Systematic & Applied Acarology 21(12): 1641–1650 (2016) http://doi.org/10.11158/saa.21.12.5 1641© Systematic & Applied Acarology Society ISSN 1362-1971 (print) ISSN 2056-6069 (online) http://zoobank.org/urn:lsid:zoobank.org:pub:10F72E8A-681A-45DB-A2CA-344C4C0F51E4 Ticks collected from Chilbal Island, Jeollanam Province, Republic of Korea, during 2014–2015 HEUNG-CHUL KIM1*, YOUNG-SOO KWON2*, MI-RAN KIM2, CHANG-UK PARK2, SEOK- MIN YUN3, WON-JA LEE3, SUNG-TAE CHONG2, TERRY A. KLEIN4 & RICHARD G. ROBBINS5,6 1 5th Medical Detachment, 168th Multifunctional Medical Battalion, 65th Medical Brigade, Unit 15247, APO AP 96205-5247, U.S.A. 2 Migratory Bird Research Center, Korea National Park Research Institute, Korea National Park Service, Jinmaeul-gil, Heuksan- myeon, Shinan-gun, Jeonnam Province 58863, Republic of Korea 3 Division of Arboviruses, Center for Immunology and Pathology, National Institute of Health, Korea Centers for Disease Control and Prevention, Cheongju-si, Chungbuk Province, 28159, Republic of Korea 4 MEDDAC-K/65th Medical Brigade, Unit 15281, APO AP 96205-5281, U.S.A. 5 Walter Reed Biosystematics Unit, Smithsonian Institution, MSC, MRC 534, 4210 Silver Hill Road, Suitland, MD 20746- 2863, U.S.A. 6 Corresponding author. E-mail: RobbinsRG@si.edu *The authors contributed equally to this work. Abstract The 65th Medical Brigade and Public Health Command District-Korea, in collaboration with the Migratory Birds Center, National Park Research Institute, conducted a migratory bird tick-borne disease surveillance program on Chilbal Island, a small, remote, uninhabited island in southwestern Jeollanam Province, Republic of Korea (ROK), during 2014–2015. Ticks were collected by dragging vegetation and from nest soil and litter of the Ancient Murrelet, Synthliboramphus antiquus, and Swinhoe’s Storm Petrel, Hydrobates monorhis, using Tullgren funnels. A total of 115 ticks belonging to three genera and three species were collected. Ornithodoros sawaii (98.3%, 113 ticks) was the most frequently collected tick species, followed by Ixodes signatus (0.9%, 1 nymph) collected from nest soil and litter, and Haemaphysalis flava (0.9%, 1 male) collected by tick drag. Key words: Ornithodoros sawaii, Ixodes signatus, Haemaphysalis flava, Synthliboramphus antiquus, Hydrobates monorhis, Korea Introduction Over the past few decades there has been a worldwide reemergence of zoonotic tick-borne pathogens that pose medical and veterinary health risks to wild and domestic animals, birds, and incidentally to humans (Jongejan & Uilenberg 2004, Heath & Hardwick 2011, Dantas-Torres et al. 2012). The role of migratory seabirds in the transportation of exotic tick species to their summer breeding and winter feeding grounds is poorly documented; most avian breeding sites are not accessible to the general public since they are often situated on remote and uninhabited or sparsely populated islands that are under government protection. It has been demonstrated that migratory birds transport ectoparasites, including ticks and associated tick-borne pathogens, along their migratory routes between breeding and overwintering grounds. These annual migrations result in the potential for exotic tick species to be transported over long distances to diverse environments in non-endemic regions (Kohls 1957, Hughes et al. 1964, Amerson 1968, Nuttall 1984, Heath 1987, 2006, Hutcheson et al. 2005, Article 1642 SYSTEMATIC & APPLIED ACAROLOGY VOL. 21 Kawabata et al. 2006, Kim et al. 2009, Dietrich et al. 2011, Kang et al. 2013). Recently, Kim et al. (2015) reported collecting ticks from nest soil and litter of the Ancient Murrelet, Synthliboramphus antiquus (Gmelin), and Swinhoe’s Storm Petrel, Hydrobates monorhis (Swinhoe), on Chilbal Island, a small, remote, uninhabited island of southwestern Jeollanam Province, Republic of Korea (ROK), using Tullgren funnels (WHO model, Yonsei University, Korea; height 60 cm, diameter 23 cm). A total of 12 Ornithodoros sawaii Kitaoka and Suzuki (2 females, 4 males, 6 nymphs) were collected