posada-florez et al. varroa vectoring of a honey bee virus · posada-florez et al. varroa vectoring...

Posada-Florez et al. Varroa vectoring of a honey bee virus

1

Title: 1

Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa 2

destructor in a non-propagative manner. 3

4

Authors: 5

Francisco Posada-Florez*, Anna K. Childers, Matthew C. Heerman, Noble I. Egekwu, Steven C. 6

Cook, Yanping Chen, Jay D. Evans, Eugene V. Ryabov* 7

8

Affiliations: 9

USDA, Agricultural Research Service, Bee Research Lab, Beltsville, MD, USA 10

11

*Corresponding authors: 12

Francisco Posada-Florez 13

Email: [email protected] ; Phone: (301) 504-5143 14

USDA, Agricultural Research Service, Bee Research Lab, BARC-East Bldg. 306, 10300 Baltimore 15

Ave., Beltsville, MD 20705, USA 16

17

Eugene V. Ryabov 18

Email: [email protected] ; Phone: (301) 504-8205 19

USDA, Agricultural Research Service, Bee Research Lab, BARC-East Bldg. 306, 10300 Baltimore 20

Ave., Beltsville, MD 20705, USA 21

22

.CC-BY-NC-ND 4.0 International licenseacertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

The copyright holder for this preprint (which was notthis version posted June 6, 2019. ; https://doi.org/10.1101/660985doi: bioRxiv preprint

https://doi.org/10.1101/660985

http://creativecommons.org/licenses/by-nc-nd/4.0/


2

Abstract 23

Honey bees, the primary managed insect pollinator, suffer considerable losses due to Deformed 24

wing virus (DWV), an RNA virus vectored by the mite Varroa destructor. Mite vectoring has 25

resulted in the emergence of virulent DWV variants. The basis for such changes in DWV is poorly 26

understood. Most importantly, it remains unclear whether replication of DWV occurs in the mite. 27

In this study, we exposed Varroa mites to DWV type A via feeding on artificially infected honey 28

bees. A significant, 357-fold increase in DWV load was observed in these mites after 2 days. 29

However, after 8 additional days of passage on honey bee pupae with low viral loads, the DWV 30

load dropped by 29-fold. This decrease significantly reduced the mites’ ability to transmit DWV 31

to honey bees. Notably, negative-strand DWV RNA, which could indicate viral replication, was 32

detected only in mites collected from pupae with high DWV levels but not in the passaged mites. 33

We also found that Varroa mites contain honey bee mRNAs, consistent with the acquisition of 34

honey bee cells which would additionally contain DWV replication complexes with negative-35

strand DWV RNA. We propose that transmission of DWV type A by Varroa mites occurs in a non-36

propagative manner. 37

38

Keywords: Honey bees, pollination, Deformed wing virus, Varroa destructor, recombination, 39

Colony Collapse Disorder, virus vector 40

41

42



https://doi.org/10.1101/660985



3

43

INTRODUCTION 44

Honey bee (Apis mellifera), the primary managed insect pollinator, suffers considerable 45

losses caused by pathogens1,2, including Deformed wing virus (DWV)3, which endanger 46

pollination service worldwide4,5. Three “master” variants of DWV were discovered including 47

DWV-A3, DWV-B (also known as Varroa destructor virus-16), and DWV-C7. In addition, 48

recombinants between DWV-A and DWV-B were reported as major DWV variants in some 49

localities in the UK, Israel and France8-10. Negative impacts of DWV infection usually manifests 50

when the virus is vectored by the ectoparasitic mite Varroa destructor11-13. Importantly, both 51

DWV-A and DWV-B variants are vectored by the mite. The Varroa mite, originally a parasite of 52

the Asian honey bee Apis cerana, spread to the Western honey bee A. mellifera in the beginning 53

of the 20th century, reaching Western Europe and subsequently North America about 30 years 54

ago14. DWV was present in honey bees prior to Varroa arrival, although at low levels15. The mite, 55

which provides an efficient horizontal transmission route for DWV, transformed this covert virus 56

into a highly virulent virus. DWV levels in Varroa-infested bees are 100 to 1000-fold higher 57

compared to Varroa-free bees16,17. Such increases of DWV virulence were accompanied by 58

genetic changes in the virus, as reported in Hawaii15 and Scotland16, then further confirmed by an 59

analysis of DWV evolution worldwide117. 60

Unraveling the mechanisms that drive genetic changes in DWV requires a comprehensive 61

understanding of the interactions between each component within the tripartite system “Honey 62

bee-DWV-Varroa mite”. Importantly, there is no agreement on whether replication of all DWV 63



https://doi.org/10.1101/660985



4

variants takes place within the mite vector17. The currently accepted view that DWV replication 64

occurs in Varroa mites is largely based mostly on the detection of negative-strand DWV 65

RNA16,18,19. DWV belongs to the genus Iflaviridae, order Picornavirales, members of which are 66

known to have virus particles containing exclusively positive strands as their genomic RNA20,21. 67

Therefore, presence of negative-strand viral RNA in the mites could indicate that replication of 68

DWV takes place in this species. Virus particles were reported in the cells of Varroa mites 69

possibly harboring DWV-B (VDV1)6. Contrary to this, an immunolocalization study of Varroa 70

mites feeding on DWV-infected pupae revealed no presence of DWV in Varroa cells. Instead, 71

DWV antigen was detected exclusively in the mites’ midgut lumen22. This apparent 72

disagreement could be a explained by the use of different DWV isolates (i.e. “master types”), 73

which could differ in respect to their replication in Varroa mites. For example, recently negative 74

strand RNA of DWV-B was detected inVarroa synganglions23.. Unfortunately, previous studies 75

which investigated DWV replication in the mites, did not include identification of the DWV 76

type18,19. Although strong positive correlation of DWV loads was reported in naturally infested 77

colonies between the Varroa mites and associated honey bee pupae, this cannot be used as an 78

argument in support of DWV replicates within Varroa mainly because DWV is vectored by mites 79

and could be acquired by the mites from the same pupae which they infect. Therefore, the 80

question of whether replication of DWV-A, the most widespread variant, occurs in the mite 81

vector remains open. 82

In this study, we investigated temporal dynamics of both positive and negative-strand DWV-A 83

RNA accumulation in Varroa mites and honey bee pupae, as well as transmission of the virus 84

between mites and pupae. The experiments were conducted in controlled conditions rather than 85



https://doi.org/10.1101/660985



5

in the honey bee colony environment, which allowed for feeding of individual mites only on 86

certain pupae for a chosen period of time. By using artificial infection of the honey bee pupae 87

with DWV-A, we could specifically monitor acquisition of the virus by the mites and transmission 88

to new pupae. Moreover, we were able to trace the DWV transmission route by using a cloned 89

DWV isolate. We found no evidence supporting DWV-A replication in the Varroa mites, 90

demonstrating that virus loads in the mite are highly dynamic, and that the mites’ ability to 91

transmit DWV significantly declines following loss of their DWV loads after feeding on the honey 92

bees with low DWV levels. 93

94

Results 95

Significant increases and decreases in DWV loads in Varroa mites following feeding on pupae 96

with different DWV levels. 97

Two experiments were carried out to investigate the effect of pupal DWV levels on mite 98

DWV loads (Figs. 1 and 2). For both experiments, phoretic mites were collected from adult honey 99

bees in colonies with DWV levels typical for US honey bee colonies. A control sample of phoretic 100

mites (V-Pho1) was immediately collected for molecular analysis. Two or three mites were placed 101

on individual honey bee pupae which had been injected 3 days earlier with either DWV or a 102

control PBS solution. The virus used was a clone-derived DWV-304(GenBank Accession Number 103

MG831200) (E. Ryabov, personal communication), which was a strain of DWV type A7 isolated 104

from a typical Varroa-infested honey bee colony. The mites and pupae were incubated in 105



https://doi.org/10.1101/660985



6

individual gelatin capsules as described earlier24 (Fig. 1B). After 2 days, all injected pupae (P0-106

PBS1, P0-DWV1, P0-PBS2 and P0-DWV2) and one Varroa mite from each pupa (V0-PBS1, V0-PBS2, 107

V0-DWV1 and V0-DWV2) were collected. The remaining one or two mites from each injected 108

pupa were transferred to fresh pupae for an additional 2 days of feeding. These two-day 109

passages were repeated four times, after which Varroa mites (V4-PBS1, V4-PBS2, V4-DWV1 and 110

V4-DWV2) and pupae (P4-PBS1 and P4-PBS1) were collected for molecular analysis. The mites’ 111

survival rates in Experiments 1 and 2 were 95.7% and 83.3%, respectively; with no significant 112

difference observed between the mites initially exposed to pupae injected with DWV or PBS 113

(Experiment 1, p = 0.32; Experiment 2, p = 0.93; Wilcoxon test). High Varroa survival in these 114

experiments demonstrates that mites were robust under in vitro conditions, allowing for reliable 115

virus accumulation and transmission results. 116

We quantified copy numbers of DWV RNA in the collected mites and pupae by using RT-117

qPCR assays which included reverse transcription using random primers and detected mainly 118

positive-strand genomic RNA. The levels of DWV in pupae injected with DWV were significantly 119

higher than in the PBS-injected pupae (Fig. 3c, Fig. 4c). In particular, we confirmed development 120

of high-level DWV infection in the DWV-injected groups, with 19 out of 20 bees in P0-DWV1, and 121

11 out of 12 bees in P0-DWV2 containing more than 109 DWV genomes copies. PBS-injected bees 122

had mainly low levels of DWV with only 1 out of 20 bees in P0-PBS1, and 1 out of 12 bees P0-PBS2 123

having more than 109 DWV genome copies. The level of 109 DWV genome copies was used as the 124

accepted threshold infection level between asymptomatic and symptomatic DWV infections (i.e. 125

“covert“ and “overt”) as reported in several studies19,25,26. Therefore, we obtained groups of 126



https://doi.org/10.1101/660985



7

honey bee pupae with significantly different DWV levels to investigate DWV acquisition and 127

transmission by the mites. 128

Quantification of DWV in the mites showed no significant difference in virus copy 129

numbers between the phoretic mites (V-Pho1) and in the mites reared for two days in the PBS-130

injected pupae in Experiment 1 (V0-PBS1), while two days feeding on pupae injected with DWV 131

(V0-DWV1) resulted in a statistically significant 357-fold increase (Fig. 3a, df = 31, F value = 46.63, 132

P < 0.0001, large effect size, Cohen's d =2.40). Importantly, in both Experiments 1 and 2, 133

significantly higher DWV levels were observed in the mites fed on the DWV-injected pupae with 134

high DWV levels, (V0-DWV1 and V0-DWV2) compared to their respective control mites fed on 135

PBS-injected pupae with significantly lower DWV levels (V0-PBS1 and V0-PBS2), a 625 fold 136

increase in Experiment 1 (Fig. 3a, df = 39, F value = 51.55, P < 0.0001; large effect size, Cohen's d 137

= 2.27), and a 51-fold increase in Experiment 2 (Fig. 4a; df = 23, F value = 12.66, P = 0.001760, 138

large effect size, Cohen's d =1.45). 139

Surprisingly, high DWV loads in the mites (V0-DWV1 and V0-DWV2) fed for 2-days on 140

pupae with high DWV levels (P0-DWV1 and P0-DWV2), were not maintained for the duration of 141

the experiment. In both experiments the levels of DWV in these mites decreased significantly 142

after four 2-day passages on un-injected pupae with low DWV levels, with a 29-fold decrease in 143

