First Detection of Honey Bee Viruses in Wax Moth

Chiang Mai J. Sci. 2016; 43(4) 695

Chiang Mai J. Sci. 2016; 43(4) : 695-698 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

First Detection of Honey Bee Viruses in Wax Moth Prapun Traiyasut [a], Wannapha Mookhploy [a], Kiyoshi Kimura [b], Mikio Yoshiyama [b], Kitiphong Khongphinitbunjong [a] and Panuwan Chantawannakul* [a] [a] Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. [b] Honeybee Research Unit, Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization (NARO), 2 Ikeno-dai, Tsukuba, Ibaraki, 305-0901, Japan. *Author for correspondence; e-mail: [email protected]

Received: 15 January 2015 Accepted: 21 September 2015

ABSTRACT Larvae of greater wax moth (Galleria mellonella) attacking the colonies of Apis cerana japonica were collected from Chiba Prefecture, Japan on December 25, 2012. By RT-PCR method, samples were tested positive for Israeli Acute Paralysis Virus (IAPV) and Black Queen Cell Virus (BQCV). These viruses were observed in both worker bees and the wax moth larvae infesting the colonies, although with a lower frequency in the worker bees. The IAPV isolates showed 97% sequence similarity to isolates from China and the BQCV isolates showed 99% sequence similarity to isolates from Korea. This is the first report of the detection of honey bee viruses in wax moth larvae infesting A. cerana japonica.

Keywords: honey bee viruses, wax moth, Apis cerana japonica, RT-PCR

1. INTRODUCTION Moth larvae attack the combs of honey honey bee viruses have an association with bees worldwide. The greater wax moth, Varroa mites, ectoparasites of honey bee, Galleria mellonella and the lesser wax moth, which act as biological vector [9]. The aim of Achroia gisella are small nondescript moths that our study was to survey the presence of honey lay eggs in cracks and crevices in bee hives bee viruses in other vectors to Apis cerana. [1]. The moths eat the comb, pollen and brood and can quickly destroy the combs. At least 2. MATERIALS AND METHODS 18 viruses that infect honey bees have been In this study, 10 larvae of G. mellonella described. These viruses have been identified and 10 worker bees of A.c. japonica were from different stages of honey bees, including: collected from Chiba Prefecture, Japan on eggs, larvae, pupae, adult workers, drones and December 25, 2012. The larvae of G. mellonella queens [2]. In general, virus transmission can were collected from a single honey bee colony occur horizontally (e.g. food and/or vectors) in an apiary of semi-feral A.c. japonica and/or vertically (viruses pass from mother colonies. Total RNA of each G. mellonella to offspring) [6]. Moreover, the majority of larvae was extracted individually using an 696 Chiang Mai J. Sci. 2016; 43(4)

