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Occurrence of multiple honeybee viruses in the ectoparasitic mites Varroa spp. in Apis cerana colonies
Journal of Invertebrate Pathology 166 (2019) 107225
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Occurrence of multiple honeybee viruses in the ectoparasitic mites Varroa spp. in Apis cerana colonies
T
Shuai Wanga, Gongwen Chena, Zheguang Linb, Yuqi Wua, Fuliang Hua, Huoqing Zhenga,
⁎
a b
College of Animal Sciences, Zhejiang University, Hangzhou 310058, China College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
ARTICLE INFO
ABSTRACT
Keywords: Honeybee viruses Varroa destructor Varroa underwoodi Apis cerana
In this study, we investigated the prevalence of honeybee viruses in Varroa destructor and Varroa underwoodi infesting Apis cerana colonies in China. Deformed wing virus (DWV) was the most prevalent virus in these two mite species, followed by Israeli acute paralysis virus (IAPV), Black queen cell virus (BQCV), Kashmir bee virus (KBV), Chronic bee paralysis virus (CBPV), Apis mellifera filamentous virus (AmFV) and Sacbrood virus (SBV) in V. destructor, while in V. underwoodi, it was followed by CBPV, AmFV, BQCV, IAPV and KBV. In addition, multiple viruses were commonly detectable in both mite species.
1. Introduction Honeybees play important roles in agriculture and natural ecosystems (Potts et al., 2016). The Western honeybee, Apis mellifera, and the Eastern honeybee, Apis cerana, are the two most important honeybee species for the beekeeping industry and share many common foraging habitats in China (Li et al., 2012). Honeybees are vulnerable to a great diversity of pathogens and parasites (Genersch, 2010) and, as reported so far, the viruses are considered to be one of the major risk factors for bee health (Tantillo et al., 2015). More than 30 honeybee viruses have been identified worldwide (Remnant et al., 2017), eight of which are widely detected in honeybee colonies. These include deformed wing virus (DWV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), Israeli acute paralysis virus (IAPV), sacbrood virus (SBV), acute bee paralysis virus (ABPV) and Varroa destructor virus-1 (VDV-1). Compared to RNA viruses, fewer studies on DNA viruses have been conducted in honeybees. A DNA virus, Apis mellifera filamentous virus (AmFV), was first reported in USA (Clark 1978) and was related to the loss of Swiss honeybee colonies (Hartmann et al., 2015). Recently, it was also detected in A. cerana and A. mellifera colonies in China (Hou et al., 2016, 2017). An ectoparasitic mite of honeybees, Varroa destructor, shifted host from A. cerana to A. mellifera with catastrophic consequences globally. In addition, Varroa underwoodi is widely distributed in Asian A. cerana colonies and is a potential threat to A. mellifera (Wang et al., 2019). V. destructor has been shown to be an effective vector of honeybee viruses
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in A. mellifera colonies (de Miranda et al., 2010; Di Prisco et al., 2011); for example, DWV infection is tightly associated with V. destructor infestation on A. mellifera (Wilfert et al., 2016). However, little is known about the effects of Varroa spp. on their original hosts, A. cerana, especially concerning the potential synergistic effects of virus infection and Varroa spp. infestation. In this study, we conducted the first investigation on the occurrence of DNA and RNA viruses in V. destructor and V. underwoodi mites collected from A. cerana colonies. 2. Materials and methods 2.1. Sample collection V. destructor samples (N = 95) were collected from six apiaries in Zhejiang Province of East China, one in Jilin Province of North China and one in Guangdong Province of South China. V. underwoodi samples (N = 34) were collected from two apiaries in Zhejiang Province, one in Jilin Province and one in Jiangxi Province of East China during March 2016–June 2019. The mites were collected from A. cerana sealed drone brood cells (Wang et al., 2019) in multiple colonies and stored separately in Eppendorf tubes with 75% EtOH at −20 °C until use. Due to the natural variations in availability of mites (Wang et al., 2019), the numbers of mites collected from each apiary were not uniform. 2.2. RNA and DNA extraction
Corresponding author. E-mail address: [email protected] (H. Zheng).
https://doi.org/10.1016/j.jip.2019.107225 Received 24 May 2019; Received in revised form 25 July 2019; Accepted 29 July 2019 Available online 29 July 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
Total RNA was extracted from individual adult female mites
Journal of Invertebrate Pathology 166 (2019) 107225 a Abbreviations of sampled apiaries: JL, Jilin; FY, Fuyang; YP, Yuanpu; ZP, Zhoupu; XH, Xihu; ZJU, Zhejiang University; JH, Jinhua; GZ, Guangzhou; CA, Chun’an; NC, Nanchang. JL in Jilin Province, NC in Jiangxi Province, GZ in Guangdong Province and the others in Zhejiang Province. b The detection limits of ABPV and BQCV was estimated to be about 1600 and 130 genome equivalents, respectively (Benjeddou et al., 2001); In the case of overt KBV infection, approximately 1–10 pg viral RNA can be detected (Stoltz et al., 1995); the detection limits of the other viruses were undetermined. c –, unavailable data. d 95% confidence intervals, the lower limits below zero were manually corrected to zeroes.
1 – – – 1 20.0 [–, –] 5 – – – 5 0 0 0 0 0 0 [0, 0] 1 0 0 0 1 3.5 [0, 3.9] JL 17 CA 6 JH2 4 NC 2 Total 29 %infected [95% CI] V. underwoodi
0 0 0 0 0 0 [0, 0]
2 3 0 0 5 17.3 [0, 38.5]
4 2 0 0 6 20.7 [0, 30.7]
11 2 4 1 18 62.1 [34.6, 89.4]
3 0 2 0 5 17.2 [0, 40.5]
0 0 0 0 0 0 [0, 0]
0 – – 1 0 – 0 0 1 4.3 [0, 11.9] 6 –c – 5 3 – 6 3 23 0 0 0 0 0 0 0 – 0 0 [0, 0] 0 1 3 5 0 2 0 – 11 15.3 [2.1, 25.9] JL 6 FY 13 YP 11 ZP 21 XH 6 ZJU 5 JH1 10 GZ – Total 72 %infected [95% CI]d V. destructor
0 0 0 0 0 0 0 – 0 0 [0, 0]
1 3 5 4 1 2 8 – 24 33.3 [17.0, 51.0]
1 2 1 3 1 0 0 – 8 11.1 [4.8, 15.2]
3 6 11 16 6 5 7 – 54 75.0 [59.2, 94.8]
1 6 3 6 2 4 7 – 29 40.3 [25.2, 60.8]
0 1 0 1 0 0 0 – 2 2.8 [0, 4.2]
No. of mites KBV IAPV DWV CBPV BQCV ABPVb No. of mites
RNA viruses Apiariesa Species
Table 1 Detection of RNA and DNA viruses in V. destructor and V. underwoodi mites from A. cerana colonies.
