title: Induction of gene silencing in Haemaphysalis longicornis ticks through immersion in double-stranded RNA.
abstract: The continuous emergence of tick-borne diseases and chemical acaricide-resistant tick strains necessitates the development of new and more effective control strategies. RNA interference through the injection of double-stranded RNA (dsRNA) has been a very useful tool in tick research for evaluating gene function. However, this technique can be sophisticated due to the required equipment and technique. Here we studied the feasibility of an immersion technique to induce gene silencing in Haemaphysalis longicornis ticks. We targeted the Hlfer1 gene, previously shown to be crucial in successful blood feeding and reproduction. Larval, nymphal, and adult female H. longicornis ticks were immersed in Hlfer1 or Luciferase dsRNA for control. The dsRNA dissolving medium, incubation temperature and time were varied to establish the optimum conditions. RT-PCR was performed to confirm gene silencing. It was found that immersing the ticks in dsRNA dissolved in nuclease-free water at 15°C for 12h resulted in clear gene silencing. The phenotypes of adult ticks immersed in dsRNA were then compared with those of adult ticks injected with dsRNA. Similar to dsRNA injection, the post-blood meal weight of ticks immersed in Hlfer1 dsRNA was significantly lower than the control group. Moreover, high post-blood meal mortality and low egg output was observed both from ticks injected with and immersed in Hlfer1 dsRNA. Our results here suggest that immersion in dsRNA can effectively induce gene silencing and not only offers an alternative method to dsRNA injection but also opens the possibility of applying dsRNA for tick control.
authors.affiliation: Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, Los Baños, Laguna 4031, Philippines; Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Remil Linggatong
authors.initials: RL
authors.lastname: Galay
authors.affiliation: Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Emmanuel Pacia
authors.initials: EP
authors.lastname: Hernandez
authors.affiliation: Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Melbourne Rio
authors.initials: MR
authors.lastname: Talactac
authors.affiliation: Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Maeda
authors.affiliation: Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Kodai
authors.initials: K
authors.lastname: Kusakisako
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Rika
authors.initials: R
authors.lastname: Umemiya-Shirafuji
authors.affiliation: Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Mochizuki
authors.affiliation: Zen-noh Institute of Animal Health, 7 Ooja, Sakura, Chiba 285-0043, Japan.
authors.forename: Kozo
authors.initials: K
authors.lastname: Fujisaki
authors.affiliation: Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. Electronic address: tetsuya@ms.kagoshima-u.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D005247:Feeding Behavior; D005260:Female; D026863:Ixodidae / Q000235:genetics*; D007814:Larva / Q000235:genetics; D009758:Nymph / Q000235:genetics; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics*; D012098:Reproduction
chemical_list: D012330:RNA, Double-Stranded
keywords: Double-stranded RNA; Ferritin; RNA interference; Ticks