Introduction

Honey bees (Apis mellifera) are essential pollinators of crops and wild plants and play an indispensable role in human life by providing important products such as honey, royal jelly, and propolis. However, the loss of honey bee colonies due to climate change and the spread of pathogens has occurred worldwide1,2,3. One of the leading causes of honey bee deaths are mites such as Varroa spp. and Tropilaelaps spp., which feed on honey bees and are vectors for disease transmission in honey bees4,5,6,7. Vector-mediated transmission significantly increases the spread of pathogens, contributing to the collapse of the infected bee colony. Recent increase in the rate of honey bee colony loss has become a great concern for beekeepers and researchers; the rate of colony loss has been as high as 36.5% in many countries8, and a loss of approximately 37.7% was reported in 2018–2019 in the United States9. In the Republic of Korea (ROK), the value of honey bee pollination has been estimated at $5.9 billion, corresponding to approximately 50% of the economic value of fruit and vegetable production10; however, the loss of about 18% of honey bee colonies in the country has been contributed to unknown reasons11. Therefore, it is crucial to identify the factors that adversely affect beekeeping in the country.

Mites of the genus Tyrophagus are distributed worldwide in a range of habitats, including natural and anthropogenic, as well as plant and animal hosts12,13,14. Several species of Tyrophagus damage economic crops such as greenhouse vegetables and ornamental flowers15,16. Tyrophagus belongs to the superorder Acariforms, family Acaridae, and it includes approximately 35 species recorded globally12,17. Tyrophagus curvipenis (Acari: Acaridea) has been reported in a variety of animal hosts in Costa Rica and Russia, 18 and in several plants in New Zealand, France, Australia, and Portugal12. Fain and Fauvel first reported T. curvipenis mites isolated from wooden structures of a greenhouse in Portugal, and they19 recorded that mites occasionally inhabit the flowers of orchids and may feed on their pollen19. Several species of Tyrophagus (such as T. putrescentiae, T. curvipenis, T. tropicus, T. debrivorus, T. similis, T. longgior, T. mixtus, T. perniciosus, T. vanheurni, and T. savasi) have been associated with bees20. Tyrophagus putrescentiae (Acari: Acaridae) was detected in dead honey bee samples21, in the body of bumblebees22, and in honey bee hives in Brazil. This suggests the potential of the mites to harm human health upon consumption of the products from contaminated honey bees23. No study reports the presence of the remaining nine species of Tyrophagus in honey bees. Acting as potential carriers of pathogens or vectors for pathogen infections, these mites can affect humans indirectly24,25,26; therefore, the role of T. curvipenis and other species of mites infecting honey bees need further studies and evaluation.

Identification of Tyrophagus mites has been traditionally based on morphology12,14,27. However, this method is laborious due to the small size of Tyrophagus mites, requires good understanding of morphological traits, and it is time-consuming. The molecular method based on the mitochondrial gene cytochrome-c oxidase subunit 1 (CO1) and the nuclear ribosomal internal transcribed spacer 2 (ITS2) was suggested as an alternative tool for species identification of microscopic mites28.

Screening for the factors responsible for the honey bee colony loss was carried out in the dead colonies collected from the regions with high colony collapse reported in winter. Here, mites were detected and collected from dead honey bee colonies in the ROK in 2022. The CO1 and ITS2 sequences were analyzed to identify the mite species. In addition, honey bee pathogens carried by the mite were also identified.

