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

SHB were diagnosed through the direct observation of whether eggs, larvae or adults were present in honey bee hives. Samples were collected according to the standard method of Neumann et al. (2013) from infested colonies between 2016 and 2018, from four apiaries in the city of Miryang where the SHB invasion was reported for the first time in South Korea and from one apiary in Damyang County, Jeollanam Province in 2021. One hive in each apiary with SHB was selected and transported to the Parasitic and Honey Bee Disease Laboratory, Animal and Plant Quarantine Agency, South Korea. Samples were used for species identification through morphological and molecular analysis. Morphological identification of A. tumida was carried out based on size, shape, colour and characteristics of the egg, larval, and adult stages of SHB (Lundie, 1940; Schmolke, 1974; Neumann et al., 2013). The specimens were observed under a stereomicroscope (M125; Leica, Wetzlar, Germany) for species identification. After species identification, the samples were stored at −20°C for further study.

Total nucleic acid was extracted with the use of the Maxwell^® RSC viral total nucleic acid purification kit (Promega, Madison, WI, USA). Two individual adult SHB or five larvae and 600 μL of PBS solution were added in a tissue-homogenizing tube with 2.381 mm diameter steel beads (SNC, Hanam, South Korea), and homogenized with the use of a Precellys 24 tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) at four 15 s cycles at 5000 rpm. Afterwards, 300 μL homogenate, 300 μL of lysis buffer, and 30 μL of proteinase K solution were added to each tube and incubated at 56°C for 10 min.

Nucleic acid was then purified automatically with the automated Maxwell^® RSC instrument (Promega, Madison, WI) according to the manufacturer’s instructions. Finally, 100 μl total nucleic acid acquired from each sample was used for the detection of honey bee pathogens. To extract DNA from SHB for the amplification of the COI gene, the FastDNA Spin Kit for Soil (Santa Ana, California, USA) was used with some modifications. Two individual adult SHB or five larvae were placed in a tissue-homogenizing tube with steel beads (SNC, Hanam, South Korea). After the addition of 978 μL sodium phosphate buffer and 122 μL MT buffer from the FastDNA Spin Kit for Soil (Santa Ana, California, USA), the samples were homogenized with the use of a Precellys 24 tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) at four 15 s cycles at 5000 rpm. The following steps for DNA purification were done according to the manufacturer’s instructions.

The mitochondrial COI gene of four adult samples from four different apiaries was used for molecular identification of SHB and for phylogenetic analysis. A 1091-bp DNA fragment COI was amplified with a primer pair AT1904S (5′-GGTGGATCTTCAGTTGATTTAGC-3′) and AT2953A (5′-TCAGCTGGGGGATAAAATTG-3′) (Evans et al., 2000) and AccuPower PCR PreMix (Bioneer, Daejeon, Korea). The 20 μl reaction mix was composed of 5 μl of total nucleic acid, 1 μl (10 pmol) of each primer, and 13 μl of ddH₂O. PCR conditions were 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 42°C for 30 s, and 72°C for 1 min, and final extension at 72°C for 5 min. After confirming the expected band in electrophoresis gel, 1.5% agarose and 1× of RedSafe^™ nucleic acid staining solution (iNtRON Biotechnology, Inc., Gyeonggi-do, South Korea), the PCR products were sent to Macrogen (Seoul, South Korea) to be sequenced with the use of the Sanger sequencing method. The generated COI sequence was deposited on NCBI with accession number MZ234080. The COI sequence was compared to deposited sequences of Aethina tumida on NCBI with the use of the basic local alignment search tool (BLAST). Consensus sequences were aligned with the use of Clustal X2 (Larkin et al., 2007), and a neighbor-joining phylogenetic tree was constructed in MEGA7 (Kumar et al., 2016) with 1000 bootstrap iterations.

Fourteen prevalent honey bee pathogens were detected in the total nucleic acid extracted from four different samples of collected adult SHB to confirm whether they were carrying these honey bee pathogens. The pathogens include the following viral pathogens: DWV type A, acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), sacbrood virus (SBV), three fungal pathogens (Nosema spp., Aspergillus flavus, and Ascosphaera apis), two bacterial pathogens (Paenibacillus larvae, which causes American foulbrood, AFB; and Melissococcus plutonius, which causes European foulbrood, EFB), tracheal mite (Acarapis woodi), and phorid fly (Apocephalus borealis). Specific primers for detection of each pathogen are shown in Tab. 1. Detection of viral pathogens with an RNA genome was done through the use of conventional reverse transcription PCR (RT-PCR) and the AccuPower^® RT-PCR premix (Bioneer, Daejeon, Korea). For the detection of other pathogens with a DNA genome, conventional PCR and the AccuPower^® ProFi Taq PCR PreMix (Bioneer, Daejeon, Korea) were used. The 20 μl reaction mix was composed of 5 μl total nucleic acid, 1 μl (10 pmol) of each primer, and 13 μl of ddH₂O. Reverse transcription was done at 42°C for 60 min prior to PCR. PCR was performed at 94°C (5 min) for pre-denaturation, followed by 35 cycles of 94°C (30 sec) for denaturation, 30 sec for annealing, 72°C (1 min) for extension, and 72°C (10 min) for final extension. The annealing temperature of each primer pair is shown in Tab. 1.

SHB infestation was detected through the observation of eggs, larvae or adults deposited in the combs of honey bee hives (Fig. 1). Morphological identification of SHB was carried out by the naked eye in the apiary and then by microscopy in the laboratory. Eggs of A. tumida were observed in egg clutches; they were around 1.4 × 0.26 mm (length × width), and two thirds the size of honey bee eggs and a pearly-white colour (Fig. 2E and 2F). The larvae were identified as being 10 mm in length and were characterized by three pairs of legs located close to the head, two rows of dorsal spines, and two larger paired spines on the posterior segment of the dorsum (Fig. 2B-2D). Adult A. tumida were identified as dark brown to black colour, having three parts of body (head, thorax, and abdomen), three pairs of legs and body size of 5~7 mm × 3~5 mm (length × width); they were oval and dorsoventrally flattened and had one pair of clubbed antennae on the head. The pronotum was identified by sharp latero-posterior tips, and the abdomen was not entirely covered elytra (Fig. 2A).

Fig. 1

Fig. 2

The COI gene of SHB collected from different apiaries in Miryang and Damyang, South Korea were 100% identitical to one another. These generated sequences also were 100% identical to reported COI sequences of SHB originating from the USA (NCBI accession no.: KC966652) and South Korea (MN023006). Phylogenetic analysis was conducted to identify the relationships among South Korean haplotypes and reported SHB haplotypes. The results showed a close relationship of the detected SHB strains to those originating from the USA, Canada and Costa Rica (Fig. 3).

Fig. 3

Four adult samples and one larval sample of SHB were used for the detection of fourteen prevalent honey bee pathogens by specific conventional PCR (Tab. 1). The results showed that two of the fourteen honey bee pathogens were detected in the adult SHB (DWV and BQCV), of which, DWV was detected in all four adult samples, and BQCV was detected in three adult samples. The result was confirmed through electrophoresis with the expected band of 479 bp and 701 bp long for DWV and BQCV detection, respectively (Fig. 4).

Fig. 4