Experiment 1 (V0-DWV1 and V4-DWV1; Fig, 3a, df = 40, F value = 32.19, P < 0.0001; large effect 144

size, Cohen's d = 1.77), and a 18-fold decrease in Experiment 2 (V0-DWV2 and V4-DWV2; Fig. 4a; 145

df = 21, F value = 28.65, P < 0.0001; large effect size, Cohen's d =2.28). After the passages, the 146

levels of DWV in the mites initially exposed to the high DWV-level pupae (V4-DWV1 and V4-147



https://doi.org/10.1101/660985



8

DWV2) were not significantly different from those in mites initially reared on control PBS-injected 148

pupae (V4-PBS1 and V4-PBS2; Fig. 3a, Fig. 4a; P>0.05). 149

150

DWV loads in Varroa mites strongly influence virus transmission to the honey bees 151

To investigate how Varroa mites’ ability to transmit DWV was influenced by their DWV 152

loads, we designed Experiment 2 (Fig. 2). Here, DWV levels were quantified in pupae incubated 153

for additional 3 days after mite removal to allow development of the DWV infection. We tested 154

Varroa-mediated transmission in the Pupae 1 and Pupae 4 groups, which were exposed to the 155

mites fed on the Pupae 0 group injected with PBS (P1-PBS-Ext2 and P4-PBS-Ext2) or with DWV 156

(P1-DWV-Ext2 and P4-DWV-Ext2) (Fig. 2). We observed significantly higher DWV copy numbers in 157

pupae exposed to mites that had directly fed on high DWV-level pupae (P1-DWV-Ext2) compared 158

to pupae exposed to mites subjected to four 2-day passages (33-fold difference) after initial 159

rearing on the DWV-infected bees (P4-DWV-Ext2) (Fig. 4c, df = 21, F value = 14.94, P = 0.000964; 160

large effect size, Cohen's d =1.79). Importantly, there were no significant differences between 161

DWV levels in pupae from group P4-DWV-Ext2, and in the control groups P1-PBS-Ext2 and P4-PBS-162

Ext2 (Fig. 4c). A significantly higher proportion of honey bees in the group P1-DWV-Ext2 pupae 163

developed a high-level DWV infection above 109 DWV genome copies per bee (7 out of 12), 164

compared to pupal group P4-DWV-Ext2 subjected to passaged mites where only 1 pupa out of 10 165

just met the minimum threshold (Fig. 4c; contingency table analysis, Fisher exact probability test, 166

P=0.0263). Notably, high DWV levels were not observed in pupae exposed to control mites 167

originally fed on PBS injected pupae (P1-PBS-Ext2 and P4-PBS-Ext2; Fig. 4c). 168



https://doi.org/10.1101/660985



9

The use of a clone-derived DWV isolate with known sequence helped trace the source of 169

DWV transmitted by the Varroa mites. Sequencing 1.2 kb-long RT-PCR fragments of the DWV 170

genome covering the most divergent leader protein-coding region, amplified from the pooled P0-171

DWV2 and P1-DWV2 pupae, showed that DWV in the both pupal groups was identical to the 172

DWV-304 used for the inoculation of the Pupae 0 (Supplementary Data Fig. S1). Sanger 173

sequencing runs showed no polymorphism in both P0-DWV2 and P1-DWV2 (Supplementary Data 174

Fig. S2), clearly indicating that only the DWV-304 isolate used for infection of the P0-DWV2 pupae 175

was transmitted. Contrary to this, the DWV RT-PCR fragment amplified from the V0-DWV2 mites 176

was highly polymorphic (Supplementary Data Fig. S2). Due to this, it was not possible to obtain a 177

complete sequence of this fragment, but the terminal sections were also identical to the DWV-178

304 (Supplementary Data Fig. S1). 179

180

Negative-strand DWV RNA detected exclusively in Varroa mites fed on high DWV-level pupae. 181

Quantification of negative-strand DWV RNA was carried out in honey bee and mite 182

samples pooled according to their treatment (in this analysis we excluded a single mite exposed 183

to pupae with high DWV levels from the pool V0-PBS1, Fig. 3a) using a tagged primer approach. 184

Two independent assays targeting different regions in the DWV genome and using different 185

primer tags were used (Supplementary Data Table S1). Accuracy of these tests was confirmed by 186

detection of negative-strand DWV RNA in the honey bee pupae pools containing individuals with 187

more than 109 DWV genome copies (Fig. 4d). In both assays, which included melting curve 188

analysis to confirm the identities of amplified fragments, the negative-strand DWV RNA was 189



https://doi.org/10.1101/660985



10

detected exclusively in the mites directly fed on the pupae with high DWV levels for 2 days. This 190

included V0-DWV1 mites from Experiment 1 (Fig. 3b) and V0-PBS2 and V0-DWV2 mites from 191

Experiment 2 (Fig. 4b). No negative-sense DWV RNA was detected in mites after four passages 192

on low DWV level pupae in both Experiments 1 and 2 or in phoretic mites, V-Pho1 (Figs. 3b and 193

4b). 194

195

Detection of honey bee mRNA in Varroa mites suggests acquisition of honeybee cells. 196

Accumulation of negative-strand DWV RNA in mites could be explained by the acquisition 197

of honey bee cells, containing the replication complexes27 including negative-strand DWV RNA, 198

rather than by replication of DWV in the mite itself. It is possible that during their feeding on the 199

honey bees, Varroa mites consume not only pure, “filtered” hemolymph plasma, but also honey 200

bee cells. Further, the digestion of these honey bee cells in the Varroa midgut could explain the 201

disappearance of the negative-strand DWV RNA following passage on low DWV-level pupae. 202

Indeed, the detection of honey bee transcripts beta-actin (BeeBase gene GB44311) and 203

Apolipophorin-III-like (BeeBase gene GB55452) in the Varroa mites using RT-qPCR supports this 204

explanation (Supplementary Data Table 2). We further detected numerous honey bee transcripts 205

in Varroa RNA-seq libraries produced in our laboratory (Supplementary Data Table 3), and a 206

different laboratory28 (Supplementary Data Table 4). We found that in the Varroa RNA-seq 207

libraries the numbers of honey bee beta-actin mRNA were as high as 3.01% ± 0.94% (average ± 208

SD) of those of Varroa beta-actin mRNA (Supplementary Data Table 3). There was variation in 209

the proportions of honey bee transcripts present in the mites reared on different honey bee life 210



https://doi.org/10.1101/660985



11

stages (pupae or adult bees), which might reflect differences in relative expression of these 211

genes in the source honey bee pupae. We also observed higher (about 8-fold) relative levels of 212

total honey bee mRNA transcripts in Varroa mites after 15 hours of feeding on the honey bee 213

pupae or adults compared to the mites which were starved for 12 hours (Supplementary Data 214

Table 3). These observed dynamics of honey bee mRNA levels in mites could be explained by a 215

gradual reduction of the ingested honey bee cells. Single-stranded mRNA transcripts are unlikely 216

to be present in hemolymph plasma, therefore their presence likely indicates ingestion of the 217

honey bee cells in addition to intake of “filtered” hemolymph plasma. Indeed, by using RT-qPCR 218

we demonstrated that following freeze-thaw disruption of hemolymph cells the level of beta-219

actin mRNA decreased about 1000-fold in 2 hours (Supplementary Data Fig. S3) suggesting it is 220

quickly degraded outside the cell. 221

222

Discussion 223

A growing body of evidence suggests that spread of the mite Varroa destructor has 224

greatly increased pathogenicity of DWV, elevating it to the most important viral pathogen of the 225

Western honey bee, Apis mellifera24. Knowledge of the interaction between DWV and its mite 226

vector is required to better understand the impact of mite vectoring on virus evolution, 227

development of accurate predictive models of Varroa mite and DWV dynamics, and design of 228

novel DWV control approaches. Surprisingly, despite the importance of DWV-Varroa 229

interactions, it is not clear if replication of all DWV types occur in Varroa mites17. The main 230

argument in support of the replication of DWV in Varroa mites remains the detection of 231



https://doi.org/10.1101/660985



12

negative-strand DWV RNA in Varroa mites, in particular, in mites associated with the pupae with 232

high DWV levels18, 19. 233

Here we used a recently developed in vitro system for maintaining Varroa mites on honey 234

bee pupae24 and a cloned isolate of DWV type A (E. Ryabov, personal communication) to 235

experimentally establish whether DWV replication occurs in Varroa mites, to determine factors 236

affecting DWV levels in the Varroa mites, and to assess the impact on DWV levels on the mites’ 237

potential for DWV vectoring. 238

We demonstrated that DWV loads in mites are highly dynamic and rapidly increase or 239

decrease depending on the levels of DWV in the honey bees on which they are feeding on. For 240

example, we used phoretic mites to show that DWV loads increased over 50-fold following 2 day 241

of feeding on pupae pre-infected with DWV, while DWV levels remained unchanged in mites fed 242

on the low-DWV PBS-injected pupae (Fig. 3a, Fig. 4a). The phoretic mites used in this experiment 243

(V-Pho1), had relatively low DWV levels (Fig. 3a) despite being collected from DWV-infected 244

colonies, which could be explained by loss of DWV loads following passage on the adult bees, 245

mostly nurses, which usually have low DWV levels. We observed a strong positive correlation 246

between levels of DWV in pupae and their associated Varroa mites (Pearson R2=0.58; Fig. 5), 247

suggesting that DWV loads in the mites could be explained by virus acquisition from the pupae 248

during feeding. This observation agrees with a previous study which also found a positive 249

correlation between DWV levels in honey bee pupae and their associated mites sourced directly 250

from capped cells from an intact colony8. Similar positive correlations between the DWV levels in 251

bees and associated mites observed during natural infestation, where mites were infecting the 252



https://doi.org/10.1101/660985



13

pupae and were feeding for 5 to 8 days, and in experiments where pupae were pre-infected with 253

DWV and feeding continued only for two days (Figs. 1a and 2) suggest that most of the DWV 254

present in mites in the course of natural infestation is also acquired from the pupae. Importantly, 255

DWV loads in the mites reared on DWV-infected pupae were significantly reduced 32- and 18-256

fold in Experiment 1 and 2, respectively, following four 2-day passages on low DWV-level pupae 257

(Figs. 3a and 4a). Moreover, after the passages, no significant differences were observed 258

between the DWV loads in mites which previously acquired high DWV doses and those initially 259

exposed to the control PBS-injected pupae (Fig. 3a, Fig. 4a). 260

Experiment 2 (Figs. 2 and 4c) clearly demonstrated transmission of DWV acquired by 261

mites during feeding on high-DWV level pupae P0-DWV2 to the recipient pupae P1-DWV-Ext2 by 262

using a cloned DWV isolate. This isolate made it possible to demonstrate mite transmission of 263

the strain that replicated to high levels in the pupae P0-DWV2 (Supplementary Data Fig. S1). We 264

further showed that the ability of the mites to vector DWV decreases following passages on low 265

DWV pupae. Significantly lower levels of DWV were observed in the P4-DWV-Ext2 pupae fed on 266

by the passaged V4-DWV2 mites compared to P1-DWV-Ext2 pupae fed on by mites transferred 267

immediately from highly infected pupae (Fig. 4c). 268

Detection of negative-strand RNA of picorna-like viruses, including DWV, is a good 269

indicator of a virus replication. It is considered the strongest evidence in support of replication of 270

DWV in Varroa mites18,19. Therefore, we sought to detect negative-strand DWV RNA in the mites 271

from this study. By using two independent RT-qPCR assays for negative-strand DWV RNA 272

quantification, we showed its accumulation exclusively in mites sampled immediately following 273



https://doi.org/10.1101/660985



14

exposure to pupae with high DWV levels (Fig. 3b, and Fig. 4b). Notably, negative-strand DWV 274

