ORIGINAL PAPER RNAi-mediated silencing of enolase confirms its biological importance in Clonorchis sinensis Xiaoyun Wang & Wenjun Chen & Yanli Tian & Yan Huang & Xuerong Li & Xinbing Yu Received: 3 January 2014 /Accepted: 10 January 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Clonorchis sinensis (C. sinensis ) infection is still a common public health problem in freshwater fish consump- tion areas in Asian countries. More molecular evidence are required to speed up the prevention strategies to control this kind of infectious disease. In the present study, to confirm the biological importance of Cs enolase followed by our previous observations of the key metabolic enzyme, we explored the RNA silence effect of the Cs enolase-derived RNA interfer- ence (RNAi) in C. sinensis . The extramembranous region aa 105–226 was selected as the target sequence of RNA silence. Cs enolase-derived double strand RNA (dsRNA-Cs enolase, 366 bp) was synthetized and delivered into C. sinensis by soaking approach. The penetration of dsRNA into adult worms and metacercariae was tracked using fluorescently labeled RNA. Western blotting and qRT-PCR experiments were performed to determine dsRNA-Cs enolase-silencing ef- fect. Our results showed that, after incubating for 120 h, dsRNA-Cs enolase could effectively target and downregulate the expression of Cs enolase in both adult worms (P<0.001) and metacercariae (P<0.01), resulting in a remarkable killing effect on C. sinensis adult worms (P<0.01). Fluorescent Cy3- labeled dsRNA was mostly deposited in the uterus and vitellarium of adult worm and in the cyst wall of metacercaria. The present study is the first report of RNAi trials in C. sinensis , allowing further applications in identifying func- tional genes in C. sinensis . Introduction In freshwater fish consumption areas, human clonorchiasis remains an important foodborne zoonosis, which may be neglected by public health agents, although accumulating evidence showed that Clonorchis sinensis infection could induce various pathological impairments in the human liver (Wang et al. 2009; Lun et al. 2005; Hong and Fang 2012). Molecular mechanisms by which C. sinensis causes patholog- ical changes are still obscure despite invaluable information of C. sinensis at genomic and transcriptional level has been available in recent years (Wang et al. 2011a). Thus, new prevention strategies are still the most urgent challenges to control C. sinensis infection (Zhou et al. 2008; Wang et al. 2012). More molecular characterizations of key molecules that are involved into crucial cellular processes could speed up the interventions of C. sinensis infection. RNA interference (RNAi) phenomenon was firstly dis- covered in the nematode worm Caenorhabditis elegans (Fire et al. 1998), since then, dsRNA-derived RNAi has been widely used to downregulate target molecules, resulting in the reduction of mRNA for protein expression. Recent advances in the design and delivery of targeting molecules allow efficient and specific gene silencing in different types of parasites, rendering this technology as a powerful tool for the identification of novel vaccine can- didates or drug targets in the near future (Manzano-Roman et al. 2012). For instance, in the past years, the RNAi technique has helped to identify key genes in various parasites, including Schistosoma mansoni (Stefanić et al. 2010), Schistosoma japonicum (Yang et al. 2012; Zou et al. 2011), and Opisthorchis viverrini (Sripa et al. 2011). X. Wang : W. Chen : Y. Tian : Y. Huang : X. Li : X. Yu Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74th Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province, People’ s Republic of China X. Wang : W. Chen : Y. Tian : Y. Huang : X. Li : X. Yu (*) Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, 74th Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province, People’ s Republic of China e-mail: yuhxteam@163.com Parasitol Res DOI 10.1007/s00436-014-3785-0