from 3/36 nest soil and litter samples obtained on 1 May and 30–31 May 2014 during the nesting season of S. antiquus and from 6/20 soil and litter samples collected on 14–15 September 2014 during the nesting season of H. monorhis. While H. monorhis occupies the same nesting sites as S. antiquus, the breeding seasons of these two species do not overlap and resident Ornithodoros larvae feed on young and adult birds of both species during their breeding season. This report summarizes tick collections from nest soil and litter conducted on Chilbal Island during 2014 (Kim et al. 2015), with additional tick records obtained from tick drags of S. antiquus and H. monorhis breeding sites on Chilbal Island during 2014–2015. Materials and methods Survey area The Migratory Birds Center, National Park Research Institute, located on Heuksan Island, Heuksan- myeon (district), Sinan-Gun (county), Jeollanam Province, ROK, conducted conservation and breeding status surveys of S. antiquus and H. monorhis during 2014–2015 on Chilbal Island (34o47' N, 125o48' E), a protected National Monument (No. 332, 14 November 1982) with a total land area of 36,900 m2 located in Goseo-ri, Bigeum-myeon, Sinan-gun, Jeollanam Province, 64 km west of Mokpo, a major mainland port city (Fig. 1 and Fig. 2A, B). Tick collections On Chilbal Island the Ancient Murrelet nests in early spring, whereas Swinhoe’s Storm Petrel nests in fall. Although the nesting seasons of the two species do not overlap, they occupy the same nests. Using a small scoop, 50–100 g of soil and nest litter were collected from nesting areas during the nesting season of the Ancient Murrelet (1 and 31 May 2014, 9–10 April, 9–10 June 2015) and Swinhoe’s Storm Petrel (15 September 2014; 9 October 2015) (Fig. 2A, B). Soil and litter samples were placed in plastic Ziploc® bags (25 x 28 cm) and sent to the 5th Medical Detachment, Yongsan U.S. Army Garrison, Seoul, ROK. Soil and litter samples from each nest site were placed separately inside Tullgren funnels equipped with a 52W incandescent light bulb (heat source) at the top and a collection bottle (120 ml urine specimen container) at the base containing 50 ml of 70% ethanol (EtOH). After 24 hours of exposure, the material in each collection bottle was examined for arthropods and the ticks removed and placed individually in 2 ml cryovials containing 70% EtOH, labeled with a unique nest identification number, and then microscopically examined to determine developmental stage and genus using taxonomic identification keys (Kohls 1957, Kitaoka & Suzuki 1973). Additional tick collections were conducted by tick drag, as previously described by Chong et al. (2013), on 30 April and 1 May 2014, and ticks removed and similarly placed in 2 ml labeled cryovials of 70% EtOH. Ticks, excluding Ornithodoros spp., were identified to species using dissecting stereomicroscopes (x100) and keys developed by Yamaguti et al. (1971). Polymerase chain reaction (PCR) and sequencing analysis Because morphological identification of Ornithodoros spp. nymphs and adults is often unreliable, samples of the Ornithodoros spp. collected on Chilbal Island were identified by PCR using partial 16432016 KIM ET AL.: TICKS COLLECTED FROM CHILBAL ISLAND, REPUBLIC OF KOREA mitochondrial 16S ribonucleic acid gene (mt-rrs) primer sets developed for the identification of soft ticks and sequence analysis of O. capensis Neumann and O. sawaii in Japan and other countries (Ushijima et al. 2003, Kim et al. 2015). Total DNA was prepared from individual ticks using a DNeasy tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with minor modification and stored at -20°C until used. The resulting product consisted of 475 base pairs, including the primer sets. PCR assays were performed using 50 μL of reaction mixture with Takara Ex Taq™ DNA polymerase (Takara, Shiga, Japan) at 94°C for 5 min, followed by 35 cycles of 10 sec at 94oC, 30 sec at 55oC, 30 sec at 72oC, and a final extension step of 5 min at 72oC. PCR products were then purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and sequenced after cloning into pCR®4-TOPO® plasmid (Invitrogen, Carlsbad, California, USA), using Applied Biosystems (ABI) (Applied Biosystems, Foster City, California, USA) Prism® BigDye™ Terminator v3.1 Cycle Sequencing Kit, with an ABI 3730xl sequencer at Cosmogenetech, Co., Ltd. (Daejeon, ROK). Sequencing results were assembled using the DNASTAR® SeqMan v5.0.6 software, (DNASTAR Inc., Madison, Wisconsin, USA) to determine consensus sequences. FIGURE 1. Collection site of Ornithodoros sawaii and Ixodes signatus (○) from Ancient Murrelet (Synthliboramphus antiquus) and Swinhoe’s Storm Petrel (Hydrobates monorhis) nest soil and litter and Haemaphysalis flava taken by tick drag on Chilbal Island, Jeollanam Province, Republic of Korea, 2014–2015 [Mokpo (●), mainland port city] (image from http://map.daum.net). Phylogenetic analysis Sequence data were analyzed using MEGA 6.0 software (http://www.megasoftware.net) (Koichiro et al. 2013). Alignment and comparisons of the partial mt-rrs segment sequences for Ornithodoros spp., amplified from specimens of previously published Ornithodoros spp., were facilitated using the CLUSTALW DNASTAR Laser gene program, version 5. For phylogenetic analysis, neighbor- joining (NJ) and bootstrap tests were carried out according to the Kimura 2-parameter distance method (Kimura 1980, Saitou & Nei 1987). Pairwise alignments were performed with an open-gap penalty of 15 and a gap extension penalty of 6.66 and multiple alignments performed using the same values. All positions containing alignment gaps and missing data were eliminated in pairwise sequence comparisons (pairwise deletion). 1644 SYSTEMATIC & APPLIED ACAROLOGY VOL. 21 FIGURE 2. Chilbal Island. Lighthouse at apex of the island (A), steep rocky slope (B), nest burrow used by Ancient Murrelet (Synthliboramphus antiquus) with eggs (C), Swinhoe’s Storm Petrel (Hydrobates monorhis) burrow under roots of Carex bootiana, predominant grass species on Chilbal Island (D), Ancient Murrelet incubating eggs during nesting season (E), Swinhoe’s Storm Petrel in rock crevices during nesting season (F). Results and discussion A total of 113 O. sawaii (7 females, 11 males, 95 nymphs) were collected from nest soil and litter samples. A total of 8/78 (10.3%) samples were positive for O. sawaii (3 females, 1 male, 6 nymphs) during the breeding season of S. antiquus in the spring, and 11/50 (22.0%) were positive for O. sawaii (4 females, 10 males, 89 nymphs) during the breeding season of H. monorhis in the fall. Additionally, one Ixodes signatus Birula nymph was collected from nest soil and litter during the nesting season of S. antiquus in June 2015, and one Haemaphysalis flava Neumann male was collected from dragging grasses near the lighthouse during May 2014 (Table 1, Fig. 3). In 2014, DNA was detected from 10/12 Ornithodoros adults and nymphs and the mt-rrs sequenced. Previously submitted GenBank accession numbers were: females, KP730690 (KOR- C1405-5), and KT372792 (KOR-C1409-20); males, KP730691 (KOR-C1409-2), KP730692 (KOR- C1409-10), KT372787 (KOR-C1405-5-2), and KT372790 (KOR-C1409-4); nymphs, KP730693 (KOR-C1405-E4), KT372788 (KOR-C1405-5-3), KT372789 (KOR-C1405-5-4), and KT372791 (KOR-C1409-18) collected from Chilbal Island (Kim et al. 2015); and larvae, KP899267 (KOR- G0908-100) collected from Gaerin Island, Jeollanam Province, ROK (Kim et al. 2016). In 2015, DNA was detected from 82/101 adults and nymphs and the mt-rrs gene sequenced and confirmed as 16452016 KIM ET AL.: TICKS COLLECTED FROM CHILBAL ISLAND, REPUBLIC OF KOREA O. sawaii. Two females, 2 males, and 6 nymphs were maintained as voucher specimens at the 5th Medical Detachment, and 9 samples were negative by PCR. Accession numbers from 9/82 O. sawaii positive samples submitted to GenBank were: females, KX227701 (KOR-C1506-A4), KX227703 (KOR-C1510-8-40), and KX227704 (KOR-C1510-8-41); males, KX227705 (KOR-C1510-8-42), KX227706 (KOR-C1510-8-43), and KX227707 (KOR-C1510-8-44); nymphs, KX227700 (KOR- C1506-A3), KX227702 (KOR-C1510-7-1), and KX227708 (KOR-C1510-9-2). Only 0–3 base differences were observed with 99.4-100% similarity in nucleotide sequences between O. sawaii collected from Gaerin Island in 2009, Chilbal Island in 2014, and other collection sites (Miyazaki, Shimane, Kyoto, Kutsujima, and Ishikawa) in Japan (Fig. 4). The mt-rrs gene sequences of ticks identified in this study collected in 2015 were deposited in GenBank under accession numbers (KX227700-KX227708). FIGURE 3. Dorsal and ventral view of Ornithodoros sawaii female (A, B), male (C, D), nymph (E, F), and Ixodes signatus nymph (G, H) collected from nest soil and litter of the Ancient Murrelet (Synthliboramphus antiquus) and Swinhoe’s Storm Petrel (Hydrobates monorhis) during June and October 2015, and Haemaphysalis flava male (I, J) collected by tick drag on May 2014 at Chilbal Island, Jeollanam Province, Republic of Korea (all scale bars, 1,000µm). In Japan, O. sawaii has been recorded from two seabird species, the Streaked Shearwater, Calonectris leucomelas (Temminck), and Swinhoe’s Storm Petrel (Kitaoka & Suzuki 1973, 1974, Kawabata et al. 2006, Takano et al. 2014). Our survey showed that O. sawaii was associated with nests occupied by both S. antiquus and H. monorhis during the spring and fall, respectively. Ancient Murrelets are medium-sized blackish seabirds that nest in colonies in rock crevices and burrow under the roots of Carex bootiana Hooker and Arnott (predominant grass species on Chilbal Island), where the females lay eggs (Fig. 2C, E). Their range extends from the Yellow Sea (islands off China and Korea), Taiwan, Hong Kong, islands in the East Sea along the Korean Peninsula, the Russian Pacific coast and the Aleutian Islands to the Haida Gwaii (Queen Charlotte) archipelago of British Columbia, Canada, and as far south as the southern coast of California, USA (Del Hoyo et al. 1996, BirdLife International 2014a). Many of the islands where Ancient Murrelets nest are uninhabited and not readily accessible and are therefore infrequently surveyed. 1646 SYSTEMATIC & APPLIED ACAROLOGY VOL. 21 FIGURE 4. Phylogenetic analysis based on mt-rrs gene of Ornithodoros sawaii collected from Ancient Murrelet (Synthliboramphus antiquus) and Swinhoe’s Storm Petrel (Hydrobates monorhis) nest soil and litter on Chilbal Island, Jeollanam Province, Republic of Korea, 2015. The PCR product consisted of 475 bp, including the primer sets. Phylogenetic trees were constructed based on NJ methods and bootstrap tests carried out according to the Kimura 2-parameter distances method. The percentages of replicate trees in which the associated taxa are clustered together in the bootstrap test (1,000 replicates) were calculated. In this analysis, the phylogenetic branches were supported with more than 70% bootstrap values. The length of the bar corresponds to the degree of sequence divergence. All positions containing alignment gaps and missing data were eliminated in pairwise sequence comparisons (pairwise deletion). Ancient Murrelets arrive on Chilbal Island from early April to June, a month before egg-laying. After rearing their chicks, they move to their coastal summer feeding grounds. Swinhoe’s Storm Petrels arrive on Chilbal and other nearby islands from early June to October and occupy the same nesting sites as the Ancient Murrelet (Park & Takeshi 2011). Swinhoe’s Storm Petrels are small blackish seabirds with white rumps and a range that extends from southern Indonesia and the Indian Ocean to northeastern Asia [Russia (south of Vladivostok), Japan, Korea, and Taiwan] (BirdLife International 2014b) (Fig. 2D, F). The population dynamics of O. sawaii in the context of this host transition merit investigation. 