RNA was not detectable in the in the phoretic V-Pho1 mites before exposure to the DWV-level 275

pupae or in the P4-DWV1 and P4-DWV2 mites with decreased overall DWV loads following 276

passage of low DWV-level pupae (Fig, 3b and Fig. 4b) . We hypothesized that such rapid 277

emergence and disappearance of negative-strand DWV RNA in the mites is explained not by 278

replication of DWV in Varroa cells, but by acquisition of honey bee cells with containing DWV 279

replication complexes with negative-strand viral RNA27. Indeed, acquisition of cells is consistent 280

with detection of honey bee genomic DNA in Varroa samples during genome sequencing30 and 281

has been recently observed during extensive feeding analyses31. 282

To estimate the extent of honey bee cell acquisition by a mite during feeding, we 283

measured honey bee mRNA levels in the mites. Detection of honey bee mRNA transcripts is an 284

indicator of intact cells acquisition by mites because we demonstrated using RT-qPCR that levels 285

of mRNA in the honey bee hemolymph decline by about 1000 times in 2 hours following cell 286

disruption (Supplementary Data Fig. S3). Therefore, the presence of honey bee mRNA revealed 287

by RT-qPCR in the mites of our experiments (Supplementary Data Table S2) and by analyzing 288

RNA-seq libraries (Supplementary Data Tables S3 and S4) strongly suggests acquisition of entire 289

honey bee cells by Varroa mites. The phenomenon of hosts undamaged cells and tissues being 290

accumulated within the midgut lumen of a blood sucking parasite has been observed in other 291

members from the superorder Parasitiformes, which includes mites and ticks. In the black-legged 292

tick Ixodes scapularis and the American dog tick Dermacentor variabilis intact erythrocytes were 293

detected up to 6 days post blood meal. It is speculated that for the subclass Acari, the midgut is 294

the primary nutrient storage site where ingested cells are kept intact32. Our honey bee cell 295



https://doi.org/10.1101/660985



15

acquisition model could explain emergence and disappearance of negative-strand DWV RNA in 296

the mites in the course of the Experiments 1 and 2 (Figs. 3b, 4b). Indeed, the lack of replication 297

of DWV-A in Varroa mites observed in our study is further supported by a previously published 298

immunolocalization study showing detection of DWV antigen exclusively in the mites midgut 299

lumen22. 300

Our finding that replication of DWV, or at least its most common type DWV-A, does not 301

take place in Varroa mites provides a novel insight into the impact of the Varroa on virus 302

evolution. The main implication is that, in the absence of replication no generation of additional 303

genetic DWV diversity occurs in Varroa mites and no adaptation of DWV for replication in mites 304

takes place. Therefore the reported effect of Varroa-mediated transmission on DWV genetics15-17 305

is more likely a consequence of selection of isolates with better non-propagative transmission 306

ability, e.g. those with higher stability in mites and/or viral variants better adapted to 307

transmission via injection into the hemolymph. Indeed, previous studies have shown that 308

selection of DWV strains could take place without Varroa mites, solely by artificial injection into 309

hemolymph16. A lack of replication of DWV in Varroa mites is also consistent with the absence of 310

the reports a negative impact of DWV on Varroa mites, which could be a result if replication of 311

DWV took place in mites. 312

In light of finding no evidence of DWV persistence in Varroa mites, it is possible that 313

development of severe Varroa and DWV-associated honey bee colony decline requires colonies 314

to reach a certain threshold level of both Varroa mite density and proportion of bees with high 315

DWV levels. Indeed, increased DWV levels were reported in colonies with high Varroa mite 316



https://doi.org/10.1101/660985



16

infestation13. Also, the lack of replication and low persistence of DWV in Varroa mites could 317

explain the absence of Varroa related damage in some island honey bee populations33. 318

It should be highlighted that the reported experiments were carried out using one type of 319

DWV-like viruses, namely DWV type A. This cloned DWV variant24 was isolated from a heavily 320

Varroa-infested colony typical for the USA and likely to be Varroa-selected, as evidenced by its 321

transmission in Experiment 2. It cannot be ruled out that other DWV-like variants, namely the 322

highly pathogenic DWV type B6, which has shown increased virulence34,35, or VDV1-DWV 323

recombinants8-10 could have replicative ability in Varroa mites, especially considering their 324

preferable transmission by the mites8,36 and the detection of VDV-1 negative strand RNA in the 325

Varroa synganglions23. Investigation of the abilities of different DWV strains to replicate in the 326

mites should be carried out using experimental procedures similar to those used in this study. 327

Taking into account the implications of a lack of replication of DWV in the Varroa mites, it 328

might be possible to control DWV solely by targeting DWV in its bee host (e.g. using an RNAi 329

approach). Data on the rate of reduction of the mites’ ability to transmit DWV following passages 330

on low DWV bees (as it likely to occur in the case of the phoretic mites) would allow for the 331

development of precise predictive model linking Varroa infestation rate and DWV load. This 332

would be important for planning measured treatments against Varroa using acaricides, which 333

may have negative health impacts on bees37. 334

Results of this study clearly suggest that DWV, or at least the most common DWV type A, 335

is transmitted between honey bees by the Varroa mite in a non-propagative manner. Significant 336

declines in the mites’ ability to transmit DWV suggest non-persistent transmission mechanism. 337



https://doi.org/10.1101/660985



17

While this does not diminish the role played by mites in DWV infection of honey bees, it does 338

clarify the selective forces acting on DWV. Namely, this virus has evolved to exploit the honey 339

bee, not adapted to replication in mite cells, and has a more intimate relationship with honey 340

bee immune defenses than those of the mite. These insights are critical in reducing the impacts 341

of the tripartite relationship among honey bees, mites, and DWV. 342

343

Methods 344

Honey bees and Varroa mites. 345

Honey bee pupae were obtained from the Beltsville USDA Bee Research Laboratory (BRL) apiary 346

from strong colonies “132”and “Nuc-L” (two boxes with more than 10 frames of brood and 347

stored food) with a low (less than 1%) Varroa mite infestation rate, and the DWV loads in the 348

pupae undetectable by qRT-PCR test17,38 in June 2018. The colonies were not subjected to any 349

Varroa control treatments. The colonies “132”and “Nuc-L” were used in Experiment 1 (July 2018) 350

and in Experiment 2 (September 2018) respectively. Pupa at the white- or pink-eyed stage were 351

pulled out of cells using soft tweezers no more than 24 hour prior their use in the experiments. 352

Varroa mites were collected from the same apiary, using colonies showing about 7% Varroa mite 353

brood infestation rate and detectable levels of DWV in brood40. Phoretic mites collected from 354

adult bees of colonies “3” were used for Experiment 1, colonies “71” and “73” were used as a 355

Varroa source in Experiment 2, where both phoretic and founder Varroa mites were collected. 356

All Varroa mite source colonies used in this study harbored only DWV-A and were free of VDV-1 357



https://doi.org/10.1101/660985



18

(DWV-B). The tests for the presence of DWV-A and DWV-B were carried out as described 358

previously17,38. The sugar roll method was used to collect Varroa mites; this included shaking 359

powdered sugar-dusted adult bees over a tray with water, collecting the released mites from the 360

water with a strainer, drying them on tissue paper, then placing the mites on honey bee pupae 361

within 2 to 3 hours after collection25. 362

The experiments were organized with a completely randomized design using two 363

treatments. Pupae were either injected as previously described16 with DWV (cDNA clone-derived 364

DWV-A variant DWV-30424, GenBank Accession Number MG831200) or PBS as a control. The 365

virus was propagated in honey bee pupae injected with infectious DWV in vitro RNA transcript 366

produced from the linearized template, plasmid DWV-304, using T7 RNA polymerase (HiScribe T7 367

kit, New England Biolabs) according to manufacturer’s instructions (E. Ryabov, personal 368

communication). For preparation of the clonal DWV extracts containing infectious DWV virus 369

particles, individual transcript-injected pupae were homogenized with 2 mL of phosphate-370

buffered saline (PBS at 3 days post injection). For each individual pupal extract, 1 mL was used 371

for preparation of the DWV inocula, which included clarification by centrifugation at 3000g for 5 372

min and filtration through 0.22 µm nylon filter (Millipore). The DWV concentration in the extract 373

was quantified by qRT-PCR and the infectivity of the virus was confirmed by development of 374

typical overt DWV infection, about 1011 genome copies per bee. The identity of clonal DWV in 375

the extract with its respective parental DWV cDNA clone was confirmed by sequencing of the RT-376

PCR fragment corresponding to the leader protein gene of DWV-A (nucleotide positions 950-377

2050 of the genomic RNA) . The DWV extract was stored at -80oC prior to use. Twenty or twelve 378

replicates were used for each treatment in Experiments 1 and 2, respectively. Each replicate 379



https://doi.org/10.1101/660985



19

consisted of one pupa and two or three Varroa mites contained inside a gelatin capsule (Veg K-380

Caps size 00 capsule from Capsuline, Pompano Beach, FL, USA). Varroa mites were tested with a 381

paint brush for mobility to be sure that they were alive. The gelatin capsule with the pupa and 382

mites were inserted into a 32/8-Place Combo Tube Rack (USA Scientific, Orlando. FL, USA) inside 383

an environmental chamber set to 32oC and about 85% of relative humidity in complete darkness. 384

The mites and pupae were checked daily for ten days and survival was recorded. Only live mites 385

were transferred to the next pupae and mite feeding was confirmed by accumulation of their 386

feces in the gelatin capsules. Mite survival was analyzed as previously25 using JMP Software (SAS, 387

Cary, NC, USA). 388

389

Quantification of RNA by RT-qPCR 390

Total RNA was extracted from individual honey bee pupae using Trizol reagent (Ambion) 391

and further purified using a RNeasy kit (QIAGEN) according to manufactures’ instructions. 392

Extraction of total RNA from individual Varroa mites was carried out using a RNeasy kit (QIAGEN) 393

and included disruption of the mites in the RLT buffer supplied with the RNeasy kit using a glass 394

micro-sized 0.2 mL tissue grinder (DWK Life Sciences, Wheaton), followed by further disruption 395

of the mite tissues using QIAShredder (QIAGEN). DWV RNA loads in individual honey bee pupae 396

and associated Varroa mites were quantified by reverse transcription quantitative PCR (RT-397

qPCR), which included cDNA synthesis using random hexanucleotides. Detection mainly included 398

the genomic positive strand DWV RNA (Supplementary Data Table S1). RT-qPCR was carried out 399

essentially as previously described28,39, DWV RNA copy numbers were log-transformed prior to 400



https://doi.org/10.1101/660985



20

statistical analyses. Negative-strand DWV RNA quantification was carried using two sets of 401

primers designed for targeting different regions in the DWV genome, Assay 1 (region 4909-5008 402

nt)16 and Assay 2 (region 7303-7503 nt)40. 403

404

mRNA degradation assay 405

Hemolymph cell suspension samples were collected from 10 pink eye honey bee pupae, 406

approximately 30 µL in total. A 5 µL sample was used to extract total RNA immediately following 407

collection. The remainder of the sample was subjected to 5 cycles of freezing and thawing (5 408

minutes each) and vortexing to disrupt cellular membranes and incubated at +33oC. Subsequent 409

