Construction of a DNA Database for Ticks Collected in Japan: Application of Molecular Identi Cation Based on the Mitochondrial 16S Rdna Gene

〔Med. Entomol. Zool. Vol. 65 No. 1 p. 13‒21 2014〕 13 reference DOI: 10.7601/mez.65.13

Construction of a DNA database for ticks collected in Japan: application of molecular identi cation based on the mitochondrial 16S rDNA gene

Ai Takano*, 1), Hiromi Fujita2), Teruki Kadosaka3), Mamoru Takahashi4), Takeo Yamauchi5), Fubito Ishiguro6), Nobuhiro Takada7), Yasuhiro Yano7), Yosaburo Oikawa 8), Toshirou Honda9), Mutsuyo Gokuden10), Takashi Tsunoda11), Miyako Tsurumi12), Shuji Ando13), Masako Andoh13), Kozue Sato14) and Hiroki Kawabata 14)

* Corresponding author: Department of Veterinary Medicine, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677‒1 Yoshida, Yamaguchi 753‒8515, Japan (E-mail: [email protected]) 1) Department of Veterinary Medicine, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677‒1 Yoshida, Yamaguchi 753‒8515, Japan 2) Mahara Institute of Medical Acarology, Anan, Tokushima 779‒1510, Japan 3) Department of Infection and Immunity, Aichi Medical University, Nagakute, Aichi 480‒1195, Japan 4) Department of Anesthesiology, Saitama Medical University, Iruma-gun, Saitama 350‒0495, Japan 5) Toyama Institute of Health, Imizu, Toyama 939‒0363, Japan 6) Fukui Prefectural Institute of Public Health and Environmental Science, Fukui, Fukui 910‒8551, Japan 7) Faculty of Medical Sciences, University of Fukui, Yoshida-gun, Fukui 910‒1193, Japan 8) Department of Medical Zoology, Kanazawa Medical University, Kahoku-gun, Ishikawa 920‒0293, Japan 9) Kaseda Public Health Center, Minamisatsuma, Kagoshima 897‒0001, Japan 10) Kagoshima Prefectural Institute for Environmental Research and Public Health, Kagoshima, Kagoshima 892‒0836, Japan 11) Department of Vector Ecology and Environment, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Nagasaki 852‒8523, Japan 12) Yamashina Institute for Ornithology, Abiko, Chiba 270‒1145, Japan 13) Department of Virology-I, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162‒8640, Japan 14) Department of Bacteriology-I, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162‒8640, Japan

(Received: 15 August 2013; Accepted: 6 December 2013)

Abstract: Tick identification is important in control of tick-born diseases because tick-borne pathogens are often transmitted by specific tick species. In this study, we determined partial DNA sequences of the mitochondrial 16S rDNA gene (mt-rrs) for ticks including 7 genera and 39 species, and these ticks were allocated to 113 sequence types. Of the 39 species of ticks, 36 species (92.3%) were distinguishable by phylogenetic analysis of mt-rrs. This result suggests that species identification of ticks based on mt-rrs is a viable alternative to morphological identification. In order to establish a DNA database for identification of ixodid and argasid ticks in Japan, we deposited all sequence data in GenBank (from AB819156 to AB819268).