Results

Morphological identification of mites

Microscopic examination of the honey bee samples revealed the presence of T. curvipenis mites within their bodies, with an infestation rate of 100%. The mites isolated from the honey bee colony were associated with different life stages of the collected honey bee samples, namely the egg, larva, nymph, and adult stages (Fig. 1a–d). The Tyrophagus genus exhibits a distinct characteristic in adult mites, with their idiosoma appearing whitish to semitransparent (Fig. 1d). Identification of the mite species T. curvipenis Fain & Fauvel (Acari: Astigmata: Acaridae) was based on female and male characteristics. The female mites were small, pale, idiosoma 415–451 µm (n = 2) long, 218–225 µm wide; chelicera 82–86 µm; prodorsal shield nearly pentagonal, eye spots prominent, 83–84 µm long, 86–84 µm wide; supracoxal seta (scx, 31–36 µm) slender, with four moderate or short pectinations; Grandjean's organ finger-like, its basal lobe with two spiniform teeth; hysterosomal setae c1, d1, and d2 relatively shorter than others; c1 (34–42 µm) a bit shorter than d2 (35–47 µm), d1 (117–109 µm) approximately 3.4 × the length of c1 and 2.6 × the length of d2; spermathecal duct narrow, base of spermathecal sac r-shaped (Fig. 2a); coxal plate II broadly triangular, extending distally beyond the apex of apodeme II, with 1/3 of posterior margin slightly concave; tarsus I ω1 slender, cylindrical and slightly widened at apex, tarsus II ω slender, almost cylindrical; setae ω and r of tarsus IV setiform (Supplementary Fig. S1).

Figure 1
figure 1

Different stages of Tyrophagus curvipenis detected in honey bees (Apis mellifera). Identity of acaroid mites was determined by analyzing morphological characteristics using a phase contrast microscope. Different living stages of the mite are presented: egg (a), larva (b), nymph (c), and adult (d).

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Figure 2
figure 2

Morphological characterization of Tyrophagus curvipenis. Identification of Tyrophagus mites was based on the position of setae ve, the shape of setae scx. (a) Female, the comparison of the size of c1, d1, and d2 setae to the distance between these setae and the posterior setae, and the shape of female spermatheca, (b) Male, S-shaped aedeagus and two suckers evenly distributed on tarsus IV.

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The male was smaller than the female, idiosoma 319 µm (n = 1) long, 196 µm wide. Chelicerae 71 µm; eyespots, coxal plates I and II, and solenidia I ω1 and II ω as in female; aedeagus curved into S-shape; two suckers evenly distributed on tarsus IV (Fig. 2b).

Genetic identification of mite species

The family to which this species belongs can be determined based on the morphological characteristics observed under the microscope. Although morphological traits are highly useful for initial identification, accurately distinguishing the exact species can be challenging. To confirm the identification, species-specific primer pairs targeting the CO1 gene and the ITS2 gene regions of Tyrophagus mites were designed. We successfully optimized the PCR reaction conditions for amplifying the CO1 and ITS2 genes. The sequences were then determined through sequence analysis. Amplification of the CO1 and ITS2 regions from adult mite and egg samples produced bands with expected length of 379 and 500 bp, respectively (Fig. 3a). Sequences of the amplified regions were 100% similar between the adult and egg samples. The search of the obtained CO1 sequences against the GenBank database (blast.ncbi.nlm.nih.gov) revealed 100% sequence identity with T. curvipenis originating from Costa Rica (NCBI accession No.: KY986270) and 86.64% similarity with T. putrescentiae (NCBI accession No.: MH262448). The IST2 sequences of T. curvipenis are not available in the NCBI database, and the search of the sequences isolated from the samples against the GenBank database indicated the highest similarity of 91.78% to T. putrescentiae in China (NCBI accession No.: GQ205623.1). In addition, non-specific band (around 1.7 kb long) was seen in the ITS2 amplification from adult sample (Fig. 3b). The unexpected band was extracted for sequencing. However, the result was unidentified due to the poor sequencing result.

Figure 3
figure 3

Amplification of CO1 and ITS2 regions from mites isolated from honey bees. (a) PCR product of CO1, (b) PCR product of ITS2. PCR products were confirmed on 1% agarose gel. Lane M is 100 bp DNA ladder; lanes 1 and 2 are PCR products of the egg and adult samples, respectively. lane “-” is negative control without a DNA template. Amplicon size from sample DNA is shown in base pair (bp).