5 µL aliquots, were collected for immediate RNA extraction after 30 min, 60 min, and 90 min of 410

incubation. The extracted RNA samples were used to quantify honey bee beta actin mRNA by RT-411

qPCR using specific primers (Supplementary Data Table 1). 412

413

Analysis of RNA-seq data 414

Pre-analysis processing of the raw Varroa RNAseq Illumina data (paired-end 150 nt reads) 415

was performed as previously described39. Cleaned data from each sample library were 416

individually aligned to a reference set containing A. mellifera official gene set 3.2 417

(amel_OGSv3.2)41, V. destructor actin-like mRNA (XR_002672520.1), V. destructor myosin heavy 418

chain mRNA (XM_022800959.1), V. destructor vitellogenin 1 mRNA (JQ974976.1) and the 419

genomic sequence of DWV RNA (GU109335.1), using Bowtie242. SAMtools43 idxstats was used 420

to identify the numbers of the reads corresponding to the A. mellifera, V. destructor and DWV 421

genomic RNA sequences. Additional, previously published Varroa RNAseq libraries28 (NCBI 422



https://doi.org/10.1101/660985



21

BioProject PRJNA392105), were screened for the presence of honey bee and Varroa mRNAs 423

using BLAST44. 424

425

REFERENCES 426

1. Vanbergen, A. J. et al. Threats to an ecosystem service: pressures on pollinators. 427

Frontiers in Ecology and the Environment 11, 251–259, doi: 10.1890/120126 (2013). 428

2. Brutscher, L. M., McMenamin A. J, & Flenniken, M. L. The Buzz about honey bee viruses. 429

PLoS Pathog 12, e1005757. doi.org/10.1371/journal.ppat.1005757 (2016). 430

3. Lanzi, G. et al. 2006. Molecular and biological characterization of deformed wing virus of 431

honeybees (Apis mellifera L.). J. Virol. 80, 4998–5009 (2006). 432

4. Gallai, N., Salles, J.-M., Settele, J. & Vaissiere, B. E., Economic valuation of the 433

vulnerability of world agriculture confronted with pollinator decline. Ecological Economics 434

68, 810–821 (2009). 435

5. Goulson, D., Nicholls, E., Botias, C. & Rotheray, E. L. Bee declines driven by combined 436

stress from parasites, pesticides, and lack of flowers. Science 347, 1255957. 437

doi:10.1126/science.1255957 (2015). 438

6. Ongus, J. R. et al. Complete sequence of a picorna-like virus of the genus Iflavirus 439

replicating in the mite Varroa destructor. J Gen. Virol. 85, 3747-3755 (2004). 440

7. Mordecai, G. J., Wilfert, L., Martin, S. J., Jones, I. M. & Schroeder, D. C. Diversity in a 441

honey bee pathogen: first report of a third master variant of the Deformed Wing Virus 442

quasispecies. ISME J. 10, 1264-1273. doi: 10.1038/ismej.2015.178. (2016). 443



https://doi.org/10.1101/660985



22

8. Moore, J. et al. Recombinants between Deformed wing virus and Varroa destructor virus-444

1 may prevail in Varroa destructor-infested honeybee colonies. J. Gen. Virol. 92, 156–161, 445

doi:10.1099/vir.0.025965-0 (2011). 446

9. Zioni, N., Soroker, V., & Chejanovsky, N. Replication of Varroa destructor virus 1 (VDV-1) 447

and a Varroa destructor virus 1-deformed wing virus recombinant (VDV-1-DWV) in the 448

head of the honey bee. Virology 417, 106-112, doi: 10.1016/j.virol.2011.05.009 (2011). 449

10. Dalmon, A. et al., Evidence for positive selection and recombination hotspots in 450

Deformed wing virus (DWV). Scientific Reports 7, 41045 (2016). 451

11. Highfield, A. C. et al. Deformed wing virus implicated in overwintering honeybee colony 452

losses. Appl. Environ. Microbiol. 75, 7212-7220 doi:10.1128/AEM.02227-09 (2009). 453

12. Le Conte, Y., Ellis, M. & Ritter, W. Varroa mites and honey bee health: can Varroa explain 454

part of the colony losses? Apidologie 41, 353-363 doi.org/10.1051/apido/2010017 (2010). 455

13. Dainat B., Evans, J. D., Chen, Y.P., Gauthier, L. & Neumann, P. Dead or Alive: Deformed 456

Wing Virus and Varroa destructor reduce the life span of winter honey bees. Appl. 457

Environ. Microbiol. 78, 981-987 (2012). 458

14. Rosenkranz, P., Aumeier, P. & Ziegelmann, B. Biology and control of Varroa destructor. 459

Journal of Invertebrate Pathology 103, S96-S119 doi.org/10.1016/j.jip.2009.07.016 460

(2010). 461

15. Martin, S. J. et al. Global honey bee viral landscape altered by a parasitic mite. Science 462

336, 1304–1306, doi:10.1126/science.1220941 (2012). 463



https://doi.org/10.1101/660985



23

16. Ryabov, E. V. et al. A Virulent strain of Deformed wing virus (DWV) of honey bees (Apis 464

mellifera) prevails after Varroa destructor-mediated, or in vitro, transmission. PLoS Path. 465

10, doi: 10.1371/journal.ppat.1004230 (2014). 466

17. Wilfert, L. et al. Deformed wing virus is a recent global epidemic in honeybees driven by 467

Varroa mites. Science 351, 594–597, doi:10.1126/science.aac9976 (2016). 468

18. Yue, C. & Genersch E. RT-PCR analysis of Deformed wing virus in honeybees (Apis 469

mellifera) and mites (Varroa destructor). J. Gen. Virol. 86, 3419-3424, doi: 470

10.1099/vir.0.81401-0. (2005). 471

19. Gisder, S., Aumeier, P. & Genersch E. Deformed wing virus: replication and viral load in 472

mites (Varroa destructor). J. Gen. Virol. 90, 463-467, doi: 10.1099/vir.0.005579-0 (2009). 473

20. Chen, Y. P. et al. Family Iflaviridae. In: Ninth Report of the International Committee on 474

Taxonomy of Viruses. (eds. King, A. M. Q., Adams, M. J., Carstens, E. B. & Lefkowitz, E. J.), 475

846-849 (Elsevier Academic Press, 2012). 476

21. Valles, S. M. et al. ICTV Virus Taxonomy Profile: Iflaviridae. J. Gen. Virol. 98, 527-528. doi: 477

10.1099/jgv.0.000757 (2017). 478

22. Santillán-Galicia, M. T., Carzaniga, R., Ball, B. B. & Alderson, P. G. Immunolocalization of 479

deformed wing virus particles within the mite Varroa destructor. J. Gen. Virol. 89, 1685-480

1689, doi: 10.1099/vir.0.83223-0 (2008). 481

23. Campbell, E. M., Budge, G. E., Watkins, M. & Bowman, A. S. Transcriptome analysis of the 482

synganglion from the honey bee mite, Varroa destructor and RNAi knockdown of neural 483

peptide targets. Insect Biochemistry and Molecular Biology 70, 116-126. 484

https://doi.org/10.1016/j.ibmb.2015.12.007 (2016). 485



https://doi.org/10.1101/660985



24

24. Egekwu, N. I., Posada, F., Sonenshine, D. E. & Cook, S. Using an in vitro system for 486

maintaining Varroa destructor mites on Apis mellifera pupae as hosts: Studies of mite 487

longevity and feeding behavior. Experimental and Applied Acarology 74, 301-315. 488

doi:10.1007/s10493-018-0236-0 (2018). 489

25. Nazzi, F. et al. Synergistic Parasite-Pathogen Interactions Mediated by Host Immunity Can 490

Drive the Collapse of Honeybee Colonies. PLoS Pathog. 8, e1002735. 491

doi:10.1371/journal.ppat.1002735 (2012). 492

26. Francis, R. M., Nielsen, S. L. & Kryger, P. Patterns of viral infection in honey bee queens. J. 493

Gen. Virol. 94, 668-676. doi: 10.1099/vir.0.047019-0 (2013). 494

27. Belov G. A. & Kuppeveld, F. J. M. (+)RNA viruses rewire cellular pathways to build 495

replication organelles. Current Opinion in Virology 2, 740-747. 496

doi.org/10.1016/j.coviro.2012.09.006 (2012). 497

28. Mondet, F. et al. Transcriptome profiling of the honeybee parasite Varroa destructor 498

provides new biological insights into the mite adult life cycle. BMC Genomics 19, 328. 499

doi.org/10.1186/s12864-018-4668-z (2018). 500

29. De Miranda, J.R. & E Genersch, E. Deformed wing virus. Journal of Invertebrate 501

pathology. 103, S48-S61. https://doi.org/10.1016/j.jip.2009.06.012 (2010). 502

30. Cornman, R. S. et al. Genomic survey of the ectoparasitic mite Varroa destructor, a major 503

pest of the honey bee Apis mellifera. BMC Genomics 11, 602. doi.org/10.1186/1471-504

2164-11-602 (2010). 505



https://doi.org/10.1101/660985



25

31. Ramsey, S. D, et al. Varroa destructor feeds primarily on honey bee fat body tissue and 506

not hemolymph. Proc. Natl. Acad. Sci. USA 116, 1792-1801. doi: 507

10.1073/pnas.1818371116 (2019). 508

32. Sonnenshine, D. E. & Anderson, J. M. Mouthparts and digestive system. Anatomy and 509

Molecular Biology of Feeding and Digestion. In “Biology of ticks”, (eds. Sonnenshine, D. E. 510

& Roe, R. M. ) 122-162 (Oxford University Press, 2014). 511

33. Brettell, L. E. & Martin, S. J. Oldest Varroa tolerant honey bee population provides insight 512

into the origins of the global decline of honey bees. Sci. Rep. 7, 45953 (2017). 513

34. McMahon, D. P. et al. Elevated virulence of an emerging viral genotype as a driver of 514

honeybee loss. Proc. R. Soc. B. 283: 20160811. DOI: 10.1098/rspb.2016.0811 (2016). 515

35. Natsopoulou, M. E. et al. The virulent, emerging genotype B of Deformed wing virus is 516

closely linked to overwinter honeybee worker loss. Sci. Rep. 7, 5242 (2017). 517

36. Gisder, S., Möckel, N., Eisenhardt, D. & Genersch, E. In vivo evolution of viral virulence: 518

switching of deformed wing virus between hosts results in virulence changes and 519

sequence shifts. Environmental Microbiology 20, 4612-4628. 520

https://doi.org/10.1111/1462-2920.14481 (2018). 521

37. Reeves, A. M. O’Neal, S. T., Fell. R. D. Brewster. C. D. & Anderson, T. D. In-hive acaricides 522

alter biochemical and morphological indicators of honey bee n utrition, immunity, and 523

development. Journal of Insect Science 18, 1–6 doi: 10.1093/jisesa/iey086 (2018). 524

38. Chen, Y., Zhao, Y. Hammond, J. Hsu, H., Evans, J. & Feldlaufer, M. Multiple virus 525

infections in the honey bee and genome divergence of honey bee viruses. Journal of 526

Invertebrate Pathology 87, 84–93 (2004). 527



https://doi.org/10.1101/660985



26

39. Ryabov, E. V. et al. Recent spread of Varroa destructor virus-1, a honey bee pathogen, in 528

the United States. Sci. Rep. 7, 17447. doi: 10.1038/s41598-017-17802-3 (2017). 529

40. Boncristiani, H. F. Di Prisco, G., Pettis, J. S. , Hamilton, M. & Chen, Y.P. Molecular 530

approaches to the analysis of deformed wing virus replication and pathogenesis in the 531

honey bee, Apis mellifera. Virology Journal 6, 221. doi: 10.1186/1743-422X-6-221 (2009). 532

