ABSTRACT

Ticks and tick-borne diseases are important issues worldwide because of their effects on animal and human health. The genus Ornithodoros, which is included in the family Argasidae, is typically associated with wild animals, including seabirds. In this study, samples from the nests of seabirds and surrounding soil were collected to investigate Ornithodoros spp. from 9 uninhabited islands in the western, eastern, and southern parts of Korea from April 2017 to October 2018. The islands are known as the breeding places of migratory and resident birds. Ticks were collected from soil and nest material of seabirds using a Tullgren funnel and identified using 16S rRNA and the cytochrome c oxidase 1 gene (COI), and host animals of soft ticks were identified using the mitochondrial DNA cytochrome b gene by a polymerase chain reaction. In the sequence identity of the 16S rRNA gene fragment of Ornithodoros sp., Ornithodoros sawaii was identified as the closest homologous sequence, and the new Ornithodoros sp. was newly identified. We found that the newly identified Ornithodoros sp. in the Republic of Korea was located in uninhabited islands used as breeding places by the black-tailed gull, Larus crassirostris.

Ticks and tick-borne diseases are a major health threat to both humans and animals and cause significant economic losses worldwide. The Family Argasidae, including the genera Argas, Ornithodoros, Nothoaspis, and Otobius, does not have a scutum and is mainly distributed in tropical and subtropical regions in seabird colonies. Ornithodoros spp., which are part of the family Argasidae, are ectoparasites, and their hosts are mainly migratory birds and mammals; their environmental distribution may be affected by bird migration (Kawabata et al., 2006), or they can be spread by bats (Anoura caudifer) or members of the family Suidae such as warthogs (Phacochoerus aethiopicus) and bushpigs (Potamochoerus porcus) (Anderson et al., 1998). Ornithodoros spp. can transmit various pathogens, including Coxiella sp. (Reeves, 2008), Rickettsia spp. (Reeves et al., 2006), and Borrelia lonestari from seabirds, African swine fever virus (ASFV) from members of Suidae, and others. Among the Ornithodoros genera, the Ornithodoros capensis complex contains a large number of described Argasidae species and is the most widespread species around the equator, affecting seabird populations along the coasts of the Pacific, Atlantic, and Indian Oceans (Guglielmone et al., 2010; Dietrich et al., 2014). Ornithodoros capensis was first reported in 1901 by Neumann and has been reported in numerous avian nests in Africa (Gómez-Díaz et al., 2012), Europe (Dupraz et al., 2016), Oceania (Ramsay, 1968; Humphery-Smith and Moorhouse, 1981), North America (Eggert and Jodice, 2008), and Asia (Kawabata et al., 2006). Ornithodoros sawaii, which is phylogenetically closely related to O. capensis, was first identified in Japan (Kitaoka and Suzuki, 1973) and subsequently reported only in Japan (Kawabata et al., 2006; Takano et al., 2009) and Korea in the nest soil and litter of Synthliboramphus antiquus, Hydrobates monorhis, and Larus crassirostris (Kim et al., 2015, 2016, 2017).

Various pathogens, including Rickettsia spp., Coxiella sp., Borrelia spp., and the Johnston Atoll and Abal viruses, have been detected in O. capensis globally distributed around the equator line (Dietrich et al., 2011). In Japan, Rickettsia was detected in O. capensis captured from the black-footed albatross (Kawabata et al., 2006). Additionally, a few pathogens, such as Rickettsia sp. and Borrelia sp., were detected in O. sawaii collected from the migratory seabirds Swinhoe's storm petrel and streaked shearwater only in Japan (Kitaoka and Suzuki, 1973; Kawabata et al., 2006; Takano et al., 2009). This study was conducted to determine the distribution and identity of soft ticks collected from the soil of nests of migratory birds that are potential hosts in Southeast Asia.

In this study, we discovered a new Ornithodoros species from the nests and soil material of L. crassirostris and detected evidence of soft tick habitation in the uninhabited (by humans) islands of the Republic of Korea.

