- Email: [email protected]
The prevalence and molecular characterization of Acarapis woodi and Varroa destructor mites in honeybees in the Tohoku region of Japan
Journal Pre-proof The prevalence and molecular characterization of Acarapis woodi and Varroa destructor mites in honeybees in the Tohoku region of Japan
Shunsuke Takashima, Yuma Ohari, Tadashi Itagaki PII:
S1383-5769(20)30002-7
DOI:
https://doi.org/10.1016/j.parint.2020.102052
Reference:
PARINT 102052
To appear in:
Parasitology International
Received date:
10 December 2019
Revised date:
27 December 2019
Accepted date:
6 January 2020
Please cite this article as: S. Takashima, Y. Ohari and T. Itagaki, The prevalence and molecular characterization of Acarapis woodi and Varroa destructor mites in honeybees in the Tohoku region of Japan, Parasitology International(2020), https://doi.org/10.1016/ j.parint.2020.102052
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
The prevalence and molecular characterization of Acarapis woodi and Varroa destructor mites in honeybees in the Tohoku region of Japan
Shunsuke Takashima, Yuma Ohari, Tadashi Itagaki*
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Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8
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Ueda, Morioka 020-8550, Japan.
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* Corresponding author:
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Tadashi Itagaki Tel./Fax: +81-19-621-6219
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Abstract
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E-mail: [email protected]
Honeybee acarapiosis and vorrosis were designated as Notifiable Infectious Diseases in the Act on Domestic Animal Infectious Diseases Control by the Minister of Agriculture, Forestry and Fisheries of Japan in 1997. However, the prevalences of A. woodi and V. destructor in Japan, especially in the Tohoku region, have not been sufficiently elucidated. This study was designed to clarify the prevalence of A. woodi and V. destructor mites in Apis cerana japonica and Apis mellifera in the Tohoku region and the characteristics of their mitochondrial cytochrome c oxidase I (COI) DNA. Acarapis woodi mites were detected from 13.5% of A. c. japonica and 0% of A. mellifera. Aomori prefecture, Japan is a new distribution locality for A. woodi. None of
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the honeybees examined showed infection by V. destructor mites. The COI sequences (1638 bp) of A. woodi were identical and phylogenetically closely related to those of A. woodi from Japan and the UK, suggesting that the mite would have been introduced into Japan with A. mellifera during the last 150 years and spread throughout the country.
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Keywords
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Acarapis woodi, Varroa destructor, COI, honeybees, Japan, prevalence
1. Introduction
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Acarapiosis is a disease caused by Acarapis woodi mites parasitizing the tracheae of
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Apis honeybees. Adult honeybees infected with these mites show a reduction in the ability to fly due to tracheal obstruction and damaged flight muscles [1, 2], which force
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them to walk around their hives [3]. Heavy infestation of the honeybee colony results in
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hive collapse and disappearance [3, 4]. Acarapis woodi infection was first reported in
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Apis mellifera from the United Kingdom in 1921 [5] and has since been reported throughout the world (excluding Australia, New Zealand and Scandinavia n Peninsula [6, 7]. Varroa destructor mites parasitize the body surface of honeybees, mainly their larvae and pupa, and deform the legs and wings of adult bees. These mites are also vectors of deformed wings viruses [8]. In Japan, honeybee acarapiosis and vorrosis were designated as Notifiable Infectious Diseases in the Act on Domestic Animal Infectious Diseases Control by the Minister of Agriculture, Forestry and Fisheries in 1997. However, the prevalences of A. woodi and V. destructor in Japan, especially in the Tohoku region, have not been sufficiently elucidated [9-11]. Further, the reports on molecular characteristics of these mites are
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limited [9, 12, 13]. This study was designed to clarify the prevalence of A. woodi and V. destructor mites in the Tohoku region and the characteristics of their mitochondrial cytochrome c oxidase I (COI) DNA.
2. Materials and Methods
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2.1 Honeybee samples Honeybees, A. c. japonica and A. mellifera, were collected from Aomori, Iwate,
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Akita, Miyagi, Fukushima and Yamagata prefectures of Japan in the period from March 2018 to June 2019 (Fig.1 and Fig. 2). For A. c. japonica, 386 honeybees from 18
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colonies of eight apiaries, 89 honeybees from six wild hives, and 109 flower-visiting
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honeybees from 15 localities were sampled. In A. mellifera, 40 honeybees from two colonies of two apiaries and 130 flower-visiting honeybees from 13 localities were
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sampled. The honeybee samples were preserved at -30℃ until analysis.