Key words: tick, mt-rrs, molecular identification

Introduction morphological discrimination of ticks is often difficult in medical fields for a number of reasons (e.g. lack of Ticks are hematophagous ectoparasites of tick parts important for identification, and/or technical vertebrates. Since they have an ability to transmit difficulties owing to insufficient training experience several pathogens to humans and livestock, there for morphological identification). We, therefore, are medical and veterinary needs for accurate tick developed an alternative method of morphological identification (Bowman and Nuttall, 2008). A total identification. We constructed a DNA database based of 47 species of ticks have been reported from Japan on the mitochondrial 16S rDNA gene (mt-rrs) using (Fujita and Takada, 2007). Of these, 21 species have ticks collected in Japan and South Korea. been shown to parasitize humans (Okino et al., 2010). Ticks have been phylogenetically analyzed using the For identification of ticks, morphological identification nuclear 18S rDNA gene, mt-rrs, mitochondrial 12S is the worldwide gold standard. However, in fact, rDNA gene, cytochrome c oxidase I (COI), internal 14 Med. Entomol. Zool. Acc. No. AB819156 AB819157 AB819158 AB819159 AB819160 AB819161 AB819162 AB819163 AB819164 AB819165 AB819166 AB819167 AB819168 AB819169 AB819170 AB819171 AB819172 AB819173 AB819174 AB819200 AB819201 AB819175 AB819176 AB819177 AB819178 AB819179 AB819180 AB819181 AB819182 AB819183 AB819184 2 2 * * 2006 2006 Sampling 2001.08.25 2004.12.05 2004.12.06 2006.07.12 2006.07.12 2006.07.12 2010.06.14 2011.04.22 2011.04.22 2008.01.13 2007.11.04 2007.11.10 2004.07.30 2010.07.29 2010.07.30 2010.07.30 2004.09.06 2004.07.29 2007.07.30 2004.06.06 2009.10.04 2006.11.08 2007.10.31 2006.09.21 2006.09.21 2004.07.29 2007.11.27 2007.10.22 2007.10.22 date (year or yyyy.mm.dd) 3 * 1 1 * * Collected source Host animals or Vegetation Vegetation Delichon urbica (Bonaparte, 1850) Delichon urbica (Bonaparte, 1850) Eptesicus japonensis Imaizumi, 1953 Vegetation Geoemyda japonica Fan, 1931 Geoemyda japonica Fan, 1931 Geoemyda japonica Fan, 1931 Cuora avomarginata evelynae (Gray, 1863) Laticauda colubrina (Schneider, 1799) Laticauda colubrina (Schneider, 1799) Vegetation Apodemus speciosus (Temminck, 1844) Vegetation Canis lupus familiaris Linnaeus, 1758 Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Emberiza spodocephala Pallas, 1776 Turdus pallidus Gmelin, 1789 Syrmaticus soemmerringii (Temminck, 1830) Syrmaticus soemmerringii (Temminck, 1830) Vegetation Turdus pallidus Gmelin, 1789 Turdus pallidus Gmelin, 1789 Turdus pallidus Gmelin, 1789 Location Haiminaka, Taketomi-cho, Yaeyama-gun, Okinawa Tahara, Tsuru-shi, Yamanashi Azumino, Matsumoto-shi, Nagano Tahara, Tsuru-shi, Yamanashi Fukai, Ishigaki-shi, Okinawa Ada, Kunigami village, Kunigami-gun, Okinawa Ada, Kunigami village, Kunigami-gun, Okinawa Ada, Kunigami village, Kunigami-gun, Okinawa Uehara, Taketomi-cho, Yaeyama-gun, Okinawa Kuroshima, Taketomi-cho, Yaeyama-gun, Okinawa Kuroshima, Taketomi-cho, Yaeyama-gun, Okinawa Ooya-cho, Yabu-shi, Hyogo Kuchinoshima, Toshima village, Kagoshima-gun, Kagoshima Aratano-cho, Anan-shi, Tokushima Gosesan, Fukui-cho, Anan-shi, Tokushima Kushiro-shi, Hokkaido Shibetsu-cho, Shibetsu-gun, Hokkaido Shibetsu-cho, Shibetsu-gun, Hokkaido Yagiumamawari, Minamiawaji-shi, Hyogo Karouji, Oowani-machi, Minamitsugaru-gun, Aomori Matsukami, Fukaura-machi, Nishitsugaru-gun, Aomori Furano-shi, Hokkaido Asyoro-cho, Asyoro-gun, Hokkaido Nimyou, Kumakougen-cho, Kamiukena-gun, Ehime Oimatsu, Echizen-cho, Nyuu-gun, Fukui Tahara, Tsuru-shi, Yamanashi Tahara, Tsuru-shi, Yamanashi Karouji, Oowani-machi, Minamitsugaru-gun, Aomori Yamada-machi, Kokurakita-ku, Kitakyusyu-shi, Fukuoka