title: RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro.
abstract: BACKGROUND
  Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).
  
  
  RESULTS
  Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200-320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.
  
  
  CONCLUSIONS
  In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.
authors.affiliation: Molecular Biosciences: Parasitology, The Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: McVeigh
authors.affiliation: Molecular Biosciences: Parasitology, The Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom.
authors.forename: Erin M
authors.initials: EM
authors.lastname: McCammick
authors.affiliation: Molecular Biosciences: Parasitology, The Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: McCusker
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, Wales, United Kingdom.
authors.forename: Russell M
authors.initials: RM
authors.lastname: Morphew
authors.affiliation: Molecular Biosciences: Parasitology, The Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom.
authors.forename: Angela
authors.initials: A
authors.lastname: Mousley
authors.affiliation: Department of Zoology, Aligarh Muslim University, Aligarh, Uttar Pradesh, India.
authors.forename: Abbas
authors.initials: A
authors.lastname: Abidi
authors.affiliation: Department of Zoology, Aligarh Muslim University, Aligarh, Uttar Pradesh, India.
authors.forename: Khalid M
authors.initials: KM
authors.lastname: Saifullah
authors.affiliation: Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India.
authors.forename: Raman
authors.initials: R
authors.lastname: Muthusamy
authors.affiliation: Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tamil Nadu, India.
authors.forename: Ravikumar
authors.initials: R
authors.lastname: Gopalakrishnan
authors.affiliation: AgriBio, the Centre for AgriBioscience, School of Life Sciences, LaTrobe University, Melbourne, Australia.
authors.forename: Terry W
authors.initials: TW
authors.lastname: Spithill
authors.affiliation: Molecular Biosciences: Parasitology, The Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom.
authors.forename: John P
authors.initials: JP
authors.lastname: Dalton
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, Wales, United Kingdom.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Brophy
authors.affiliation: Molecular Biosciences: Parasitology, The Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom.
authors.forename: Nikki J
authors.initials: NJ
authors.lastname: Marks
authors.affiliation: Molecular Biosciences: Parasitology, The Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom.
authors.forename: Aaron G
authors.initials: AG
authors.lastname: Maule
mesh_terms: D000818:Animals; D005210:Fasciola hepatica / Q000235:genetics*; D034622:RNA Interference; D018510:RNA, Helminth / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D018510:RNA, Helminth; D034741:RNA, Small Interfering