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2.2 Detection of mites and total DNA extraction
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The honeybees were examined under a stereoscopic microscope for detecting the presence of V. destructor mites on their surface; they were then dissected using forceps, and their tracheae were examined for A. woodi mites [14]. The detected mites were cleared by Gater solution [15] and morphologically identified [16]. Total DNA was extracted from the tracheae of 30 honeybees infected with A. woodi mites using High Pure PCR Template Kit (Roche Diagnostics, Indianapolis, USA). 2.3 DNA analysis For determining partial COI nucleotide sequences (441 bp), PCR was carried out in a total volume of 10 μL including 0.2 μL of Tks Gflex DNA Polymerase (Takara, Shiga, Japan), 5 μL of 2 × Gflex buffer, 0.2 μL of the primer #1F and R [12], 3.9 μL of MQ
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water and 0.5 μL of DNA template. The thermocycler program was set as follows: 94 0 C for 60 s, followed by 40 cycles of 980 C for 10 s, 550 C for 15 s and 680 C for 30 s. The amplicon was purified using NucleoSpin Gel and PCR Clean-up (Takara) and directly sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) and ABIPRISM 3500 Genetic Analyzer (Applied Biosystems).
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The amplicons of approximately complete length of mitochondrial DNA were obtained from seven honeybees. PCR was performed using the same reaction mixture as that for COI amplicon described above, except for using Aw-mito1F and mito1R
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primers (Table 1). The thermal conditions were as follows: 940 C for 60 s, followed by
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25 cycles of 980 C for 10 s, 600 C for 15 s and 680 C for 7 min. The amplicon was purified
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and directly sequenced using the primers, Aw-mito F2 and R2 and Aw-mito F3 and R3 (Table 1), and BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems),
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The obtained sequences (approximately 2,800 bp) were compared to complete
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mitochondrial DNA sequences of Tetranychus urticae (EU345430) and Tetranychus
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truncatus (MG518337) belonging to Prostigmata. The COI sequences (677 bp) forming a part of the sequences were assembled using ATGC ver. 6.0.3 (Genetyx, Tokyo, Japan) and aligned with reference COI sequences of A. woodi (AB634838 and HQ162656), A. dorsalis (HQ162658) and A. externus (HQ162662). A phylogenetic tree was constructed based on T92+G model using the Maximum Likelihood method of MEGA 6.0 software [17]. The nucleotide sites with base deletion were eliminated from the sequences for phylogenetic analysis. Bootstrap support was calculated using 1,000 replicates.
3. Results 3.1 Mite detection
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None of the honeybees examined showed infection by V. destructor mites. In A. c. japonica, mites were detected in the tracheas of 79 honeybees (13.5%), which included 68 honeybees (17.6%) from five apiaries (62.5 %, the locality numbers 4, 6 and 24-26 in Table 2) in Aomori and Iwate, 10 honeybees (11.2%) from 2 wild hives (33.3%, the locality numbers 22 and 23) in Iwate, and one flower-visiting
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honeybee (0.92%) from one locality (6.3%, the locality number 3) in Aomori (Table 2 and Fig. 1). In the apiaries where the mites were found, most (10/13) of the colonies
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examined were positive for tracheal mites. No mites were detected in the tracheae of A. mellifera (Table 3 and Fig. 2).
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The female mite detected had the short and stubby leg IV with five setae. The
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posterior margin of coxal plate IV was distinctively notched but not bilobed. The anterior median apodeme was not developed posteriorly and not joining with the
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transverse apodeme. The male was lacking the claws on leg Ⅳ. Based on these
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3.2 COI analyses
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morphological characteristics, the mites were identified as A. woodi (Fig. 3).
The partial COI sequences (441 bp) of A. woodi obtained from 30 honeybees were identical and were also identical to those of A. woodi from honeybees from Japan (AB634838) and UK (HQ162656). The COI sequences (1,638 bp) of A. woodi obtained from seven honeybees in Aomori and Iwate (the locality numbers 3 and 22) were also completely identical and represented as the haplotype, AW-H1. The sequences determined in this study are available in GenBank under accession numbers LC510136 and LC512730.
4. Discussion
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In this study, Varroa mite infection was not found in either, A. c. japonica or A. mellifera. Varroa mites were also rarely found in A. c. japonica [11, 18]. Adult A. cerana bees frequently groom their body surface and maintain hygiene in their hives, and this behavior seems to be useful towards removing mites. The Varroa-infected and deformed adult bees cannot fly from the hives to visit flowers for collecting nectar [19,
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20]. In this study, A. mellifera honeybees examined were mostly flower-visiting, which may be one of the reasons why V. destructor mites were not detected from A. mellifera
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in this study.
