Present address: Section for Organismal Biology, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg, Denmark. ⁸Present address: Centre for Ecology and Conservation, University of Exeter, Penryn, UK. Myrsini E. Natsopoulou and Dino P. McMahon contributed equally to this work. Correspondence and requests for materials should be addressed to M.E.N. (email: myrsini.natsopoulou@plen.ku.dk)</footnote>
A major source of honey bee decline is colony depopulation during the winter. Colonies suffering elevated winter worker losses are more prone to collapse, resulting in overwinter colony losses (OCL)¹⁸. High rates of OCL are a continuing concern in temperate zones, both in Europe and North America, where 15–20% and 25–30% of colonies respectively have been consistently lost overwinter in recent years¹⁹⁻²². Pathogens may be major contributors to OCL. Among those that have been implicated in colony decline are several positive ssRNA viruses: *Kashmir bee virus* (KBV)¹²; *Slow bee paralysis virus* (SBPV)²³; *Israeli acute paralysis virus* (IAPV)¹¹; *Acute bee paralysis virus* (ABPV)²⁴, ²⁵; *Deformed wing virus* (DWV)¹², ²⁴⁻²⁶; microsporidia, particularly *Nosema ceranae*, an emerging gut parasite from Asia¹³, ²⁷; and *V. destructor*, due to its association with several of the viruses²⁸.

One virus in particular, DWV, is considered an important predictor of colony failure²⁵, ²⁶, ²⁸⁻³⁰. *Varroa destructor* has altered the dynamics of DWV dramatically, reducing within-host genetic DWV strain diversity, and leading to the global spread and dominance of genetic variants related to DWV genotype A (DWV-A)¹⁷. We have recently shown that another recently described genotypic variant of DWV, genotype B (DWV-B, also known as *Varroa destructor virus-1* or VDV-1¹⁷, ³¹, ³², is more virulent than classic DWV-A³³. Although DWV-B has been recorded in Europe, Africa and Asia³⁴⁻³⁷ and is widespread in UK³³, its role in OCL is currently unknown.

To date, field studies on the role of pathogens in OCL have been largely based on correlations between pathogens discovered at the colony-level and qualitative assessments of colony health (i.e. stable or collapsed colony). Quantitative field measures of intra-colony disease dynamics provide a better insight into their role in OCL. For example, measurements of pathogen prevalence and workforce mortality within colonies would permit a more precise understanding of the change in disease and overall health status of overwintering colonies. We address this shortfall by assessing the pathogen landscape in 28 colonies in two apiaries before and after the winter season while experimentally controlling *V. destructor* infestation levels. We then determined whether there is an explicit association between DWV-B and overwinter reduction of the honeybee colony workforce.

## Results

**Pathogen detection.** To identify pathogens that may be responsible for overwinter honeybee mortality, we conducted an apiary study to assess the overwinter change in pathogen composition in 28 managed honeybee colonies. Sampling was performed once in autumn, at the end of brood rearing, and once the following spring, before commencement of major brood rearing, allowing us to exclude birth rate and directly to estimate loss of workers per colony. We initially used multiple ligation probe amplification (MLPA) to target a wide range of RNA viruses and RT-PCR to identify two microsporidian gut pathogens: *Nosema apis* and *Nosema ceranae*. For these analyses, we pooled 30 honeybee workers per colony and analysed one total RNA extract per colony.

Apart from chronic bee paralysis virus (CBPV), which occurred rarely in both autumn and spring, only viruses belonging to the DWV complex and *Black queen cell virus* (BQCV) could be detected by MLPA (Supplementary Table S1). We subsequently employed qRT-PCR on the same pooled samples to gain a more accurate understanding of the two prominent viruses: BQCV and DWV. In addition to BQCV and the original DWV-A genotype, we screened for DWV-B which shares 84% nucleotide identity with DWV-A¹⁴. DWV-A and -B were screened for by genotype-specific qRT-PCR amplification of Leader polypeptide (Lp) and RNA-dependent RNA polymerase (RdRp) genes; these are located at the 5' and 3' parts of the viral genome, respectively¹⁴.

We found that 96% (N = 27) of colonies in the autumn contained significant quantities of DWV-B (Supplementary Table S2). Compared with the autumn, prevalence of DWV-B in spring colonies was significantly reduced, with 21% (N = 6) of colonies containing DWV-B ($\chi^2_1$ = 19.1, p < 0.0001; Fig. 1). Some colonies in both autumn and spring contained very low viral loads of the DWV-A genotype, but these fell below an *a priori* detection threshold, and were only detected using the RdRp primer pair (Supplementary Table S2). This indicated that DWV-A was not (or at best, very rarely) a cause of infection in these colonies. DWV-B titres were also significantly reduced in spring colonies compared to autumn levels (paired Wilcoxon signed rank test V = 378, p < 0.00001) (Fig. 2). Potential caveats are that we may have missed very low titres of DWV-A because of pooling 30 bees per extract and that the qPCR primers for DWV-A could have been of lower sensitivity than those for DWV-B. To address the first caveat, we subsequently sampled individual honeybees (15 individuals per colony) but still did not detect DWV-A (see 'Overwinter mortality and pathogen prevalence'). In relation to the second caveat, primer sensitivity was similar for DWV-A and DWV-B primers (Supplementary Table S4).

Interestingly, no recombinant viruses were detected (i.e. samples containing a mix of DWV-A and DWV-B fragments, Supplementary Table S2). However, a few samples positive for DWV-B in the RdRp region (N = 3 in autumn and N = 5 in spring) could not be amplified in the Lp region (Supplementary Table S2). This was attributed to the low amount of DWV-B detected coupled with the lower sensitivity of the DWV-B primer in this region (Supplementary Table S4).

The prevalence of BQCV was also high in autumn colonies (82%, N = 22; Supplementary Table S2), and then significantly reduced in spring to 18% (N = 5; $\chi^2_1$ = 15.1, p = 0.0001; Fig. 1). BQCV titres were significantly lower in spring compared to autumn colonies (V = 272, p < 0.00001), also following the same pattern as for DWV-B. However, DWV-B titres in autumn sampling were significantly higher than those of BQCV (V = 377, p < 0.00001; see Fig. 2).

The Microsporidia *N. ceranae* and *N. apis* were detected in 54% and 25% of colonies in the autumn, respectively. Prevalences were also significantly lower in the spring, with 18% (N = 5) and 0% of colonies carrying each pathogen, respectively ($\chi^2_1$ = 8.1, 5.1; p = 0.004, 0.023 respectively; Fig. 1).

**Overwinter mortality and pathogen prevalence.** To understand the link between pathogens and overwinter workforce mortality in finer detail, we estimated the prevalence of pathogens within colonies by sampling 15 additional individual honeybees from each colony in autumn, using MLPA for RNA viruses and RT-PCR for *Nosema* spp. To verify the rarity of DWV-A across the colonies in the pooled samples (0% true prevalence from 30 pooled individuals per colony (N = 840), 95% confidence intervals (CIs): 0–0.45%), a subset of 37 individual
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Proportional overwinter change in pathogen prevalence among colonies (qPCR screen of pooled worker samples, N = 30 bees per colony). Black = pathogen present, white = pathogen absent. \*Significant comparisons: paired McNemar's χ² test p < 0.05.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 2.** Histogram of DWV-B and BQCV viral titre in pooled colony samples (N = 30 bees per colony). Values represent average titre per bee. Black: viral titres in autumn; grey: viral titres in spring.</figcaption></figure>

workers found positive for DWV in MLPA (representing ca. 37% of DWV MLPA-positive samples, distributed randomly across 7 colonies) was subjected to qRT-PCR using the DWV-A specific primers. DWV-A was not present in any of the samples tested, supporting our previous analyses and suggesting that, if present, DWV-A had an extremely low titre. Therefore, DWV-positive individual honeybees screened by MLPA are treated as DWV-B, and are henceforth reported as such.

