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Pathogen Detection and Phylogenetic Analysis of Aethina tumida Murray in South Korea

Mi-Sun Yoo

Mi-Sun Yoo

Department of Animal and Plant Health Research, Animal and Plant Quarantine AgencyKorea (South)
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Yoo, Mi-Sun
, 
A-Tai Truong

A-Tai Truong

Department of Animal and Plant Health Research, Animal and Plant Quarantine AgencyKorea (South)
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Truong, A-Tai
, 
Yong-Soo Choi

Yong-Soo Choi

Department of Agricultural Biology, National Institute of Agricultural ScienceKorea (South)
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Choi, Yong-Soo
, 
Ki-Jeong Hong

Ki-Jeong Hong

Department of Plant Medicine, Sunchon National UniversityKorea (South)
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Hong, Ki-Jeong
, 
Tae Jun Hwang

Tae Jun Hwang

Department of Animal and Plant Health Research, Animal and Plant Quarantine AgencyKorea (South)
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Hwang, Tae Jun
, 
Soo Kyoung Seo

Soo Kyoung Seo

Department of Animal and Plant Health Research, Animal and Plant Quarantine AgencyKorea (South)
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Seo, Soo Kyoung
, 
Hyun-Ji Seo

Hyun-Ji Seo

Department of Animal and Plant Health Research, Animal and Plant Quarantine AgencyKorea (South)
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Seo, Hyun-Ji
, 
Sukchan Jung

Sukchan Jung

Department of Animal and Plant Health Research, Animal and Plant Quarantine AgencyKorea (South)
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Jung, Sukchan
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Soon-Seek Yoon

Soon-Seek Yoon

Department of Animal and Plant Health Research, Animal and Plant Quarantine AgencyKorea (South)
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Yoon, Soon-Seek
 y 
Yun Sang Cho

Yun Sang Cho

Department of Animal and Plant Health Research, Animal and Plant Quarantine AgencyKorea (South)
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Cho, Yun Sang
  
22 jun 2022
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Categoría del artículo: Original Article
Publicado en línea: 22 jun 2022
Páginas: 45 - 55
Recibido: 25 jun 2021
Aceptado: 17 feb 2022
DOI: https://doi.org/10.2478/jas-2022-0004
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© 2022 Mi-Sun Yoo et al, published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Fig. 1

Infestation of small hive beetle in honeybee hives. Aethina tumida in apiaries were diagnosed through the observation of the presence of adults (A), and larvae in supplied pollen (B) and honeycomb of infested hive (C).
Infestation of small hive beetle in honeybee hives. Aethina tumida in apiaries were diagnosed through the observation of the presence of adults (A), and larvae in supplied pollen (B) and honeycomb of infested hive (C).

Fig. 2

Identification of small hive beetle based on morphological characteristics. Appearance of adults observed with 5~7 mm × 3~5 mm (length × width) of body size, dark brown colour, one pair of clubbed antennae on the head, and body shape of oval and dorsoventrally flattened (A). The larvae were characterized by three pairs of legs located close to the head, two rows of dorsal spines, two larger paired spines on the posterior segment of the dorsum, and body size was around 10 mm in length (B-D). Eggs of SHB were seen in capped bee brood with pearly-white colour and size of around 1.4 mm × 0.26 mm (length × width) (E and F).
Identification of small hive beetle based on morphological characteristics. Appearance of adults observed with 5~7 mm × 3~5 mm (length × width) of body size, dark brown colour, one pair of clubbed antennae on the head, and body shape of oval and dorsoventrally flattened (A). The larvae were characterized by three pairs of legs located close to the head, two rows of dorsal spines, two larger paired spines on the posterior segment of the dorsum, and body size was around 10 mm in length (B-D). Eggs of SHB were seen in capped bee brood with pearly-white colour and size of around 1.4 mm × 0.26 mm (length × width) (E and F).