MATERIALS AND METHODS

Tick collection

The sites of tick collection were selected based on the habitat of migratory birds in uninhabited islands (Nan-do, 36°39′34.01″N, 125°49′24.58″E, in Chungcheongnam-do province; Chilsan-do, 35°19′20.20″N, 126°16′37.01″E; Chilbal-do, 34°47′16.46″N, 125°47′18.99″E, and Gugul-do, 34°6′59.44″N, 125°5′7.39″E; Gaerin-do, 34°6′57.81″N, 125°5′37.80″E; Sogukhol-do, 34°7′4.60″N, 125°4′40.53″E in Jeollanam-do province; Sasu-do, 33°55′15.08″N, 126°38′16.85″E, in Jejudo province; Hong-do, 34°32′14.35″N, 128°43′58.14″E, in Gyeongsangnam-do province; and Dok-do, 37°14′24.38″N, 131°52′13.75″E in Gyeongsangbuk-do) from July to October 2017 and April to October 2018 (Fig. 1). Among these islands, Chilbal-do, Nan-do, Hong-do, and Chilsan-do are known as breeding sites of L. crassirostris (Fig. 2a, b), and Chilbal-do, Chilsan-do, Gaerin-do, Sogukhol-do, and Gugul-do are known as breeding sites of H. monorhis (Fig. 2c, d). Samples were collected in zipper bags from nest litter and soil material of migratory birds on the islands. The collected nests with soil were moved to the laboratory. Soil and litter samples from each nest site were placed separately into Tullgren funnels using a scoop. A Tullgren funnel with an incandescent heating light bulb was set up with an Erlenmeyer flask below containing 70% ethanol (Fig. 2e, f) (Kim et al., 2015). After 24 hr of radiating heat, the soft ticks entered the flask. These ticks were removed from the flask using a filter and forceps, placed in 2-ml tubes, and labeled with their collection data. Subsequently, all ticks were identified to determine their genus levels and developmental stages using taxonomic identification keys based on microscopic examination (Yamaguti et al., 1971). After identification, the ticks were placed individually into 1.5-ml tubes containing 70% ethanol.

Scanning electron microscopy (SEM)

For morphological analysis, collected tick specimens were placed in Karnovsky's fixative for 4 hr for primary fixation. The ticks were washed with 0.05-M sodium cacodylate buffer 3 times for 10 min each time. After fixation, the ticks were treated with 9.1-M cacodylate buffer containing 2% osmium tetraoxide for 2 hr and then dehydrated by immersion in increasing concentrations of ethanol from 30% to 100%. Specimens were dried using a Critical Point Dryer (Leica, Wetzlar, Germany) and examined by field-emission SEM (Carl Zeiss, Oberkochen, Germany) (Fig. 3).

Molecular identification of tick species and host gene

DNA extraction:

To perform molecular identification of tick species, the ticks were homogenized using a Beadbeater TissueLyser II (Qiagen, Hilden, Germany) according to the manufacturer's instructions with 200 μl of lysis buffer, 40 μl of proteinase K, and 2-mm-diameter stainless beads at 30 Hz/sec for 7 min, followed by lysis at 56 C for 60 min. Genomic DNA was extracted according to the protocol of the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland) and stored at −20 C until use.

Polymerase chain reaction (PCR) amplification:

PCR was performed using primer sets based on the mitochondrial 16S rRNA gene (mt-rrs) fragment (Black and Piesman, 1994) and using 3 primer sets designed in this study (GenBank accession number KJ133587) based on the mitochondrial 16S rRNA full-length gene (Fig. 4). For precise molecular identification, mitochondrial gene fragments of cytochrome c oxidase 1 (COI) were also amplified by PCR (Table I).