41. Honey Bee Genome Sequencing Consortium. Finding the missing honey bee genes: 533

lessons learned from a genome upgrade. BMC Genomics 15, 86. doi: 10.1186/1471-2164-534

15-86 (2014). 535

42. Langmead, B., & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nature 536

Methods. 9, 357-359. doi:10.1038/nmeth.1923 (2012). 537

43. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–538

2079. doi: 10.1093/bioinformatics/btp352 (2009). 539

44. Altschul, S.F., Gish, W., Miller, W., Myers, E. W. &Lipman, D.J. Basic local alignment search 540

tool. J. Mol. Biol.215: 403–410 (1990). 541

542

543

Acknowledgements 544

This research used resources provided by the SCINet project of the USDA - Agricultural Research 545

Service, ARS project number 0500-00093-001-00-D and was supported by the USDA National 546

Institute of Food and Agriculture grant 2017-06481 to EVR, YPC and JDE. USDA is an equal 547

opportunity provider and employer. 548



https://doi.org/10.1101/660985



27

549

Author Contributions 550

F.P.-F. and E.V.R., the two corresponding authors, conceived the study and contributed equally to 551

this research. Y.C. supervised honeybee colony monitoring. F.P.-F and E.V.R. carried work with 552

bees and mites. E.V.R. carried out the laboratory molecular work and analyzed virus 553

quantification. M.H. analyzed accumulation of the honey bee transcripts. A.K.C. analyzed the 554

NGS data. F.P.-F. A.K.C., Y. C, J.D.E. and E.V.R., wrote the manuscript. All co-authors contributed 555

to data interpretation, and to the writing of the manuscript. 556

557

Competing interests 558

The authors declare no competing interests. Mention of trade names or commercial products in 559

this publication is solely for the purpose of providing specific information and does not imply 560

recommendation or endorsement by the U.S. Department of Agriculture. 561

562

563

564

565

566

567

568

569

570



https://doi.org/10.1101/660985



28

Figure legends 571

572

Figure 1. Experiment 1 design: “Varroa mite DWV acquisition experiment”. (a) Honey bee and 573

the mite treatment groups are shown in bold. Times of sampling (days of the experiment) are 574

indicated. (b) In vitro incubation of honey bee pupa with Varroa mites in gelatin capsule. 575

Figure 2. Experiment 2 design: “Varroa-mediated DWV transmission experiment”. Honey bee 576

and the mite treatment groups are shown in bold. Times of sampling (days of the experiment) 577

are indicated. 578

Figure 3. Dynamics of DWV RNA accumulation in Varroa destructor mites and honey bee pupae 579

from Experiment 1: “Varroa mite DWV acquisition experiment”. (a, c) Average DWV RNA copies 580

per (a) Varroa destructor mites, and (b) honey bee pupae, ±SD. Red letters above the bars 581

indicate significantly and non-significantly different groups (ANOVA). Treatment groups are 582

named as in the Fig. 1. Individual mite and pupa copy numbers indicated by black dots. 583

Corresponding mite (a) and pupae (c) groups are aligned vertically. (b) Detection of negative 584

strand DWV RNA in Varroa mite groups. The Ct values shown are as follows: Assay 1 / Assay 2. 585


from Experiment 2: “Varroa-mediated DWV transmission experiment”. (a, c) Average DWV RNA 587

copies per (a) Varroa destructor mites, and (b) honey bee pupae, ±SD. Red letters above the bars 588


named as in Fig. 2. Individual mite and pupa copy numbers indicated by black dots. 590

Corresponding mite (a) and pupae (c) groups are aligned vertically. The threshold levels of 109 591



https://doi.org/10.1101/660985



29

DWV genomic RNA copies per honey bee pupae is shown as a red line in (c). Detection of the 592

negative strand DWV RNA in (b) Varroa mite groups and (d) honey bee pupae. The Ct values 593

shown are as follows: Assay 1/ Assay 2. 594

Figure 5. Prevalence of DWV in honey bees (X axis) and associated Varroa mites (Y-axis) in 595

simultaneously sampled pupae and mites. Positive correlation between DWV copy numbers 596

(Pearson R2 = 0.58068, n=51, p<0.0001). Only mites and pupae with DWV levels above the 597

detection thresholds were used. 598

599

600

601



https://doi.org/10.1101/660985



30

602

603

604

605

Figure 1. Experiment 1 design: “Varroa mite DWV acquisition experiment”. (a) Honey bee and 606

the mite treatment groups are shown in bold. Times of sampling (days of the experiment) are 607

indicated. (b) In vitro incubation of honey bee pupa with Varroa mites in gelatin capsule. 608

609

610



https://doi.org/10.1101/660985



31

611

612

613

Figure 2. Experiment 2 design: “Varroa-mediated DWV transmission experiment”. Honey bee 614

and the mite treatment groups are shown in bold. Times of sampling (days of the experiment) 615

are indicated. 616

617



https://doi.org/10.1101/660985



32

618


from Experiment 1: “Varroa mite DWV acquisition experiment” . (a, c) Average DWV RNA copies 620

per (a) Varroa destructor mites, and (b) honey bee pupae, ±SD. Red letters above the bars 621


named as in the Fig. 1. Individual mite and pupa copy numbers indicated by black dots. 623

Corresponding mite (a) and pupae (c) groups are aligned vertically. (b) Detection of negative 624

strand DWV RNA in Varroa mite groups. The Ct values shown are as follows: Assay 1 / Assay 2. 625



https://doi.org/10.1101/660985



33

626


from Experiment 2: “Varroa-mediated DWV transmission experiment”. (a, c) Average DWV RNA 628

copies per (a) Varroa destructor mites, and (b) honey bee pupae, ±SD. Red letters above the bars 629


named as in Fig. 2. Individual mite and pupa copy numbers indicated by black dots. 631

Corresponding mite (a) and pupae (c) groups are aligned vertically. The threshold levels of 109 632

DWV genomic RNA copies per honey bee pupae is shown as a red line in (c). Detection of the 633

negative strand DWV RNA in (b) Varroa mite groups and (d) honey bee pupae. The Ct values 634

shown are as follows: Assay 1/ Assay 2. 635

636

637



https://doi.org/10.1101/660985



34

638

639

Figure 5. Prevalence of DWV in honey bees (X axis) and associated Varroa mites (Y-axis) in 640

simultaneously sampled pupae and mites. Positive correlation between DWV copy numbers 641

(Pearson R2 = 0.58068, n=51, p<0.0001). Only mites and pupae with DWV levels above the 642

detection thresholds were used. 643

644



https://doi.org/10.1101/660985


Supplementary Information

Title:

Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa destructor in a non-propagative manner.

Authors:

Francisco Posada-Florez*, Anna K. Childers, Matthew C. Heerman, Noble I. Egekwu, Steven C. Cook, Yanping Chen, Jay D. Evans, Eugene V. Ryabov*

Affiliation:

USDA, Agricultural Research Service, Bee Research Laboratory, Beltsville Agricultural Research Center, 10300 Baltimore Avenue, Bldg. 306, Beltsville MD 20705, USA

This PDF file includes:

Supplementary Table 1. Primers used in this study.

Supplementary Table 2. Detection of honey bee mRNA in Varroa mites by RT-qPCR.

Supplementary Table 3. Detection and quantification of honey bee mRNAs in RNAseq libraries from phoretic Varroa mites following starvation or feeding.

Supplementary Table 4. Detection of honey bee mRNAs in RNAseq libraries from different Varroa mite developmental stages.

Supplementary Figure S1. Alignments of consensus DWV RNA nucleotide sequences from honey bees and mites in Experiment 2: “Varroa-mediated DWV transmission experiment”.

Supplementary Figure S2. DWV polymorphisms in honey bees and mites in Experiment 2: “Varroa-mediated DWV transmission experiment”.

Supplementary Figure S3. Single stranded RNA degradation in disrupted honey bee hemolymph cells.



https://doi.org/10.1101/660985


Supplementary Table 1. Primers used in this study.

Primer ID Nucleotide sequence (5' to 3')

Target: GenBank accession number, (position in the nucleotide sequence),

polarity

Applications

DWV-qPCR-F GAGATTGAAGCGCATGAACA AY292384, (6471- 6490),

sense

DWV RT-qPCR

DWV-qPCR-R tgaattcagtgtcgcccata AY292384, (6600 - 6581),

complement

DWV RT-qPCR

Varr-qbACT-R TGAAGGTAGTCTCATGGATAC XR_002672520 , (142 - 122)

complement

Varroa actin mRNA RT-qPCR

Varr-qbACT-F GTCTCTGTTCCAGCCCTCGTTC XR_002672520, (81 - 102),

sense

Varroa actin mRNA qRT-PCR

Amel-qbACT-F AGGAATGGAAGCTTGCGGTA NM_001185146, (919 -

938), sense

Honey bee actin mRNA RT-

qPCR

Amel-qbACT-R AATTTTCATGGTGGATGGTGC NM_001185146, (1099 -

1079), complement

Honey bee actin mRNA RT-

qPCR

DWV900F GTTAATGTCTCATAGCCCAGACG AY292384, (899 - 921),

sense

RT-PCR, 1.2 kbp fragment,

DWV strain identification

DWV2050Rev CATATAGCATCAGAATTAGCCTC AY292384, (2071 - 2049),

complement

RT-PCR, 1.2 kbp fragment,

DWV strain identification

APO3Q-F1 GCGAAATCTCTGAGCCAAC NM_001114198, (473 - 491),

sense

Honey bee Apo3 mRNA RT-

qPCR

APO3Q-R1 GAGTTGCGGCAGTTTGAAG NM_001114198 (599 - 581),

complement

Honey bee Apo3 mRNA RT-

qPCR

Tag1-DWV-For CTTGGTTAGCTGTGTTGCAGTTGCTGTAGTCAAGCGGTTACTTGAG

AY292384, (4909 - 4934),

sense

Negative-strand DWV RNA

detection (Assay 1), RT

DWV-1-rev GGAGCTTCTGGAACGGCAGAT AY292384, (5008 - 4988),

complement


detection (Assay 1), qPCR

Tag1 CTTGGTTAGCTGTGTTGCAGTTG not applicable Negative-strand DWV RNA


Tag2-DWV-2-For AGCCTGCGCACGTGGcgaaaccaacttct

gaggaa

AY292384, (7330 - 7349),

sense


detection (Assay 2), RT

DWV-2-rev GTGTTGATCCCTGAGGCTTA AY292384, (7503 - 7484),

complement



Tag2 AGCCTGCGCACGTGG not applicable Negative-strand DWV RNA




https://doi.org/10.1101/660985


Supplementary Table 2. Detection of honey bee mRNA in Varroa mites by RT-qPCR. Ct values for honey bee actin (BeeBase gene identifier GB44311) and Apolipophorin-III-like, Apo3 (BeeBase gene identifier GB55452) mRNAs, Varroa destructor actin (GenBank Accession XR_002672520) mRNA, and DWV genomic RNA in pooled Varroa mite and control honey bee pupae.

Pooled samples DWV RNA Varroa actin mRNA

Honey bee actin mRNA

Honey bee Apo3 mRNA

Varroa mitesV-Pho1 25.8 19.7 26.8 36.6

V0-PBS1 nd 21.9 31.0 ndV0-DWV1 14.7 22.8 36.4 41.2V4-PBS1 25.1 24.4 34.2 nd

V4-DWV1 21.5 22.8 33.5 37.9

Honey bee pupaeP0-DWV1 11.0 nd 17.3 23.6P4-DWV1 18.0 nd 24.3 31.3

Water control nd nd nd nd

nd - not detected



https://doi.org/10.1101/660985


Supplementary Table 3. Detection and quantification of honey bee mRNAs in RNAseq libraries from phoretic Varroa mites following starvation or feeding. Numbers of NGS reads corresponding a set of Varroa and honey bee mRNAs as well as DWV genomic RNA were determined in Varroa destructor RNAseq libraries (Egekwu & Cook, submitted).