In this study, A. woodi mites were found in 79 honeybees of A. c. japonica from
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apiaries, wild hives and flower-visiting in Aomori and Iwate prefectures of Tohoku
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region, Japan. Acarapis woodi has been detected in A. c. japonica honeybees in some prefectures including Iwate, Akita and Miyagi of Tohoku [10, 14]. However, Aomori
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prefecture is a new distribution locality for A. woodi. The detection rate of A. woodi was
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the highest (62.5 %, 5/8 apiaries) in honeybees from apiaries, although honeybees were
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collected from apiaries, wild hives, and wild flowers. Mites parasitize the tracheae and air sacs of adult honeybees. Sexually mature female mites mate in the trachea of honeybees and move outside to invade other honeybees [21, 22]. Horizontal transmission of the mite seems to occur from an infected to a free colony when the infected mites accidentally returned to another colony of the apiary [23]. Transmission experiments revealed that A. woodi infection was transferred to the mite - free colony from the infected colony after 10 months of the commencement [24]. These might explain the high prevalence of A. woodi in apiaries. On the other hand, the prevalence was very low (6.7%, 1/15 localities) in flower-visiting bees. The infected hives might not occur near the flower - visiting localities because honeybees commonly collect
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flower nectar around 2 - 3 km distance from their hives [25]. The other probable explanation is the reduced flight ability of the infected honeybees, since it has been reported that infected honeybees suffer from tracheal obstruction and their flight muscles are damaged [1, 2]. The mitochondrial COI sequences of A. woodi obtained from Aomori and Iwate
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were completely identical, indicating that the A. woodi isolates are genetically very close each other. Furthermore, the results of phylogenetic tree indicated that the present
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isolates are also closely related to A. woodi detected from A. c. japonica in Japan (AB634838) and from A. mellifera in UK (HQ162656). Acarapis woodi was first
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reported in 1921 in the UK [5] and expanded throughout the world, except for Australia,
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New Zealand, and the Scandinavian Peninsula [6, 7]. Apis mellifera was first introduced to Japan in 1877 [26]; thereafter, it has been imported from USA, Canada, and
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Colombia, where A. woodi infection was detected [27-29]. From these findings, we
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consider that the mite would have been introduced into Japan with A. mellifera during
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the last 150 years and spread throughout the country. Cepero et al. [30] reported that genetic variants based on the COI sequence occur within A. woodi from A. mellifera honeybees in Spain, and that the variants belong to different clades in the phylogenetic tree. Therefore, the limited A. woodi isolate might be introduced into Japan. Mitogenome analyses of A. woodi mites isolated from different geographical regions including Japan are needed to elucidate the precise origin of the mite existing in Japan. Acarapis woodi mites were not detected from any honeybees of A. mellifera in this study. There are also no reports on the detection of the mite from A. mellifera in Japan in 2015 [10], although A. woodi DNA was found in A. mellifera in 2011 [9, 12]. However, the reason why no or only a very few mites occur in A. mellifera has
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remained unclear.
Acknowledgements We gratefully appreciate the members of the Japanese Original Honeybee
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Organization and apiarists for helping us to collect honeybee samples.
Declaration of Interest:
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none
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References
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[1] F.A. Eischen, D. Cardoso-Tamez, W.T. Wilson, A. Dietz, Honey production of honey bee colonies infested with Acarapis woodi (Rennie), Apidologie 20 (1989) 1 – 8.
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[2] T.P. Liu, Ultrastructure of the flight muscle of worker honey bees heavily infested by
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the tracheal mite Acarapis woodi, Apidologie 21 (1990) 537 – 540.
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[3] T. Maeda, Y. Sakamoto, K Okabe, H Taki, M. Yoshiyama, K. Goka, K. Kimura, Tracheal Mite, Acarapis woodi (Acari: Tarsonemidae), of Honey Bees: Biology, Impact on Honey Bees and Occurrence in Japan, Jpn. J. Appl. Entomol. Zool. 59 (2015) 109 – 126 (in Japanese). [4] J.B. McMullan, M.J. Brown, A qualitative model of mortality in honey bee (Apis mellifera) colonies infested with tracheal mites (Acarapis woodi), Exp. Appl. Acarol. 47 (2009) 225. [5] J. Rennie, Isle of wight disease in hive bees—acarine disease: the organism associated with the disease—Tarsonemus woodi, n. sp., Earth Environ. Sci. Trans. Royal Soc. Edinburgh 52 (1921) 768 – 779.
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[6] L. Bailey, B.V. Ball, Acarapis woodi: Parasitic mites, in: Honey Bee Pathology, 2nd ed., Academic Press, London, London, 1991, pp. 78 – 94. [7] D. Sammataro, U. Gerson, G. Needham, Parasitic mites of honey bees: life history, implications, and impact, Ann. Rev. Entomol. 45 (2000) 519 – 548. [8] S. Gisder, P. Aumeier, E. Generdch, Deformed wing virus: replication and viral load
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in mites (Varroa destructor), J. Gen. Virol. 90 (2009) 463 – 467. [9] Y. Kojima, T. Toki, T. Morimoto, M. Yoshiyama, K. Kimura, T. Kadowaki, Infestation of Japanese native honey bees by tracheal mite and virus from
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non-native European honey bees in Japan, Micro. Ecol. 62 (2011) 895 – 906.
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[10] T. Maeda, Infestation of honey bees by tracheal mites, Acarapis woodi, in Japan, J.