Fig. 3

Phylogenetic tree of mitochondrial cytochrome c oxidase I (COI) gene sequences from small hive beetle (SHB). Neighbor-joining phylogenetic tree was created based on mitochondrial COI gene of SHB with 1000 bootstrap iterations in MEGA7. Aethina tumida detected in this study with NCBI accession number MZ234080 was written in bold. Other reference strains of A. tumida with NCBI accession numbers and country names are shown. Cratonura rufithorax was used as an outgroup. Number above line represents bootstrap percentages.
Phylogenetic tree of mitochondrial cytochrome c oxidase I (COI) gene sequences from small hive beetle (SHB). Neighbor-joining phylogenetic tree was created based on mitochondrial COI gene of SHB with 1000 bootstrap iterations in MEGA7. Aethina tumida detected in this study with NCBI accession number MZ234080 was written in bold. Other reference strains of A. tumida with NCBI accession numbers and country names are shown. Cratonura rufithorax was used as an outgroup. Number above line represents bootstrap percentages.

Fig. 4

Detection of honeybee pathogens in small hive beetle (SHB). Positive results of black queen cell virus (A) and deformed wing virus (B) detection were confirmed with expected band, 701 bp and 479 bp long, respectively, in electrophoresis. Lanes 1 to 4 were for four SHB samples. “M” is a 100-bp DNA marker. “+” and “-” are positive using recombinant DNA and negative control without DNA template, respectively.
Detection of honeybee pathogens in small hive beetle (SHB). Positive results of black queen cell virus (A) and deformed wing virus (B) detection were confirmed with expected band, 701 bp and 479 bp long, respectively, in electrophoresis. Lanes 1 to 4 were for four SHB samples. “M” is a 100-bp DNA marker. “+” and “-” are positive using recombinant DNA and negative control without DNA template, respectively.

Specific primers used for detection of honey bee pathogens

No. Pathogen Primer sequence (5′-3′) Amplicon size (bp) Annealing temp (°C) Reference
1 SBV F: ACCAACCGATTCCTCAGTAGR: CCTTGGAACTCTGCTGTGTA 487 57 Grabensteiner et al., 2001
2 ABPV F: TTATGTGTCCAGAGACTGTATCCAR: GCTCCTATTGCTCGGTTTTTCGGT 901 55 Benjeddou et al., 2001
3 CBPV F: AGTTGTCATGGTTAACAGGATACGAGR: TCTAATCTTAGCACGAAAGCCGAG 455 55 Ribiere et al., 2002
4 Virus DWV F: TCATCTTCAACTCGGCTTTCTACGR: CGAATCATTTTCACGGGACG 479 62 Lee et al., 2005a
5 BQCV F: TGGTCAGCTCCCACTACCTTAAACR: GCAACAAGAAGAAACGTAAACCAC 701 55 Benjeddou et al., 2001
6 KBV F: GATGAACGTCGACCTATTGAR: TGTGGGTGGCTATGAGTCA 415 50 Stoltz et al., 1995
7 IAPV F: GATTTGAGAGATGTATTTCCTTCTGCGGR: ACACTTGCGTTGGTCCTGAATGTTAATGG 725 52 This study
8 Bacteria Paenibacillus larvae F: GTGTTTCCTTCGGGAGACGR: CTCTAGGTCGGCTACGCATC 232 55 Lee et al., 2004
9 Melissococcus plutonius F: AAGAGTAACTGTTTTCCTCGR: AAACCTTATCTCTAAGGCGT 583 52 Ha et al., 2005
10 Ascosphaera apis F: GGCTGTAGGGGGGAACCAGGAR: CGGGTGGTCGTTTCCAGCCTC 995 62 Lee et al., 2005b
11 Fungus Aspergillus flavus F: ATCGGGCGGTGTTTCTATGR: ACCGGGCTATTTAAGGGCCG 311 55 Lee et al., 2004
12 Nosema sp. F: CTGCCTGACGTAGACGCTATR: CTTCGATCCTCTAGCTTACG 592 50 Yoo et al., 2008
13 Parasite Acarapis woodi F: CAGTAGGGCTAGATATCGATACCCGAGCTTR: TGAGCTACAACATAATATCTGTCATGAAGA 247 55 This study
14 Apocephalus borealis F: GTACACCTATACATTGGGTTCGTACATTACR: GAGRGCCATAAAAGTAGCTACACC 500 57 This study
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