Ticks and Tick-borne Diseases 7 (2016) 813–816 Contents lists available at ScienceDirect Ticks and Tick-borne Diseases j ourna l h o me page: w ww.elsevier.com/locate/ttbdis Short communication Induction of gene silencing in Haemaphysalis longicornis ticks through immersion in double-stranded RNA Remil Linggatong Galay a,b , Emmanuel Pacia Hernandez b , Melbourne Rio Talactac b , Hiroki Maeda b , Kodai Kusakisako b , Rika Umemiya-Shirafuji c , Masami Mochizuki b , Kozo Fujisaki d , Tetsuya Tanaka b,∗ a Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los Ba˜ nos, Los Ba˜ nos, Laguna 4031, Philippines b Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan c National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan d Zen-noh Institute of Animal Health, 7 Ooja, Sakura, Chiba 285-0043, Japan a r t i c l e i n f o Article history: Received 17 February 2016 Received in revised form 30 March 2016 Accepted 30 March 2016 Available online 1 April 2016 Keywords: Ticks RNA interference Double-stranded RNA Ferritin a b s t r a c t The continuous emergence of tick-borne diseases and chemical acaricide-resistant tick strains neces- sitates 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 feed- ing 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 var- ied 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 12 h 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. © 2016 Elsevier GmbH. All rights reserved. 1. Introduction Ticks continue to threaten human and animal health world- wide with their role in the transmission of various pathogens through their obligatory blood-feeding habit (Dantas-Torres et al., 2012; Jonjegan and Uilenberg, 2004). Indeed, vector ticks should be efficiently controlled to effectively prevent the spread of tick- borne diseases. However, despite the immense effort to control ticks primarily through the application of chemical acaricides, a big challenge is posed by the emergence of resistant tick strains and environmental contamination (Domingos et al., 2013). ∗ Corresponding author. E-mail address: tetsuya@ms.kagoshima-u.ac.jp (T. Tanaka). RNA interference (RNAi) has advanced from being a mere research tool to various applications in human therapy, plant dis- ease control and pest control. RNAi offers several advantages as a method of pest control, such as species specificity, absence of side effects on crops, and no or negligible environmental pollution (Xue et al., 2012). In tick research, RNAi has been a very useful and convenient tool in evaluating gene function. Through RNAi, more than a hundred genes and proteins with functions in blood feeding, reproduction, immune response, and pathogen interac- tion in several tick species have been already characterized to date (Karim and Adamson, 2012). More importantly, RNAi aided in the identification of potential antigens for the development of anti-tick vaccines (de la Fuente and Merino, 2013). The injection of double-stranded RNA (dsRNA) has been the most commonly employed method of inducing RNAi in ticks (de la Fuente et al., 2007; Karim and Adamson, 2012). However, this method may be http://dx.doi.org/10.1016/j.ttbdis.2016.03.018 1877-959X/© 2016 Elsevier GmbH. All rights reserved.