SBV
VDV-1
DNA virus
AmFV
S. Wang, et al.
according to the manufacturer’s protocol (Aidlab, China). For brood cells that were infested with more than one mite foundress, only one adult female mite was used. Total DNA was extracted from different individual adult female mites following the manufacturer’s protocol (Tiangen, China). RNA and DNA samples were stored at −80 °C until use. 2.3. RT-PCR and sequencing RNA samples were used for the detection of eight bee viruses by qualitative RT-PCR using specific PCR primers from previous reports: ABPV and BQCV (Benjeddou et al., 2001), CBPV (Ribière et al., 2002), DWV and KBV (Tentcheva et al., 2004), IAPV (Maori et al., 2007), SBV (Chen et al., 2004) and VDV-1 (Gauthier et al., 2011). cDNA synthesis and amplification were performed using the manufacturer’s recommended protocol (TOYOBO, Japan). DNA samples were checked for the presence of AmFV with the specific primer (Hartmann et al., 2015). Normal PCR (40 cycles) and gel electrophoresis were performed as described previously (Wang et al., 2019). Positive PCR products were sequenced by the ABI 3730XL automatic sequencing system (Sangon, China). The sequence of each virus fragment was confirmed using the BLAST server at the NCBI website (https://blast.ncbi.nlm.nih.gov/). Part of the sequences (N = 77) were uploaded to GenBank (https:// www.ncbi.nlm.nih.gov/genbank/) with accession numbers of MN114541 - MN114617. 3. Results and discussion Seven RNA viruses (ABPV, BQCV, CBPV, DWV, IAPV, KBV and SBV) were previously detected in A. cerana colonies in Asia (Kojima et al., 2011; Ai et al., 2012; Choe et al., 2012; Li et al., 2012; Yañez et al., 2015; Thu et al., 2016; Chanpanitkitchote et al., 2018). We found that six of the viruses (except ABPV) were detected in V. destructor and five (all but ABPV and SBV) were detected in V. underwoodi infesting A. cerana colonies (Table 1). VDV-1 was not found in either mite species. BQCV was detected in V. destructor from all apiaries while DWV was detected in both V. destructor and V. underwoodi from all apiaries (Table 1). DWV is one of the most prevalent viruses in honeybees in Asia (Kojima et al., 2011; Ai et al., 2012; Li et al., 2012; Yañez et al., 2015), and its prevalence was tightly linked to the infestation of V. destructor in A. mellifera colonies (Martin et al., 2012; Wilfert et al., 2016). Here we showed that DWV was the most prevalent virus in both V. destructor (75.0%; 95% confidence intervals (CI), [59.2%, 94.8%]) and V. underwoodi (62.1%; 95% CI, [34.6%, 89.4%]) samples collected from A. cerana colonies, suggesting an association between DWV and both V. destructor and V. underwoodi in A. cerana colonies. IAPV infects A. mellifera, A. cerana, Bombus terrestris and V. destructor in A. mellifera colonies (Maori et al., 2007; Singh et al., 2010; Di Prisco et al., 2011; Ai et al., 2012; Niu et al., 2014). Our results showed that IAPV was the second most prevalent virus in V. destructor (40.3%; 95% CI, [25.2%, 60.8%]) and the fourth most prevalent in V. underwoodi (17.2%; 95% CI, [0, 40.5%]) in A. cerana colonies. CBPV rarely has been detected in A. cerana (0–7%, Ai et al., 2012; Li et al., 2012; Yañez et al., 2015) or in A. mellifera (0–9%, Ai et al., 2012; Peng et al., 2015; Yañez et al., 2015) in China. Interestingly, it occurred in V. destructor and V. underwoodi mites of A. cerana colonies at a relatively high level (11.1%; 95% CI, [4.8%, 15.2%] and 20.7%; 95% CI, [0, 30.7%], respectively). BQCV was detected in A. cerana colonies in Japan, Korea, China and Vietnam (Kojima et al., 2011; Choe et al., 2012; Ai et al., 2012; Li et al., 2012; Yañez et al., 2015; Thu et al., 2016). We identified BQCV as the third most prevalent virus in both Varroa mites (V. destructor: 33.3%; 95% CI, [17.0%, 51.0%] and V. underwoodi: 17.3%; 95% CI, [0, 38.5%]) in A. cerana colonies. KBV was not reported in China previously (Ai et al., 2012; Yañez et al., 2015); however, our results revealed its presence in both V. destructor (15.3%; 95% CI, [2.1%, 25.9%]) and V. underwoodi 2