The analysis of individual honeybees confirmed the disease patterns detected in pools of 30 worker honeybees per colony (above), with DWV-B featuring most frequently among autumn bees (Supplementary Table S2).
<table><caption>**Table 1.** Best models explaining overwinter colony workforce mortality using GLMs and QAICc for model selection. Model 1: DWV-B as explanatory variable ($r^2$ = 0.16), Model 2: *V. destructor* infestation as explanatory variable ($r^2$ = 0.25). Viral and *Nosema* spp. prevalence was based on 15 individuals per colony while *V. destructor* infestation was estimated on a sample of 150 individuals per colony.</caption><thead><tr><th>Response (Model)</th><th>Model</th><th>Parameters</th><th>Estimate</th><th>SE</th><th>t-value</th><th>P-value</th></tr></thead><tbody><tr><td rowspan="4">Mortality</td><td rowspan="2">1</td><td>Intercept</td><td>-0.384</td><td>0.133</td><td>-2.893</td><td rowspan="2">0.033*</td></tr><tr><td>DWV-B</td><td>0.297</td><td>0.132</td><td>2.257</td></tr><tr><td rowspan="2">2</td><td>Intercept</td><td>-0.401</td><td>0.125</td><td>-3.203</td><td rowspan="2">0.006*</td></tr><tr><td>V. destructor</td><td>0.348</td><td>0.116</td><td>3.001</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Prevalence of DWV-B and BQCV within colonies plotted against overwinter workforce mortality. Each point represents the prevalence of a virus in 15 individually MLPA-screened worker bees sampled from each colony in autumn. Spearman's $\rho$ and significance levels are shown for correlations.</figcaption></figure>

Generalized linear models (GLMs) were conducted to infer whether within-colony pathogen prevalence (including *V. destructor* infestation estimated from 150 honeybees per colony) were important predictors of overwinter workforce mortality. GLMs were conducted either with DWV-B prevalence (model 1) or *V. destructor* infestation (model 2) in addition to all other detected pathogens and apiary site as explanatory variables. Simplified final models either contained only DWV-B or only *V. destructor* but not BQCV as significant predictors of overwinter colony workforce mortality (Table 1).

The relationships between pathogen prevalence and overwinter mortality for the DWV-B and BQCV viruses (Fig. 3), CBPV and Microsporidian pathogens (Supplementary Fig. S1), and *V. destructor* (Fig. 4) were tested using GLMs (Table 1) and Spearman's correlations (Figs 3 and 4), which indicated that DWV-B and *V. destructor* were closely tied to overwinter colony decline but BQCV was not. In colonies suffering >50% adult worker mortality (N = 10 colonies), DWV-B prevalence and *V. destructor* infestation averaged (standard deviation, SD): 33% (SD 21%) and 13.3% (SD 11.3%) and in the remaining colonies (N = 18), 14.4% (SD 16.1%) and 1.4% (SD 2.5%) respectively.

*Varroa destructor* infestation and acaricide treatment were negatively associated (Wilcoxon signed rank test, W = 195, p < 0.0001), demonstrating that intensive acaricide treatment had worked effectively to remove *V. destructor* mites from colonies (Fig. 4b). Additionally, *V. destructor* and within-colony DWV-B prevalence were positively correlated (GLM, t = 2.486, p = 0.02), while BQCV, CBPV and *Nosema* spp. were not found to be affected by the mite infestation levels or acaricide treatment (p > 0.05 in all cases).

**DWV composition.** In order to verify the DWV-B variant detected in our study, a viral propagation derived from a DWV-B positive colony extract was subjected to ultra-deep sequencing. Results (presented in ref. 33) confirmed our qRT-PCR finding; DWV was the only virus detected in our field extract, with DWV-B being the main genetic variant detected. A very low background level of DWV-A was also present but accounted for only 0.13% of all DWV reads compared to 99.87% of DWV-B reads. A third DWV variant, DWV-C³⁸, was not detected (Supplementary Table S3).
<figure><img src="image_7.png" /><figcaption>**Figure 4.** (a) Relationship between *V. destructor* infestation and overwinter mortality within each colony. Spearman's ρ and significance levels are shown. (b) Levels of *V. destructor* infestation and overwinter mortality across the two acaricide treatments (in the periodic treatment, acaricides were removed from 26th July until 18th October 2011).</figcaption></figure>

An important caveat is that, though we did not detect any DWV-A/B recombinants in our samples, either by qPCR or by ultra-deep sequencing, we cannot exclude the possibility that one or more colonies harboured a DWV-A/B recombinant that caused high mortality. If this is the case, then the recombinant comprises predominantly DWV-B at both 5' (Lp gene) and 3' (RdRp gene) parts of its genome and DWV-A somewhere in its centre.

## Discussion

Colony losses are a major source of ongoing honeybee population decline in temperate zones<sup>21, 22, 39</sup>. The *V. destructor* mite and its association with viruses have together been implicated as a possible cause of widespread losses of honeybee colonies globally<sup>9, 11, 24, 26, 29, 40</sup>. But determining which among several potential factors of OCL are more important has remained elusive. In a field study, we now find the prevalence of DWV-B, an emerging variant of DWV<sup>17, 31, 32</sup>, and *V. destructor* infestation to be highly correlated with overwinter honeybee worker loss. Low virus titres in bees surviving to spring further suggested that individuals infected with high DWV-B titres had died overwinter. To our knowledge, our study is the first to provide strong correlative evidence of this distinct genotypic variant of DWV and OCL in the absence of the classical DWV-A.

Colony losses have been linked to the presence of other positive ssRNA viruses, in particular to the ABPV-KBV-IAPV complex<sup>11, 25</sup> and to SBPV<sup>9</sup> that, in association with *V. destructor*, have been linked to high virulence and individual-level bee mortality<sup>41, 42</sup>. Our data are in line with the view that DWV, although less virulent when vectored by *V. destructor* than viruses such as ABPV and SBPV<sup>40, 43</sup>, may be more widespread and damaging at the colony and population level<sup>44</sup>. Unlike ABPV and SBPV, honeybee pupae (and their *V. destructor* ectoparasites) infected with DWV typically do not die prior to eclosion. Survival of infected pupae into adulthood permits the ongoing spread of infection by co-emerging *V. destructor* mites, and therefore increases the persistence of infections within colonies. Field evidence shows that, of the documented honeybee viruses, DWV occurs most frequently, and at high levels globally wherever *V. destructor* is also present<sup>17</sup>. In accordance, a recent study based on phylogenetic analysis of DWV strongly suggested that the dispersal of DWV around the globe followed a similar pattern to the spread of *V. destructor* mites<sup>45</sup>.

Although levels of *V. destructor* infestation in colonies were significantly correlated with the prevalence of DWV-B, making it difficult to disentangle their effects in OCL, reduced virus detection in the spring suggests that colony depopulation during winter can be attributed to the death of heavily DWV-B infected individuals. In addition, we have recently shown that, under lab conditions, DWV-B is more virulent that DWV-A, leading to significantly reduced honeybee worker survival even in the absence of *V. destructor*<sup>33</sup>. Taken together, these results suggest that the variance in overwinter honeybee loss can be largely accounted for by differences among colonies in autumn DWV-B loads.
Interestingly, we only detected the DWV-B genotype among our honeybee samples, and found little evidence of either the classic DWV-A, or recombinant forms of DWV-A and -B. An increasing number of studies report the presence of recombinants between DWV-A/-B in nature⁴⁶⁻⁴⁹, and it has been suggested that recombinants may be more virulent than DWV-A or -B, at least in honeybee pupae⁴⁸. In the present study, we did not detect any mixed infections in our field samples to indicate the presence of recombinants. In addition, recombinant DWV-A/B viruses so far reported by others⁴⁶⁻⁴⁹ all comprise DWV-A at their 3' end (including the RdRp gene), whereas colonies in our study contained only DWV-B RdRp; we can therefore exclude the possibility that we oversaw previously detected recombinant variants. Yet we cannot exclude the possibility that one or more colonies in which we detected DWV-B may have harboured a novel recombinant DWV-A/B virus comprising DWV-B at its 5' and 3' parts, where respectively our Lp and RdRp primers bound to the DWV genome. Alternatively, for a small number of samples with low virus load, the Lp region remained uncharacterized (no amplification with either the DWV-A or DWV-B Lp primers), possibly due to the lower sensitivity of the Lp primers. Given a recent study by Dalmon *et al.*⁴⁹, which suggests the presence of a recombination breakpoint located in the region where our Lp primers bind to the DWV genome, we cannot exclude the possibility of recombinants being present in some of our colonies.