RNeasy Kit (QIAGEN, USA), following the The virus nucleotide sequences were analyzed manufacturer’s instructions. Total RNA (1μg) using BLAST search, NCBI website. was used for reverse transcription reaction by using first strand cDNA synthesis (Invitrogen, 3. RESULTS Germany) to synthesize cDNA, following The results showed that worker bees the manufacturer’s instructions. The reverse (3/10) and the wax moth larvae (5/10) in the transcription products were then used bee colony were both found with IAPV. for PCR with KOD FX DNA polymerase BQCV was found in one only sample (out (TOYOBO, Japan). PCR amplification was of 10 each) of worker bee and wax moth performed with the specific primer for larvae (Figure 1). The IPAV and BQCV Acute bee paralysis virus (ABPV), Kashmir sequences derived from both worker bees bee virus (KBV), BQCV, Deformed wing and wax moth larvae were identical. The virus virus (DWV), Sacbrood virus (SBV) [10], nucleotide sequences found both in the Chronic bee paralysis virus (CBPV) [13] and wax moths and the bees are similar. The IAPV [7]. The thermal cycling conditions were sequence of IAPV (GenBank Accession as follows: one cycle of initial denaturation at No. KT717337) from this study showed 97% 94 °C for 2 min; 40 cycles of denaturation at similarity to an isolate from China (GenBank 94 °C for 40 s; annealing at 55 °C for (ABPV, Accession No. HQ897161.1). BQCV positive BQCV, IAPV, SBV and DWV), 50 °C for samples (GenBank Accession No.KT717338) (KBV), 60 °C for (CBPV), all for 30 s; showed 99% similarity to BQCV from Korea and extension at 68 °C for 45 s. A negative (GenBank Accession No. JN542439.1). The control lacking template RT product was relationships between these sequences are performed for each PCR reaction. Positive represented in the phylogenetic tree, with the identification was confirmed by sequencing confidence values associated with each node the PCR products. The PCR product was based on 1,000 bootstrap replicates. The virus electrophoresed in a 1.5% agarose gel, stained sequences from those wax moth larvae with ethidium bromide and photographed and worker bee were same clade (Figure 2). under UV light. The PCR products were We did not find any samples of honey bees purified with QIAEX®II Gel Extraction or wax moth larvae infected with ABPV, Kit (150) (Qiagen) and sequenced directly. DWV, KBV, SBV or CBPV.

Figure 1. Electrophoresis PCR products of honey bee viruses in wax moth larvae (Galleria mellonella) and workers of Apis cerana japonica of the same colony. MW: 1kb DNA ladder (Invitrogen). Lane 1-3: Positive amplification IAPV of worker bees (400 bp). Lane 4-8: Positive amplification IAPV of wax moth larvae (400 bp). Lane 9: Positive amplification of BQCV in a worker bee (700 bp). Lane 10: Positive amplification of BQCV in wax moth larvae (700 bp). Chiang Mai J. Sci. 2016; 43(4) 697

Figure 2. Phylogram of the IAPV isolated based on RNA-dependent RNA polymerase (RdRp) sequences constructed using the Neighbor-Joining method in MEGA5 [11]. The statistical support of the nodes shows as the percentage of correct partition in a 1,000-replicate bootstrap analysis.

4. DISCUSSION AND CONCLUSION (Bombus terrestris and B. pascuorum) [8], KBV In our case, the honey bee virus may have has been reported to infect the German wasp present in the wax moth larvae via shared (Vespa germanica) [3], ABPV has been reported food source / brood consumption in the bee in many Bombus spp. [4] in Britain and Meeus hive. Singh (2010) [14] reported that RNA (2010) [12] detected ABPV, KBV and DWV viruses could be transmitted via pollen or bee in bumble bees in Europe. In addition to bread and infect Hymenopteran pollinators. hymenoptera, CBPV has been reported in They also demonstrated that viruses could be two species of ants (Camponotus vagus and transmitted from honey bee to bumble bee Formica rufa) in France [5]. These results report and from infected bumble bee to honey bee. not only the interspecific transmission of Our results showed that IAPV and BQCV BQCV and IAPV, but also a potential route - are present in the wax moth larvae and orally horizontal transmission - of viral correlated with the worker bees in the colony. infection. Eventually, the viruses may be Kojuma (2011) [10] reported the presence of infected to other hosts than honey bee (e.g. four honey bee viruses (DWV, BQCV, IAPV wax moth larvae), which contain honey bee and SBV) infecting A. c. japonica colonies in in its food chain. Japan, but not in the wax moth. Our observation is the first to demonstrate the ACKNOWLEDGEMENTS evidence of honey bee viruses in host other We acknowledge Chiang Mai University than honey bees. It appears that the honey bee for financial support, JSPS for bilateral project viruses are not restricted to the honey bee, and the Royal Golden Jubilee Ph.D. Program, but have a broader host range, for example, the Thailand Research Fund for financial DWV has also been detected in bumble bees support to Prapun Traiyasut. We would like 698 Chiang Mai J. Sci. 2016; 43(4)

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