Title Establishment of a laboratory colony of taiga tick Ixodes persulcatus for tick-borne pathogen transmission studies Konnai, Satoru; Saito, Yoichi; Nishikado, Hideto; Yamada, Shinji; Imamura, Saiki; Mori, Akina; Ito, Takuya; Onuma, Author(s) Misao; Ohashi, Kazuhiko Citation Japanese Journal of Veterinary Research, 55(2-3), 85-92 Issue Date 2008-02-09 DOI 10.14943/jjvr.55.2-3.85 Doc URL http://hdl.handle.net/2115/32368 Type bulletin (article) File Information 55(2-3)_85-92.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Japanese Journal of Veterinary Research 55(2-3): 85-92, 2008 FULL PAPER Establishment of a laboratory colony of taiga tick Ixodes persulcatus for tick-borne pathogen transmission studies Satoru Konnai1)＊,Yoichi Saito1), Hideto Nishikado1),Shinji Yamada1), Saiki Imamura1),Akina Mori1),TakuyaIto2),Misao Onuma1) and Kazuhiko Ohashi1) 1)Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan 2)Hokkaido Institute of Public Health, Sapporo, 060-0819, Japan Received for publication, December 6, 2007; accepted, January 18, 2008 Abstract Ixodes persulcatus Schulze (I. persulcatus)isdistributed in Russia and Far East Asia including Japan, and has been implicated as the vector of several human pathogens. In particular, I. persulcatus acts as the only tick vector for human lyme borreliosis in Japan. In order to elu- cidate the mechanism of transmission of I. persulcatus-borne pathogens, we developed a labo- ratory colony of I. persulcatus. Ticks were fed on Syrian hamster and engorged ticks that had dropped off the animals were collected and maintained to allow them to molt. Tick rearing was performed in incubator at 20℃ with 95% relative humidity and 12-hour light/dark photo-period regimen. We found out that adult females fed for 8±2days and had a pre-oviposition period lasting for 7±2days. The minimum egg incubation period was 1 month with the hatched lar- vae feeding for 3±1days and molting to nymphs 3-4 months thereafter. Meanwhile, the nymphs fed for 4±1days and molted to adult 2-3 months thereafter. For future analysis of gene expres- sion profiles in I. persulcatus, we cloned and sequenced the actin gene (a housekeeping gene), and found that it is 92.7% to 98.6% homologous to the published sequences of related ixodid ticks. This laboratory colony of I. persulcatus will facilitate investigations on the role of tick- derived molecules on the transmission of I. persulcatus-borne pathogens and will be important for identification of potential anti-tick vaccine and acaricide target molecules. Key Words: ixodid tick, I. persulcatus, laboratory colony, life cycle, actin gene, vector Introduction the widely distributed ixodid ticks3,10).Theixodid tick found in Russia and in North and Far East Lyme disease and tick-borne encephalitis are Asia, belongs to the species Ixodes persulcatus two emerging tick-borne diseases transmitted by (I. persulcatus), whereas in Europe and the *Corresponding author: Satoru Konnai, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan Phone: +81-11-706-5215. Fax: +81-11-706-5217. E-mail: [email protected] ８６ Establishment of a laboratory colony of I. persulcatus Americas, ixodid tick species include Ixodes scapu- ity of the ticks in transit. Tick species were deter- laris (I. scapularis), Ixodes pacificus (I. pacificus) mined in the laboratory by examining the mor- and Ixodes ricinus (I. ricinus). Lyme disease is a phology of the ticks under a dissection light micro- multi-systemic disorder caused by infection with scope (Fig. 1B). I. persulcatus ticks were then at least three spirochete species including Borrelia maintained in 50 ml centrifuge tubes packed with burgdorfeli (B. burgdorfeli), B. garinii and B. afzelii. 5mlof10% plaster mixture (Wako, Tokyo) includ- There are mainly four types of vector ticks for hu- ing 0.5 g activated charcoal (Wako) at 4℃ to pre- man lyme borreliosis, namely I. scapularis, vent bacterial and fungal spoilage until use. I. ricinus, I. pacificus and I. persulcatus. In Japan, Lyme disease in humans is due to infection with Maintenance and development of various stages of B. garinii or B. afzelii, which are biologically and I. persulcatus tick specifically transmitted by I. persulcatus 13). The stored ticks were reactivated by incuba- Recently, there have been several reports on tion for 1 h atroom temperature. Feeding was in- the identification and characterization of tick- itiated by placing 10-20 adult ticks in a net and at- derived molecules from the closely related ixodid taching them to a pathogen-free Syrian hamster tick species, I. scapularis and I. ricinus. Some of (Japan SLC Inc., Shizuoka). Adult I. persulcatus the functions associated with the