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Phylogenetic analysis based on the CO1 gene sequences placed the collected mite sister with T. curvipenis isolate AD2025 collected from a bird nest in Costa Rica and both were in the same clade with T. curvipenis isolated from plants (peach and chayote) in Russia and Costa Rica (Fig. 4).

Figure 4
figure 4

Phylogenetic neighbor-joining tree based on sequences of CO1 gene. Species name, NCBI accession numbers, and origin of the accession are given for each sequence. The mite identified in honey bee samples in this study is in bold.

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Detection of honey bee pathogens in mites

Pathogen detection from 15 honey bee samples showed that the bee samples were infected with DWV, BQCV, and Trypanosoma (Supplementary Table S1). Therefore, to identify the possibility of the honey bee pathogen infection in the mites, we conducted pathogen detection using total nucleic acids extracted from the mites. Two honey bee pathogens (DWV and Trypanosoma) were detected in eggs and adult samples of the mite. Additionally, the two pathogens were present in the honey bee samples from the same colonies where the mites were collected. The identity of the pathogens was confirmed using reverse transcription real-time PCR (RT-qPCR), which produced bands of 199 bp and 276 bp for DWV and Trypanosoma, respectively (Figs. 5 and 6).

Figure 5
figure 5

Detection of deformed wing virus (DWV) from honey bee and mite samples. Positive detection of DWV was identified in amplification curves of RT-qPCR (a). Results were confirmed by visualizing the expected band (199 bp long) on agarose gel (b). Lane M is 100 bp DNA marker. Lanes 1 and 2 are bands amplified from egg and adult samples of Tyrophagus curvipenis, respectively. Lanes 3 and 4 indicate the results from honey bee samples collected from two colonies. Lane “−” is negative control without DNA template, and lane ‘+’is positive control using recombinant DNA of DWV.

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Figure 6
figure 6

Detection of Trypanosoma from honey bee and mite samples. Positive detection of Trypanosoma in real-time PCR (a) was confirmed with the expected band (276 bp) on electrophoresis agarose gel (b). Lanes 1 and 2 indicate the results of detection from egg and adult samples of T. curvipenis, respectively. Lanes 3 and 4 are the result of Trypanosoma detection from honey bee samples collected from two different colonies. Line ‘−’ is negative control without DNA template, and line ‘+’ is positive control using Trypanosoma recombinant DNA.

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Discussion

In this study, Tyrophagus mites were detected and identified in the colonies of five apiaries in Gongju, Chungcheongnam Province, Republic of Korea. This is the first report of T. curvipenis in A. mellifera colonies in the country. Morphological identification and genetic identification using CO1 gene revealed that the mite belongs to the species T. curvipenis, and phylogenetic analysis of the obtained sequence indicated a close relationship between the T. curvipenis from the Republic of Korea and the isolate from a bird’s nest in Costa Rica (Fig. 4). In addition, the species phylogeny of Tyrophagus based on nucleotide similarity in the CO1 gene region showed that the T. curvipenis detected in honey bee in this study was in the same cluster as that identified in plants such as peach and chayote from Russia and Costa Rica. The result suggests that the source of T. curvipenis identified in this study could be a flower visited by the honeybee for pollen and nectar collection. Four of the 35 identified species in the genus Tyrophagus have been reported to date in the Republic of Korea: T. putrescentiae, T. similis, T. neiswanderi, and T. longior29,30,31. Tyrophagus curvipenis reported here is thus a newly recorded species in the ROK. Similar to other species, T. curvipenis has been found in honey bee hives in New Zealand32. It was reported that T. putrescentiae in Korea (Wanju-gun, Jeonnam) inhabits beehives and feeds on debris and pollen33. However, the relationship between honey bee health and the presence of T. curvipenis mite in the hive remains unknown.

