The 65th Medical Brigade and Medical Department Activity-Korea, in collaboration with the Migratory Birds Research Center, National Park Research Institute, conducted a migratory bird tick-borne disease surveillance program during 2014–2015 on two small, remote, uninhabited islands, Hong (Gull) Island, southern Gyeongnam Province, and Nan Island, western Chungnam Province, Republic of Korea (ROK). Argasid ticks were collected from Black-tailed Gull (Larus crassirostris) nestlings that had recently died and associated nest soil/litter, and all tick life history stages were identified morphologically. Because morphological keys are unreliable for the identification of adult and nymphal argasid ticks, identifications were confirmed by genotyping using polymerase chain reaction techniques. A total of 29 Ornithodoros capensis larvae and 2 Ornithodoros sawaii larvae were collected from 4 of 7 (57.1%) Black-tailed Gull nestlings that had recently died. An additional five O. capensis (2 males, 1 nymph, and 2 larvae) were collected from nest soil/litter. Only O. sawaii larvae (2/41, 4.9%) were collected from dead Black-tailed Gull nestlings on Nan Island. This is the first report of O. capensis from these seabird breeding islands.
Introduction
Migratory seabirds are hosts to both soft (argasid) and hard (ixodid) ticks and may transport exotic tick species and associated tick-borne pathogens to non-endemic areas during their long migrations between summer breeding and winter feeding grounds (Kohls 1957; Hughes et al. 1964; Amerson 1968; Nuttall 1984; Heath 1987, 2006, Hutcheson et al. 2005; Kawabata et al. 2006; Kim et al. 2009; Takano et al. 2009; Dietrich et al. 2011; Kang et al. 2013). However, tick ecology at seabird nesting sites is poorly understood because such sites are often located on remote, uninhabited or sparsely populated islands that are under government protection, making them inaccessible to the general public.
The genus Ornithodoros Koch contains the largest number of described argasid species (112/193), and many of these are commonly associated with various seabird hosts throughout the world (Asanuma et al. 1955; Asanuma 1960, 1965; Denmark & Clifford 1962; Yamaguti et al. 1971; Jonkers et al. 1973; Kitaoka & Suzuki 1973, 1974; Hoogstraal et al. 1976; Heath 1987, 2006; Murray et al. 1990; Keirans et al. 1992; Kawabata et al. 2006; Guglielmone et al. 2010; Gomez-Diaz et al. 2012; Vander Velde & Vander Velde 2013; Dupraz et al. 2016; Muñoz-Leal et al. 2017), including Korea (Kim et al. 2015, 2016a, b). However, the taxonomy of argasid ticks is confused because reliable morphological characters for identifying nymph and adult stages are often obscure or nonexistent. This report presents new host and distribution records for two ornithodorine ticks, Ornithodoros capensis Neumann, 1901, and Ornithodoros sawaii Kitaoka and Suzuki, 1973, based on collections from soil/litter at nesting sites of the Black-tailed Gull, Larus crassirostris Vieillot, 1818, and from dead chicks of this gull at two seabird breeding islands in the Republic of Korea(ROK).
Materials and methods
Survey area
The Migratory Birds Research Center, National Park Research Institute, conducted tick surveillance during the breeding season of Black-tailed Gull colonies at Hong (Gull) Island on 14 June and 17 July 2014, and 22 Jun 2015, and at Nan Island on 7 July 2015. Hong Island (Maejuk-ri, Hansan-myeon, Tongyeong-si, Gyeongnam Province; 34° 30′ N, 128° 50′ E) is a small (98,380 m2), remote, uninhabited island 50 km southeast of Tongyeong, a major mainland port city, and has been protected as a National Monument (No. 335) since 4 November 1982 (Fig. 1A, B). Nan Island (Gauido-ri, Geunheung-myeon, Taean-gun, Chungnam Province; 36°41′ N, 126°6′ E) is also a small (47,603 m2), remote, uninhabited island located 30 km west of Mohang, a major mainland port city, and has been a protected National Monument (No. 334) since 16 November 1982.