Var_C333_VaD_WaS_12H Var_C3_VaD_WaF_15H Var_C3_VaD_WpF_15H

Phoretic mites starved for 12 hours

Phoretic mites fed on adult honey bees for 15 hours

Phoretic mites fed on honey bee pupae for 15 hours

59560566 49673302 65165306

38870677 18964968 24572362

0.017 0.039 0.034

2.540 24.152 19.066

A. mellifera beta- actin (GB44311) NM_001185146

626 674 836

A. mellifera myosin heavy chain (GB51653)

1428 4094 5231

A. mellifera apolipophorin-III-like protein (GB55452)

1 240 220

92604 414034 470256

XR_002672520, V. destructor beta actin

36462 17143 24665

V. destructor myosin heavy chain

2246 3318 4859

V. destructor vitellogenin 1 JQ974976

1901 773 1289

Varroa mite treatment

Total A. mellifera mRNAs reads

Varroa destructor

mRNA

Varroa RNA-seq library ID

Total number of reads in the library

DWV reads

Reads numbers ratio, honey bee beta-actin mRNA to Varroa beta-actin Reads numbers ratio, total honey bee mRNAs to Varroa beta-actin mRNA

Apis mellifera

mRNA



https://doi.org/10.1101/660985


Supplementary Table 4. Detection of honey bee mRNAs in RNAseq libraries from different Varroa mite developmental stages. Numbers of NGS reads corresponding a set of Varroa and honey bee mRNAs were determined in Varroa destructor RNAseq libraries23 (NCBI BioProject PRJNA392105).

SRX2960636 Emerging

mites (collected

from P8 to P9 brood cells

SRX29606371 Young mites

(collected from P8 to P9 brood cells)

SRX2960647 Young mites

(collected from P8 to P9 brood cells)

SRX2960634 Laying mites

(collected from sealed brood cells

containing pre-pupae)

SRX2960649 Phoretic mites (collected on adult bees)

SRX2960670 Phoretic mites

artificially reared in

cages with adult bees

SRX2960669 Phoretic mites (collected on adult bees)

SRX2960632 Arresting

mites (collected in unsealed L5 brood cells)

26531852 27083570 33433690 26670037 17467280 29446920 32563456 28709594

NM_001185146 A. mellifera actin related protein 1 (Arp1)

140 473 683 174 93 206 88 87

XM_02644216 A. mellifera myosin heavy chain

122 758 355 36 56 92 57 14

NM_001114198 A. mellifera apolipophorin-III-like protein

3 51 77 11 1 0 0 2

XR_002672520, V. destructor actin-like protein

>20000 >20000 >20000 8606 17838 >20000 13931 9678

XM_022800959 V. destructor myosin heavy chain

2314 2523 4080 1847 1494 2920 1017 1061

JQ974976 V. destructor vitellogenin 1

1649 524 1182 >20000 848 1446 756 3405

Apis mellifera mRNAs

Varroa destructor

mRNAs

Total number of reads in the library

Varroa destructor RNA-seq library, accession number, mite description



https://doi.org/10.1101/660985


Supplementary Figure S1. Alignments of consensus DWV RNA nucleotide sequences from honey bees and mites in Experiment 2: “Varroa-mediated DWV transmission experiment”. RT-PCR fragments corresponding to the DWV LP region were amplified using RNA extracted from bees and mites pooled according to their treatment (Experiment 2, Figs. 2 and 4). (a, b) Complete identity between the DWV sequences amplified from honey bee pupae P0-DWV2, P1-DWV2, and the cloned isolate DWV-304. (c, d) Complete nucleotide identity between the sequenced terminal portions of the RT-PCR fragment amplified from V0-DWV2 mites, the sequences from honey bee pupae P0-DWV2, P1-DWV2, and the cloned isolate DWV-304. Due to high polymorphism in the mite DWV load only the terminal portions of V0-DWV2 were sequenced. Cloned DWV isolate DWV-304 (GenBank accession number MG831200, isolated in the USA in 2015) was used for pupae P0-DWV2 injection. The type DWV isolate DWV-PA isolated the USA in 2006 (GenBank Accession number AY292384), was included with alignments (b) and (c) to show distribution of polymorphic sites in the LP region of DWV RNA.

(a)

DWV-304 agacatcatattttattttaatgctgtctttattgctgatttattttgctgtttttattt 1020 Pupae-P0-DWV-2 -------------TATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 47 Pupae-P1-DWV—Ext-2 ------CATATTTTATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 54 *********************************************** DWV-304 gctattttatatttgctaattttcattattgcgaaatatattacattgctatttttatta 1080 Pupae-P0-DWV-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 107 Pupae-P1-DWV—Ext-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 114 ************************************************************ DWV-304 tatacgctagattcaattttatttttcctatattttcaatttaattttgattttgaaggt 1140 Pupae-P0-DWV-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 167 Pupae-P1-DWV—Ext-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 174 ************************************************************ DWV-304 aaatatatataattaattattaaaaatggccttcagttgtggaactctctcctactctgc 1200 Pupae-P0-DWV-2 AAATATATATAATTAATTATTAAAAATGGCCTTCAGTTGTGGAACTCTCTCCTACTCTGC 227 Pupae-P1-DWV—Ext-2 AAATATATATAATTAATTATTAAAAATGGCCTTCAGTTGTGGAACTCTCTCCTACTCTGC 234 ************************************************************ DWV-304 tgtcgcccaagctccgtccgttgcccatgcacctcgtacatgggaagttgatgaagccag 1260 Pupae-P0-DWV-2 TGTCGCCCAAGCTCCGTCCGTTGCCCATGCACCTCGTACATGGGAAGTTGATGAAGCCAG 287 Pupae-P1-DWV—Ext-2 TGTCGCCCAAGCTCCGTCCGTTGCCCATGCACCTCGTACATGGGAAGTTGATGAAGCCAG 294 ************************************************************ DWV-304 gcggcgccgagtcatcaaacgtttggcgctggagcaagaacgtattcgcaacgttcttga 1320 Pupae-P0-DWV-2 GCGGCGCCGAGTCATCAAACGTTTGGCGCTGGAGCAAGAACGTATTCGCAACGTTCTTGA 347 Pupae-P1-DWV—Ext-2 GCGGCGCCGAGTCATCAAACGTTTGGCGCTGGAGCAAGAACGTATTCGCAACGTTCTTGA 354 ************************************************************ DWV-304 cgccggcgtctatgaccaggcgacatgggaacaggaggacgcgcgcgataatgagttcct 1380 Pupae-P0-DWV-2 CGCCGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 407 Pupae-P1-DWV—Ext-2 CGCCGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 414 ************************************************************ DWV-304 aacggaacaattaaacaatttatatactatttattcgatcgccgaacgttgcacgcgtcg 1440 Pupae-P0-DWV-2 AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCCGAACGTTGCACGCGTCG 467 Pupae-P1-DWV—Ext-2 AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCCGAACGTTGCACGCGTCG 474 ************************************************************ DWV-304 acctattaaagagcactctcctatatcagtttcgaataggtttgctccattggaatccct 1500 Pupae-P0-DWV-2 ACCTATTAAAGAGCACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCATTGGAATCCCT 527 Pupae-P1-DWV—Ext-2 ACCTATTAAAGAGCACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCATTGGAATCCCT 534 ************************************************************ DWV-304 caaagtcgaggtcggtcaagaagcaggcgaatgtatatttaagaaacctaaatacacgcg 1560 Pupae-P0-DWV-2 CAAAGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATACACGCG 587 Pupae-P1-DWV—Ext-2 CAAAGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATACACGCG 594 ************************************************************



https://doi.org/10.1101/660985


DWV-304 cgtttgcaagaaagtgaagcgtgttgcaacccgcttcgttcgtgaaaaagttgttcgtcc 1620 Pupae-P0-DWV-2 CGTTTGCAAGAAAGTGAAGCGTGTTGCAACCCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 647 Pupae-P1-DWV—Ext-2 CGTTTGCAAGAAAGTGAAGCGTGTTGCAACCCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 654 ************************************************************ DWV-304 tatgtgttctagatctcctatgctattatttaagcttaagaaaattatttatgatttgca 1680 Pupae-P0-DWV-2 TATGTGTTCTAGATCTCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 707 Pupae-P1-DWV—Ext-2 TATGTGTTCTAGATCTCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 714 ************************************************************ DWV-304 cttatatagattaagaaaacagattaggattttgagacgtcaaaaacagcgcgagtatga 1740 Pupae-P0-DWV-2 CTTATATAGATTAAGAAAACAGATTAGGATTTTGAGACGTCAAAAACAGCGCGAGTATGA 767 Pupae-P1-DWV—Ext-2 CTTATATAGATTAAGAAAACAGATTAGGATTTTGAGACGTCAAAAACAGCGCGAGTATGA 774 ************************************************************ DWV-304 gttagagtgtgtcactaatctgttacaattatcgaatccggtgcaggcaaaaccagagat 1800 Pupae-P0-DWV-2 GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 827 Pupae-P1-DWV—Ext-2 GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 834 ************************************************************

DWV-304 ggataaccctaatccaggacctgatggcgagggtgaagttgaattagaaaaggatagtaa 1860 Pupae-P0-DWV-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 887 Pupae-P1-DWV—Ext-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 894 ************************************************************

DWV-304 tgttgttttaacaactcagcgagatcctagtacatctattccagcgccggtgagcgtaaa 1920 Pupae-P0-DWV-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 947 Pupae-P1-DWV—Ext-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 954 ************************************************************ DWV-304 atggagtagatggactagtaatgatgtagtagatgattacgccacaatcacatctcgatg 1980 Pupae-P0-DWV-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1007 Pupae-P1-DWV—Ext-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1014 ************************************************************ DWV-304 gtatcagattgctgaatttgtttggtcgaaggatgatccatttgataaggagttagcacg 2040 Pupae-P0-DWV-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1067 Pupae-P1-DWV—Ext-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1074 ************************************************************