Knowledge of the distribution of DWV genotypes across Europe and more widely across the globe is largely lacking. In a recent survey across Great Britain, we found that DWV-B is widespread across the landscape³³. Preliminary data suggest that DWV-B in addition to recombinant DWV-A/-B forms are currently more prevalent in Europe than in North America⁴⁶, ⁴⁸, ⁵⁰. Our findings highlight the significant risk posed by this emerging virus genotype to the wellbeing of managed honeybee populations; understanding the global distribution of DWV genotypes is an urgent necessity.

In conclusion, our findings support the view that DWV-B poses a particular problem in temperate zones, when queens cease egg laying during winter and the workforce is not replenished by newly emerging workers for several months. Given its putatively higher virulence, this specific genetic variant of DWV can be considered a sufficient cause of premature death in otherwise healthy adult worker bees, even in the absence of its vector the *V. destructor* mite. However, the extent and rate of overwinter decline of a standing population of honeybee workers in a colony, and therefore the chance of that colony's survival into spring, will be a function of initial workforce size, virus load and prevalence, in addition to other factors such as availability of overwinter resources. Models of honeybee colony dynamics that incorporate *Varroa*-virus interactions explicitly (reviewed in ref. 51) seem especially appropriate in light of our data.

## Methods

**Field Experiment.** Fourteen honeybee colonies from an apiary at Kenzingen (48°11'30"N 7°46'6"E) and 14 colonies at an apiary in Simonswald (48°6'1"N 8°3'21"E), both in Baden-Württemberg, Germany, were monitored overwinter from 2011–2012. All colonies were treated with the acaricides flumethrin (Bayvarol®) and coumaphos (CheckMite+™) except from 26th July until 18th October 2011, when acaricides was removed from half of the colonies at each site⁴⁷ Worker bees from each colony were freeze-killed on dry-ice on 26th September 2011 and 9th April 2012 and subsequently preserved at -80°C for pathogen analysis. To quantify levels of overwinter worker mortality we applied the Liebefeld method⁵² to estimate numbers of bees in each colony in autumn (17th October 2011), after most brood rearing had stopped, and early spring (28th February 2012), before new workers had enclosed. These data allowed us to quantify precisely overwinter mortality of each colony (Supplementary Table S2). Levels of *V. destructor* infestation were assessed by counting the number of mites associated with samples of 150 adult workers per colony (17th October 2011)⁵³ (Supplementary Table S2).

**RNA extraction and pathogen detection.** For each colony, 30 adult worker bees from each sampling period (September 2011 and April 2012) were pooled in RNAse free mesh bags (Bioreba, Reinach, Switzerland) and crushed in 6mL DEPC treated (RNAse free) water while snap-frozen in liquid nitrogen, 150µl of which was used for RNA isolation. In addition, 15 adults per colony (collected during exactly the same sampling times as the pooled samples above) were individually crushed in 500µl RLT buffer plus 1% b-mercaptoethanol, 100µl of which was used for RNA isolation. RNA was obtained using the RNeasy mini kit in a QiaCube robot (Qiagen, Hilden, Germany). We thereby aimed to capture all RNA viruses in our extracts, including those with low titre.

We targeted the amplification of 10 widespread viruses and 2 Microsporidia (*N. ceranae*, *N. apis*). As we conducted an investigation of overwinter mortality when brood production largely ceases, we did not screen for brood diseases such as European and American Foulbrood (*Paenibacillus larvae*, *Melissococcus plutonius* respectively) or Chalkbrood (*Ascosphaera apis*). For virus screens of pooled and individual samples, we employed multiple ligation-dependent probe amplification (MLPA) using the RT-MLPA® kit (MRC-Holland, Netherlands) according to manufacturer's instructions, using probes designed for the positive strand of the following RNA virus complexes: DWV-VDV-KV; CBPV; ABPV-IAPV-KBV; BQCV; SBPV; SBV, and β-actin (host housekeeping gene)⁵⁴. Amplified fragments were viewed on a QIAxcel capillary fragment analyzer (Qiagen), using an acceptance threshold of 0.1 relative fluorescence units.

As MLPA probes were unable to distinguish between DWV-A and DWV-B, pooled samples were re-analyzed using qRT-PCR to gain higher resolution data on DWV and BQCV, the main RNA viruses found among colonies. Total cDNA was synthesized using M-MLV Revertase (Promega) following manufacturer's instructions, using 800 ng of sample RNA. For absolute viral load, separate qPCRs were performed for DWV-A, DWV-B, BQCV and *A. mellifera* RP49 (housekeeping gene) in a Bio-Rad C1000 Thermal Cycler (Bio-Rad) using 2 x SensiMix SYBR and Fluorescein (Bioline), 0.2 µM of each primer and 1 µl of 1:10 diluted cDNA in a final volume of 10 µl. Negative controls comprising all reaction components except cDNA template were included in each run. Each reaction was performed in duplicate and the average quantification cycle (Ct) value was used (the maximum accepted Ct difference between replicates was set to one Ct). Amplification was performed using the following thermal
profile: 5 min at 95°C, followed by 40 cycles of 10 sec at 95°C and 30 sec at 57°C (including a read at each cycle). Post amplification melting curve analysis was used to check for non-specific amplification (50–90 °C with an increment of 0.5 °C s⁻¹). To minimize risk of false positives, an upper threshold of 35 Ct for detection of DWV-A, DWV-B and BQCV was applied⁵⁵.

For DWV-A/-B we conducted qRT-PCR amplification in two regions at either end of the genome: the leader polypeptide (Lp, 5' end of genome) and RNA-dependent RNA polymerase (RdRp, 3' end of genome), in order to screen for possible recombinants. Samples positive for DWV-B in both RdRp and Lp regions that showed negative amplification (or Ct > 35) for DWV-A (Lp and RpRd region) were classified as DWV-B positive. In the case of samples (n = 3 autumn, n = 5 spring samples) that showed positive amplification only in the DWV-B RdRp region and not in the DWV-B Lp region, samples were also considered to be DWV-B positive as no amplification was detected in the DWV-A Lp or DWV-A RdRp regions to indicate the presence of a recombinant. Absolute quantification of DWV-A, DWV-B and BQCV was calculated using duplicate DNA standard curves of purified PCR products (DWV-A/-B) or plasmids (BQCV, RP49), with efficiencies between 90% to 97% and correlation coefficients (R²) from 0.985–0.999. Pooled sample virus loads are reported as average viral titre per bee, calculated by the mean viral load of 30 pooled bees and taking into account the qRT-PCR starting quantities (Supplementary Table S2). Absolute quantities and prevalence of DWV-A and -B in pooled colony samples were calculated using the RdRp primers. Additionally, we also calculated normalized viral titres to the amount of RP49 mRNA per sample to account for differences in RNA integrity across samples (Supplementary Table S2). *Nosema* spp. detection was performed from total cDNA by RT-PCR using species-specific primers. All (q)RT-PCR primers used in the present study are listed in Supplementary Table S4. The high specificity of DWV primers spanning in the RdRp region has been previously verified in McMahon *et al.*³³ (see Supplementary Table S3 in ref. 33).

**Virus verification.** In order to verify the identity of DWV genotypes detected in our samples, one of the DWV-B positive pooled-colony samples, devoid of BQCV and CBPV, namely colony ts3, was used to produce a virus extract and was subsequently subjected to ultra-deep sequencing on an Illumina platform (GATC Biotech, Konstanz, Germany). Details of the method are available in McMahon *et al.*³³, where exactly the same viral extract was used for experimental purposes.