To analyze the host animals of soft ticks, the mitochondrial DNA cytochrome b gene was amplified by conventional PCR (Kocher et al., 1989). PCR amplification of the host animal DNA was conducted using primer sets L14841: 5′-AAA AAG CTT CCA TCC AAC ATC TCA GCA TGA TGA AA-3′, and H15149: 5′-AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTC A-3′. PCR assays were performed in a 50-μl reaction mixture with Takara Taq DNA polymerase (Takara, Shiga, Japan) at 94 C for 1 min, followed by 30 cycles for 45 sec at 94 C, 45 sec at 50 C, and 45 sec at 72 C, and a final extension step of 5 min at 72 C.

Cloning, sequencing, and phylogenetic analysis

The amplified PCR products were purified using a Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. After purification, the PCR products were cloned into the pGEM®-T Easy Vector Systems (Promega, Madison, Wisconsin), followed by transformation into Escherichia coli DH5a cells, and then the cells were plated onto LB agar containing 100 μg/ml of ampicillin. Recombinant clones were selected by blue-white screening. Plasmid DNA for sequencing was purified using the MG™ Plasmid SV Miniprep kits (Macrogen, Seoul, Korea). Purified recombinant plasmid DNA was sequenced using a T7 and SP6 promoter primer set by dideoxy termination with an automatic sequencer (ABI 3730xl capillary DNA sequencer, Applied Biosystems, Foster City, California). The obtained sequences were evaluated with Chromas software (Ver. 2.6.2, http://technelysium.com.au/wp/chromas/) and aligned by Clustal X2 (Ver 2.0, http://www.clustal.org/). Phylogenetic trees were constructed using the maximum-likelihood method in the Kimura 2-parameter model with the MEGA 7 program (1,000 bootstrap replicates) (Kimura, 1980; Kumar et al., 2016). The nucleotide sequences generated were deposited into GenBank under accession numbers MK389598–MK389641, MK605995–MK606024, MK606031–MK606060, and MK613790–MK613833 for the 16S rRNA gene and MK836055–MK836059 and MT040624 for COI.

RESULTS

In this study, tick surveillance was conducted on Korean islands during the breeding season of migratory birds, including L. crassirostris and H. monorhis, from July to October 2017 and from April to October 2018. Nests and soil material of migratory birds were collected to capture argasid ticks from uninhabited islands. In total, 77 ticks were collected from 410 nests with soil: 30 ticks from Chilsan-do, 19 ticks from Chilbal-do, 8 ticks from Gugul-do, 2 ticks from Sogukhol-do, 16 ticks from Gaerin-do, 1 tick from Nan-do, and 1 tick from Hong-do (Table II).

To morphologically identify the argasid ticks, we evaluated a total of 77 Argasidae ticks by optical microscopy; however, significant differences between species were not observed in the images. Therefore, three randomly selected soft tick specimens were used for SEM analysis. SEM images of the argasid tick specimens were acquired, but no significant differences were observed between species. Thus, PCR was conducted using 74 argasid ticks, and phylogenetic analysis was conducted for the mitochondrial gene 16S rRNA by PCR. To determine the host animals of ticks, mitochondrial DNA cytochrome b gene sequences were amplified from the tick samples (Suppl. Data, Fig. S1).