Tick collections
Dead Black-tailed Gull nestlings were individually sealed in plastic Ziploc® bags (30 × 40 cm) at the collection site (Fig. 2A, B), then placed in a Styrofoam cooler and transported to the Migratory Birds Research Center. Approximately 24 hrs after the nestlings were placed in the Ziploc bags, they were removed, leaving the detached ticks in the Ziploc bags, which were returned with the ticks to the Styrofoam cooler. In addition, Black-tailed Gull nest soil/litter (50–100 g) was collected using a small scoop and samples placed separately in Ziploc bags (25 × 28 cm) that were then transferred to a Styrofoam cooler and returned to the Migratory Birds Research Center. All ticks from the dead nestlings and the soil/litter samples were transported to the 5th Medical Detachment, Yongsan U.S. Army Garrison, Seoul, ROK. The soil and litter samples from each nest site were placed separately, according to date of collection, in 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 hrs, the ticks were removed, placed individually in 2 ml cryovials containing 80% EtOH, and labeled with a unique nest identification number, as described by Kim et al. (2015). Ticks from the dead nestlings also were placed in 70% EtOH and labeled with a unique collection number. All ticks were subsequently identified to stage of development and species using standard keys (Kohls 1957, Yamaguti et al. 1971, Kitaoka & Suzuki 1973). Three larvae (2014) and one male and eight larvae (2015) were submitted to the Korea National Institute of Health for specific identification by polymerase chain reaction (PCR).
FIGURE 1.
(A) Collection sites (•) of Ornithodoros capensis from nest soil/litter and recently dead nestlings of the Black-tailed Gull (Larus crassirostris Vieillot, 1818) on Hong (Gull) Island, Gyeongnam province, and Nan Island, Chungnam province, Republic of Korea [Tongyeong and Mohang (•), mainland port city]. (B) Collection records of O. capensis in Japan and Korea [previously reported (○) and this survey (•)]. (C) Distribution map of the Black-tailed gull (Larus crassirostris) (image from http://maps.iucnredlist.org/map.html?id=22694289).
PCR and sequencing analysis
Total DNA was prepared from individual ticks using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, with minor modification, and stored at -20°Cuntil used. PCR was performed using primer sets (mt-rrs1: 5-CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3 and mt-rrs2: 5-CCG GTC TGA ACT CAG ATC AAG TA-3) based on the mitochondrial 16S rDNA gene (mt-rrs) fragment, as previously described by Black and Piesman (1994) and Ushijima et al. (2003). PCR assays were performed using 50 µL of reaction mixture with TaKaRa ExTaq™ DNA polymerase (Takara, Shiga, Japan) at 94°C for 5 min, followed by 35 cycles for 10 sec at 94°C, 30 sec at 55°C, and 30 sec at 72°C, and final extension for 5 min at 72°C. PCR products were then purified using a QIAquick® Gel Extraction Kit (Qiagen, Hilden, Germany), cloned using pCR4-TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA, USA), and sequenced using ABI Prism BigDye™ Terminator v3.1Cycle Sequencing Kits and an ABI 3730xl sequencer (Applied Biosystems®, Foster City, CA, USA) at Macrogen, Inc. (Daejeon, ROK). Sequencing results were assembled using the SeqMan program implemented in DNASTAR software (version 5.0.6; DNASTAR Inc., Madison, WI, USA) to determine consensus sequences. Sequence data for the mt-rrs gene fragment were deposited in GenBank under Accession Numbers KY825206-KY825217.
Phylogenetic analysis
Sequence data for amplicons of the mt-rrs gene fragment were analyzed using MEGA 6.0 software ( http://www.megasoftware.net) (Tamura et al. 2013). Sequences for Ornithodoros spp. collected from nesting sites were aligned and compared with previously published Ornithodoros spp. and facilitated using the CLUSTALW method (Lasergene program version 5, DNASTAR Inc. Madison, WI). 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. Multiple alignments were performed using the same values. All positions containing alignment gaps and missing data were eliminated in pairwise sequence comparisons (pairwise deletion).
Results and discussion
Black-tailed Gulls build their nests on the ground on isolated islands that are difficult to access in the ROK. They are moderately large birds with a white body, gray wings, black tail, and yellow bill with red and black spots near the tip (Fig. 2A, B). They are distributed throughout much of East Asia, including the Democratic People's Republic of Korea, ROK, Japan, maritime Russia and eastern China (Won & Kim 2012, BirdLife International 2016) (Fig. 1C), but have also infrequently been observed in North America (Heinl 1997), Mexico (Garrett & Molina 1998) and the Philippines (Redman 1993). Although they are resident seabirds that are often seen in coastal seashore areas, they mate and raise their chicks from April–July on nearby isolated islands (Kwon et al. 2006), where they feed on small marine fish (Kwon et al. 2013).