Oimatsu, Echizen-cho, Nyuu-gun, Fukui Oimatsu, Echizen-cho, Nyuu-gun, Fukui Male Male Male Male Larva Larva Larva Larva Larva Larva Larva Larva Female Female Female Female Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Tick stage type Tick AJ-B HJ-B AJ-A HJ-A Hfl-F Hfl-E Hfl-B AG-F DT-B AG-E AG-B Hfl-C AT-A Hfl-A AN-B DT-A Hfl-G Hfl-D AG-C AV-A AG-A Hfl-H HD-B AN-A AG-D HD-A Hca-A Hcor-A Hcon-B Hcon-C Hcon-A sequence Tick species [synonym] Argas vespertilionis (Latreille, 1796) [ Carios vespertilionis ] Amblyomma geoemydae (Cantor, 1847) Argas japonicus Yamaguti, Clifford and Tipton, 1968 Amblyomma nitidum Hirst and Hirst, 1910 Amblyomma testudinarium Koch, 1844 Dermacentor taiwanensis Sugimoto, 1935 Haemaphysalis campanulata Warburton, 1908 Haemaphysalis concinna Koch, 1844 Haemaphysalis cornigera Neumann, 1897 Haemaphysalis japonica Warburton, 1908 Haemaphysalis japonica douglasi Nuttall and Warburton, 1915 [ Haemaphysalis douglasi ] Haemaphysalis ava Neumann, 1897 Table 1. List of tick species and sequence types. 2 3 1 4 5 6 7 8 9 10 11 12 No Vol. 65 No. 1 2014 15 Acc. No. AB819209 AB819216 AB819210 AB819211 AB819217 AB819185 AB819212 AB819186 AB819213 AB819187 AB819214 AB819188 AB819215 AB819189 AB819190 AB819191 AB819192 AB819193 AB819194 AB819195 AB819196 AB819197 AB819198 AB819199 AB819202 AB819203 AB819204 AB819205 AB819206 AB819207 AB819208 Sampling Unknown Unknown 2008.07.23 1999.05.01 2004.12.07 2007.07.25 1999.05.01 2007.07.25 2007.07.25 2007.07.25 2004.06.26 2007.07.25 2004.06.26 2007.07.25 2007.07.25 2007.07.26 2007.07.26 2007.10.05 2004.09.29 2008.04.16 2004.09.28 2004.09.28 2004.09.28 2007.06.24 2004.06.05 2004.06.05 2004.06.05 2008.07.23 2004.09.01 2004.09.01 2004.09.01 date (year or yyyy.mm.dd) Collected source Host animals or Vegetation Unknown Unknown Vegetation Unknown Vegetation Vegetation Unknown Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Ficedula narcissina (Temminck, 1835) Vegetation Vegetation Vegetation Capra aegagrus Erxleben, 1777 Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Location Chiba Chiba Mageshima, Nishinoomote-shi, Kagoshima Shiriya, Higashidoori village, Shimokita-gun, Aomori Uchiura, Kamogawa-shi, Chiba Akusekijima, Toshima village, Kagoshima-gun, Kagoshima Mageshima, Nishinoomote-shi, Kagoshima Akusekijima, Toshima village, Kagoshima-gun, Kagoshima Akusekijima, Toshima village, Kagoshima-gun, Kagoshima Akusekijima, Toshima village, Kagoshima-gun, Kagoshima Hirauchi, Yakushima-cho, Kumage-gun, Kagoshima Akusekijima, Toshima village, Kagoshima-gun, Kagoshima Hirauchi, Yakushima-cho, Kumage-gun, Kagoshima Akusekijima, Toshima village, Kagoshima-gun, Kagoshima Akusekijima, Toshima village, Kagoshima-gun, Kagoshima Nakanoshima, Toshima village, Kagoshima-gun, Kagoshima Nakanoshima, Toshima village, Kagoshima-gun, Kagoshima Kouchi, Yamamoto-cho, Mitoyo-shi, Kagawa Minami-cho, Kaifu-gun, Tokushima Mugiura, Mugi-cho, Kaifu-gun, Tokushima Mihonoseki-cho, Matsue-shi, Simane Mugiura, Mugi-cho, Kaifu-gun, Tokushima Otoutojima, Ogasawara village, Tokyo Mugiura, Mugi-cho, Kaifu-gun, Tokushima Nishiide, Ooizumi-cho, Hokuto-shi, Yamanashi Tennyosan, Nishiide, Ooizumi-cho, Hokuto-shi, Yamanashi Nishiide, Ooizumi-cho, Hokuto-shi, Yamanashi Shiriya, Higashidoori village, Shimokita-gun, Aomori Yagiumamawari, Minamiawaji-shi, Hyogo Jindaiurakabe, Minamiawaji-shi, Hyogo Jindaiurakabe, Minamiawaji-shi, Hyogo 4 4 * * Male Male Larva Larva Larva Female Female Female Female Female Female Female Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Female Female Tick stage type Tick Hfl-J Hfl-I HL-F Hfl-L Hfl-P HL-E HL-B Hfl-K HL-C HL-A HK-B Hfl-N Hfl-O Hfl-Q HL-D HK-C HH-B HK-A Hfo-B Hfl-M HH-C HH-A Hfo-A HH-D Hmag-B Hmag-C Hmag-A sequence Hmegaspi-B Hmegaspi-C Hmegaspi-A Hmegaspi-D Tick species [synonym] Haemaphysalis