Phylogenetic analysis showed that the Argasidae ticks detected in this study were grouped in 2 clades, which showed 89.1% identity to O. sawaii (GR4) and a new Ornithodoros sp. (CS1) in the 16S rRNA full gene (Fig. 5). Table III shows 88.4 to 89.0% identity in the 16S rRNA full gene when comparing Ornithodoros sp. (domestic isolate, MK613790), O. sawaii (domestic isolate, MK606044), O. capensis (South Africa, KR907245), O. capensis (South Africa, KJ133586), and O. capensis (Japan, AB075953) sequences. The sequence identity matrix in Table IV was generated from the 74.4 to 94.4% range of identities between the sequences of the new Ornithodoros sp. and Ornithodoros spp. from the GenBank database, where only more than 80.0% identities were selected. Table IV shows 80.1 to 94.4% identity in partial 16S rRNA when comparing Ornithodoros sp. (domestic isolate, MK613790), O. sawaii (domestic isolate, MK606044), and Ornithodoros spp. from NCBI. The identity between the new Ornithodoros sp. and O. capensis ranged from 90.0 to 94.4% (Table V). All sequences of O. capensis were analyzed, and the overlapping data were deleted. Thirty O. sawaii (40.5%) samples were collected from the H. monorhis nests with soil from Chilbal-do, Chilsan-do, Gaerin-do, Sogukhol-do, and Gugul-do, and 44 new Ornithodoros sp. (59.5%) samples were collected from L. crassirostris nests with soil from Chilbal-do, Nan-do, Hong-do, and Chilsan-do. To evaluate the genetic relationship between O. sawaii and the new Ornithodoros sp., both 16S rRNA gene nucleotide sequences were independently analyzed with other reference sequences available in the GenBank database. Additionally, randomly selected soft tick specimens were molecularly analyzed for COI by PCR for more precise identification.

Phylogenetic analysis of COI revealed 2 clusters with new Ornithodoros sp. and O. sawaii (Fig. 6). Table VI shows 73.3 to 84.4% identity in COI when comparing new Ornithodoros sp. (domestic isolate, MK836055) and Ornithodoros spp. from the GenBank database, and Table VII shows 86.8 to 88.1% identity in COI when comparing new Ornithodoros sp. (domestic isolate, MK836055) and O. capensis from the GenBank database. All sequences of O. capensis were analyzed, and the overlapping data were deleted. Ornithodoros sawaii could not be analyzed due to the lack of sequence. Tables VIII and IX show the alignment of the variable regions V1 and V2 of the 16S rRNA gene sequences from the new Ornithodoros sp. and other Ornithodoros species, respectively. Aligned sequences were edited to the overlapped length, and a sequence identity matrix was generated for the alignments. The percentage of sequence identity was calculated on the alignments from the 332-bp segment of the 16S rRNA gene containing both the V1 and V2 regions (Fig. 4).

The cytochrome b gene fragment was sequenced to identify the host animals. In total, 16 sequences were acquired from 74 samples, including 9 samples from Gaerin-do, 1 sample from Chilbal-do, and 6 samples from Chilsan-do. According to sequencing analysis of the 16 amplicons, black-tailed gulls (L. crassirostris = 6) were detected in samples from Chilsan-do, and Swinhoe's storm petrel (H. monorhis = 10) were detected in samples from Gaerin-do and Chilbal-do. The results showed that the sequences collected from Gaerin-do and Chilbal-do had 100% identity with the H. monorhis isolate from the United Kingdom (HG975295), and the sequences collected from Chilsan-do had 99% identity with L. crassirostris from South Korea (KM507782). The sequence analyses of the new Ornithodoros sp. and O. sawaii were aligned using multiple sequence alignment with hierarchical clustering (http://multalin.toulouse.inra.fr/multalin/) to compare their differences (Fig. 7) (Corpet, 1988). Figure 7 shows invariant sequences except for positions at A529, G589, and C659 in the all-new Ornithodoros sp. from this study. This shows the exact locality of the invariant but unique sites for the new Ornithodoros sp. and O. sawaii. The sequence analyses of the new Ornithodoros sp., O. sawaii, and O. capensis were also aligned to compare their differences, and these results are shown in Figure 8.

DISCUSSION

As described above, Gaerin-do, Sogukhol-do, Gugul-do, Chilbal-do, Chilsan-do, Hong-do (Hallyeohaesang), and Nan-do are uninhabited islands that are used as mass breeding places for migratory birds. Chilsan-do, Hong-do (Hallyeohaesang), and Nan-do are well known as the mass breeding place for L. crassirostris colonies, which are common resident birds in the western, southern, and eastern seas around the Republic of Korea and distributed mostly in the Far East, including in Japan, southwestern Russia, and eastern China (Kwon et al., 2006); these birds have also been infrequently observed in Mexico (Garrett and Molina, 1998). Although these resident seabirds are often observed in mainland coastal areas, they mate and raise their chicks from early April to late August on nearby uninhabited islands (Lee and Yoo, 2005; Kwon et al., 2006).