Journal of Invertebrate Pathology 166 (2019) 107225
S. Wang, et al.
Table 2 Frequencies of multiple RNA viral infections in Varroa spp. samples. Species
No. of viruses
V. destructor
0 1 2
3
4
V. underwoodi
0 1
2
3
Type of infection
No. of mites
Rate (%)
Origina
BQCV DWV IAPV BQCV, SBV BQCV, DWV CBPV, DWV DWV, IAPV DWV, KBV IAPV, KBV BQCV, CBPV, DWV BQCV, CBPV, IAPV BQCV, DWV, IAPV DWV, IAPV, KBV BQCV, DWV, IAPV, KBV CBPV, DWV, IAPV, KBV CBPV, DWV, IAPV, SBV
8 5 17 1 1 4 3 10 3 1 2 1 8 3 3 1 1
11.1 6.9 23.6 1.4 1.4 5.6 4.2 13.9 4.2 1.4 2.8 1.4 11.1 4.2 4.2 1.4 1.4
2JL, 4FY, 2ZP 1FY, 3JH, 1ZP 2JL, 2FY, 3YP, 3XH, 6ZP, 1ZJU 1FY 1ZP 3YP, 1ZP 1YP, 2ZP 1JL, 1FY, 2XH, 2ZP, 2ZJU, 2JH 1YP, 2ZP 1ZP 1JL, 1XH 1FY 1FY, 1YP, 1ZP, 5JH 1FY, 1YP, 1ZP 1YP, 2ZJU 1ZP 1FY
CBPV DWV IAPV KBV BQCV, CBPV BQCV, DWV CBPV, DWV CBPV, IAPV DWV, IAPV BQCV, CBPV, DWV
5 2 10 1 1 1 3 1 1 3 1
17.2 6.9 34.5 3.4 3.4 3.4 10.3 3.4 3.4 10.3 3.4
2JL, 1JL, 7JL, 1JL 1JL 1CA 1JL, 1JL 1JL 1JL, 1JL
1NC, 2CA 1CA 2JH, 1NC
2CA 2JH
a Abbreviations of sampled apiaries: FY, Fuyang; YP, Yuanpu; ZP, Zhoupu; XH, Xihu; ZJU, Zhejiang University; JH, Jinhua; JL, Jilin; CA, Chun’an; NC, Nanchang. The numbers of infested mites were indicated before the apiary locations.
mites, as well as to show if they are transmitted to the workers and if they have negative effects on the colonies.
(3.5%; 95% CI, [0, 3.9%]), suggesting that this virus may present in A. cerana in China and deserves further researches, both on detection of the virus in A. cerana and on the relationships between KBV and Varroa spp. in A. cerana colonies. To our surprise, the prevalence of SBV was quite low in V. destructor (2.8%; 95% CI, [0, 4.2%]) and was not detectable in V. underwoodi despite being the most prevalent virus in A. cerana populations in China, Korea and Vietnam (Ai et al., 2012; Choe et al., 2012; Thu et al., 2016). It is possible that SBV primarily affects honeybee larvae and even causes mortality (Ai et al., 2012), reducing the potential for transmission to mites. Alternatively, but not mutually exclusive, the low prevalence of this virus in mites could be due to its inability to replicate in the mites. This is in accordance with the study on V. destructor mites collected from A. mellifera colonies, which revealed that the prevalence of SBV in the parasites was lower than that in their honeybee hosts (Tentcheva et al., 2004). AmFV, a DNA virus, was confirmed to be widely present in A. cerana and A. mellifera populations in China (Hou et al., 2016, 2017). Although the prevalence was low in our study, we showed that V. destructor and V. underwoodi mites could be potential hosts of the DNA virus in A. cerana colonies. Multiple RNA viruses were detected in our Varroa spp. samples and were common in both mite species (Table 2). In V. destructor, 30.7% of samples were infected with two viruses, 19.5% with three viruses, and 7.0% with four viruses. Multiple infections with two viruses and three viruses were 30.8% and 3.4%, respectively, in V. underwoodi. In both mite species, the most common dual infection of RNA viruses was DWV and IAPV. Owing to the low infestation rates of Varroa spp. in A. cerana colonies (Wang et al., 2019), the sample sizes were limited, especially for V. underwoodi, which may cause bias in calculating the prevalence of the tested viruses. Further studies with more samples are needed to provide more precise results. Moreover, it is important to quantify the detected viruses and to determine whether they replicate in the Varroa
4. Conclusions We investigated the presence of the eight RNA viruses and one DNA honeybee virus in Varroa spp. collected from A. cerana colonies. DWV, IAPV, BQCV, KBV, CBPV, SBV and AmFV were detected in V. destructor and DWV, CBPV, AmFV, BQCV, IAPV and KBV were detected in V. underwoodi. The high occurrence of honeybee viruses in the two species of Varroa mites suggested that the mites, despite a low infestation rate in A. cerana colonies, act as vectors of honeybee viruses and may affect the health of A. cerana. Consequently, the relationships/interactions between the Varroa spp. mites and honeybee viruses in A. cerana colonies deserve further studies, which may shed new light on hostparasite-virus interactions. Acknowledgements The work was supported by National Natural Science Foundation of China (31672498, H.Z.), Science and Technology Department of Zhejiang Province, China (2016C02054-11, F.H.) and the Modern Agroindustry Technology Research System of China (CARS-44, F.H. and H.Z.). Declaration of Competing Interest None. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jip.2019.107225.
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S. Wang, et al.