ava Neumann, 1897 Haemaphysalis mageshimaensis Saito and Hoogstraal, 1973 Haemaphysalis megaspinosa Saito, 1969 Haemaphysalis formosensis Neumann, 1913 Haemaphysalis hystricis Supino, 1897 Haemaphysalis kitaokai Hoogstraal, 1969 Haemaphysalis longicornis Neumann, 1901 12 17 18 13 14 15 16 No 16 Med. Entomol. Zool. Acc. No. AB819218 AB819219 AB819220 AB819221 AB819222 AB819223 AB819224 AB819225 AB819226 AB819227 AB819228 AB819229 AB819230 AB819231 AB819232 AB819233 AB819234 AB819235 AB819236 AB819237 AB819238 AB819239 AB819240 AB819241 AB819242 AB819243 AB819244 AB819245 AB819246 AB819247 Sampling Unknown Unknown 2003.09.28 2003.09.28 2007.05.04 2007.04.29 2004.06.26 2008.08.08 2008.07.23 2008.06.14 2007.07.26 2007.07.26 2007.07.26 2006.12.17 2008.01.13 2008.01.13 2008.01.13 2007.11.03 1985.11.21 2007.07.30 2007.09.23 2007.06.04 2007.06.04 2004.06.05 2004.06.05 2004.06.05 2004.07.29 2008.08.10 2008.08.10 2008.08.10 date (year or yyyy.mm.dd) Collected source Host animals or Vegetation Pentalagus furnessi (Stone, 1900) Pentalagus furnessi (Stone, 1900) Emberiza chrysophrys Pallas, 1776 Emberiza variabilis Temminck, 1835 Vegetation Canis lupus familiaris Linnaeus, 1758 Vegetation Apodemus speciosus (Temminck, 1844) Ateuchosaurus pellopleurus (Hallowell, 1861) Ateuchosaurus pellopleurus (Hallowell, 1861) Ateuchosaurus pellopleurus (Hallowell, 1861) Turdus pallidus Gmelin, 1789 Vegetation Vegetation Vegetation Vegetation Vegetation Rattus rattus (Linnaeus, 1758) Rattus rattus (Linnaeus, 1758) Rattus rattus (Linnaeus, 1758) Vegetation Locustella ochotensis (Middendorff, 1853) Locustella ochotensis (Middendorff, 1853) Vegetation Vegetation Vegetation Vegetation Turdus cardis Temminck, 1831 Turdus cardis Temminck, 1831 Turdus cardis Temminck, 1831 Location Amamiooshima, Kagoshima Amamiooshima, Kagoshima Shnan-ri, Haenam gun, South Korea Mihonoseki, Mihonoseki-cho, Matsue-shi, Simane Anbou, Yakushima-cho, Kumage-gun, Kagoshima Koseda, Yakushima-cho, Kumage-gun, Kagoshima Sai, Sai village, Shimokita-gun, Aomori Minato, Wakkanai-shi, Hokkaido Takarajima, Toshima village, Kagoshima-gun, Kagoshima Takarajima, Toshima village, Kagoshima-gun, Kagoshima Takarajima, Toshima village, Kagoshima-gun, Kagoshima Nazekominato, Amami-shi, Kagoshima Kuchinoshima, Toshima village, Kagoshima-gun, Kagoshima Kuchinoshima, Toshima village, Kagoshima-gun, Kagoshima Kuchinoshima, Toshima village, Kagoshima-gun, Kagoshima Oozasou, Fukushima-shi, Fukushima Niimachi, Kawamata-machi, Date-gun, Fukushima Okinawa Okinawa Chichijima, Ogasawara village, Tokyo Sai, Sai village, Shimokita-gun, Aomori Koyamaike, Tottori-shi, Tottori Koyamaike, Tottori-shi, Tottori Nishiide, Ooizumi-cho, Hokuto-shi, Yamanashi Tennyosan, Nishiide, Ooizumi-cho, Hokuto-shi, Yamanashi Nishiide, Ooizumi-cho, Hokuto-shi, Yamanashi Karouji, Oowani-machi, Minamitsugaru-gun, Aomori Takaoka, Tomakomai-shi, Hokkaido Takaoka, Tomakomai-shi, Hokkaido Takaoka, Tomakomai-shi, Hokkaido 4 4 5 * * * Male Male Male Male Larva Larva Female Female Female Female Female Female Female Female Female Female Female Male Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Nymph Female Female Tick stage type Tick IC-B IG-B Ias-F IN-B IO-B IC-A Ias-E Ias-B IG-C IG-A IO-C IN-A IO-A Ias-C Ias-A IO-D Ias-G Ias-D Iac-A IM-A HY-B Ian-A HY-A Ipav-B HW-A Ipav-C Ipav-A Hpen-B Hpen-A Hpha-A sequence Tick species [synonym] Haemaphysalis phasiana Saito, Hoogstraal and Wassef, 1974 Haemaphysalis wellingtoni Nuttall and Warburton, 1908 Haemaphysalis pentalagi Pospelova-Shtrom, 1935 Haemaphysalis yeni Toumanoff, 1944 Ixodes acutitarsus (Karsch, 1880) Ixodes angustus Neumann, 1899 Ixodes asanumai Kitaoka, 1973 Ixodes columnae Takada and Fujita, 1992 Ixodes granulatus Supino, 1897 Ixodes monospinosus Saito, 1968 Ixodes nipponensis Kitaoka and Saito, 1967 Ixodes ovatus Neumann, 1899 Ixodes pavlovskyi Pomerantzev, 1946 20 21 19 22 23 24 25 26 27 28 29 30 31 No Vol. 65 No. 1 2014 17 Acc. No. AB819248 AB819249 AB819250 AB819251 AB819252 AB819253 AB819254 AB819255 AB819256 AB819257 AB819258 AB819259 AB819260 AB819261 AB819262 AB819263 AB819264 AB819265 AB819266 AB819267 AB819268 Engorged ticks. 