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Acarol. Soc. Japan 24 (2015) 9 – 17 (in Japanese). [11] Y. Kittaka, T. Yoshida, Reproduction of Varroa jacobsoni parasitizing newly –
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emerged Apis mellifera drones introduced into Apis cerana japonica colony,
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Honeybee Sci. 22 (2001) 31 – 36 (in Japanese).
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[12] Y. Kojima, M. Yoshiyama, K. Kimura, T. Kadowaki, PCR-based detection of a tracheal mite of the honey bee Acarapis woodi, J. Invertebr. Pathol. 108 (2011) 135 – 137.
[13] D. L. Anderson, J. W. H. Trueman, Varroa jacobsoni (Acari: Varroidae) is more than one species, Exp. Appl. Acarol. 24 (2000) 165 – 189. [14] T. Maeda, Y. Sakamoto, Tracheal mites, Acarapis woodi, greatly increase overwinter mortality in colonies of the Japanese honeybee, Apis cerana japonica, Apidologie 47 (2016) 762 – 770. [15] S. Imai, K. Fujisaki, T. Itagaki, T. Morita, [Veterinary Sanitary Zoology], p. 300, Koudansya, Tokyo, Japan, 2009 (in Japanese).
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[16] M. Delfinado-Baker, E.W Baker, Notes on honey bee mites of the genus Acarapis Hirst (Acari: Tarsonemidae), Int. J. Acarol. 8 (1982) 211 – 226. [17] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0, Mol. Biol. Evol. 30 (2013) 2725 – 2729.
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[18] T. Yoshida, [Japanese honeybee, ecology and its rearing methods] Tamagawa Uni. Press, Tokyo, Japan, 2000 (in Japanese).
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[19] Y. S. Peng, Y. Fang, S. Xu, L. Ge, The resistance mechanism of the Asian honey
Invertebr. Pathol. 49 (1987) 54 – 60.
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bee, Apis cerana Fabr., to an ectoparasitic mite, Varroa jacobsoni Oudemans, J.
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[20] W. Rath, Co-adaptation of Apis cerana Fabr. and Varroa jacobsoni Oud., Apidologie 30 (1999) 97 – 110.
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[21] N. E. Gary, R.E. Page, Phenotypic variation in susceptibility of honey bees, Apis
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(1987) 291 – 305.
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mellifera, to infestation by tracheal mites, Acarapis woodi, Exp. Appl. Acarol. 3
[22] D. Sammataro, G.R. Needham, Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on honey bees (Hymenoptera: Apidae), Exp. Appl. Acarol. 20 (1996) 121 – 136.
[23] J.E. Eckert, Acarapis mites of the honey bee, Apis mellifera Linnaeus, J. Insect Pathol. 3 (1961) 409 – 425. [24] A. B. Komeili, J,T. Ambrose, Biology, ecology and damage of tracheal mites on honey bees (Apis mellifera), Am. Bee J. 130 (1990) 423 – 429. [25] M. Sasaki, U. Takahashi, S. Sato, Comparison of the dance dialect and foraging range between Apis mellifera and northern most subspecies of A. cerana in Japan,
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Honeybee Sci. 14 (1993) 49 – 54. [26] T. Sakai, I. Okada, The present beekeeping in Japan, Gleaning. Bee Culture 101 (1973) 356 –357. [27] M. Delfinado-Baker, Acarapis woodi in the United States, Am. bee J. (USA) 124 (1984) 805 – 806.
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[28] D. Peer, J. Gruszka, A. Tremblay, Preliminary observations on the impact of Acarapis woodi on the development of package bee colonies, Saskatchewan
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Beekeepers Assoc. 81 (1987) 1 – 8.
[29] D.M. Menapace, W.T. Wilson, Acarapis woodi mites found in honey bees from
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Colombia, South America, Am. Bee J. 120 (1980) 761 – 765.
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[30] A. Cepero, R. Martin-Hernandez, L. Prieto, T. Gomez-Moracho, A. Martinez-Salvador, C. Bartolome, X. Maside, A. Meana, M. Higes, Is Acarapis
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woodi a single species? A new PCR protocol to evaluate its prevalence, Parasitol.
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Res. 114 (2015) 651-658.
Figure legends
Fig. 1. Localities where Apis cerana japonica samples were collected. White circles represent the localities where A. woodi was not detected from honeybees. Black circles represent the localities where A. woodi was detected from honeybees. The numbers around the circles are corresponding to the locality numbers in Table 2.
Fig. 2. Localities where Apis mellifera samples were collected. White circles represent the localities where A. woodi was not detected from honeybees. The numbers around the circles are corresponding to the locality numbers in Table 3.
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Fig. 3. Acarapis woodi detected from A. c. japonica. 1: the trachea infected with mites. A scale bar shows 100 m. 2: A matured female. A scale bar shows 50 m. 3: A matured male. A scale bar shows 50 m.