Journal of Invertebrate Pathology 166 (2019) 107225
References
parasitic mite. Science 336 (6086), 1304–1306. Maori, E., Lavi, S., Mozes-Koch, R., Gantman, Y., Peretz, Y., Edelbaum, O., Tanne, E., Sela, I., 2007. Isolation and characterization of Israeli acute paralysis virus, a dicistrovirus affecting honeybees in Israel: evidence for diversity due to intra and inter-species recombination. J. Gen. Virol. 88 (12), 3428–3438. Niu, J., Cappelle, K., de Miranda, J.R., Smagghe, G., Meeus, I., 2014. Analysis of reference gene stability after Israeli acute paralysis virus infection in bumblebees Bombus terrestris. J. Invertebr. Pathol. 115, 76–79. Peng, W., Li, J., Zhao, Y., Cen, Y., Zeng, Z., 2015. A descriptive study of the prevalence of parasites and pathogens in Chinese black honeybees. Parasitology 142 (11), 1364–1374. Potts, S.G., Imperatriz-Fonseca, V., Ngo, H.T., Aizen, M.A., Biesmeijer, J.C., Breeze, T.D., Dicks, L.V., Garibaldi, L.A., Hill, R., Settele, J., Vanbergen, A.J., 2016. Safeguarding pollinators and their values to human well-being. Nature 540 (7632), 220–229. Remnant, E.J., Shi, M., Buchmann, G., Blacquière, T., Holmes, E.C., Beekman, M., Ashe, A., 2017. A diverse range of novel RNA viruses in geographically distinct honey bee populations. J. Virol. 91 (16), e00158–e217. Ribière, M., Triboulot, C., Mathieu, L., Aurières, C., Faucon, J.-P., Pépin, M., 2002. Molecular diagnosis of chronic bee paralysis virus infection. Apidologie 33 (3), 339–351. Singh, R., Levitt, A.L., Rajotte, E.G., Holmes, E.C., Ostiguy, N., vanEngelsdorp, D., Lipkin, W.I., dePamphilis, C.W., Toth, A.L., Cox-Foster, D.L., 2010. RNA viruses in hymenopteran pollinators: evidence of inter-taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species. PLoS One 5 (12), e14357. Stoltz, D., Shen, X.R., Boggis, C., Sisson, G., 1995. Molecular diagnosis of Kashmir bee virus infection. J. Apic. Res. 34 (3), 153–160. Tantillo, G., Bottaro, M., Di Pinto, A., Martella, V., Di Pinto, P., Terio, V., 2015. Virus infections of honeybees Apis mellifera. Italian J. Food Saf. 4 (3), 5364. Tentcheva, D., Gauthier, L., Zappulla, N., Dainat, B., Cousserans, F., Colin, M.E., Bergoin, M., 2004. Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France. Appl. Environ. Microbiol. 70 (12), 7185–7191. Thu, H.T., Thi Kim Lien, N., Thuy Linh, M., Le, T.H., Hoa, N.T., Hong Thai, P., Reddy, K.E., Yoo, M.S., Kim, Y.H., Cho, Y.S., Kang, S.W., Quyen, D.V., 2016. Prevalence of bee viruses among Apis cerana populations in Vietnam. J. Apic. Res. 55 (5), 379–385. Wang, S., Lin, Z., Dietemann, V., Neumann, P., Wu, Y., Hu, F., Zheng, H., 2019. Ectoparasitic Mites Varroa underwoodi (Acarina: Varroidae) in Eastern Honeybees, but not in Western Honeybees. J. Econ. Entomol. 112 (1), 25–32. Wilfert, L., Long, G., Leggett, H.C., Schmid-Hempel, P., Butlin, R., Martin, S.J.M., Boots, M., 2016. Deformed wing virus is a recent global epidemic in honeybees driven by Varroa mites. Science 351 (6273), 594–597. Yañez, O., Zheng, H.Q., Su, X.L., Hu, F.L., Neumann, P., Dietemann, V., 2015. Potential for virus transfer between the honey bees Apis mellifera and A. cerana. J. Apic. Res. 54 (3), 179–191.
Ai, H., Yan, X., Han, R., 2012. Occurrence and prevalence of seven bee viruses in Apis mellifera and Apis cerana apiaries in China. J. Invertebr. Pathol. 109 (1), 160–164. Benjeddou, M., Leat, N., Allsopp, M., Davison, S., 2001. Detection of acute bee paralysis virus and black queen cell virus from honeybees by reverse transcriptase PCR. Appl. Environ. Microbiol. 67 (5), 2384–2387. Chanpanitkitchote, P., Chen, Y., Evans, J.D., Li, W., Li, J., Hamilton, M., Chantawannakul, P., 2018. Acute bee paralysis virus occurs in the Asian honey bee Apis cerana and parasitic mite Tropilaelaps mercedesae. J. Invertebr. Pathol. 151, 131–136. Chen, Y., Zhao, Y., Hammond, J., Hsu, H., Evans, J., Feldlaufer, M., 2004. Multiple virus infections in the honey bee and genome divergence of honey bee viruses. J. Invertebr. Pathol. 87 (2-3), 84–93. Choe, S.E., Nguyen, L.T.K., Noh, J.H., Koh, H.B., Jean, Y.H., Kweon, C.H., Kang, S.W., 2012. Prevalence and distribution of six bee viruses in Korean Apis cerana populations. J. Invertebr. Pathol. 109 (3), 330–333. Clark, T.B., 1978. A filamentous virus of the honey bee. J. Invertebr. Pathol. 32 (3), 332–340. de Miranda, J.R., Cordoni, G., Budge, G., 2010. The Acute bee paralysis virus–Kashmir bee virus–Israeli acute paralysis virus complex. J. Invertebr. Pathol. 103 (Suppl.), S30–S47. Di Prisco, G., Pennacchio, F., Caprio, E., Boncristiani, H.F., Evans, J.D., Chen, Y., 2011. Varroa destructor is an effffective vector of Israeli acute paralysis virus in the honeybee, Apis mellifera. J. Gen. Virol. 92 (1), 151–155. Gauthier, L., Ravallec, M., Tournaire, M., Cousserans, F., Bergoin, M., Dainat, B., de Miranda, J.R., 2011. Viruses associated with ovarian degeneration in Apis mellifera L. queens. PLoS One 6 (1), e0016217. Genersch, E., 2010. Honey bee pathology: current threats to honey bees and beekeeping. Appl. Microbiol. Biotechnol. 87 (1), 87–97. Hartmann, U., Forsgren, E., Charrière, J.D., Neumann, P., Gauthier, L., 2015. Dynamics of Apis mellifera Filamentous Virus (AmFV) infections in honey bees and relationships with other parasites. Viruses 7 (5), 2654–2667. Hou, C., Li, B., Luo, Y., Deng, S., Diao, Q., 2016. First detection of Apis mellifera filamentous virus in Apis cerana cerana in China. J. Invertebr. Pathol. 138, 112–115. Hou, C., Li, B., Deng, S., Chu, Y., Diao, Q., 2017. Diagnosis and distribution of the Apis mellifera filamentous virus (AmFV) in honey bees (Apis mellifera) in China. Insect. Soc. 64 (4), 597–603. Kojima, Y., Toki, T., Morimoto, T., Yoshiyama, M., Kimura, K., Kadowaki, T., 2011. Infestation of Japanese native honey bees by tracheal mite and virus from non-native European honey bees in Japan. Microb. Ecol. 62 (4), 895–906. Li, J., Qin, H., Wu, J., Sadd, B.M., Wang, X., Evans, J.D., Peng, W., Chen, Y., 2012. The prevalence of parasites and pathogens in Asian honeybees Apis cerana in China. PLoS One 7 (11), e47955. Martin, S.J., Highfield, A.C., Brettell, L., Villalobos, E.M., Budge, G.E., Powell, M., Nikaido, S., Schroeder, D.C., 2012. Global honey bee viral landscape altered by a
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Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Occurrence of multiple honeybee viruses in the ectoparasitic mites Varroa spp. in Apis cerana colonies
T
Shuai Wanga, Gongwen Chena, Zheguang Linb, Yuqi Wua, Fuliang Hua, Huoqing Zhenga,
⁎
a b
College of Animal Sciences, Zhejiang University, Hangzhou 310058, China College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
ARTICLE INFO
ABSTRACT
Keywords: Honeybee viruses Varroa destructor Varroa underwoodi Apis cerana
In this study, we investigated the prevalence of honeybee viruses in Varroa destructor and Varroa underwoodi infesting Apis cerana colonies in China. Deformed wing virus (DWV) was the most prevalent virus in these two mite species, followed by Israeli acute paralysis virus (IAPV), Black queen cell virus (BQCV), Kashmir bee virus (KBV), Chronic bee paralysis virus (CBPV), Apis mellifera filamentous virus (AmFV) and Sacbrood virus (SBV) in V. destructor, while in V. underwoodi, it was followed by CBPV, AmFV, BQCV, IAPV and KBV. In addition, multiple viruses were commonly detectable in both mite species.