4 * Sampling 2004.06.05 2004.06.05 2004.08.02 2004.08.02 2004.08.02 2008.08.14 2004.08.25 2007.06.12 2003.02.23 2002.11.05 2008.01.13 2008.04.09 2007.09.29 2008.05.13 2008.04.30 2007.12.16 2004.01.05 2004.01.05 2008.08.22 2007.08.25 2007.04.24 date (year or yyyy.mm.dd) Collected source Host animals or Vegetation Vegetation Vegetation Vegetation Vegetation Vegetation Turdus cardis Temminck, 1831 Calonectris leucomelas (Temminck, 1835) Larus schistisagus Stejneger, 1884 Mustela itatsi Temminck, 1844 Nyctereutes procyonoides viverrinus Temminck, 1838 Vegetation Parus major Linnaeus, 1758 Turdus pallidus Gmelin, 1789 Turdus cardis Temminck, 1831 Turdus cardis Temminck, 1831 Turdus pallidus Gmelin, 1789 Turdus naumanni Temminck, 1820 Turdus naumanni Temminck, 1820 Punus pacicus (Gmelin, 1789) Oceanodroma monorhis (Swinhoe, 1867) Capra aegagrus Erxleben, 1777 Ticks were collected from under the nest of Eptesicus japonensis . 3 * Location Nishiide, Ooizumi-cho, Hokuto-shi, Yamanashi Nishiide, Ooizumi-cho, Hokuto-shi, Yamanashi Imaizumi, Nakadomari-machi, Kitatsugaru-gun, Aomori Imaizumi, Nakadomari-machi, Kitatsugaru-gun, Aomori Imaizumi, Nakadomari-machi, Kitatsugaru-gun, Aomori Takaoka, Tomakomai-shi, Hokkaido Minamigou, Mikurajima village, Tokyo Hamamatsu, Nemuro-shi, Hokkaido Ookubo, Higashinagura, Shitara-cho, Kitashitara-gun, Aichi Kuroda-cho, Toyota-shi, Aichi Kuchinoshima, Tomakomai-shi, Hokkaido Sekiya, Tyuuou-ku, Niigata-shi, Niigata Nara, Kihoku-cho, Kitauwa-gun, Ehime Takaoka, Tomakomai-shi, Hokkaido Hidaka-cho, Hidaka-gun, Wakayama Sekiya, Tyuuou-ku, Niigata-shi, Niigata Fusaheiwadai, Abiko-shi, Chiba Fusaheiwadai, Abiko-shi, Chiba Mukojima, Ogasawara village, Tokyo Kutsujima, Maizuru-shi, Kyoto Otoutojima, Ogasawara village, Tokyo Male Male Male Male Larva Female Female Female Female Female Female Female Female Female Female Female Nymph Nymph Nymph Nymph Nymph Tick stage Ticks were collected from August to September of 2006. 2 * type Tick Ita-B Ita-A OS-A Itur-F OC-A Itur-E Isig-A Itur-B Iper-F Itur-C RM-A Iper-E Itur-A Iper-B Itur-G Itur-D Iper-C Itur-H Iper-A Iphi-A Iper-D sequence Tick species [synonym] Molted ticks. 5 Ixodes persulcatus Schulze, 1930 Ixodes philipi Keirans and Kohls, 1970 Ixodes signatus Birula, 1895 Ixodes tanuki Saito, 1964 Ixodes turdus Nakatsuji, 1942 Ornithodoros capensis Neumann, 1901 [ Carios capensis ] Ornithodoros sawaii Kitaoka and Suzuki, 1973 [ Carios sawaii ] Rhipicephalus microplus (Canestrini, 1888) [ Boophilus microplus ] Ticks were collected from the nest of Delichon urbica . * 1 * 32 33 34 35 36 37 38 39 No 18 Med. Entomol. Zool. transcribed spacer 2 (ITS2), and the large subunit of “RM-A” in Table 1) were detected from 39 species of RNA polymerase II (Pol II) (Black and Piesman, 1994; ticks and all sequence data were deposited in GenBank Black et al., 1997; Mangold et al., 1998; Fukunaga et al., as listed in Table 1. The length of amplicons ranged 2000; Beati and Keirans, 2001; Murrell et al., 2001). In from 401 to 416 bp. In this study, sequence data were this study, we used mt-rrs as the target gene because the collected from more than one specimen of each species mt-rrs is variable enough to differentiate species, and except in the case of Haemaphysalis cornigera Neumann, the registered sequences of this gene in GenBank were Haemaphysalis phasiana Saito, Hoogstraal and Wassef, more abundant than those of other genes. and