The molecular method was successfully used to identify Tyrophagus species. The result of the molecular method based on the CO1 gene sequence was consistent with the identification based on morphological characteristics. However, due to the limited availability of Tyrophagus sequences in the NCBI database, species identification of the Tyrophagus mite could not be confirmed using the IST2 region. In addition, the IST2 primer pair amplified a non-specific band using the total DNA extracted from adult mites (Fig. 3b). The CO1 gene, owing to a larger deposited sequence database, is potentially more beneficial for species identification of Tyrophagus mites.

Infestation of honey bees with hemolymph-feeding mites such as Varroa and Tropilaelaps increased colony loss over the winter season and transmission of honey bee diseases4,6,7,34,35,36. The viral, bacterial, and fungal honey bee pathogens harbored and transmitted by Varroa and Tropilaelaps mites have been demonstrated36,37. The T. curvipenis mite detected in this study was positive for a honey bee viral pathogen (DWV) and protozoal pathogen (Trypanosoma). The infection with these pathogens weakens the colony and increases the winter mortality of honey bees38,39,40. Additionally, these pathogens were detected in the honey bee samples where the mites were collected, suggesting a high possibility of mite transmitting such pathogens in the hives. Furthermore, Tyrophagus mites, commonly known as storage mites, feed on mold found in food41,42. However, the food sources of T. curvipenis in honey bee hives remain unknown. Therefore, understanding the influence of T. curvipenis mite on honey bees would help to develop appropriate methods for mite prevention and control. In addition, it is necessary to minimize the potential effects of the mites on humans who consume the honey bee products collected from infested colonies, because some Tyrophagus mites were identified as allergens in animals and humans43,44. With an observed infestation rate of 100% and the potential to serve as intermediate disease vectors within honey bee colonies in the five apiaries studied, Tyrophagus mites potentially play an important role in the observed colony losses. The findings from this study have the potential to contribute to the development of targeted management strategies aimed at minimizing the impact of Tyrophagus mite infestation on honey bee health and improving overall colony survival rates. By understanding the significance of Tyrophagus mites as potential contributors to colony decline, proactive measures can be implemented to mitigate their negative effects and safeguard the well-being of honey bee populations.

This is the first record of T. curvipenis mites in honey bee colonies in the Republic of Korea, and this study demonstrated the presence of honey bee pathogens (DWV and Trypanosoma) in mites. The result suggests that the mite could have an important role in spreading the honey bee pathogens. However, whether T. curvipenis mite contributed to the death of honey bee colonies has not been confirmed in this study; therefore, further study is necessary to understand the influence of this enemy and biological vector of honey bee pathogens in apiculture, and to reveal the potential risk of mites to humans consuming the products collected from infested honey bee colonies.

Materials and methods

Mite collection and morphological identification

Honey bee samples were collected from dead colonies in apiaries in Gongju-si, Chungcheongnam Province, ROK, in November 2022. A total of 45 honey bee samples were collected from 15 colonies in five different apiaries for mite examination. The presence of mites was confirmed under a dissecting microscope. The mites were collected from the body surface and hairs of honey bees. Mites were mounted on slides with Hoyer's medium. The specimens were identified and measured according to Fan and Zhang12. All the measurements used herein are in micrometers. After species identification, the mites were used for genetic analysis and pathogen detection.

Genomic DNA extraction and total nucleic acid extraction

Honey bee samples and collected mites were washed three times using UltraPure™ distilled water (Invitrogen, USA) and used for total nucleic acid extraction. The Maxwell RSC viral total nucleic acid purification Kit (Promega, USA) was used for nucleic acid extraction according to the manufacturer’s instructions. The extracted nucleic acids were used for the detection of honey bee pathogens. Extraction of the mite genomic DNA (gDNA) was done using a QIAamp DNA Mini Kit (Qiagen, UK) according to the manufacturer’s instructions with some modifications45. Ten adult mites or four mite eggs collected from each honey bee colony were pooled and placed in a 1.5 mL tube containing 200 µL ATL buffer and 2.381 mm steel beads (Hanam, ROK). After homogenizing at 5000 rpm for 15 s, 20 µL proteinase K (50 µg/mL) was added, and the mixture was incubated at 56 °C for 1 h. Exactly 200 µL of lysis buffer was added to the solution, and the solution was incubated for 10 min at 70 °C. Next, 200 µL of 98% ethanol was added, and the mixture was mixed by vortexing. The solution was transferred to the AIAamp mini spin column and centrifuged at 12,000×g for 1 min. After washing the spin column twice with the washing buffer, the genomic DNA of the mites was eluted into a new tube using the elution buffer and centrifuged at 12,000×g for 2 min. The isolated DNA was used for PCR amplification of the CO1 and ITS2 regions.