In this study, tick surveillance was conducted on Hong and Nan Islands during the Black-tailed Gull breeding season (Fig. 2). A total of 29 Ornithodoros capensis larvae and two O. sawaii larvae were collected from 4/7 (57.1%) Black-tailed Gull nestlings that had recently died. Additionally, five O. capensis (2 males, 1 nymph, and 2 larvae) were collected from associated nest soil and litter (Table 1). While the morphological identification of Ornithodoros spp. nymphs and adults is often difficult or impossible, larval morphological characters are reported to be reliable (Jones & Clifford 1972, Kitaoka & Suzuki 1973). Larvae were morphologically determined as O. capensis based on the following taxonomic characters: dorsal plate pyriform, widest posteriorly, dorsal setae 22–25 pairs, dorsolateral setae 18–21 pairs, central setae 4 pairs, posthypostomal setae 2 pairs, hypostome with apex blunt and dentition 5/5 in the anterior third, 4/4 near mid-length, and 2/2 toward base.Because similarly reliable characters and keys for the identification of Ornithodoros nymphs and adults are unavailable, these stages were identified using PCR and amplicon cloning and sequencing for comparison with O. capensis and O. sawaii specimens collected in Japan, Korea, and other countries (Fig. 3).
TABLE 1.
Number and stage of development of Ornithodoros capensis and O. sawaii collected from recently dead nestlings and associated nest soil and litter of the Black-tailed Gull (Larus crassirostris) on Hong Island, Gyeongnam Province and Nan Island, Chungnam Province, Republic of Korea, 2014–2015.
FIGURE 2.
(A) Black-tailed Gull (Larus crassirostris) nest, and (B) breeding site on Hong Island, Gyeongnam Province, Republic of Korea (photographed by Chang-uk Park).
Sequence analysis showed that O. capensis larvae [KOR-H1406Y-1-8 (KY825206), KOR-H1406Y-2-2 (KY825207), KOR-H1407Y-53-1 (KY825208), and KOR-H1506-14 (KY825209)] collected from Black-tailed Gull nestlings on Hong Island and nymphs [KOR-N1507Y-14 (KY825215)] and larvae [KOR-N1507Y-6-6 (KY825210), KOR-N1507Y-6-7 (KY825211), KOR-N1507Y-6-9 (KY825212), KOR-N1507Y-6-11 (KY825213), KOR-N1507Y-6-13 (KY825114)] collected from Black-tailed Gull nestlings and nest soil and litter on Nan Island aligned most closely with O. capensis in GenBank. Ornithodoros capensis from Korea demonstrated 22–25 base pair (bp) differences and 94.1–95.4% identity in nucleotide (nt) sequences with O. capensis from breeding bird colonies in Japan (423 or 475 bp) (Table 2). These results constitute new distribution and host records for O. capensis in the ROK.
Sequence analysis of O. capensis collected in Korea, compared with O. capensis collected in other regions of the world (e.g., Japan, which is geographically close to Korea, the Pacific Ocean, Indian Ocean, Atlantic Ocean, Mediterranean Sea, and South America), demonstrates genetic differences that may be due to geographic isolation or preferred/available hosts. However, Dupraz et al. (2016) demonstrated that morphological variation, analyzed genetically and on the basis of tick size and shape, of at least five different species within the O. capensis complex varied strongly in relation to five different host types (shearwater, booby, gull, tern, and penguin) and weakly with geographical distribution. Ornithodoros capensis was collected only from the Black-tailed Gull in Korea, but in Japan it has been collected from this species and three others: Streaked Shearwater [Calonectris leucomelas (Temminck, 1835)], Black-footed Albatross [Phoebastria (=Diomedea) nigripes (Audubon, 1849)], and Ancient Murrelet [Synthliboramphus antiquus (Gmelin, 1789)] (Asanuma et al. 1955, Yamaguti et al. 1971, Tsurumi et al. 2002).