1. Introduction Honeybees play important roles in agriculture and natural ecosystems (Potts et al., 2016). The Western honeybee, Apis mellifera, and the Eastern honeybee, Apis cerana, are the two most important honeybee species for the beekeeping industry and share many common foraging habitats in China (Li et al., 2012). Honeybees are vulnerable to a great diversity of pathogens and parasites (Genersch, 2010) and, as reported so far, the viruses are considered to be one of the major risk factors for bee health (Tantillo et al., 2015). More than 30 honeybee viruses have been identified worldwide (Remnant et al., 2017), eight of which are widely detected in honeybee colonies. These include deformed wing virus (DWV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), Israeli acute paralysis virus (IAPV), sacbrood virus (SBV), acute bee paralysis virus (ABPV) and Varroa destructor virus-1 (VDV-1). Compared to RNA viruses, fewer studies on DNA viruses have been conducted in honeybees. A DNA virus, Apis mellifera filamentous virus (AmFV), was first reported in USA (Clark 1978) and was related to the loss of Swiss honeybee colonies (Hartmann et al., 2015). Recently, it was also detected in A. cerana and A. mellifera colonies in China (Hou et al., 2016, 2017). An ectoparasitic mite of honeybees, Varroa destructor, shifted host from A. cerana to A. mellifera with catastrophic consequences globally. In addition, Varroa underwoodi is widely distributed in Asian A. cerana colonies and is a potential threat to A. mellifera (Wang et al., 2019). V. destructor has been shown to be an effective vector of honeybee viruses
⁎
in A. mellifera colonies (de Miranda et al., 2010; Di Prisco et al., 2011); for example, DWV infection is tightly associated with V. destructor infestation on A. mellifera (Wilfert et al., 2016). However, little is known about the effects of Varroa spp. on their original hosts, A. cerana, especially concerning the potential synergistic effects of virus infection and Varroa spp. infestation. In this study, we conducted the first investigation on the occurrence of DNA and RNA viruses in V. destructor and V. underwoodi mites collected from A. cerana colonies. 2. Materials and methods 2.1. Sample collection V. destructor samples (N = 95) were collected from six apiaries in Zhejiang Province of East China, one in Jilin Province of North China and one in Guangdong Province of South China. V. underwoodi samples (N = 34) were collected from two apiaries in Zhejiang Province, one in Jilin Province and one in Jiangxi Province of East China during March 2016–June 2019. The mites were collected from A. cerana sealed drone brood cells (Wang et al., 2019) in multiple colonies and stored separately in Eppendorf tubes with 75% EtOH at −20 °C until use. Due to the natural variations in availability of mites (Wang et al., 2019), the numbers of mites collected from each apiary were not uniform. 2.2. RNA and DNA extraction
Corresponding author. E-mail address: [email protected] (H. Zheng).
https://doi.org/10.1016/j.jip.2019.107225 Received 24 May 2019; Received in revised form 25 July 2019; Accepted 29 July 2019 Available online 29 July 2019 0022-2011/ © 2019 Elsevier Inc. All rights reserved.
Total RNA was extracted from individual adult female mites
Journal of Invertebrate Pathology 166 (2019) 107225 a Abbreviations of sampled apiaries: JL, Jilin; FY, Fuyang; YP, Yuanpu; ZP, Zhoupu; XH, Xihu; ZJU, Zhejiang University; JH, Jinhua; GZ, Guangzhou; CA, Chun’an; NC, Nanchang. JL in Jilin Province, NC in Jiangxi Province, GZ in Guangdong Province and the others in Zhejiang Province. b The detection limits of ABPV and BQCV was estimated to be about 1600 and 130 genome equivalents, respectively (Benjeddou et al., 2001); In the case of overt KBV infection, approximately 1–10 pg viral RNA can be detected (Stoltz et al., 1995); the detection limits of the other viruses were undetermined. c –, unavailable data. d 95% confidence intervals, the lower limits below zero were manually corrected to zeroes.
1 – – – 1 20.0 [–, –] 5 – – – 5 0 0 0 0 0 0 [0, 0] 1 0 0 0 1 3.5 [0, 3.9] JL 17 CA 6 JH2 4 NC 2 Total 29 %infected [95% CI] V. underwoodi
0 0 0 0 0 0 [0, 0]
2 3 0 0 5 17.3 [0, 38.5]
4 2 0 0 6 20.7 [0, 30.7]
11 2 4 1 18 62.1 [34.6, 89.4]
3 0 2 0 5 17.2 [0, 40.5]
0 0 0 0 0 0 [0, 0]
0 – – 1 0 – 0 0 1 4.3 [0, 11.9] 6 –c – 5 3 – 6 3 23 0 0 0 0 0 0 0 – 0 0 [0, 0] 0 1 3 5 0 2 0 – 11 15.3 [2.1, 25.9] JL 6 FY 13 YP 11 ZP 21 XH 6 ZJU 5 JH1 10 GZ – Total 72 %infected [95% CI]d V. destructor
0 0 0 0 0 0 0 – 0 0 [0, 0]
1 3 5 4 1 2 8 – 24 33.3 [17.0, 51.0]
1 2 1 3 1 0 0 – 8 11.1 [4.8, 15.2]
3 6 11 16 6 5 7 – 54 75.0 [59.2, 94.8]
1 6 3 6 2 4 7 – 29 40.3 [25.2, 60.8]
0 1 0 1 0 0 0 – 2 2.8 [0, 4.2]
No. of mites KBV IAPV DWV CBPV BQCV ABPVb No. of mites
RNA viruses Apiariesa Species
Table 1 Detection of RNA and DNA viruses in V. destructor and V. underwoodi mites from A. cerana colonies.