16472016 KIM ET AL.: TICKS COLLECTED FROM CHILBAL ISLAND, REPUBLIC OF KOREA Ornithodoros sawaii is closely related to O. capensis, a species with a broad distribution that includes North Atlantic, Pacific, and Indian Ocean islands as well as coastal areas of South Africa, New Zealand, and southern Australia (Kohls 1957, Heath 1987). Ornithodoros sawaii was first described in 1973, but its known distribution includes only Japan (Hanmya Island, Miyazaki, Shimane, Kyoto, Kutsujima, and Ishikawa) (Kitaoka & Suzuki 1973, 1974, Kawabata et al. 2006, Takano et al. 2014) and the Republic of Korea (Kim et al. 2015). TABLE 1. Records of ticks collected by tick drag and from nest soil and litter (Tullgren funnel) of the Ancient Murrelet (Synthliboramphus antiquus) and Swinhoe’s Storm Petrel (Hydrobates monorhis) on Chilbal Island, Jeollanam Province, Republic of Korea, 2014–2015. *One male of Haemaphysalis flava collected by dragging grasses near the lighthouse located at the apex of Chilbal Island on 1 May 2015. Haemaphysalis flava, originally described from Japan (Yamaguti et al. 1971), has long been known from mainland Korea and its islands (Kishida 1936). One male H. flava was collected on 1 May 2015 while dragging grasses near a lighthouse located at the apex of Chilbal Island. Ixodes signatus was first reported in 1968–1969 (4 females, 1 male); later in 1982 (21 females, 1 male, 4 larvae) from the Daurian Starling, Sturnia sturnia (Pallas), Japanese Cormorant, Phalacrocorax capillatus (Temminck and Schlegel), and Pelagic Cormorant, P. pelagicus Pallas, captured in Seoul Metropolitan City, and at Namhae, Koejae, Gyeongnam Province (Noh 1969, 1983); and in 1987 (3 nymphs) from the White-tailed Sea Eagle, Haliaeetus albicilla L., captured at Seongsan, Jeju-do (Province) (Kang et al. 1987). In our survey, one nymph was collected from nest soil on Chilbal Island during the nesting period of the Ancient Murrelet (Fig. 3). The role of migratory seabirds as reservoirs of tick-borne diseases and in transporting exotic tick species and associated pathogens to avian summer breeding and winter feeding grounds is poorly documented. Ornithodoros spp. have been collected from seabirds, and nymphs and adults can survive without feeding for long periods between nesting seasons, resulting in the infestation of naïve birds that return to nesting sites, as well as their chicks. Additionally, birds may become infected with novel pathogens that are transmitted while at their feeding and/or breeding sites. Recent surveys have demonstrated Rickettsia and Borrelia spp. in O. sawaii collected from C. leucomelas and H. monorhis in Japan (Kawabata et al. 2006, Takano et al. 2009). More recently, Kang et al. (2013) reported Anaplasma, Bartonella, and Borrelia spp. from Ixodes spp. collected Year Host bird Month Infested/ tested soil samples Infestation rates (%) Ornithodoros sawaii Ixodes signatus Haemaphysalis flava* Nymph Male Female Nymph Male 2014 Ancient Murrelet (Synthliboramphus antiquus) MAY 3/36 8.3 4 1 1 Swinhoe’s Storm Petrel (Hydro- bates monorhis) SEP 6/21 28.6 2 3 1 Drag MAY 1 2015 Ancient Murrelet (Synthliboramphus antiquus) APR 0/3 0 JUN 5/39 12.8 2 - 2 1 Swinhoe’s Storm Petrel (Hydro- bates monorhis) OCT 5/29 17.2 87 7 3 Total 19/128 14.8 95 11 7 1 1 113 (98.26%) 1 (0.87%) 1 (0.87%) 1648 SYSTEMATIC & APPLIED ACAROLOGY VOL. 21 from migratory birds on Hong Island (34o 41'N, 125o 11'E), Jeollanam Province, located near Chilbal Island, during 2008–2009. Such results do not necessarily mean that migratory birds are reservoirs of these pathogens or responsible for the introduction of exotic ticks and associated pathogens into non-endemic areas, but they hint at the potential role of migratory birds in the dispersal of ticks and tick-borne microbial agents in northeastern Asia. Further studies are needed in order to define the geographical distribution, host range, and specific pathogens associated with ticks collected from resident and migratory seabirds inhabiting islands and coastal areas of the ROK. Acknowledgments We thank the staff of the Migratory Birds Center, National Park Research Institute, Korea National Park Service, on Heuksan Island, Jeollanam Province, Republic of Korea, for collecting nest soil and litter during seabird conservation and breeding surveys. 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