Sequence analysis (Table 2) of O. capensis collected on Hong and Nan Islands of Korea demonstrated 94.1–95.5% (mean 94.8%) identity with O. capensis from other areas and from the Japanese Islands. However, O. capensis from Peru, the Galapagos Islands (No. 28–30 in Table 2), and the Cape Verde and Canary Islands (No. 34–39) demonstrated 90.3–91.7% identity. These differences may be due to geographic isolation or preferred/available hosts. Additional molecular and morphological studies of immature and mature specimens are needed to determine the status of O. capensis throughout its geographic and host range (Fig. 3, Table 2).
Ornithodoros sawaii larvae [KOR-N1507Y-6-8 (KY825216), and KOR-N1507Y-6-10 (KY825217)], collected from a single dead Black-tailed Gull nestling on Nan Island, aligned most closely with O. sawaii. Comparatively, there were only 0–7 nt differences with 98.5–100% nt sequence identity between O. sawaii collected from Chilbal and Gugul Islands, ROK (Kim et al. 2015, 2016a, b, 2017), and 98.3–100% identity (0–7 nt differences) with O. sawaii collected from seabirds and nest soils in Japan (Tsurumi et al. 2002; Kawabata et al. 2006; Takano et al. 2014) (Fig. 3). Our collection therefore represents a new distribution record for O. sawaii in the ROK.
As seabirds migrate to the mainland, there is potential for the dispersal of soft ticks and associated pathogens. Rickettsia, Borrelia, and Coxiella spp. have been identified from O. capensis collected from C. leucomelas and L. crassirostris in Japan (Kawabata et al. 2006), from Brown Pelican (Pelecanus occidentalis Linnaeus, 1766) in the U.S. (Reeves et al. 2006), and from Sooty Tern [Onychoprion fuscatus (Linnaeus, 1766)], Wedge-tailed Shearwater [Puffinus (= Ardenna) pacificus (Gmelin, 1789)], Masked Booby (Sula dactylatra Lesson, 1831), and Red-footed Booby [S. sula (Linnaeus, 1766)] on Indian Ocean Islands (Dietrich et al. 2014). Recently, Kang et al. (2013) reported Anaplasma, Bartonella, and Borrelia spp. from Ixodes spp. collected from migratory birds on Hong (Red) Island (34o 41′ N, 125o 11′ E) 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 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 to determine the geographical distribution and host range of these ticks and, importantly, the specific pathogens carried by ticks associated with resident and migratory seabirds inhabiting islands and coastal areas of the ROK.
TABLE 2.
Matrix of genetic similarity for the 16S rRNA partial sequence of Ornithodoros capensis used in this survey.
FIGURE 3.
Phylogenetic analysis based on mt-rrs gene fragments of Ornithodoros spp. collected from nest soil/litter and recently dead nestlings of the Black-tailed Gull (Larus crassirostris). The phylogenetic tree was constructed based on NJ methods and bootstrap tests carried out according to the Kimura 2-parameter distances method. The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (1,000 replicates) was calculated. The phylogenetic branches were supported with more than 70% bootstrap values in this analysis. 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). The red and blue dots respectively denote the O. capensis and O. sawaii ticks in this study. The label includes country (KOR, the Republic of Korea), collection site (N for Nan Island, H for Hong Island), collection year, month and collection source (e.g., 1507Y for 2015 July from dead young bird, and 1507 for 2015 July from nest soil/litter sample), subject ID number for each Black-tailed Gull (e.g., Y-6-10) and developmental stage of the ticks.
Acknowledgments
We thank Dr. Jong-Gil Park, Chief, Migratory Birds Research Center, National Park Research Institute, Korea National Park Service, on Hong (Gull) Island, Gyeongnam Province and Nan Island, Chungnam Province, Republic of Korea, for collecting recently dead nestlings and nest soil and litter during seabird conservation and breeding surveys. The seabird survey was supported by “Bird Survey and Research,” Korea National Park Research Institute; the Armed Forces Health Surveillance Branch-Global Emerging Infections Surveillance and Response System (AFHSB-GEIS), Silver Spring, MD, USA; and the 65th Medical Brigade. The opinions expressed herein are those of the authors and are not to be construed as official or reflecting the views of the US Departments of the Army or Defense.