**Statistical analysis.** All analyses were performed in R v 3.1.3⁵⁶. We compared pathogen presence in autumn and spring colonies using McNemar's χ² (repeated colony measure) and tested for differences in pathogen load in autumn and spring using paired Wilcoxon signed rank tests. The latter analysis was performed using either absolute viral titre derived from standard curves or viral titre normalized to the host housekeeping gene (RP49). As both approaches yielded the same results, reported test statistics refer to absolute viral titres. Bee mortality at apiaries was analyzed in a generalized linear model as a two-vector response variable (number of dying and surviving bees). *Varroa destructor* infestation and treatment were negatively associated but as *V. destructor* was a significantly better fit (in terms of residual deviance) than acaricide treatment, it was retained in the model. *Varroa destructor* infestation and DWV-B were positively associated (see results). While *V. destructor* was superior to DWV-B in terms of model fit, we conducted parallel models with either DWV-B (model 1) or *V. destructor* infestation (model 2) because the causal relationship between DWV-B or *V. destructor* and mortality is unclear. All quantitative predictors were standardized to a mean of zero and standard deviation of one prior to analysis.

Models containing either DWV-B or *V. destructor* in addition to BQCV, CBPV, *N. ceranae*, *N. apis* and apiary site were simplified by performing automated model selection using the 'dredge' function of the R-package 'MuMln'⁵⁷. Inspection of residual deviance indicated overdispersion, which was accommodated by using a quasibinomial error structure. We used the Akaike information criterion adjusted for overdispersion and corrected for small sample sizes (QAICc⁵⁸) calculated by the 'dredge' function for all plausible subsets of model, to select explanatory variables driving overwinter worker mortality. Model residuals were explored graphically, while Shapiro-Wilk's and White's Tests were used to examine normality ('shapiro.test' function) and heteroskedasticity ('het.test' function), respectively. McFadden's pseudo *r*² values are reported. The Spearman's rank correlation test was applied to assess the relationship between the prevalence of each pathogen (%) and overwinter worker mortality (%).

**Compliance with ethical standards.** All experiments conducted and presented in the manuscript comply with the laws and rules of the institution and country in which they were performed.

**Data availability statement.** The datasets generated during the current study are included in this published article (and its Supplementary Information files).

**References**

1. Klein, A.-M. *et al.* Importance of pollinators in changing landscapes for world crops. *Proc. R. Soc. B Biol. Sci.* **274**, 303–313 (2007).

2. Gallai, N., Salles, J. M., Settele, J. & Vaissière, B. E. Economic valuation of the vulnerability of world agriculture confronted with pollinator decline. *Ecol Econ* **68**, 220–229 (2009).

3. Potts, S. G. *et al.* Safeguarding pollinators and their values to human well-being. *Nature.* **540**, 220–229 (2016).

4. Ollerton, J., Winfree, R. & Tarrant, S. How many flowering plants are pollinated by animals? *Oikos.* **120**, 321–326 (2011).

5. Morse, R. A. & Calderone, N. W. The value of honey bees as pollinators of U.S. crops in 2000. *Bee Cult* 1–15 (2000).

6. Oldroyd, B. P. What's killing American honey bees? *PLoS Biol.* **5**, 1195–1199 (2007).

7. Stokstad, E. The case of the empty hives. *Science.* **316**, 970–972 (2007).

8. Moritz, R. F. A. *et al.* Research strategies to improve honeybee health in Europe *. *Apidologie*. **41**, 227–242 (2010).

9. Neumann, P. & Carreck, N. L. Honey bee colony losses. *J. Apic. Res.* **49**, 1–6 (2010).

10. Potts, S. G. *et al.* Global pollinator declines: trends, impacts and drivers. *Trends Ecol. Evol.* **25**, 345–53 (2010).

11. Cox-Foster, D. L. *et al.* A metagenomic survey of microbes in honey bee colony collapse disorder. *Science.* **318**, 283–287 (2007).

12. Johnson, R. M., Evans, J. D., Robinson, G. E. & Berenbaum, M. R. Changes in transcript abundance relating to colony collapse disorder in honey bees (*Apis mellifera*). *Proc. Natl. Acad. Sci. USA* **106**, 14790–5 (2009).
13. Paxton, R. Does infection by *Nosema ceranae* cause 'Colony Collapse Disorder' in honey bees (*Apis mellifera*)? *J. Apic. Res.* **49**, 80 (2010).

14. de Miranda, J. R. & Genersch, E. Deformed wing virus. *J. Invertebr. Pathol.* **103**, S48–S61 (2010).

15. Chen, Y. P. & Siede, R. Honey bee viruses. *Adv. Virus Res.* **70**, 33–80 (2007).

16. Genersch, E. & Aubert, M. Emerging and re-emerging viruses of the honey bee (*Apis mellifera* L.). *Vet. Res.* **41**, 54 (2010).

17. Martin, S. J. et al. Global honey bee viral landscape altered by a parasitic mite. *Science*. **336**, 1304–1306 (2012).

18. Russell, S., Barron, A. B. & Harris, D. Dynamic modelling of honey bee (*Apis mellifera*) colony growth and failure. *Ecol. Modell.* **265**, 158–169 (2013).

19. van der Zee, R. et al. Managed honey bee colony losses in Canada, China, Europe, Israel and Turkey, for the winters of 2008–9 and 2009–10. *J. Apic. Res.* **51**, 100–114 (2012).

20. Spleen, A. M. et al. A national survey of managed honey bee 2011–12 winter colony losses in the United States: results from the Bee Informed Partnership. *J. Apic. Res.* **52**, 44–53 (2013).

21. Seitz, N. et al. A national survey of managed honey bee 2014–2015 annual colony losses in the USA. *J. Apic. Res.* **54**, 292–304 (2015).

22. Laurent, M., Hendrikx, P., Ribiere-Chabert, M. & Chauzat, M.-P. A pan-European epidemiological study on honeybee colony losses 2012–2014. **2013**, (2014).

23. Carreck, N. & Neumann, P. Honey bee colony losses. *J. Apic. Res.* **49**, 1 (2010).

24. Genersch, E. et al. The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies. *Apidologie*. **41**, 332–352 (2010).

25. Francis, R. M., Nielsen, S. L. & Kryger, P. Varroa-Virus interaction in collapsing honey bee colonies. *PLoS One* **8** (2013).

26. Highfield, A. C. et al. Deformed Wing Virus implicated in overwintering honeybee colony losses. *Appl. Environ. Microbiol.* **75**, 7212–7220 (2009).

27. Higes, M. et al. How natural infection by *Nosema ceranae* causes honeybee colony collapse. *Environ. Microbiol.* **10**, 2659–2669 (2008).

28. Rosenkranz, P., Aumeier, P. & Ziegelmann, B. Biology and control of *Varroa destructor*. *J. Invertebr. Pathol.* **103**, S96–S119 (2010).

29. Dainat, B., Evans, J. D., Chen, Y. P., Gauthier, L. & Neumann, P. Dead or alive: Deformed wing virus and *Varroa destructor* reduce the life span of winter honeybees. *Appl. Environ. Microbiol.* **78**, 981–987 (2012).

30. van Dooremalen, C. et al. Winter survival of individual honey bees and honey bee colonies depends on level of *Varroa destructor* infestation. *PLoS One* **7**, e36285 (2012).

31. Ongus, J. R. et al. Complete sequence of a picorna-like virus of the genus Iflavirus replicating in the mite *Varroa destructor*. *J. Gen. Virol.* **85**, 3747–3755 (2004).

32. Mordecai, G. J. et al. Superinfection exclusion and the long-term survival of honey bees in Varroa-infested colonies. *ISME J.* **10**, 1–10 (2016).

33. McMahon, D. P. et al. Elevated virulence of an emerging viral genotype as a driver of honeybee loss. *Proc. R. Soc. B Biol. Sci.* doi:10.1098/rspb.2016.0811 (2016).

34. Benaets, K. et al. Covert deformed wing virus infections have long-term deleterious effects on honeybee foraging and survival. doi:10.1098/rspb.2016.2149 (2017).

35. Zioni, N., Soroker, V. & Chejanovsky, N. Replication of *varroa destructor* virus 1 (VDV-1) and a *varroa destructor* virus 1-deformed wing virus recombinant (VDV-1-DWV) in the head of the honey bee. *Virology.* **417**, 106–112 (2011).

36. Radzevičiūtė, R. et al. Replication of honey bee-associated RNA viruses across multiple bee species in apple orchards of Georgia, Germany and Kyrgyzstan. *J. Invertebr. Pathol.* doi:10.1016/j.jip.2017.04.002 (2017).