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Fig. 4. The phylogenetic tree based on the COI sequences (677bp) of Acarapis spp.
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Fig. 1. Localities where Apis cerana japonica samples were collected. White circles represent the localities where A. woodi was not detected from honeybees. Black circles represent the localities where A. woodi was detected from honeybees. The numbers around the circles are corresponding to the locality numbers in Table 2.
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Fig. 2. Localities where Apis mellifera samples were collected. White circles represent the localities where A. woodi was not detected from to the locality numbers in Table 3.
honeybees. The numbers around the circles are corresponding
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Fig. 3. Acarapis woodi detected from A. c. japonica. 1: the trachea infected with the mites. A scale bar shows 100 m. 2: A matured female. A scale bar shows 50 m. 3: A matured male. A scale bar shows 50 m.
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Fig. 4. Phylogenetic tree based on the COI sequences (677bp) of Acarapis spp.
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Graphical abstract
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Highlights • Acarapis woodi mites were detected from 13.5% of A. c. japonica and 0% of A. mellifera. • Aomori prefecture, Japan is a new distribution locality for A. woodi. • None of the honeybees examined showed infection by V. destructor mites. • Acarapis woodi mites would have been introduced into Japan with A. mellifera during
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the last 150 years and spread throughout the country.
Shunsuke Takashima, Yuma Ohari, Tadashi Itagaki PII:
S1383-5769(20)30002-7
DOI:
https://doi.org/10.1016/j.parint.2020.102052
Reference:
PARINT 102052
To appear in:
Parasitology International
Received date:
10 December 2019
Revised date:
27 December 2019
Accepted date:
6 January 2020
Please cite this article as: S. Takashima, Y. Ohari and T. Itagaki, The prevalence and molecular characterization of Acarapis woodi and Varroa destructor mites in honeybees in the Tohoku region of Japan, Parasitology International(2020), https://doi.org/10.1016/ j.parint.2020.102052
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier.
Journal Pre-proof
The prevalence and molecular characterization of Acarapis woodi and Varroa destructor mites in honeybees in the Tohoku region of Japan
Shunsuke Takashima, Yuma Ohari, Tadashi Itagaki*
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Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8
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Ueda, Morioka 020-8550, Japan.
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* Corresponding author:
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Tadashi Itagaki Tel./Fax: +81-19-621-6219
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Abstract
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E-mail: [email protected]
Honeybee acarapiosis and vorrosis were designated as Notifiable Infectious Diseases in the Act on Domestic Animal Infectious Diseases Control by the Minister of Agriculture, Forestry and Fisheries of Japan in 1997. However, the prevalences of A. woodi and V. destructor in Japan, especially in the Tohoku region, have not been sufficiently elucidated. This study was designed to clarify the prevalence of A. woodi and V. destructor mites in Apis cerana japonica and Apis mellifera in the Tohoku region and the characteristics of their mitochondrial cytochrome c oxidase I (COI) DNA. Acarapis woodi mites were detected from 13.5% of A. c. japonica and 0% of A. mellifera. Aomori prefecture, Japan is a new distribution locality for A. woodi. None of
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the honeybees examined showed infection by V. destructor mites. The COI sequences (1638 bp) of A. woodi were identical and phylogenetically closely related to those of A. woodi from Japan and the UK, suggesting that the mite would have been introduced into Japan with A. mellifera during the last 150 years and spread throughout the country.
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Keywords
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Acarapis woodi, Varroa destructor, COI, honeybees, Japan, prevalence
1. Introduction
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Acarapiosis is a disease caused by Acarapis woodi mites parasitizing the tracheae of
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Apis honeybees. Adult honeybees infected with these mites show a reduction in the ability to fly due to tracheal obstruction and damaged flight muscles [1, 2], which force
al
them to walk around their hives [3]. Heavy infestation of the honeybee colony results in
rn
hive collapse and disappearance [3, 4]. Acarapis woodi infection was first reported in
Jo u
Apis mellifera from the United Kingdom in 1921 [5] and has since been reported throughout the world (excluding Australia, New Zealand and Scandinavia n Peninsula [6, 7]. Varroa destructor mites parasitize the body surface of honeybees, mainly their larvae and pupa, and deform the legs and wings of adult bees. These mites are also vectors of deformed wings viruses [8]. In Japan, honeybee acarapiosis and vorrosis were designated as Notifiable Infectious Diseases in the Act on Domestic Animal Infectious Diseases Control by the Minister of Agriculture, Forestry and Fisheries in 1997. However, the prevalences of A. woodi and V. destructor in Japan, especially in the Tohoku region, have not been sufficiently elucidated [9-11]. Further, the reports on molecular characteristics of these mites are
Journal Pre-proof
limited [9, 12, 13]. This study was designed to clarify the prevalence of A. woodi and V. destructor mites in the Tohoku region and the characteristics of their mitochondrial cytochrome c oxidase I (COI) DNA.