SBV
VDV-1
DNA virus
AmFV
S. Wang, et al.
according to the manufacturer’s protocol (Aidlab, China). For brood cells that were infested with more than one mite foundress, only one adult female mite was used. Total DNA was extracted from different individual adult female mites following the manufacturer’s protocol (Tiangen, China). RNA and DNA samples were stored at −80 °C until use. 2.3. RT-PCR and sequencing RNA samples were used for the detection of eight bee viruses by qualitative RT-PCR using specific PCR primers from previous reports: ABPV and BQCV (Benjeddou et al., 2001), CBPV (Ribière et al., 2002), DWV and KBV (Tentcheva et al., 2004), IAPV (Maori et al., 2007), SBV (Chen et al., 2004) and VDV-1 (Gauthier et al., 2011). cDNA synthesis and amplification were performed using the manufacturer’s recommended protocol (TOYOBO, Japan). DNA samples were checked for the presence of AmFV with the specific primer (Hartmann et al., 2015). Normal PCR (40 cycles) and gel electrophoresis were performed as described previously (Wang et al., 2019). Positive PCR products were sequenced by the ABI 3730XL automatic sequencing system (Sangon, China). The sequence of each virus fragment was confirmed using the BLAST server at the NCBI website (https://blast.ncbi.nlm.nih.gov/). Part of the sequences (N = 77) were uploaded to GenBank (https:// www.ncbi.nlm.nih.gov/genbank/) with accession numbers of MN114541 - MN114617. 3. Results and discussion Seven RNA viruses (ABPV, BQCV, CBPV, DWV, IAPV, KBV and SBV) were previously detected in A. cerana colonies in Asia (Kojima et al., 2011; Ai et al., 2012; Choe et al., 2012; Li et al., 2012; Yañez et al., 2015; Thu et al., 2016; Chanpanitkitchote et al., 2018). We found that six of the viruses (except ABPV) were detected in V. destructor and five (all but ABPV and SBV) were detected in V. underwoodi infesting A. cerana colonies (Table 1). VDV-1 was not found in either mite species. BQCV was detected in V. destructor from all apiaries while DWV was detected in both V. destructor and V. underwoodi from all apiaries (Table 1). DWV is one of the most prevalent viruses in honeybees in Asia (Kojima et al., 2011; Ai et al., 2012; Li et al., 2012; Yañez et al., 2015), and its prevalence was tightly linked to the infestation of V. destructor in A. mellifera colonies (Martin et al., 2012; Wilfert et al., 2016). Here we showed that DWV was the most prevalent virus in both V. destructor (75.0%; 95% confidence intervals (CI), [59.2%, 94.8%]) and V. underwoodi (62.1%; 95% CI, [34.6%, 89.4%]) samples collected from A. cerana colonies, suggesting an association between DWV and both V. destructor and V. underwoodi in A. cerana colonies. IAPV infects A. mellifera, A. cerana, Bombus terrestris and V. destructor in A. mellifera colonies (Maori et al., 2007; Singh et al., 2010; Di Prisco et al., 2011; Ai et al., 2012; Niu et al., 2014). Our results showed that IAPV was the second most prevalent virus in V. destructor (40.3%; 95% CI, [25.2%, 60.8%]) and the fourth most prevalent in V. underwoodi (17.2%; 95% CI, [0, 40.5%]) in A. cerana colonies. CBPV rarely has been detected in A. cerana (0–7%, Ai et al., 2012; Li et al., 2012; Yañez et al., 2015) or in A. mellifera (0–9%, Ai et al., 2012; Peng et al., 2015; Yañez et al., 2015) in China. Interestingly, it occurred in V. destructor and V. underwoodi mites of A. cerana colonies at a relatively high level (11.1%; 95% CI, [4.8%, 15.2%] and 20.7%; 95% CI, [0, 30.7%], respectively). BQCV was detected in A. cerana colonies in Japan, Korea, China and Vietnam (Kojima et al., 2011; Choe et al., 2012; Ai et al., 2012; Li et al., 2012; Yañez et al., 2015; Thu et al., 2016). We identified BQCV as the third most prevalent virus in both Varroa mites (V. destructor: 33.3%; 95% CI, [17.0%, 51.0%] and V. underwoodi: 17.3%; 95% CI, [0, 38.5%]) in A. cerana colonies. KBV was not reported in China previously (Ai et al., 2012; Yañez et al., 2015); however, our results revealed its presence in both V. destructor (15.3%; 95% CI, [2.1%, 25.9%]) and V. underwoodi 2
Journal of Invertebrate Pathology 166 (2019) 107225
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Table 2 Frequencies of multiple RNA viral infections in Varroa spp. samples. Species
No. of viruses
V. destructor
0 1 2
3
4
V. underwoodi
0 1
2
3
Type of infection
No. of mites
Rate (%)
Origina
BQCV DWV IAPV BQCV, SBV BQCV, DWV CBPV, DWV DWV, IAPV DWV, KBV IAPV, KBV BQCV, CBPV, DWV BQCV, CBPV, IAPV BQCV, DWV, IAPV DWV, IAPV, KBV BQCV, DWV, IAPV, KBV CBPV, DWV, IAPV, KBV CBPV, DWV, IAPV, SBV
8 5 17 1 1 4 3 10 3 1 2 1 8 3 3 1 1
11.1 6.9 23.6 1.4 1.4 5.6 4.2 13.9 4.2 1.4 2.8 1.4 11.1 4.2 4.2 1.4 1.4
2JL, 4FY, 2ZP 1FY, 3JH, 1ZP 2JL, 2FY, 3YP, 3XH, 6ZP, 1ZJU 1FY 1ZP 3YP, 1ZP 1YP, 2ZP 1JL, 1FY, 2XH, 2ZP, 2ZJU, 2JH 1YP, 2ZP 1ZP 1JL, 1XH 1FY 1FY, 1YP, 1ZP, 5JH 1FY, 1YP, 1ZP 1YP, 2ZJU 1ZP 1FY
CBPV DWV IAPV KBV BQCV, CBPV BQCV, DWV CBPV, DWV CBPV, IAPV DWV, IAPV BQCV, CBPV, DWV
5 2 10 1 1 1 3 1 1 3 1
17.2 6.9 34.5 3.4 3.4 3.4 10.3 3.4 3.4 10.3 3.4
2JL, 1JL, 7JL, 1JL 1JL 1CA 1JL, 1JL 1JL 1JL, 1JL
1NC, 2CA 1CA 2JH, 1NC
2CA 2JH
a Abbreviations of sampled apiaries: FY, Fuyang; YP, Yuanpu; ZP, Zhoupu; XH, Xihu; ZJU, Zhejiang University; JH, Jinhua; JL, Jilin; CA, Chun’an; NC, Nanchang. The numbers of infested mites were indicated before the apiary locations.