37. Strauss, U. et al. Seasonal prevalence of pathogens and parasites in the savannah honeybee (*Apis mellifera scutellata*). *J. Invertebr. Pathol.* **114**, 45–52 (2013).

38. Mordecai, G. J., Wilfert, L., Martin, S. J., Jones, I. M. & Schroeder, D. C. Diversity in a honey bee pathogen: first report of a third master variant of the Deformed Wing Virus quasispecies. *ISME J.* **10**, 1–10 (2016).

39. van der Zee, R. et al. Results of international standardised beekeeper surveys of colony losses for winter 2012-2013: analysis of winter loss rates and mixed effects modelling of risk factors for winter loss. *J. Apic. Res.* **53**, 19–34 (2014).

40. Salvy, M. et al. Modifications of the cuticular hydrocarbon profile of *Apis mellifera* worker bees in the presence of the ectoparasitic mite *Varroa jacobsoni* in brood cells. *Parasitology.* **122**, 145–159 (2001).

41. Bailey, L. & Woods, R. D. Three previously undescribed viruses from the honey bee. *J. Gen. Virol.* **25**, 175–186 (1974).

42. Martin, S. J. The role of varroa and viral pathogens in the collapse of honeybee colonies: A modelling approach. *J. Appl. Ecol* **38**, 1082–1093 (2001).

43. Bailey, L. & Gibbs, a. J. Acute infection of bees with Paralysis Virus. *J. Insect Pathol.* **6**, 395–407 (1964).

44. Schroeder, D. C. & Martin, S. J. Deformed wing virus: the main suspect in unexplained honeybee deaths. *Virulence.* **3**, 589–598 (2012).

45. Wilfert, L. et al. Deformed Wing Virus is a recent global epidemic in honeybees driven by Varroa mites. *Science.* **351**, 594–597 (2016).

46. Moore, J. et al. Recombinants between Deformed wing virus and *Varroa destructor* virus-1 may prevail in *Varroa destructor*-infested honeybee colonies. *J. Gen. Virol.* **92**, 156–61 (2011).

47. Wang, H. et al. Sequence recombination and conservation of *Varroa destructor* virus-1 and Deformed wing virus in field collected honey bees (*Apis mellifera*). *PLoS One* **8**, 1–9 (2013).

48. Ryabov, E. V. et al. A virulent strain of deformed wing virus (DWV) of honeybees (*Apis mellifera*) prevails after Varroa destructor-mediated, or *in vitro*, transmission. *PLoS Pathog.* **10**, e1004230 (2014).

49. Dalmon, A. et al. Evidence for positive selection and recombination hotspots in *Deformed wing virus* (DWV). *Sci. Rep.* **7**, 41045, doi:10.1038/srep41045 (2017).

50. Cornman, R. S. et al. Population-genomic variation within RNA viruses of the Western honey bee, *Apis mellifera*, inferred from deep sequencing. *BMC Genomics.* **14**, 154 (2013).

51. Becher, M. A., Osborne, J. L., Thorbek, P., Kennedy, P. J. & Grimm, V. Towards a systems approach for understanding honeybee decline: A stocktaking and synthesis of existing models. *J. Appl. Ecol.* **50**, 868–880 (2013).

52. Imdorf, A., Buehlmann, G., Gerig, L., Kilchenmann, V. & Wille, H. Überprüfung der Schätzmethode zur Ermittlung der Brutfläche und der Anzahl Arbeiterinnen in Freifliegenden Bienenvölkern. *Apidologie.* **18**, 137–146 (1987).

53. Frey, E. & Rosenkranz, P. Autumn invasion rates of *Varroa destructor* (Mesostigmata: Varroidae) into honey bee (Hymenoptera: Apidae) colonies and the resulting increase in mite populations. *J. Econ. Entomol.* **107**, 508–515 (2014).

54. de Smet, L. et al. BeeDoctor, a versatile MLPA-based diagnostic tool for screening bee viruses. *PLoS One* **7** (2012).

55. de Miranda, J. R. et al. Standard methods for virus research in *Apis mellifera*. *J. Apic. Res.* **52**, 1–48 (2013).

56. R Core Team R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing (2014).

57. Barton, K. MuMIN: Multi-model inference. R package version 1.15.6. Available at: https://cran.r-project.org/web/packages/MuMIN/index.html (2016).

58. Burnham, K. & Anderson, D. Model selection and multimodel inference: a practical information-theoretic approach. (Springer, 2002).
Acknowledgements

We thank S. Erler for assistance during the sampling, J.R. de Miranda and L. De Smet for supplying qRT-PCR and MLPA positive controls respectively, in the form of plasmids, and the anonymous referees for their valuable comments, which helped strengthen the manuscript. This work was supported by funds from BBSRC, NERC, Defra, the Scottish Government and the Wellcome Trust as part of the UK Insect Pollinators Initiative (IPI grant BB/1000100/1), the Federal Ministry of Food, Agriculture and Consumer Protection (Germany): Fit Bee project (grant 511-06.01-28-1-71.007-10), and the EU: BeeDoc (grant 244956).

Author Contributions

M.E.N., D.P.M., V.D. and R.J.P. conceived the study. E.F. and P.R. conducted and designed the field experiment. M.E.N., D.P.M. and V.D. carried out the laboratory molecular work. M.E.N. and D.P.M. analysed the data and drafted the manuscript; all co-authors contributed to data interpretation and to the writing of the manuscript.

**Additional Information**

**Supplementary information** accompanies this paper at doi:10.1038/s41598-017-05596-3

**Competing Interests:** The authors declare that they have no competing interests.

**Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_8.png" /></figure>

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2017
Supplementary information: The virulent, emerging genotype B of
*Deformed wing virus* is closely linked to overwinter honeybee worker loss

Myrsini E. Natsopoulou<fnref n="1" />,†‡, Dino P. McMahon<fnref n="2" />,<fnref n="3" />,<fnref n="4" />†, Vincent Doublet<fnref n="1" />,<fnref n="5" />¤, Eva Frey<fnref n="6" />, Peter
Rosenkranz<fnref n="6" />, Robert J. Paxton<fnref n="1" />,<fnref n="2" />,<fnref n="5" />

<footnote n="1">¹Institute for Biology, Martin-Luther-University Halle-Wittenberg, Hoher Weg 8, 06120 Halle (Saale), Germany</footnote>

<footnote n="2">²School of Biological Sciences, MBC, Queen's University Belfast, Belfast BT9 7BL, UK</footnote>

<footnote n="3">³Institute of Biology, Free University Berlin, Schwendenerstr. 1, 14195 Berlin, Germany</footnote>

<footnote n="4">⁴Department for Materials and Environment, BAM Federal Institute for Materials Research and Testing, Unter den Eichen 87, 12205 Berlin, Germany</footnote>

<footnote n="5">⁵German Centre for Integrative Biodiversity Research Halle-Jena-Leipzig (iDiv), Deutscher Platz 5e, 04103 Leipzig, Germany</footnote>

<footnote n="6">⁶Apicultural State Institute, University of Hohenheim, 70599 Stuttgart, Germany</footnote>

<footnote>†These authors contributed equally</footnote>

<footnote>‡Current address: Section for Organismal Biology, Department of Plant and Environmental Sciences, University of Copenhagen, Denmark</footnote>

<footnote>¤Current address: Centre for Ecology and Conservation, University of Exeter, Penryn, UK</footnote>
<table><caption>**Table S1.** Virus and host housekeeping gene (beta-Actin) presence/absence (MLPA) from pooled samples.</caption><thead><tr><th rowspan="2">Colony</th><th colspan="2">DWV complex</th><th colspan="2">BQCV</th><th colspan="2">CBPV</th><th colspan="2">Actin</th></tr><tr><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th></tr></thead><tbody><tr><td>cs1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>cs2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>cs3</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>cs4</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>cs5</td><td>1</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>cs6</td><td>1</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>cs7</td><td>1</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ck1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ck2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ck3</td><td>1</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ck4</td><td>1</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ck5</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ck6</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ck7</td><td>1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ts1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ts2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>1</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ts3</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ts4</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ts5</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>ts6</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td><td>1</td></tr><tr><td>ts7</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>tk1</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>tk2</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>tk3</td><td>1</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>tk4</td><td>0</td><td>0</td><td>1</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>tk5</td><td>1</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>tk6</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr><tr><td>tk7</td><td>1</td><td>0</td><td>0</td><td>0</td><td>0</td><td>0</td><td>1</td><td>1</td></tr></tbody></table>
<caption>**Table S2. A.** Virus loads (qRT-PCR) from pooled samples as Ct values for viruses detected by MLPA (see Table S1). **B.** Ct values transformed into virus genome equivalents, averaged per bee, for those two viruses with Ct < 35, along with the normalized values relative to the absolute quantity of the host housekeeping gene RP49. **C.** Overwinter worker mortality (%) along with viral and *Nosema* spp. prevalence per colony. Pathogen prevalence was based on 15 individuals per colony while *V. destructor* infestation was estimated on a sample of 150 individuals per colony.</caption>