Ixodes signatus Birula. Of the 39 species, 36 (92.3%) were distinguishable by phylogenetic analysis Materials and Methods based on mt-rrs sequences (Fig. 1). On the other hand, Ticks three Haemaphysalis spp. (Haemaphysalis japonica From 1985 to 2011, 39 species of ticks were collected Warburton, Haemaphysalis japonica douglasi Nuttall from vegetation or vertebrates, including mammals, and Warburton and Haemaphysalis megaspinosa Saito) birds, and reptiles (Table 1). After morphological could not be differentiated by this analysis. Sequence identification, total DNA was prepared from whole ticks types of H. japonica (HJ-A and HJ-B) and H. japonica using a DNA extraction kit (DNeasy tissue kit; Qiagen, douglasi (HD-A and HD-B) showed 100% sequence Hilden, Germany) according to the manufacturer’s identity, and sequence types of H. japonica and H. instructions with minor modification (The column was megaspinosa (from Hmegaspi-A to Hmegaspi-D) incubated for 10 min at 70°C after adding elution buffers showed 99.3%∼99.8% sequence similarity. In this study, to it). The DNA was stored at −30°C until use. The tick each of 6 tick species, Amblyomma geoemydae (Cantor), species were described according to Guglielmone et al. Haemaphysalis ava Neumann, Haemaphysalis (2010). More than one tick was used for each species longicornis Neumann, Ixodes asanumai Kitaoka, Ixodes where possible. persulcatus Schulze, and Ixodes turdus Nakatsuji, had over 6 sequence types, and the intraspecific sequence PCR and sequencing analysis similarity within these 6 species was 92.9%∼99.8%, PCR was performed using a set of primers (mt-rrs1: 5′- 97.3%∼99.8%, 99%∼99.8%, 99%∼99.8%, 99.3%∼ CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-3′, 99.8% and 98.8%∼99.8%, respectively. mt-rrs2: 5′-CCG GTC TGA ACT CAG ATC AAG TA-3′), Discussion previously described by Ushijima et al. (2003). The PCR assays were performed using 25 µL of reaction mixture In this study, we used mt-rrs as the target gene for tick for 30 cycles (10 s at 94°C, 30 s at 55°C, and 30 s at 72°C) identification which had high variation among species with puRe Taq Ready-To-Go PCR Beads (GE Healthcare and a large number of registered sequences in GenBank. UK, Ltd., Buckinghamshire, UK) or Takara Ex Taq Of the ticks examined, 92.3% were distinguishable by (Takara Bio Inc., Otsu, Japan). Subsequently, the PCR the phylogenetic analysis based on mt-rrs sequences. products were purified using a High Pure PCR Product Even this analysis could not differentiate 3 tick species Purification Kit (Roche Diagnostics, Basel, Switzerland), (or subspecies), H. japonica, H. japonica douglasi and and all PCR products were directly sequenced using H. megaspinosa. Fukunaga (2007) suggested H. japonica an ABI Prism 3130 or 3130xl Genetic Analyzer (Life and H. megaspinosa also could not be differentiated by Technologies Corporation, Gaithersburg, MD). NADH dehydrogenase subunit 2 (ND2) gene sequences, which was one of variable genes on tick mitochondrial Phylogenetic analysis genomes. On the other hand, the registered sequence Sequence data were analyzed using MEGA5.2 data of other variable genes such as ITS2, mitochondrial software (http://www.megasoftware.net) (Tamura et 12S rDNA gene and COI of the Haemaphysalis ticks al., 2011). After alignment using the CLUSTAL-W were quite few. Thus, for improvement in sensitivity (ver. 1.6) software package, the neighbor joining (NJ) for the Haemaphysalis ticks, further enrichment of the phylogenetic tree construction and bootstrap tests database is necessary. were carried out according to the Kimura 2-parameter The database we developed covered 83% (39/47), and distance method (Kimura, M., 1980; Saitou and Nei, identified 77% (36/47) of the tick species previously 1987). Pairwise alignments