2. Materials and Methods
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2.1 Honeybee samples Honeybees, A. c. japonica and A. mellifera, were collected from Aomori, Iwate,
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Akita, Miyagi, Fukushima and Yamagata prefectures of Japan in the period from March 2018 to June 2019 (Fig.1 and Fig. 2). For A. c. japonica, 386 honeybees from 18
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colonies of eight apiaries, 89 honeybees from six wild hives, and 109 flower-visiting
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honeybees from 15 localities were sampled. In A. mellifera, 40 honeybees from two colonies of two apiaries and 130 flower-visiting honeybees from 13 localities were
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sampled. The honeybee samples were preserved at -30℃ until analysis.
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2.2 Detection of mites and total DNA extraction
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The honeybees were examined under a stereoscopic microscope for detecting the presence of V. destructor mites on their surface; they were then dissected using forceps, and their tracheae were examined for A. woodi mites [14]. The detected mites were cleared by Gater solution [15] and morphologically identified [16]. Total DNA was extracted from the tracheae of 30 honeybees infected with A. woodi mites using High Pure PCR Template Kit (Roche Diagnostics, Indianapolis, USA). 2.3 DNA analysis For determining partial COI nucleotide sequences (441 bp), PCR was carried out in a total volume of 10 μL including 0.2 μL of Tks Gflex DNA Polymerase (Takara, Shiga, Japan), 5 μL of 2 × Gflex buffer, 0.2 μL of the primer #1F and R [12], 3.9 μL of MQ
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water and 0.5 μL of DNA template. The thermocycler program was set as follows: 94 0 C for 60 s, followed by 40 cycles of 980 C for 10 s, 550 C for 15 s and 680 C for 30 s. The amplicon was purified using NucleoSpin Gel and PCR Clean-up (Takara) and directly sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) and ABIPRISM 3500 Genetic Analyzer (Applied Biosystems).
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The amplicons of approximately complete length of mitochondrial DNA were obtained from seven honeybees. PCR was performed using the same reaction mixture as that for COI amplicon described above, except for using Aw-mito1F and mito1R
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primers (Table 1). The thermal conditions were as follows: 940 C for 60 s, followed by
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25 cycles of 980 C for 10 s, 600 C for 15 s and 680 C for 7 min. The amplicon was purified
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and directly sequenced using the primers, Aw-mito F2 and R2 and Aw-mito F3 and R3 (Table 1), and BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems),
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The obtained sequences (approximately 2,800 bp) were compared to complete
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mitochondrial DNA sequences of Tetranychus urticae (EU345430) and Tetranychus
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truncatus (MG518337) belonging to Prostigmata. The COI sequences (677 bp) forming a part of the sequences were assembled using ATGC ver. 6.0.3 (Genetyx, Tokyo, Japan) and aligned with reference COI sequences of A. woodi (AB634838 and HQ162656), A. dorsalis (HQ162658) and A. externus (HQ162662). A phylogenetic tree was constructed based on T92+G model using the Maximum Likelihood method of MEGA 6.0 software [17]. The nucleotide sites with base deletion were eliminated from the sequences for phylogenetic analysis. Bootstrap support was calculated using 1,000 replicates.
3. Results 3.1 Mite detection
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None of the honeybees examined showed infection by V. destructor mites. In A. c. japonica, mites were detected in the tracheas of 79 honeybees (13.5%), which included 68 honeybees (17.6%) from five apiaries (62.5 %, the locality numbers 4, 6 and 24-26 in Table 2) in Aomori and Iwate, 10 honeybees (11.2%) from 2 wild hives (33.3%, the locality numbers 22 and 23) in Iwate, and one flower-visiting
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honeybee (0.92%) from one locality (6.3%, the locality number 3) in Aomori (Table 2 and Fig. 1). In the apiaries where the mites were found, most (10/13) of the colonies
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examined were positive for tracheal mites. No mites were detected in the tracheae of A. mellifera (Table 3 and Fig. 2).
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The female mite detected had the short and stubby leg IV with five setae. The
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posterior margin of coxal plate IV was distinctively notched but not bilobed. The anterior median apodeme was not developed posteriorly and not joining with the
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transverse apodeme. The male was lacking the claws on leg Ⅳ. Based on these
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3.2 COI analyses
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morphological characteristics, the mites were identified as A. woodi (Fig. 3).
The partial COI sequences (441 bp) of A. woodi obtained from 30 honeybees were identical and were also identical to those of A. woodi from honeybees from Japan (AB634838) and UK (HQ162656). The COI sequences (1,638 bp) of A. woodi obtained from seven honeybees in Aomori and Iwate (the locality numbers 3 and 22) were also completely identical and represented as the haplotype, AW-H1. The sequences determined in this study are available in GenBank under accession numbers LC510136 and LC512730.