**A.**

<table><thead><tr><th rowspan="2">Colony</th><th colspan="2">DWV-B (RdRp)</th><th colspan="2">DWV-B (Lp)</th><th colspan="2">DWV-A (RdRp)</th><th colspan="2">DWV-A (Lp)</th><th colspan="2">BQCV (Cp)</th><th colspan="2">RP49</th></tr><tr><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th></tr></thead><tbody><tr><td>cs1</td><td>34.79</td><td>na</td><td>na</td><td>na</td><td><strong>39.02</strong></td><td><strong>36.41</strong></td><td>na</td><td>na</td><td>32.73</td><td>33.02</td><td>35.96</td><td>34.43</td></tr><tr><td>cs2</td><td>22.06</td><td>na</td><td>33.76</td><td>na</td><td><strong>38.15</strong></td><td>na</td><td>na</td><td>na</td><td><strong>35.66</strong></td><td><strong>38.45</strong></td><td>31.48</td><td>30.14</td></tr><tr><td>cs3</td><td>19.21</td><td>na</td><td>28.44</td><td>na</td><td><strong>38.69</strong></td><td>na</td><td>na</td><td>na</td><td>35.35</td><td>na</td><td>34.08</td><td>31.97</td></tr><tr><td>cs4</td><td>22.03</td><td>na</td><td>35.02</td><td>na</td><td><strong>36.43</strong></td><td>na</td><td>na</td><td>na</td><td><strong>37.47</strong></td><td>na</td><td>34.72</td><td>29.47</td></tr><tr><td>cs5</td><td>28.39</td><td>na</td><td><strong>39.59</strong></td><td>na</td><td><strong>38.14</strong></td><td>na</td><td>na</td><td>na</td><td><strong>36.00</strong></td><td><strong>37.12</strong></td><td>32.83</td><td>27.96</td></tr><tr><td>cs6</td><td>17.19</td><td>na</td><td>29.17</td><td>na</td><td><strong>38.78</strong></td><td><strong>39.18</strong></td><td>na</td><td>na</td><td>32.51</td><td>na</td><td>34.61</td><td>35.76</td></tr><tr><td>cs7</td><td>22.01</td><td>na</td><td>32.21</td><td>na</td><td><strong>38.34</strong></td><td>na</td><td>na</td><td>na</td><td>19.38</td><td>na</td><td>28.04</td><td>28.90</td></tr><tr><td>ck1</td><td>21.21</td><td>34.04</td><td>31.07</td><td>na</td><td><strong>36.34</strong></td><td>na</td><td>na</td><td>na</td><td>32.22</td><td>na</td><td>29.46</td><td>32.16</td></tr><tr><td>ck2</td><td>20.13</td><td>32.14</td><td>28.90</td><td>na</td><td><strong>37.68</strong></td><td><strong>38.25</strong></td><td>na</td><td>na</td><td>31.16</td><td><strong>38.80</strong></td><td>28.45</td><td>30.95</td></tr><tr><td>ck3</td><td>19.87</td><td>20.24</td><td>28.78</td><td>32.77</td><td>na</td><td><strong>38.81</strong></td><td>na</td><td>na</td><td>28.13</td><td>34.40</td><td>33.46</td><td>28.18</td></tr><tr><td>ck4</td><td>23.06</td><td>34.57</td><td>32.00</td><td>na</td><td><strong>38.35</strong></td><td>na</td><td>na</td><td>na</td><td>33.49</td><td>33.22</td><td>30.79</td><td>30.84</td></tr><tr><td>ck5</td><td>20.45</td><td>28.82</td><td>35.48</td><td>na</td><td><strong>37.52</strong></td><td>na</td><td>na</td><td>na</td><td>31.24</td><td><strong>38.74</strong></td><td>32.49</td><td>31.78</td></tr><tr><td>ck6</td><td>21.09</td><td>na</td><td>31.19</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td><strong>36.20</strong></td><td><strong>38.34</strong></td><td>34.76</td><td>32.62</td></tr><tr><td>ck7</td><td>26.34</td><td>na</td><td>34.98</td><td><strong>38.53</strong></td><td>na</td><td>na</td><td>na</td><td>na</td><td>31.45</td><td><strong>38.92</strong></td><td>33.04</td><td>29.57</td></tr><tr><td>ts1</td><td>30.12</td><td>na</td><td>33.03</td><td>na</td><td><strong>37.60</strong></td><td>na</td><td>na</td><td>na</td><td>35.21</td><td>na</td><td>30.31</td><td>34.30</td></tr><tr><td>ts2</td><td>29.67</td><td>na</td><td><strong>36.63</strong></td><td>na</td><td><strong>37.13</strong></td><td>na</td><td>na</td><td>na</td><td>34.79</td><td>na</td><td>31.03</td><td>33.99</td></tr><tr><td>ts3</td><td>22.5</td><td>na</td><td>30.73</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td><strong>36.56</strong></td><td>na</td><td>33.03</td><td>32.69</td></tr><tr><td>ts4</td><td>22.94</td><td>na</td><td>31.57</td><td>na</td><td>na</td><td><strong>35.55</strong></td><td>na</td><td>na</td><td>34.93</td><td><strong>38.17</strong></td><td>33.51</td><td>32.61</td></tr><tr><td>ts5</td><td>20.78</td><td>na</td><td>27.78</td><td>na</td><td><strong>37.50</strong></td><td>na</td><td>na</td><td>na</td><td><strong>35.59</strong></td><td>na</td><td>34.22</td><td>35.49</td></tr><tr><td>ts6</td><td>18.44</td><td>na</td><td>28.53</td><td>na</td><td><strong>36.02</strong></td><td><strong>36.28</strong></td><td>na</td><td>na</td><td>35.40</td><td>na</td><td>30</td><td>35.34</td></tr><tr><td>ts7</td><td>21.54</td><td>na</td><td>29.51</td><td>na</td><td><strong>37.35</strong></td><td>na</td><td>na</td><td>na</td><td>30.93</td><td>na</td><td>28.1</td><td>33.31</td></tr><tr><td>tk1</td><td>20.6</td><td>na</td><td>28.39</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>27.20</td><td><strong>37.79</strong></td><td>34.45</td><td>31.03</td></tr><tr><td>tk2</td><td>22.7</td><td>na</td><td>31.81</td><td>na</td><td>na</td><td><strong>35.62</strong></td><td>na</td><td>na</td><td>32.92</td><td>na</td><td>33.2</td><td>30.79</td></tr><tr><td>tk3</td><td>18.75</td><td>na</td><td>28.82</td><td>na</td><td><strong>37.79</strong></td><td>na</td><td>na</td><td>na</td><td>32.15</td><td><strong>35.75</strong></td><td>30.46</td><td>33.88</td></tr><tr><td>tk4</td><td><strong>35.79</strong></td><td>na</td><td>na</td><td>na</td><td><strong>36.79</strong></td><td>na</td><td>na</td><td>na</td><td>27.65</td><td>34.68</td><td>31.57</td><td>31.69</td></tr><tr><td>tk5</td><td>20.81</td><td>na</td><td>30.44</td><td>na</td><td><strong>39.10</strong></td><td>na</td><td>na</td><td>na</td><td>29.81</td><td>33.84</td><td>34.5</td><td>32.30</td></tr><tr><td>tk6</td><td>21.02</td><td>34.47</td><td>29.82</td><td>na</td><td><strong>37.43</strong></td><td><strong>38.68</strong></td><td>na</td><td>na</td><td>31.83</td><td><strong>37.96</strong></td><td>34.2</td><td>32.17</td></tr><tr><td>tk7</td><td>21.24</td><td>na</td><td>30.05</td><td>na</td><td>na</td><td>na</td><td>na</td><td>na</td><td>33.27</td><td>na</td><td>32.97</td><td>32.95</td></tr></tbody></table>

c = control treated; t = pesticide treated; s = Simonswald; k = Kenzingen. Bold/italic Ct values > acceptance threshold (Ct = 35). na = not detected; RdRp = RNA-dependent RNA polymerase; Lp = Leader polypeptide; Cp = Capsid protein
<caption>Table S2. Cont.</caption>

B.