There are currently 12 described species of soft ticks associated specifically with seabirds, all of which belong to the Argas and Ornithodoros genera (Dietrich et al., 2011), including O. sawaii collected in this survey and the newly identified new Ornithodoros sp., which is associated with migratory birds including seabirds. The samples of nests with soil containing Ornithodoros spp. soft ticks were collected from June to October 2017 and April to October 2018. Therefore, the nests formed on the ground by migratory birds may create a habitat for Ornithodoros spp. Among the uninhabited islands investigated, the new Ornithodoros sp. was identified on the western islands, which are the breeding sites of L. crassirostris. We selected western and southern islands because migratory birds migrate to these islands from East Asia and North America to breed and raise their chicks.

In Japan, O. sawaii has been recorded from 2 seabird species, the streaked shearwater, Calonectris leucomelas (Temminck), and Swinhoe's storm petrel, H. monorhis (Kitaoka and Suzuki, 1973; Kawabata et al., 2006; Takano et al., 2014). In South Korea, O. sawaii has been reported from ancient murrelet, Synthliboramphus antiquus, H. monorhis (Kim et al., 2015, 2016), and L. crassirostris (Kim et al., 2017; this study). As the feeding time of soft ticks is shorter than that of hard ticks, their repletion time can be less than 1 hr (Dietrich et al., 2011), and thus the dispersal of soft ticks through the host may be more limited and occur only in a very restricted space (Heath, 1987). This suggests that the new Ornithodoros sp. collected in this study has lived on these uninhabited islands and formed colonies for a long time. Ornithodoros sawaii, however, was detected on Gugul-do and Chilbal-do near Japan and may have been transported by Japanese migratory birds. It is well known that ticks are ectoparasites; however, this does not mean that they transmit pathogens, as their locations on the uninhabited islands are not accessible to the public. The species L. crassirostris breeds and raises its chicks on uninhabited islands, where it remains throughout the coldest season. It then moves to the coast for feeding, living near humans; therefore, the possibility that migratory birds function as vectors of pathogens cannot be completely excluded.

This study has some limitations. Studies are needed to evaluate tick-borne pathogens from soft ticks collected in this study such as Borrelia spp. and Rickettsia spp., among others. Whole-genome sequencing of the new Ornithodoros sp. and O. sawaii has not been conducted in South Korea; only full-length 16S rRNA gene sequencing was conducted. Thus, additional studies are needed.

In this study, we found that the new Ornithodoros sp. inhabited the western islands Chilsan-do, Hong-do, and Nan-do and were mainly bred and reared by the black-tailed gull. This indicates that black-tailed gull (Chilsan-do and Nan-do) and H. monorhis (Chilbal-do) dominate these islands (respectively). This is the first record of the new species that was found in 44 soft ticks collected from nests (with soil material) of L. crassirostris on the breeding islands Nan-do, Chilsan-do, Chilbal-do, and Hong-do (Hallyeohaesang). We found molecular differences for the new Ornithodoros sp. via a significantly different sequence from the 16S rRNA gene and COI gene, but not morphological differences. Significant variability was found in the nucleotide sequences of O. capensis, the new Ornithodoros sp., and O. sawaii in the partial sequence of 16S rRNA. We also found that the host of the new Ornithodoros sp. was L. crassirostris. Further studies are needed to investigate the pathogens of ticks and additional inhabited western and eastern islands to collect ticks to obtain more detailed information.

ACKNOWLEDGMENT

This research was supported by a fund (no. Z-1543085-2017-18-01) from the Research of Animal and Plant Quarantine Agency, the Republic of Korea.

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