(b) DWV-PA AGACATCATATTTTATTTTAATGCTGTCTTTATTGCTGATTTATCTTGCTGTTTTTATTT 994 DWV-304 agacatcatattttattttaatgctgtctttattgctgatttattttgctgtttttattt 1020 Pupae-P0-DWV-2 -------------TATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 47 Pupae-P1-DWV—Ext-2 ------CATATTTTATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 54 ******************************* *************** DWV-PA GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 1054 DWV-304 gctattttatatttgctaattttcattattgcgaaatatattacattgctatttttatta 1080 Pupae-P0-DWV-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 107 Pupae-P1-DWV—Ext-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 114 ************************************************************ DWV-PA TATACGCTAGATTCAATTTTATTTTTTCTATATTTTCAATTTAATTTTGATTTCGAAGGT 1114 DWV-304 tatacgctagattcaattttatttttcctatattttcaatttaattttgattttgaaggt 1140 Pupae-P0-DWV-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 167 Pupae-P1-DWV—Ext-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 174 ************************** ************************** ****** DWV-PA AAATATATATAATTAATTATTAAAAATGGCCTTTAGTTGTGGAACTCTTTCTTACTCTGC 1174 DWV-304 aaatatatataattaattattaaaaatggccttcagttgtggaactctctcctactctgc 1200 Pupae-P0-DWV-2 AAATATATATAATTAATTATTAAAAATGGCCTTCAGTTGTGGAACTCTCTCCTACTCTGC 227 Pupae-P1-DWV—Ext-2 AAATATATATAATTAATTATTAAAAATGGCCTTCAGTTGTGGAACTCTCTCCTACTCTGC 234 ********************************* ************** ** ******** DWV-PA CGTCGCCCAAGCTCCGTCTGTCGCCTATGCACCTCGTACATGGGAAGTTGATGAAGCTAG 1234 DWV-304 tgtcgcccaagctccgtccgttgcccatgcacctcgtacatgggaagttgatgaagccag 1260 Pupae-P0-DWV-2 TGTCGCCCAAGCTCCGTCCGTTGCCCATGCACCTCGTACATGGGAAGTTGATGAAGCCAG 287 Pupae-P1-DWV—Ext-2 TGTCGCCCAAGCTCCGTCCGTTGCCCATGCACCTCGTACATGGGAAGTTGATGAAGCCAG 294 ***************** ** *** ******************************* **



https://doi.org/10.1101/660985


DWV-PA GCGGCGCCGAGTCATTAAACGTTTGGCGCTGGAGCAAGAACGTATTCGTAACGTTCTTGA 1294 DWV-304 gcggcgccgagtcatcaaacgtttggcgctggagcaagaacgtattcgcaacgttcttga 1320 Pupae-P0-DWV-2 GCGGCGCCGAGTCATCAAACGTTTGGCGCTGGAGCAAGAACGTATTCGCAACGTTCTTGA 347 Pupae-P1-DWV—Ext-2 GCGGCGCCGAGTCATCAAACGTTTGGCGCTGGAGCAAGAACGTATTCGCAACGTTCTTGA 354 *************** ******************************** *********** DWV-PA CGTTGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 1354 DWV-304 cgccggcgtctatgaccaggcgacatgggaacaggaggacgcgcgcgataatgagttcct 1380 Pupae-P0-DWV-2 CGCCGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 407 Pupae-P1-DWV—Ext-2 CGCCGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 414 ** ******************************************************** DWV-PA AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCTGAACGTTGTACGCGTCG 1414 DWV-304 aacggaacaattaaacaatttatatactatttattcgatcgccgaacgttgcacgcgtcg 1440 Pupae-P0-DWV-2 AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCCGAACGTTGCACGCGTCG 467 Pupae-P1-DWV—Ext-2 AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCCGAACGTTGCACGCGTCG 474 ****************************************** ******** ******** DWV-PA GCCTATCAAAGAGTACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCACTGGAATCCCT 1474 DWV-304 acctattaaagagcactctcctatatcagtttcgaataggtttgctccattggaatccct 1500 Pupae-P0-DWV-2 ACCTATTAAAGAGCACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCATTGGAATCCCT 527 Pupae-P1-DWV—Ext-2 ACCTATTAAAGAGCACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCATTGGAATCCCT 534 ***** ****** *********************************** ********** DWV-PA CAAGGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATATACGCG 1534 DWV-304 caaagtcgaggtcggtcaagaagcaggcgaatgtatatttaagaaacctaaatacacgcg 1560 Pupae-P0-DWV-2 CAAAGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATACACGCG 587 Pupae-P1-DWV—Ext-2 CAAAGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATACACGCG 594 *** ************************************************** ***** DWV-PA CGTTTGCAAGAAAGTGAAGCGTGTTGCAACTCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 1594 DWV-304 cgtttgcaagaaagtgaagcgtgttgcaacccgcttcgttcgtgaaaaagttgttcgtcc 1620 Pupae-P0-DWV-2 CGTTTGCAAGAAAGTGAAGCGTGTTGCAACCCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 647 Pupae-P1-DWV—Ext-2 CGTTTGCAAGAAAGTGAAGCGTGTTGCAACCCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 654 ****************************** ***************************** DWV-PA TATGTGTTCTAGATCCCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 1654 DWV-304 tatgtgttctagatctcctatgctattatttaagcttaagaaaattatttatgatttgca 1680 Pupae-P0-DWV-2 TATGTGTTCTAGATCTCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 707 Pupae-P1-DWV—Ext-2 TATGTGTTCTAGATCTCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 714 *************** ******************************************** DWV-PA CTTATATAGATTAAGAAAACAGATTAGGATGTTGAGACGTCAAAAACAGCGCGATTACGA 1714 DWV-304 cttatatagattaagaaaacagattaggattttgagacgtcaaaaacagcgcgagtatga 1740 Pupae-P0-DWV-2 CTTATATAGATTAAGAAAACAGATTAGGATTTTGAGACGTCAAAAACAGCGCGAGTATGA 767 Pupae-P1-DWV—Ext-2 CTTATATAGATTAAGAAAACAGATTAGGATTTTGAGACGTCAAAAACAGCGCGAGTATGA 774 ****************************** *********************** ** ** DWV-PA GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 1774 DWV-304 gttagagtgtgtcactaatctgttacaattatcgaatccggtgcaggcaaaaccagagat 1800 Pupae-P0-DWV-2 GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 827 Pupae-P1-DWV—Ext-2 GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 834 ************************************************************ DWV-PA GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 1834 DWV-304 ggataaccctaatccaggacctgatggcgagggtgaagttgaattagaaaaggatagtaa 1860 Pupae-P0-DWV-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 887 Pupae-P1-DWV—Ext-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 894 ************************************************************ DWV-PA TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 1894 DWV-304 tgttgttttaacaactcagcgagatcctagtacatctattccagcgccggtgagcgtaaa 1920 Pupae-P0-DWV-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 947 Pupae-P1-DWV—Ext-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 954 ************************************************************ DWV-PA ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1954 DWV-304 atggagtagatggactagtaatgatgtagtagatgattacgccacaatcacatctcgatg 1980 Pupae-P0-DWV-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1007 Pupae-P1-DWV—Ext-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1014 ************************************************************

DWV-PA GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 2014 DWV-304 gtatcagattgctgaatttgtttggtcgaaggatgatccatttgataaggagttagcacg 2040 Pupae-P0-DWV-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1067 Pupae-P1-DWV—Ext-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1074 ************************************************************ -



https://doi.org/10.1101/660985


(c) V0-DWV-2 -------------TCATATTTTGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 55 DWV-304 agacatcatattttattttaatgctgtctttattgctgatttattttgctgtttttattt 1020 Pupae-P0-DWV-2 -------------TATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 47 Pupae-P1-DWV—Ext-2 ------CATATTTTATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 54 *********************************************** V0-DWV-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 115 DWV-304 gctattttatatttgctaattttcattattgcgaaatatattacattgctatttttatta 1080 Pupae-P0-DWV-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 107 Pupae-P1-DWV—Ext-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 114 ************************************************************ V0-DWV-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 175 DWV-304 tatacgctagattcaattttatttttcctatattttcaatttaattttgattttgaaggt 1140 Pupae-P0-DWV-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 167 Pupae-P1-DWV—Ext-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 174 ************************************************************ V0-DWV-2 AAATATATATAATTAATTATTAAAAATGGCCTnnnnnnnnnnnnnnnnnnnnnnnnnnnn 235 DWV-304 aaatatatataattaattattaaaaatggccttcagttgtggaactctctcctactctgc 1200 Pupae-P0-DWV-2 AAATATATATAATTAATTATTAAAAATGGCCTTCAGTTGTGGAACTCTCTCCTACTCTGC 227 Pupae-P1-DWV—Ext-2 AAATATATATAATTAATTATTAAAAATGGCCTTCAGTTGTGGAACTCTCTCCTACTCTGC 234 ******************************** V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 295 DWV-304 tgtcgcccaagctccgtccgttgcccatgcacctcgtacatgggaagttgatgaagccag 1260 Pupae-P0-DWV-2 TGTCGCCCAAGCTCCGTCCGTTGCCCATGCACCTCGTACATGGGAAGTTGATGAAGCCAG 287 Pupae-P1-DWV—Ext-2 TGTCGCCCAAGCTCCGTCCGTTGCCCATGCACCTCGTACATGGGAAGTTGATGAAGCCAG 294 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 355 DWV-304 gcggcgccgagtcatcaaacgtttggcgctggagcaagaacgtattcgcaacgttcttga 1320 Pupae-P0-DWV-2 GCGGCGCCGAGTCATCAAACGTTTGGCGCTGGAGCAAGAACGTATTCGCAACGTTCTTGA 347 Pupae-P1-DWV—Ext-2 GCGGCGCCGAGTCATCAAACGTTTGGCGCTGGAGCAAGAACGTATTCGCAACGTTCTTGA 354 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 415 DWV-304 cgccggcgtctatgaccaggcgacatgggaacaggaggacgcgcgcgataatgagttcct 1380 Pupae-P0-DWV-2 CGCCGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 407 Pupae-P1-DWV—Ext-2 CGCCGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 414 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 475 DWV-304 aacggaacaattaaacaatttatatactatttattcgatcgccgaacgttgcacgcgtcg 1440 Pupae-P0-DWV-2 AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCCGAACGTTGCACGCGTCG 467 Pupae-P1-DWV—Ext-2 AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCCGAACGTTGCACGCGTCG 474 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 535 DWV-304 acctattaaagagcactctcctatatcagtttcgaataggtttgctccattggaatccct 1500 Pupae-P0-DWV-2 ACCTATTAAAGAGCACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCATTGGAATCCCT 527 Pupae-P1-DWV—Ext-2 ACCTATTAAAGAGCACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCATTGGAATCCCT 534 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 594 DWV-304 caaagtcgaggtcggtcaagaagcaggcgaatgtatatttaagaaacctaaatacacgcg 1560 Pupae-P0-DWV-2 CAAAGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATACACGCG 587 Pupae-P1-DWV—Ext-2 CAAAGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATACACGCG 594 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 654 DWV-304 cgtttgcaagaaagtgaagcgtgttgcaacccgcttcgttcgtgaaaaagttgttcgtcc 1620 Pupae-P0-DWV-2 CGTTTGCAAGAAAGTGAAGCGTGTTGCAACCCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 647 Pupae-P1-DWV—Ext-2 CGTTTGCAAGAAAGTGAAGCGTGTTGCAACCCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 654 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 714 DWV-304 tatgtgttctagatctcctatgctattatttaagcttaagaaaattatttatgatttgca 1680 Pupae-P0-DWV-2 TATGTGTTCTAGATCTCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 707 Pupae-P1-DWV—Ext-2 TATGTGTTCTAGATCTCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 714 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 774 DWV-304 cttatatagattaagaaaacagattaggattttgagacgtcaaaaacagcgcgagtatga 1740 Pupae-P0-DWV-2 CTTATATAGATTAAGAAAACAGATTAGGATTTTGAGACGTCAAAAACAGCGCGAGTATGA 767 Pupae-P1-DWV—Ext-2 CTTATATAGATTAAGAAAACAGATTAGGATTTTGAGACGTCAAAAACAGCGCGAGTATGA 774



https://doi.org/10.1101/660985


V0-DWV-2 nnnnnnnnnnnnnnnTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 834 DWV-304 gttagagtgtgtcactaatctgttacaattatcgaatccggtgcaggcaaaaccagagat 1800 Pupae-P0-DWV-2 GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 827 Pupae-P1-DWV—Ext-2 GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 834 ********************************************* V0-DWV-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 894 DWV-304 ggataaccctaatccaggacctgatggcgagggtgaagttgaattagaaaaggatagtaa 1860 Pupae-P0-DWV-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 887 Pupae-P1-DWV—Ext-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 894 ************************************************************ V0-DWV-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 954 DWV-304 tgttgttttaacaactcagcgagatcctagtacatctattccagcgccggtgagcgtaaa 1920 Pupae-P0-DWV-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 947 Pupae-P1-DWV—Ext-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 954 ************************************************************ V0-DWV-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1014 DWV-304 atggagtagatggactagtaatgatgtagtagatgattacgccacaatcacatctcgatg 1980 Pupae-P0-DWV-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1007 Pupae-P1-DWV—Ext-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1014 ************************************************************ V0-DWV-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1074 DWV-304 gtatcagattgctgaatttgtttggtcgaaggatgatccatttgataaggagttagcacg 2040 Pupae-P0-DWV-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1067 Pupae-P1-DWV—Ext-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1074 ************************************************************