<table><thead><tr><th rowspan="2">Colony</th><th colspan="2">DWV-B (RdRp)</th><th colspan="2">BQCV (Cp)</th><th colspan="2">DWV-B (RdRp)<br>Normalized to RP49</th><th colspan="2">BQCV (Cp)<br>Normalized to RP49</th></tr><tr><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th><th>Autumn</th><th>Spring</th></tr></thead><tbody><tr><td>cs1</td><td>301358</td><td></td><td>2E+06</td><td>2E+06</td><td>0.004509128</td><td></td><td>0.06355</td><td>0.01954</td></tr><tr><td>cs2</td><td>5E+08</td><td></td><td></td><td></td><td>0.896240745</td><td></td><td></td><td></td></tr><tr><td>cs3</td><td>9E+09</td><td></td><td>335537</td><td></td><td>29.86662299</td><td></td><td>0.00319</td><td></td></tr><tr><td>cs4</td><td>1E+09</td><td></td><td></td><td></td><td>7.35743038</td><td></td><td></td><td></td></tr><tr><td>cs5</td><td>8E+06</td><td></td><td></td><td></td><td>0.036566367</td><td></td><td></td><td></td></tr><tr><td>cs6</td><td>1E+10</td><td></td><td>1E+06</td><td></td><td>153.2693408</td><td></td><td>0.03099</td><td></td></tr><tr><td>cs7</td><td>2E+09</td><td></td><td>2E+10</td><td></td><td>0.101596447</td><td></td><td>3.37688</td><td></td></tr><tr><td>ck1</td><td>2E+09</td><td>31858</td><td>3E+06</td><td></td><td>0.422940383</td><td>1.54068E-05</td><td>0.00137</td><td></td></tr><tr><td>ck2</td><td>2E+09</td><td>116216</td><td>3E+06</td><td></td><td>0.442219063</td><td>2.98097E-05</td><td>0.00148</td><td></td></tr><tr><td>ck3</td><td>1E+10</td><td>3E+08</td><td>8E+07</td><td>428361</td><td>13.13280105</td><td>2.667018767</td><td>0.28887</td><td>0.00014</td></tr><tr><td>ck4</td><td>8E+08</td><td>21225</td><td>1E+06</td><td>2E+06</td><td>0.304060351</td><td>2.63357E-05</td><td>0.00136</td><td>0.00169</td></tr><tr><td>ck5</td><td>4E+09</td><td>908275</td><td>6E+06</td><td></td><td>4.839704616</td><td>0.004003648</td><td>0.01873</td><td></td></tr><tr><td>ck6</td><td>2E+09</td><td></td><td></td><td></td><td>13.82242841</td><td></td><td></td><td></td></tr><tr><td>ck7</td><td>8E+07</td><td></td><td>4E+06</td><td></td><td>0.156514321</td><td></td><td>0.02309</td><td></td></tr><tr><td>ts1</td><td>8E+06</td><td></td><td>370572</td><td></td><td>0.002384866</td><td></td><td>0.00031</td><td></td></tr><tr><td>ts2</td><td>5E+06</td><td></td><td>253119</td><td></td><td>0.00506875</td><td></td><td>0.00066</td><td></td></tr><tr><td>ts3</td><td>1E+09</td><td></td><td></td><td></td><td>1.834678945</td><td></td><td></td><td></td></tr><tr><td>ts4</td><td>8E+08</td><td></td><td>455627</td><td></td><td>1.879646072</td><td></td><td>0.00295</td><td></td></tr><tr><td>ts5</td><td>3E+09</td><td></td><td></td><td></td><td>11.90782037</td><td></td><td></td><td></td></tr><tr><td>ts6</td><td>1E+10</td><td></td><td>304670</td><td></td><td>3.546638523</td><td></td><td>0.00022</td><td></td></tr><tr><td>ts7</td><td>1E+09</td><td></td><td>4E+06</td><td></td><td>0.142624919</td><td></td><td>0.00138</td><td></td></tr><tr><td>tk1</td><td>3E+09</td><td></td><td>9E+07</td><td></td><td>15.50489468</td><td></td><td>1.02962</td><td></td></tr><tr><td>tk2</td><td>1E+09</td><td></td><td>2E+06</td><td></td><td>1.803011696</td><td></td><td>0.00947</td><td></td></tr><tr><td>tk3</td><td>1E+10</td><td></td><td>3E+06</td><td></td><td>3.914529268</td><td></td><td>0.00274</td><td></td></tr><tr><td>tk4</td><td></td><td></td><td>6E+07</td><td>850053</td><td></td><td></td><td>0.11841</td><td>0.00108</td></tr><tr><td>tk5</td><td>3E+09</td><td></td><td>1E+07</td><td>1E+06</td><td>13.98768012</td><td></td><td>0.18009</td><td>0.00285</td></tr><tr><td>tk6</td><td>3E+09</td><td>20489</td><td>4E+06</td><td></td><td>10.06830247</td><td>0.000282625</td><td>0.03783</td><td></td></tr><tr><td>tk7</td><td>2E+09</td><td></td><td>1E+06</td><td></td><td>3.967555442</td><td></td><td>0.00642</td><td></td></tr></tbody></table>
<table><caption>**Table S2. Cont.**
**C.**</caption><thead><tr><th rowspan="2">Colony</th><th rowspan="2">overwinter worker mortality (%)</th><th rowspan="2">Varroa infestation (%)</th><th>DWV-B prevalence (%)</th><th>BQCV prevalence (%)</th><th>CBPV prevalence (%)</th><th>N. ceranae prevalence (%)</th><th>N. apis prevalence (%)</th></tr><tr><th>Autumn</th><th>Autumn</th><th>Autumn</th><th>Autumn</th><th>Autumn</th></tr></thead><tbody><tr><td>cs1</td><td>43.66</td><td>5.3</td><td>6.7</td><td>6.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>cs2</td><td>6.51</td><td>3.8</td><td>6.7</td><td>26.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>cs3</td><td>34.78</td><td>2.2</td><td>33.3</td><td>40</td><td>0</td><td>6.7</td><td>0</td></tr><tr><td>cs4</td><td>52.93</td><td>6.2</td><td>40</td><td>20</td><td>6.7</td><td>0</td><td>0</td></tr><tr><td>cs5</td><td>45.47</td><td>2.8</td><td>60</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>cs6</td><td>13.08</td><td>8.9</td><td>6.7</td><td>0</td><td>6.7</td><td>0</td><td>0</td></tr><tr><td>cs7</td><td>40.55</td><td>0.5</td><td>13.3</td><td>0</td><td>6.7</td><td>0</td><td>0</td></tr><tr><td>ck1</td><td>55.57</td><td>31.6</td><td>73.3</td><td>26.7</td><td>0</td><td>6.7</td><td>0</td></tr><tr><td>ck2</td><td>55.6</td><td>20</td><td>26.7</td><td>13.3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>ck3</td><td>52.38</td><td>4.1</td><td>20</td><td>13.3</td><td>0</td><td>6.7</td><td>0</td></tr><tr><td>ck4</td><td>72.9</td><td>20.2</td><td>13.3</td><td>13.3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>ck5</td><td>55.75</td><td>10.8</td><td>33.3</td><td>26.7</td><td>6.7</td><td>0</td><td>0</td></tr><tr><td>ck6</td><td>51.9</td><td>28.5</td><td>40</td><td>40</td><td>0</td><td>13.3</td><td>0</td></tr><tr><td>ck7</td><td>63.9</td><td>10.7</td><td>6.7</td><td>26.7</td><td>6.7</td><td>0</td><td>0</td></tr><tr><td>ts1</td><td>22.91</td><td>0</td><td>6.7</td><td>0</td><td>6.7</td><td>6.7</td><td>6.7</td></tr><tr><td>ts2</td><td>33.67</td><td>0</td><td>20</td><td>20</td><td>13.3</td><td>0</td><td>0</td></tr><tr><td>ts3</td><td>32.34</td><td>0</td><td>13.3</td><td>0</td><td>13.3</td><td>6.7</td><td>0</td></tr><tr><td>ts4</td><td>74.38</td><td>0.4</td><td>13.3</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>ts5</td><td>59.92</td><td>0</td><td>60</td><td>6.