Soft ticks, Family Argasidae, are commonly associated with remote rocky nesting grounds of seabirds and for that reason have not been thoroughly investigated [3,4]. The genus Ornithodoros consists of the largest number of described species (112 of 193) in the Family Argasidae [5] and is commonly associated with various sea bird hosts throughout the New and Old World, including Pacific Islands [6], North and South Americas [7-9], New Zealand [10-12], Australia [12], Antarctic [12], Japan [13-19], Russia [20], and Africa [3].

The Migratory Bird Research Center, National Park Research Institute, located on Heuksan Island, conducted a monitoring program on breeding seabirds at Sogugul (34˚11' N, 125˚07' E) and Gaerin Islands (34˚11' N, 125˚09' E) located 136 km southwest of Mokpo, Jeollanam-do (Province), Republic of Korea on 30 July and 1 August 2009 (Fig. 1A, B). Sogugul and Gaerin Islands are remote uninhabited islands that are protected as National Monuments (Sogugul and Gaerin Islands, No. 341, 13 August 1984) with a total land mass of 18,400 m2 , and 49,000 m2 , respectively. These islands are known to host breeding colonies of several seabirds such as Hydrobates monorhis (Swinhoe) (Swinhoe’s storm petrel), Synthliboramphus antiguues (Gmelin) (ancient murrelet), S. wumizusume Temminck (crested murrelet), and Calonectris leucomelas Temminck (streaked shear-water).

Nesting seabirds were captured by hand from their nest burrows under vegetation covers. Breeding adults captured from nests were carefully removed, identified to species, and then banded with a unique identification number. Prior to release, they were examined for ticks, particularly around the head, neck, wings, and abdomen, and ticks were carefully removed with a forceps to prevent injury to the bird. After removal, ticks were placed individually in cryovials containing 70% ethanol and labeled with a unique identification number that corresponded to the migratory bird collection data. Ticks were microscopically examined, developmental stages determined, and then identified to genus and species using morphological keys [16,17,21-23]. Field data that included the unique bird identification number, species, and other pertinent information were archived for future references.

PCR was performed using mt-rrs primer sets based on the mitochondrial 16S rDNA gene (mt-rrs) fragment previously described by Black and Piesman [24], Ushijima et al. [25], and Kim et al. [26]. The resulting product consisted of 475 base pairs, including the primer sets. Sequencing results were assembled using the SeqMan program implemented in DNASTAR software (version 5.0.6; DNASTAR Inc., Madison, Wisconsin, USA) to determine the consensus sequences. Sequence data were analyzed using the MEGA 6.0 software (http://www.megasoftware.net) [27]. For phylogenetic analysis, the neighbor-joining (NJ) and bootstrap tests were carried out according to the Kimura’s 2-parameter distance method [28,29].

A total of 9 larvae of Ornithodoros sawaii Kitaok and Suzuki were removed from the wings and abdomen of 2 adult H. monorhis captured on 31 July and 1 August in 2009 at Sogugul Island (6 larvae) and 2 adult H. monorhis at Gaerin Island (3 larvae) (Fig. 2A, B). O. sawaii and O. capensis Neumann are closely related to each other, and morphological differentiation is based on the shape of the dorsal plate, dentition of the hypostome, and outline of the basis capituli in the ventral aspect (Fig. 2C, D). To confirm these identifications, the ticks were also identified by PCR using partial mitochondrial 16S ribonucleic acid gene primer sets designed for the identification of soft ticks, and products were cloned and sequenced, and then compared with 16S rDNA gene fragments of O. capensis and O. sawaii in Japan and other countries (Fig. 3). Ornithodoros larvae collected from H. monorhis from Gaerin (KOR-G0908-100) Island most closely aligned with O. sawaii with 0-1 base differences and 99.8-100% identity compared to female, male, and nymph stages of O. sawaii collected from Chilbal Island [26], and other collection sites of Japan (Miyazaki, Shimane, Maizuru, Kyoto, and Ishikawa).

H. monorhis breeding pairs nest in colonies among rock crevices and burrows under Carex bootiana Hook grass roots during the breeding season. H. monorhis are small blackish seabirds with white rumps and distributed from southern Indonesia and Indian Ocean to northeast Asia, including the ROK [30,31]. Nesting populations on Gugul Islets (Sogugul, Gugul, and Gaerin Islands) make up approximately 75% of the global populations [31]. Nesting sites (e.g., Sogugul and Gaerin Islands) are remote small rocky volcanic outcroppings that are difficult to access and are therefore infrequently surveyed. Nesting soil and litter of the ancient murrelet, Synthliboramphus antiquus (Gmelin) and H. monorhis were collected at Chilbal Island, a small remote uninhabited island of south-western Jeollanam-do Province, ROK, and ticks were collected from the nest soil and litter using the Tullgren funnel [26]. A total of 12 H. sawaii (2 females, 4 males, and 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. antiquusi and from 6/20 soil and litter samples collected on 14-15 September 2014 during the nesting season of H. monorhis [26]. While H. monorhis occupies the same nesting sites as S. antiquusi, the breeding seasons of these 2 species do not overlap, and resident Ornithodoros larvae feed on young and adult birds of both species during their breeding season. This is the first collection report of O. sawaii larvae recovered from the wing and abdomen of H. monorhis in the ROK in this survey.

In Japan, O. sawaii was recorded from burrow nest of hosts, Calonectris leucomelas (Temminck) (streaked shear-water), and H. monorhis at Hanmya, Miyazaki, Shimane, Maizuru, Kyoto, and Ishikawa [17,19,32,33] (Fig. 1A).

Recently, Kang et al. [34] reported tick-borne pathogens belonging to the genera Anaplasma, Bartonella, and Borrelia from Ixodes spp. collected during 2008-2009 from migratory birds on Hong Island, Jeollanam-do Province. Hong Island is near Sogugul and Gaerin Islands where O. sawaii were collected from H. monorhis. These results do not conclusively identify the role of migratory birds as reservoirs, the introduction of exotic ticks in non-endemic areas, or the role of soft ticks in the potential maintenance of tick- borne pathogens, but do provide an insight into the potential role of migratory birds in the dispersal and infectious cycles of tick-borne diseases in Northeast Asia.

This report summarizes the first collection record of O. sawaii larvae, based on morphological and molecular identification, from 2 remote islands in the ROK. Further studies are needed to define the geographical distribution, host range, and pathogens present in ticks collected from nesting soil and litter, and resident and migratory seabirds inhabiting islands and coastal areas of the ROK.