were performed with an recorded in Japan. In addition, there were the sequence open-gap penalty of 15, and a gap extension penalty of data for mt-rrs of Rhipicephalus sanguineus (Latreille), 6.66. Multiple alignments were also performed using Ixodes uriae White and Ixodes vespertilionis Koch in the same values. All positions containing alignment GenBank (Accession Numbers were L34302, AB030017 gaps and missing data were eliminated in pairwise and U95910, respectively). Taken together, 83% (39/47) sequence comparisons (pairwise deletion). of ticks found in Japan could be identified by this analysis when morphological identification proved Results difficult (Fig. 1). In total, 113 sequence types (designated as “AJ-A” to Our analysis detected intraspecific variation among Vol. 65 No. 1 2014 19

Fig. 1. Phylogenetic analysis based on mt-rrs of ticks in Japan. The phylogenetic trees were 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). Phylogenetic analyses were conducted in MEGA5.2. 20 Med. Entomol. Zool.

A. geoemydae (Fig. 1). Several A. geoemydae individuals We also thank Y. Ito (Yamaguchi Univ.), A. Sakata and were collected from tortoises on the Okinawa islands M. Muto (NIID) for their excellent technical assistance. and Yaeyama Archipelago in Okinawa Prefecture We appreciate Dr. Kyle R. Taylor (University of Florida, in the southern part of Japan. The ticks collected Department of Infectious Diseases and Pathology) for from the Okinawa islands and Yaeyama Archipelago his critical comments on this manuscript. were considered to belong to the same species The authors would like to acknowledge the technical morphologically. However, genetic analysis revealed expertise of The DNA Core facility of the Center for that ticks originated from the two regions formed Gene Research, Yamaguchi University, supported distinct monophyletic groups. Further analysis will by a Grant-in-Aid from the Ministry of Education, be needed to resolve this discrepancy between DNA Science, Sports and Culture of Japan. This work was sequence variation and morphological similarity. supported in part by a grant for research on emerging In contrast, although the Okinawa islands and and reemerging infectious diseases from the Japanese Ogasawara Archipelago are separated by over 1,500 km Ministry of Health, Labor, and Welfare, and Program over ocean, Ixodes granulatus Supino collected from each to Disseminate Tenure Tracking System, MEXT, Japan. location formed a monophyletic group. Since I. granulatus References often infested black rats [Rattus rattus (Linnaeus, 1758)], this result might be the consequence of the artificial or Beati, L. and Keirans, J. E. 2001. Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: non-artificial diﬀuseness of the host animals. Ixodidae) based on mitochondrial 12S ribosomal DNA gene Though DNA sequence-based analysis, with such as sequences and morphological characters. J. Parasitol., 87: 32‒48. mt-rrs in this study, sometimes did not correlate with Black, W. C. 4th, Klompen, J. S. and Keirans, J. E. 1997. Phylogenetic morphology-based analysis for phylogeny (Black et relationships among tick subfamilies (Ixodida: Ixodidae: al., 1997), we believe that the former could be a useful Argasidae) based on the 18S nuclear rDNA gene. Mol. Phylogenet. Evol., 7: 129‒144. alternative to the latter for identification of tick species Black, W. C. 4th and Piesman, J. 1994. Phylogeny of hard- and soft- (Jizhou et al., 2013). tick taxa (Acari: Ixodida) based on mitochondrial 16S rDNA In this study, we were not able to collect specimens sequences. Proc. Natl. Acad. Sci. U.S.A., 91: 10034‒10038. of the remaining five tick species of Japan, Dermacentor Bowman, A. S. and Nuttall, P. A. 2008. Ticks: biology, disease and silvarum Olenev, Haemaphysalis fujisana Kitaoka, control. 