4. Discussion
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In this study, Varroa mite infection was not found in either, A. c. japonica or A. mellifera. Varroa mites were also rarely found in A. c. japonica [11, 18]. Adult A. cerana bees frequently groom their body surface and maintain hygiene in their hives, and this behavior seems to be useful towards removing mites. The Varroa-infected and deformed adult bees cannot fly from the hives to visit flowers for collecting nectar [19,
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20]. In this study, A. mellifera honeybees examined were mostly flower-visiting, which may be one of the reasons why V. destructor mites were not detected from A. mellifera
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in this study.
In this study, A. woodi mites were found in 79 honeybees of A. c. japonica from
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apiaries, wild hives and flower-visiting in Aomori and Iwate prefectures of Tohoku
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region, Japan. Acarapis woodi has been detected in A. c. japonica honeybees in some prefectures including Iwate, Akita and Miyagi of Tohoku [10, 14]. However, Aomori
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prefecture is a new distribution locality for A. woodi. The detection rate of A. woodi was
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the highest (62.5 %, 5/8 apiaries) in honeybees from apiaries, although honeybees were
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collected from apiaries, wild hives, and wild flowers. Mites parasitize the tracheae and air sacs of adult honeybees. Sexually mature female mites mate in the trachea of honeybees and move outside to invade other honeybees [21, 22]. Horizontal transmission of the mite seems to occur from an infected to a free colony when the infected mites accidentally returned to another colony of the apiary [23]. Transmission experiments revealed that A. woodi infection was transferred to the mite - free colony from the infected colony after 10 months of the commencement [24]. These might explain the high prevalence of A. woodi in apiaries. On the other hand, the prevalence was very low (6.7%, 1/15 localities) in flower-visiting bees. The infected hives might not occur near the flower - visiting localities because honeybees commonly collect
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flower nectar around 2 - 3 km distance from their hives [25]. The other probable explanation is the reduced flight ability of the infected honeybees, since it has been reported that infected honeybees suffer from tracheal obstruction and their flight muscles are damaged [1, 2]. The mitochondrial COI sequences of A. woodi obtained from Aomori and Iwate
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were completely identical, indicating that the A. woodi isolates are genetically very close each other. Furthermore, the results of phylogenetic tree indicated that the present
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isolates are also closely related to A. woodi detected from A. c. japonica in Japan (AB634838) and from A. mellifera in UK (HQ162656). Acarapis woodi was first
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reported in 1921 in the UK [5] and expanded throughout the world, except for Australia,
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New Zealand, and the Scandinavian Peninsula [6, 7]. Apis mellifera was first introduced to Japan in 1877 [26]; thereafter, it has been imported from USA, Canada, and
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Colombia, where A. woodi infection was detected [27-29]. From these findings, we
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consider that the mite would have been introduced into Japan with A. mellifera during
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the last 150 years and spread throughout the country. Cepero et al. [30] reported that genetic variants based on the COI sequence occur within A. woodi from A. mellifera honeybees in Spain, and that the variants belong to different clades in the phylogenetic tree. Therefore, the limited A. woodi isolate might be introduced into Japan. Mitogenome analyses of A. woodi mites isolated from different geographical regions including Japan are needed to elucidate the precise origin of the mite existing in Japan. Acarapis woodi mites were not detected from any honeybees of A. mellifera in this study. There are also no reports on the detection of the mite from A. mellifera in Japan in 2015 [10], although A. woodi DNA was found in A. mellifera in 2011 [9, 12]. However, the reason why no or only a very few mites occur in A. mellifera has
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remained unclear.
Acknowledgements We gratefully appreciate the members of the Japanese Original Honeybee
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Organization and apiarists for helping us to collect honeybee samples.
Declaration of Interest:
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none
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References
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[2] T.P. Liu, Ultrastructure of the flight muscle of worker honey bees heavily infested by
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[3] T. Maeda, Y. Sakamoto, K Okabe, H Taki, M. Yoshiyama, K. Goka, K. Kimura, Tracheal Mite, Acarapis woodi (Acari: Tarsonemidae), of Honey Bees: Biology, Impact on Honey Bees and Occurrence in Japan, Jpn. J. Appl. Entomol. Zool. 59 (2015) 109 – 126 (in Japanese). [4] J.B. McMullan, M.J. Brown, A qualitative model of mortality in honey bee (Apis mellifera) colonies infested with tracheal mites (Acarapis woodi), Exp. Appl. Acarol. 47 (2009) 225. [5] J. Rennie, Isle of wight disease in hive bees—acarine disease: the organism associated with the disease—Tarsonemus woodi, n. sp., Earth Environ. Sci. Trans. Royal Soc. Edinburgh 52 (1921) 768 – 779.