mites, as well as to show if they are transmitted to the workers and if they have negative effects on the colonies.
(3.5%; 95% CI, [0, 3.9%]), suggesting that this virus may present in A. cerana in China and deserves further researches, both on detection of the virus in A. cerana and on the relationships between KBV and Varroa spp. in A. cerana colonies. To our surprise, the prevalence of SBV was quite low in V. destructor (2.8%; 95% CI, [0, 4.2%]) and was not detectable in V. underwoodi despite being the most prevalent virus in A. cerana populations in China, Korea and Vietnam (Ai et al., 2012; Choe et al., 2012; Thu et al., 2016). It is possible that SBV primarily affects honeybee larvae and even causes mortality (Ai et al., 2012), reducing the potential for transmission to mites. Alternatively, but not mutually exclusive, the low prevalence of this virus in mites could be due to its inability to replicate in the mites. This is in accordance with the study on V. destructor mites collected from A. mellifera colonies, which revealed that the prevalence of SBV in the parasites was lower than that in their honeybee hosts (Tentcheva et al., 2004). AmFV, a DNA virus, was confirmed to be widely present in A. cerana and A. mellifera populations in China (Hou et al., 2016, 2017). Although the prevalence was low in our study, we showed that V. destructor and V. underwoodi mites could be potential hosts of the DNA virus in A. cerana colonies. Multiple RNA viruses were detected in our Varroa spp. samples and were common in both mite species (Table 2). In V. destructor, 30.7% of samples were infected with two viruses, 19.5% with three viruses, and 7.0% with four viruses. Multiple infections with two viruses and three viruses were 30.8% and 3.4%, respectively, in V. underwoodi. In both mite species, the most common dual infection of RNA viruses was DWV and IAPV. Owing to the low infestation rates of Varroa spp. in A. cerana colonies (Wang et al., 2019), the sample sizes were limited, especially for V. underwoodi, which may cause bias in calculating the prevalence of the tested viruses. Further studies with more samples are needed to provide more precise results. Moreover, it is important to quantify the detected viruses and to determine whether they replicate in the Varroa
4. Conclusions We investigated the presence of the eight RNA viruses and one DNA honeybee virus in Varroa spp. collected from A. cerana colonies. DWV, IAPV, BQCV, KBV, CBPV, SBV and AmFV were detected in V. destructor and DWV, CBPV, AmFV, BQCV, IAPV and KBV were detected in V. underwoodi. The high occurrence of honeybee viruses in the two species of Varroa mites suggested that the mites, despite a low infestation rate in A. cerana colonies, act as vectors of honeybee viruses and may affect the health of A. cerana. Consequently, the relationships/interactions between the Varroa spp. mites and honeybee viruses in A. cerana colonies deserve further studies, which may shed new light on hostparasite-virus interactions. Acknowledgements The work was supported by National Natural Science Foundation of China (31672498, H.Z.), Science and Technology Department of Zhejiang Province, China (2016C02054-11, F.H.) and the Modern Agroindustry Technology Research System of China (CARS-44, F.H. and H.Z.). Declaration of Competing Interest None. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jip.2019.107225.