506 pp., Cambridge University Press, Cambridge. Fujita, H. and Takada, N. 2007. Identification of immature ticks in Haemaphysalis megalaimae Rajagopalan, Ixodes lividus Japan. In: Acari and Emerging/Reemerging Infectious Diseases Koch, and Ixodes simplex Neumann. These ticks were (ed. Yanagihara, Y.), pp. 53‒68, Zenkoku Noson Kyoiku Kyokai considered to be rare species in Japan. In fact, ticks Publishing, Tokyo (In Japanese). collected from human cases are possible to be identified Fukunaga, M. 2007. Molecular phylogeny of ticks. In: Acari and by the method presented in this study, which uses the Emerging / Reemerging Infectious Diseases (ed. Yanagihara, Y.), pp. 69‒75, Zenkoku Noson Kyoiku Kyokai Publishing, Tokyo (In database as ours (Kawabata et al., 2011). This suggests Japanese). that our method is helpful for surveillance on tick- Fukunaga, M., Yabuki, M., Hamase, A., Oliver, J. H. Jr. and Nakao, M. borne diseases for humans and livestock. In reference 2000. Molecular phylogenetic analysis of ixodid ticks based on the to this study, there is currently worldwide development ribosomal DNA spacer, internal transcribed spacer 2, sequences. of DNA barcoding database, such as The Barcode of J. Parasitol., 86: 38‒43. Guglielmone, A. A., Robbins, R. G., Apanaskevich, D. A., Petney, T. Life Data System (BOLD) (Ratnasingham and Hebert, N., Estrada-Peña, A., Horak, I. G., Shao, R. and Barker, S. C. 2010. 2007). Further examination for updating the DNA The Argasidae, Ixodidae and Nuttalliellidae (Acari: Ixodida) of the database would be beneficial for public health to control world: a list of valid species names. Zootaxa, 2528: 1‒28. the tick-borne infectious diseases through the process Jizhou, L., Wu, S., Zhang, Y., Zhang, T., Feng, C., Jia, G. and Lih, of identification of ticks to be involved. Z. 2013. Development of a barcode system for the Ixodida (Acari: Ixodida). Mitochondrial DNA, [Internet] Early online Acknowledgements in Journal home [accessed Sep 13, 2013]. Available from: http:// informahealthcare.com/doi/abs/10.3109/19401736.2013.792052. We would like to thank K. Miyamoto (Asahikawa Kawabata, H., Takano, A., Ohnishi, M., Ando, S., Ogasawara, Y., Medical University), M. Takashi and H. Torikai Fujita, H., Kadosaka, T., Wada, Y., Baba, S. and Seishima, M. 2011. (Amami Wild Bird Society of Japan), S. Yamamoto Investigation of tick bite cases that visited medical facilities in Japan, from 2005 to June 2011. IASR, 32: 226‒228, In Japanese. (Kagoshima University), N. Koizumi (National Institute Kimura, M. 1980. A simple method for estimating evolutionary rates of Infectious Diseases: NIID), Y. Nitta (Okinawa of base substitutions through comparative studies of nucleotide Prefectural Institute of Animal Health), T. Kasuya sequences. J. Mol. Evol., 16: 111‒120. (University of Tsukuba), T. Kuramoto (Kagoshima Mangold, A. J., Bargues, M. D. and Mas-Coma, S. 1998. 18S rRNA Prefectural Institute for Environmental Research and gene sequences and phylogenetic relationships of European hard- tick species (Acari: Ixodidae). Parasitol. Res., 84: 31‒37. Public Health), A. Nakamoto (Univ. of the Ryukyus), Murrell, A., Campbell, N. J. and Barker, S. C. 2001. A total-evidence S. Kawada (National Museum of Nature and Science) phylogeny of ticks provides insights into the evolution of life cycles and K. Ozaki (Yamashina Institute for Ornithology) for and biogeography. Mol. Phylogenet. Evol., 21: 244‒258. tick collection. We are also grateful to the members of Okino, T., Ushirogawa, H., Matoba, K. and Hatsushika, R. 2010. Bird banders of Yamashina Institute for Ornithology. Bibliographical Studies on Human Cases of Hard Tick (Acarina: Vol. 65 No. 1 2014 21

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