Detection of honey bee pathogens

The mite and honey bee samples from each colony were tested for the presence of honey bee pathogens using the total nucleic acid of honey bee and mite samples. The honey bee pathogens targeted for detection included American foulbrood (AFB), European foulbrood (EFB), Ascosphaera apis (ASCO), Aspergillus flavus (ASP), Nosema, Acarapis woodi (ACAR), Apocephalus borealis (PHORID), Sacbrood virus (SBV), DWV, Black queen cell virus (BQCV), Chronic bee paralysis virus (CBPV) Kashmir bee virus (KBV), Acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), Apis mellifera filamentous virus (AmFV), clades of Lake Sinai virus (LSV1, LSV2, LSV3, LSV4), Trypanosoma, Varroa destructor virus-1 (VDV-1; DWV-B), and recombinant Deformed wing virus-Varroa destructor virus-1 (DWV-VDV-1). Detection of pathogens was conducted in honey bee samples using RT-qPCR Kits (LiliF ABPV/KBV/IAPV/CBPV Real-time RT-PCR Kit, LifiF SBV/KSBV/DWV/BQCV Real-time RT-PCR Kit [iNtRON Biotechnology, Inc., ROK], Pobgen bee pathogen detection Kit [Postbio, ROK], and iTaq Universal SYBR green one-step Kit [Bio-Rad, USA]). Primers used for the detection of honey bee pathogens are presented in Supplementary Tables S2 and S3. The pathogens detected in honey bee samples were targeted for detection in mite samples using the PCR kits.

PCR amplification and sequencing of mite DNA

The CO1 and ITS2 regions of mites were amplified using universal primer sets CO1-forward (5ʹ-GTTTTGGGATATCTCTCATAC-3ʹ) and CO1-reverse (5ʹ-GAGCAACAACATAATAAGTATC-3ʹ)46; and ITS2-forward (5ʹ-CGACTTTCGAACGCATATTGC-3ʹ) and ITS2-reverse (5ʹ-GCTTAAATTCAGGGGGTAATCTCG-3ʹ)47. Each reaction mixture (20 µL) was composed of 20 ng of gDNA template, 1 µL of each primer (10 pmol), AccuPower® PCR preMix and master Mix (Bioneer, Korea), and ddH2O (to make up to 20 µL). The PCR thermal cycler protocol was optimized as follows: 94 °C (5 min); 5 cycles of 94 °C (20 s), 52 °C (30 s), and 68 °C (30 s); followed by 5 cycles of 94 °C (20 s), 50 °C (30 s), and 68 °C (30 s); 30 cycles of 94 °C (20 s), 48 °C (30 s), 68 °C (30 s); and the final 30 cycles of 94 °C (20 s), 46 °C (30 s), 68 °C (30 s); and the final extension step at 68 °C (5 min). After confirming the bands of CO1 (379 bp) and ITS2 (500 bp) using agarose gel electrophoresis, the bands were extracted and purified using a QIAquick gel extraction Kit (Qiagen). The purified PCR products were sequenced by Cosmogenetech Co, Ltd. (ROK).

Phylogenetic analysis

Identified nucleotide sequences from different adult and egg samples were subjected to BLASTn search against the NCBI nucleotide database. The nucleotide sequences were aligned using BioEdit and Cluster W software48,49. The phylogenetic tree based on the CO1 sequence was created using the neighbor-joining method with 1000 bootstrap replications50 in MEGA7 software51.