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>ts6</td><td>36.54</td><td>0</td><td>26.7</td><td>6.7</td><td>0</td><td>0</td><td>0</td></tr><tr><td>ts7</td><td>37.91</td><td>0</td><td>6.7</td><td>0</td><td>0</td><td>0</td><td>0</td></tr><tr><td>tk1</td><td>25.17</td><td>0</td><td>40</td><td>40</td><td>0</td><td>6.7</td><td>0</td></tr><tr><td>tk2</td><td>17.79</td><td>0</td><td>6.7</td><td>13.3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>tk3</td><td>25.09</td><td>0</td><td>6.7</td><td>53.3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>tk4</td><td>22.68</td><td>0</td><td>0</td><td>20</td><td>0</td><td>0</td><td>0</td></tr><tr><td>tk5</td><td>39.06</td><td>0</td><td>6.7</td><td>40</td><td>0</td><td>0</td><td>0</td></tr><tr><td>tk6</td><td>29.82</td><td>0</td><td>0</td><td>40</td><td>0</td><td>0</td><td>6.7</td></tr><tr><td>tk7</td><td>10</td><td>1.2</td><td>0</td><td>13.3</td><td>0</td><td>0</td><td>0</td></tr></tbody></table>
<table><caption>**Table S3.** Genome of origin for Illumina sequence reads from the DWV-B field extract (from McMahon et al.¹).</caption><thead><tr><th>Origin</th><th>DWV-B reads</th></tr></thead><tbody><tr><td><em>Apis</em></td><td>15,276,447 (74.5%)</td></tr><tr><td>Viruses</td><td></td></tr><tr><td>DWV-B</td><td>4,547,491 (22.2%)</td></tr><tr><td>DWV-A</td><td>6,157 (0.03%)</td></tr><tr><td>Bacteria</td><td>1,011 (0.0049%)</td></tr><tr><td>Overhead</td><td></td></tr><tr><td>phiX</td><td>26,355 (0.13%)</td></tr><tr><td>sequencing adapters</td><td>3,555 (0.017%)</td></tr><tr><td>low complexity</td><td>602,276 (2.9%)</td></tr><tr><td>Unknown</td><td>47,587 (0.23%)</td></tr><tr><td>Total</td><td>20,510,879</td></tr></tbody></table>
<table><caption>**Table S4.** Primers used in this study. The position of DWV-A and DWV-B primers across the genome are indicated in brackets (Accession number in Genbank: DWV-A ref: NC_004830; DWV-B ref: NC_006494). Minimum detection limit is based upon standard curves of viral dilutions run simultaneously with the samples.</caption><thead><tr><th>Target</th><th>Name</th><th>Sequence</th><th>Application</th><th>Amplicon size (bp)</th><th>Reference</th><th>Minimum detection limit (ct=35) in viral genome equivalents per reaction</th></tr></thead><tbody><tr><td>DWV-A (Lp)</td><td>DWV-F1425</td><td>CGTCGGCCTATCAAAG</td><td>qPCR</td><td>417</td><td>[2]</td><td rowspan="2">~1.7x 10<sup>3</sup></td></tr><tr><td>DWV-A (Lp)</td><td>DWV-B1806</td><td>CTTTTCTAATTCAACTTCACC</td><td>qPCR</td><td>position[1415...1831]</td><td>"</td></tr><tr><td>DWV-B (Lp)</td><td>VDV1-F1409</td><td>GCCCTGTTCAAGAACATG</td><td>qPCR</td><td>413</td><td>"</td><td rowspan="2">~1.5x10<sup>3</sup></td></tr><tr><td>DWV-B (Lp)</td><td>DWV-B1806</td><td>CTTTTCTAATTCAACTTCACC</td><td>qPCR</td><td>position[1392...1804]</td><td>"</td></tr><tr><td>DWV-A (RdRp)</td><td>DWV-F2</td><td>TGTCTTCATTAAAGCCACCTGGAA</td><td>qPCR</td><td>140</td><td>[1]</td><td rowspan="2">~4.9</td></tr><tr><td>DWV-A (RdRp)</td><td>DWV-R2a</td><td>TTTCCTCATTAACCTGTGTCGTTGAT</td><td>qPCR</td><td>position[8646...8785]</td><td>"</td></tr><tr><td>DWV-B (RdRp)</td><td>VDV-F2</td><td>TATCTTCATTAAAACCGCCAGGCT</td><td>qPCR</td><td>140</td><td>"</td><td rowspan="2">~11.33</td></tr><tr><td>DWV-B (RdRp)</td><td>VDV-R2a</td><td>CTTCCTCATTAACCTGAGTTGTTGTC</td><td>qPCR</td><td>position[8619...8758]</td><td>"</td></tr><tr><td>BQCV</td><td>BQCV-F7893</td><td>AGTGGCGGAGATGTATGC</td><td>qPCR</td><td>294</td><td>[2]</td><td rowspan="2">~3x10<sup>2</sup></td></tr><tr><td>BQCV</td><td>BQCV-B8150</td><td>GGAGGTGAAGTGGCTATATC</td><td>qPCR</td><td></td><td>"</td></tr><tr><td>RP49</td><td>RP49-qF</td><td>AAGTTCATTCGTCACCAGAG</td><td>qPCR</td><td>205</td><td>[3]</td><td rowspan="2">~3.3x10<sup>4</sup></td></tr><tr><td>RP49</td><td>RP49-qB</td><td>CTTCCAGTTCCTTGACATTATG</td><td>qPCR</td><td></td><td>"</td></tr><tr><td>N. ceranae</td><td>Ncer-F</td><td>CAATATTTTATTATTTTGAGAGA</td><td>RT-PCR</td><td>232</td><td>[4]</td><td rowspan="2">NA</td></tr><tr><td>N. ceranae</td><td>Ncer-R</td><td>TATATTTATTGTATTGCGCGTGCA</td><td>RT-PCR</td><td></td><td>"</td></tr><tr><td>N. apis</td><td>Napis-F</td><td>CAATATTTTATTGTTCTGCGAGG</td><td>RT-PCR</td><td>208</td><td>modified from [4]</td><td rowspan="2">NA</td></tr><tr><td>N. apis</td><td>Napis-R</td><td>AAAGTCTATTGTATTGCGCGTGCT</td><td>RT-PCR</td><td></td><td></td></tr></tbody></table>
<figure><img src="image_9.png" /></figure>

**Figure S1.** Relationship between CBPV, *N. ceranae* and *N. apis* prevalence within colonies in autumn (estimated from 15 individually screened honeybees) and overwinter mortality. Spearman's *ρ* and significance levels are shown.

References

1. McMahon, D. P. et al. Elevated virulence of an emerging viral genotype as a driver of honeybee loss. *Proc. R. Soc. B Biol. Sci.* (2016). doi:10.1098/rspb.2016.0811

2. Locke, B., Forsgren, E., Fries, I. & de Miranda, J. R. Acaricide treatment affects viral dynamics in *Varroa destructor*-infested honey bee colonies via both host physiology and mite control. *Appl. Environ. Microbiol.* **78**, 227–235 (2012).

3. Yañez, O. et al. Deformed wing virus and drone mating flights in the honey bee (*Apis mellifera*): Implications for sexual transmission of a major honey bee virus. *Apidologie* **43**, 17–30 (2012).

4. VanEngelsdorp, D. et al. Colony collapse disorder: a descriptive study. *PLoS One* **4**, e6481 (2009).