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Journal of Invertebrate Pathology 166 (2019) 107225
References
parasitic mite. Science 336 (6086), 1304–1306. Maori, E., Lavi, S., Mozes-Koch, R., Gantman, Y., Peretz, Y., Edelbaum, O., Tanne, E., Sela, I., 2007. Isolation and characterization of Israeli acute paralysis virus, a dicistrovirus affecting honeybees in Israel: evidence for diversity due to intra and inter-species recombination. J. Gen. Virol. 88 (12), 3428–3438. Niu, J., Cappelle, K., de Miranda, J.R., Smagghe, G., Meeus, I., 2014. Analysis of reference gene stability after Israeli acute paralysis virus infection in bumblebees Bombus terrestris. J. Invertebr. Pathol. 115, 76–79. Peng, W., Li, J., Zhao, Y., Cen, Y., Zeng, Z., 2015. A descriptive study of the prevalence of parasites and pathogens in Chinese black honeybees. Parasitology 142 (11), 1364–1374. Potts, S.G., Imperatriz-Fonseca, V., Ngo, H.T., Aizen, M.A., Biesmeijer, J.C., Breeze, T.D., Dicks, L.V., Garibaldi, L.A., Hill, R., Settele, J., Vanbergen, A.J., 2016. Safeguarding pollinators and their values to human well-being. Nature 540 (7632), 220–229. Remnant, E.J., Shi, M., Buchmann, G., Blacquière, T., Holmes, E.C., Beekman, M., Ashe, A., 2017. A diverse range of novel RNA viruses in geographically distinct honey bee populations. J. Virol. 91 (16), e00158–e217. Ribière, M., Triboulot, C., Mathieu, L., Aurières, C., Faucon, J.-P., Pépin, M., 2002. Molecular diagnosis of chronic bee paralysis virus infection. Apidologie 33 (3), 339–351. Singh, R., Levitt, A.L., Rajotte, E.G., Holmes, E.C., Ostiguy, N., vanEngelsdorp, D., Lipkin, W.I., dePamphilis, C.W., Toth, A.L., Cox-Foster, D.L., 2010. RNA viruses in hymenopteran pollinators: evidence of inter-taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species. PLoS One 5 (12), e14357. Stoltz, D., Shen, X.R., Boggis, C., Sisson, G., 1995. Molecular diagnosis of Kashmir bee virus infection. J. Apic. Res. 34 (3), 153–160. Tantillo, G., Bottaro, M., Di Pinto, A., Martella, V., Di Pinto, P., Terio, V., 2015. Virus infections of honeybees Apis mellifera. Italian J. Food Saf. 4 (3), 5364. Tentcheva, D., Gauthier, L., Zappulla, N., Dainat, B., Cousserans, F., Colin, M.E., Bergoin, M., 2004. Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France. Appl. Environ. Microbiol. 70 (12), 7185–7191. Thu, H.T., Thi Kim Lien, N., Thuy Linh, M., Le, T.H., Hoa, N.T., Hong Thai, P., Reddy, K.E., Yoo, M.S., Kim, Y.H., Cho, Y.S., Kang, S.W., Quyen, D.V., 2016. Prevalence of bee viruses among Apis cerana populations in Vietnam. J. Apic. Res. 55 (5), 379–385. Wang, S., Lin, Z., Dietemann, V., Neumann, P., Wu, Y., Hu, F., Zheng, H., 2019. Ectoparasitic Mites Varroa underwoodi (Acarina: Varroidae) in Eastern Honeybees, but not in Western Honeybees. J. Econ. Entomol. 112 (1), 25–32. Wilfert, L., Long, G., Leggett, H.C., Schmid-Hempel, P., Butlin, R., Martin, S.J.M., Boots, M., 2016. Deformed wing virus is a recent global epidemic in honeybees driven by Varroa mites. Science 351 (6273), 594–597. Yañez, O., Zheng, H.Q., Su, X.L., Hu, F.L., Neumann, P., Dietemann, V., 2015. Potential for virus transfer between the honey bees Apis mellifera and A. cerana. J. Apic. Res. 54 (3), 179–191.
Ai, H., Yan, X., Han, R., 2012. Occurrence and prevalence of seven bee viruses in Apis mellifera and Apis cerana apiaries in China. J. Invertebr. Pathol. 109 (1), 160–164. Benjeddou, M., Leat, N., Allsopp, M., Davison, S., 2001. Detection of acute bee paralysis virus and black queen cell virus from honeybees by reverse transcriptase PCR. Appl. Environ. Microbiol. 67 (5), 2384–2387. Chanpanitkitchote, P., Chen, Y., Evans, J.D., Li, W., Li, J., Hamilton, M., Chantawannakul, P., 2018. Acute bee paralysis virus occurs in the Asian honey bee Apis cerana and parasitic mite Tropilaelaps mercedesae. J. Invertebr. Pathol. 151, 131–136. Chen, Y., Zhao, Y., Hammond, J., Hsu, H., Evans, J., Feldlaufer, M., 2004. Multiple virus infections in the honey bee and genome divergence of honey bee viruses. J. Invertebr. Pathol. 87 (2-3), 84–93. Choe, S.E., Nguyen, L.T.K., Noh, J.H., Koh, H.B., Jean, Y.H., Kweon, C.H., Kang, S.W., 2012. Prevalence and distribution of six bee viruses in Korean Apis cerana populations. J. Invertebr. Pathol. 109 (3), 330–333. Clark, T.B., 1978. A filamentous virus of the honey bee. J. Invertebr. Pathol. 32 (3), 332–340. de Miranda, J.R., Cordoni, G., Budge, G., 2010. The Acute bee paralysis virus–Kashmir bee virus–Israeli acute paralysis virus complex. J. Invertebr. Pathol. 103 (Suppl.), S30–S47. Di Prisco, G., Pennacchio, F., Caprio, E., Boncristiani, H.F., Evans, J.D., Chen, Y., 2011. Varroa destructor is an effffective vector of Israeli acute paralysis virus in the honeybee, Apis mellifera. J. Gen. Virol. 92 (1), 151–155. Gauthier, L., Ravallec, M., Tournaire, M., Cousserans, F., Bergoin, M., Dainat, B., de Miranda, J.R., 2011. Viruses associated with ovarian degeneration in Apis mellifera L. queens. PLoS One 6 (1), e0016217. Genersch, E., 2010. Honey bee pathology: current threats to honey bees and beekeeping. Appl. Microbiol. Biotechnol. 87 (1), 87–97. Hartmann, U., Forsgren, E., Charrière, J.D., Neumann, P., Gauthier, L., 2015. Dynamics of Apis mellifera Filamentous Virus (AmFV) infections in honey bees and relationships with other parasites. Viruses 7 (5), 2654–2667. Hou, C., Li, B., Luo, Y., Deng, S., Diao, Q., 2016. First detection of Apis mellifera filamentous virus in Apis cerana cerana in China. J. Invertebr. Pathol. 138, 112–115. Hou, C., Li, B., Deng, S., Chu, Y., Diao, Q., 2017. Diagnosis and distribution of the Apis mellifera filamentous virus (AmFV) in honey bees (Apis mellifera) in China. Insect. Soc. 64 (4), 597–603. Kojima, Y., Toki, T., Morimoto, T., Yoshiyama, M., Kimura, K., Kadowaki, T., 2011. Infestation of Japanese native honey bees by tracheal mite and virus from non-native European honey bees in Japan. Microb. Ecol. 62 (4), 895–906. Li, J., Qin, H., Wu, J., Sadd, B.M., Wang, X., Evans, J.D., Peng, W., Chen, Y., 2012. The prevalence of parasites and pathogens in Asian honeybees Apis cerana in China. PLoS One 7 (11), e47955. Martin, S.J., Highfield, A.C., Brettell, L., Villalobos, E.M., Budge, G.E., Powell, M., Nikaido, S., Schroeder, D.C., 2012. Global honey bee viral landscape altered by a
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