(d) V0-DWV-2 -----TCATATTTTATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 55 DWV-PA AGACATCATATTTTATTTTAATGCTGTCTTTATTGCTGATTTATCTTGCTGTTTTTATTT 994 DWV-304 agacatcatattttattttaatgctgtctttattgctgatttattttgctgtttttattt 1020 Pupae-P0-DWV-2 -------------TATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 47 Pupae-P1-DWV—Ext-2 ------CATATTTTATTTTAATGCTGTCTTTATTGCTGATTTATTTTGCTGTTTTTATTT 54 ******************************* *************** V0-DWV-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 115 DWV-PA GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 1054 DWV-304 gctattttatatttgctaattttcattattgcgaaatatattacattgctatttttatta 1080 Pupae-P0-DWV-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 107 Pupae-P1-DWV—Ext-2 GCTATTTTATATTTGCTAATTTTCATTATTGCGAAATATATTACATTGCTATTTTTATTA 114 ************************************************************ V0-DWV-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 175 DWV-PA TATACGCTAGATTCAATTTTATTTTTTCTATATTTTCAATTTAATTTTGATTTCGAAGGT 1114 DWV-304 tatacgctagattcaattttatttttcctatattttcaatttaattttgattttgaaggt 1140 Pupae-P0-DWV-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 167 Pupae-P1-DWV—Ext-2 TATACGCTAGATTCAATTTTATTTTTCCTATATTTTCAATTTAATTTTGATTTTGAAGGT 174 ************************** ************************** ****** V0-DWV-2 AAATATATATAATTAATTATTAAAAATGGCCTnnnnnnnnnnnnnnnnnnnnnnnnnnnn 235 DWV-PA AAATATATATAATTAATTATTAAAAATGGCCTTTAGTTGTGGAACTCTTTCTTACTCTGC 1174 DWV-304 aaatatatataattaattattaaaaatggccttcagttgtggaactctctcctactctgc 1200 Pupae-P0-DWV-2 AAATATATATAATTAATTATTAAAAATGGCCTTCAGTTGTGGAACTCTCTCCTACTCTGC 227 Pupae-P1-DWV—Ext-2 AAATATATATAATTAATTATTAAAAATGGCCTTCAGTTGTGGAACTCTCTCCTACTCTGC 234 ******************************** V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 295 DWV-PA CGTCGCCCAAGCTCCGTCTGTCGCCTATGCACCTCGTACATGGGAAGTTGATGAAGCTAG 1234 DWV-304 tgtcgcccaagctccgtccgttgcccatgcacctcgtacatgggaagttgatgaagccag 1260 Pupae-P0-DWV-2 TGTCGCCCAAGCTCCGTCCGTTGCCCATGCACCTCGTACATGGGAAGTTGATGAAGCCAG 287 Pupae-P1-DWV—Ext-2 TGTCGCCCAAGCTCCGTCCGTTGCCCATGCACCTCGTACATGGGAAGTTGATGAAGCCAG 294

V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 355 DWV-PA GCGGCGCCGAGTCATTAAACGTTTGGCGCTGGAGCAAGAACGTATTCGTAACGTTCTTGA 1294 DWV-304 gcggcgccgagtcatcaaacgtttggcgctggagcaagaacgtattcgcaacgttcttga 1320 Pupae-P0-DWV-2 GCGGCGCCGAGTCATCAAACGTTTGGCGCTGGAGCAAGAACGTATTCGCAACGTTCTTGA 347 Pupae-P1-DWV—Ext-2 GCGGCGCCGAGTCATCAAACGTTTGGCGCTGGAGCAAGAACGTATTCGCAACGTTCTTGA 354

V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 415 DWV-PA CGTTGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 1354 DWV-304 cgccggcgtctatgaccaggcgacatgggaacaggaggacgcgcgcgataatgagttcct 1380 Pupae-P0-DWV-2 CGCCGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 407 Pupae-P1-DWV—Ext-2 CGCCGGCGTCTATGACCAGGCGACATGGGAACAGGAGGACGCGCGCGATAATGAGTTCCT 414



https://doi.org/10.1101/660985


V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 475 DWV-PA AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCTGAACGTTGTACGCGTCG 1414 DWV-304 aacggaacaattaaacaatttatatactatttattcgatcgccgaacgttgcacgcgtcg 1440 Pupae-P0-DWV-2 AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCCGAACGTTGCACGCGTCG 467 Pupae-P1-DWV—Ext-2 AACGGAACAATTAAACAATTTATATACTATTTATTCGATCGCCGAACGTTGCACGCGTCG 474 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 535 DWV-PA GCCTATCAAAGAGTACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCACTGGAATCCCT 1474 DWV-304 acctattaaagagcactctcctatatcagtttcgaataggtttgctccattggaatccct 1500 Pupae-P0-DWV-2 ACCTATTAAAGAGCACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCATTGGAATCCCT 527 Pupae-P1-DWV—Ext-2 ACCTATTAAAGAGCACTCTCCTATATCAGTTTCGAATAGGTTTGCTCCATTGGAATCCCT 534 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn-nnnnnnnnnnnnnnnnnnnnn 594 DWV-PA CAAGGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATATACGCG 1534 DWV-304 caaagtcgaggtcggtcaagaagcaggcgaatgtatatttaagaaacctaaatacacgcg 1560 Pupae-P0-DWV-2 CAAAGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATACACGCG 587 Pupae-P1-DWV—Ext-2 CAAAGTCGAGGTCGGTCAAGAAGCAGGCGAATGTATATTTAAGAAACCTAAATACACGCG 594 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 654 DWV-PA CGTTTGCAAGAAAGTGAAGCGTGTTGCAACTCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 1594 DWV-304 cgtttgcaagaaagtgaagcgtgttgcaacccgcttcgttcgtgaaaaagttgttcgtcc 1620 Pupae-P0-DWV-2 CGTTTGCAAGAAAGTGAAGCGTGTTGCAACCCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 647 Pupae-P1-DWV—Ext-2 CGTTTGCAAGAAAGTGAAGCGTGTTGCAACCCGCTTCGTTCGTGAAAAAGTTGTTCGTCC 654 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 714 DWV-PA TATGTGTTCTAGATCCCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 1654 DWV-304 tatgtgttctagatctcctatgctattatttaagcttaagaaaattatttatgatttgca 1680 Pupae-P0-DWV-2 TATGTGTTCTAGATCTCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 707 Pupae-P1-DWV—Ext-2 TATGTGTTCTAGATCTCCTATGCTATTATTTAAGCTTAAGAAAATTATTTATGATTTGCA 714 V0-DWV-2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 774 DWV-PA CTTATATAGATTAAGAAAACAGATTAGGATGTTGAGACGTCAAAAACAGCGCGATTACGA 1714 DWV-304 cttatatagattaagaaaacagattaggattttgagacgtcaaaaacagcgcgagtatga 1740 Pupae-P0-DWV-2 CTTATATAGATTAAGAAAACAGATTAGGATTTTGAGACGTCAAAAACAGCGCGAGTATGA 767 Pupae-P1-DWV—Ext-2 CTTATATAGATTAAGAAAACAGATTAGGATTTTGAGACGTCAAAAACAGCGCGAGTATGA 774

V0-DWV-2 nnnnnnnnnnnnnnnTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 834 DWV-PA GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 1774 DWV-304 gttagagtgtgtcactaatctgttacaattatcgaatccggtgcaggcaaaaccagagat 1800 Pupae-P0-DWV-2 GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 827 Pupae-P1-DWV—Ext-2 GTTAGAGTGTGTCACTAATCTGTTACAATTATCGAATCCGGTGCAGGCAAAACCAGAGAT 834 ********************************************* V0-DWV-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 894 DWV-PA GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 1834 DWV-304 ggataaccctaatccaggacctgatggcgagggtgaagttgaattagaaaaggatagtaa 1860 Pupae-P0-DWV-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 887 Pupae-P1-DWV—Ext-2 GGATAACCCTAATCCAGGACCTGATGGCGAGGGTGAAGTTGAATTAGAAAAGGATAGTAA 894 ************************************************************ V0-DWV-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 954 DWV-PA TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 1894 DWV-304 tgttgttttaacaactcagcgagatcctagtacatctattccagcgccggtgagcgtaaa 1920 Pupae-P0-DWV-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 947 Pupae-P1-DWV—Ext-2 TGTTGTTTTAACAACTCAGCGAGATCCTAGTACATCTATTCCAGCGCCGGTGAGCGTAAA 954 ************************************************************

V0-DWV-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1014 DWV-PA ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1954 DWV-304 atggagtagatggactagtaatgatgtagtagatgattacgccacaatcacatctcgatg 1980 Pupae-P0-DWV-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1007 Pupae-P1-DWV—Ext-2 ATGGAGTAGATGGACTAGTAATGATGTAGTAGATGATTACGCCACAATCACATCTCGATG 1014 ************************************************************ V0-DWV-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1074 DWV-PA GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 2014 DWV-304 gtatcagattgctgaatttgtttggtcgaaggatgatccatttgataaggagttagcacg 2040 Pupae-P0-DWV-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1067 Pupae-P1-DWV—Ext-2 GTATCAGATTGCTGAATTTGTTTGGTCGAAGGATGATCCATTTGATAAGGAGTTAGCACG 1074 ************************************************************



https://doi.org/10.1101/660985


Supplementary Figure S2. DWV polymorphisms in honey bees and mites in Experiment 2: “Varroa-mediated DWV transmission experiment”. Electropherograms of direct Sanger sequencing of RT-PCR fragments corresponding to the DWV LP region were amplified using RNA extracted from bees and mites pooled according to their treatment (Experiment 2, Figs. 2 and 4). (a) Honey bee pupae P0-DWV2, infected with a clone-derived DWV isolate served as the “source bees”; (b) Varroa mites V0-DWV2, which acquired DWV from the “source bees”; (c) Honey bee pupae P1-DWV-Ext2 infected with DWV strains transmitted by the Varroa mites fed first on the “source bees”. Clonal DWV accumulated in the honey bee pupae and divergent DWV accumulated in the Varroa mites, as evidenced by the presence of double peaks indicated by arrows. (a) Pupae P0-DWV2 – clonal DWV

(b) Varroa mites V0-DWV2 - polymorphic DWV

(c) Pupae P1-DWV-Ext2 – clonal DWV



https://doi.org/10.1101/660985


Supplementary Figure S3. Single stranded RNA degradation in disrupted honey bee hemolymph cells.



https://doi.org/10.1101/660985


posada-florez et al. varroa vectoring of a honey bee virus · posada-florez et al. varroa vectoring...

Documents