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[6] L. Bailey, B.V. Ball, Acarapis woodi: Parasitic mites, in: Honey Bee Pathology, 2nd ed., Academic Press, London, London, 1991, pp. 78 – 94. [7] D. Sammataro, U. Gerson, G. Needham, Parasitic mites of honey bees: life history, implications, and impact, Ann. Rev. Entomol. 45 (2000) 519 – 548. [8] S. Gisder, P. Aumeier, E. Generdch, Deformed wing virus: replication and viral load
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[13] D. L. Anderson, J. W. H. Trueman, Varroa jacobsoni (Acari: Varroidae) is more than one species, Exp. Appl. Acarol. 24 (2000) 165 – 189. [14] T. Maeda, Y. Sakamoto, Tracheal mites, Acarapis woodi, greatly increase overwinter mortality in colonies of the Japanese honeybee, Apis cerana japonica, Apidologie 47 (2016) 762 – 770. [15] S. Imai, K. Fujisaki, T. Itagaki, T. Morita, [Veterinary Sanitary Zoology], p. 300, Koudansya, Tokyo, Japan, 2009 (in Japanese).
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[16] M. Delfinado-Baker, E.W Baker, Notes on honey bee mites of the genus Acarapis Hirst (Acari: Tarsonemidae), Int. J. Acarol. 8 (1982) 211 – 226. [17] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0, Mol. Biol. Evol. 30 (2013) 2725 – 2729.
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[18] T. Yoshida, [Japanese honeybee, ecology and its rearing methods] Tamagawa Uni. Press, Tokyo, Japan, 2000 (in Japanese).
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[19] Y. S. Peng, Y. Fang, S. Xu, L. Ge, The resistance mechanism of the Asian honey
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[23] J.E. Eckert, Acarapis mites of the honey bee, Apis mellifera Linnaeus, J. Insect Pathol. 3 (1961) 409 – 425. [24] A. B. Komeili, J,T. Ambrose, Biology, ecology and damage of tracheal mites on honey bees (Apis mellifera), Am. Bee J. 130 (1990) 423 – 429. [25] M. Sasaki, U. Takahashi, S. Sato, Comparison of the dance dialect and foraging range between Apis mellifera and northern most subspecies of A. cerana in Japan,
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Honeybee Sci. 14 (1993) 49 – 54. [26] T. Sakai, I. Okada, The present beekeeping in Japan, Gleaning. Bee Culture 101 (1973) 356 –357. [27] M. Delfinado-Baker, Acarapis woodi in the United States, Am. bee J. (USA) 124 (1984) 805 – 806.
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[28] D. Peer, J. Gruszka, A. Tremblay, Preliminary observations on the impact of Acarapis woodi on the development of package bee colonies, Saskatchewan
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[29] D.M. Menapace, W.T. Wilson, Acarapis woodi mites found in honey bees from
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[30] A. Cepero, R. Martin-Hernandez, L. Prieto, T. Gomez-Moracho, A. Martinez-Salvador, C. Bartolome, X. Maside, A. Meana, M. Higes, Is Acarapis
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Figure legends
Fig. 1. Localities where Apis cerana japonica samples were collected. White circles represent the localities where A. woodi was not detected from honeybees. Black circles represent the localities where A. woodi was detected from honeybees. The numbers around the circles are corresponding to the locality numbers in Table 2.
Fig. 2. Localities where Apis mellifera samples were collected. White circles represent the localities where A. woodi was not detected from honeybees. The numbers around the circles are corresponding to the locality numbers in Table 3.
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Fig. 3. Acarapis woodi detected from A. c. japonica. 1: the trachea infected with mites. A scale bar shows 100 m. 2: A matured female. A scale bar shows 50 m. 3: A matured male. A scale bar shows 50 m.
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Fig. 4. The phylogenetic tree based on the COI sequences (677bp) of Acarapis spp.
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Fig. 1. Localities where Apis cerana japonica samples were collected. White circles represent the localities where A. woodi was not detected from honeybees. Black circles represent the localities where A. woodi was detected from honeybees. The numbers around the circles are corresponding to the locality numbers in Table 2.
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Fig. 2. Localities where Apis mellifera samples were collected. White circles represent the localities where A. woodi was not detected from to the locality numbers in Table 3.
honeybees. The numbers around the circles are corresponding
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Fig. 3. Acarapis woodi detected from A. c. japonica. 1: the trachea infected with the mites. A scale bar shows 100 m. 2: A matured female. A scale bar shows 50 m. 3: A matured male. A scale bar shows 50 m.
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Fig. 4. Phylogenetic tree based on the COI sequences (677bp) of Acarapis spp.
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Graphical abstract
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Highlights • Acarapis woodi mites were detected from 13.5% of A. c. japonica and 0% of A. mellifera. • Aomori prefecture, Japan is a new distribution locality for A. woodi. • None of the honeybees examined showed infection by V. destructor mites. • Acarapis woodi mites would have been introduced into Japan with A. mellifera during
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the last 150 years and spread throughout the country.