identified mole- males were placed with females at 1:1 ratio to en- cules include acting as specific receptors for patho- sure mating and feeding. The tick-infested ani- gens14),anti-coagulants12),pathogen-associated pro- mals were maintained in an isolated experimental teins15,16) and immunosuppressants1,4,5,7,11).However, animal facility which is under a strict acaricidal no sequence information and function analysis of control regimen of the Hokkaido University, follow- such molecules has been reported for I. persulcatus mainly because of the lack of an established labo- ratory tick colony. In spite of this, reports on the prevalence and diversity of pathogens associated with I. persulcatus are increasing. Here, we report the successful establishment of an I. persulcatus laboratory colony that will aid to elucidate the tick’s biology at molecular level as well as to understand the events of I. persulcatus- borne pathogens transmission. Materials and Methods Tick collection Host-seeking I. persulcatus adult ticks (Fig. 1 A) were collected from lower vegetation at periph- eral and inner parts of deciduous and mixed for- ests in Nissho area located in Hidaka district, Hok- kaido, Japan. These samples were gathered by Fig. 1. Ixodes persulcatus. (A) Adult ticks (dorsal view). (B) Photography and diagram il- flagging with cotton flannel. After collection, the lustrating coxae I-IV (ventral view of female ticks were placed in 50 mlcentrifugetubescon- tick). Arrow indicates the long internal spur on taining damped sterile filter paper and packed in a coax I, indicating one of morphological charac- cooler box containing ice packs to maintain viabil- teristics of I. persulcatus. Satoru Konnai et al. ８７ ing guidelines of the Institutional Animal Care were carried out by the Clustal X program. The and Use Committee of the Graduate School of Vet- EMBL/GenBank accession numbers are: AJ889837 erinary Medicine, Hokkaido University. Engorged (Ixodes ricinus), AY254898 (Haemaphysalis longi- adult ticks were collected daily starting from day 5 cornis), AY255624 (Rhipicephalus (Boophilus) mi- post-attachment, placed individually in clean con- croplus), AY254899 (Rhipicephalus appendicula- tainers with dampened sterile filter paper and in- tus), AF426178 (Ixodes scapularis), X05185 (Bom- cubated at 20℃ to initiate oviposition. Hatched byx mori), NM079076 (Drosophila melanogaster), larval and nymphal ticks were fed to repletion on AF358264 (Drosophila virilis), AF300705 (Li- hamsters as described above to allow them to molt topenaeus vannamei), L13764 (Manduca sexta), into succeeding stages. All tick rearing was per- AB055975 ( Marsupenaeus japonicus )and formed in an incubator (Sanyo, Osaka) at 20℃ and AY547732 (Ornithodoros moubata). with a 12-hour light/darkphotoperiod regimen. Cloning of I. persulcatus actin gene Results and Discussion Total RNA was extracted from salivary glands of pathogen free-adult females (day 4 post-blood The life cycle of I. persulcatus under laboratory con- feeding) by using TRIZOLTM reagent according to ditions the manufacturer’s protocol(Invitrogen, Carisbad, To initiate reproduction, adult male and fe- CA, USA). cDNA was synthesized with the reverse male ticks were kept together during feeding as transcriptase-MMLV (Takara, Otsu) according to well as during storage (Fig. 2). The life cycle (exact the manufacture’s instructions. One microliter of pre-feeding period not determined) had a duration the cDNA template was added to a total volume of of approximately 250 days (Fig. 3). We found out 20 µlcontaining PCR buffer, oligonucleotide prim- that adult female ticks fed for 6-10 days and had a ers (at 0.2 µMeachoftickactin sense: 5’-ATGTGT pre-oviposition period lasting for 5-9 days. The GACGACGAGGTTGCCG-3’ and tick actin anti- minimum egg incubation period was 30-40 days sense: 5’-TTAGAAGCACTTGCGGTGGACAATG- with the hatched larvae feeding for 2-4 days and 3’)2),and2.5units of rTaq polymerase (Takara). molting to nymph, 90-120 days thereafter. The The PCR product was purified by the Gene-clean- nymphs fed for 3-5 days and molted to adult, 60-90 III Kit (MP Biomedicals, Solon, OH, USA) and days thereafter (Table 1). Troughton and Levin cloned into the pGEM T-Easy-vector (Promega have recently documented the biological data and Corp., Madison, WI, USA) for sequencing. The pu- life cycle for several Ixodid tick species, including I. rified plasmids were sequenced using the CEQ scapularis, I. pacificus, Amblyomma americanum, 2000 Dye Terminator Cycle Sequencing method Dermacentor occidentalis, Dermacentor variabilis, with the Quick Start kit (Beckman Coulter, Inc., Hemaphysalis leporispalustris and Rhipicepalus Fullerton,