title: RNA interference in a cestode reveals specific silencing of selected highly expressed gene transcripts.
abstract: Evolving RNA interference (RNAi) platforms are providing opportunities to probe gene function in parasitic helminths using reverse genetics. Although relatively robust methods for the application of RNAi in parasitic flatworms have been established, reports of successful RNAi are confined to three genera and there are no known reports of the application of RNAi to the class Cestoda. Here we report the successful application of RNAi to a cestode. Our target species was the common ruminant tapeworm, Moniezia expansa which can significantly impact the health/productivity of cattle, sheep and goats. Initial efforts aimed to silence the neuronally expressed neuropeptide F gene (Me-npf-1), which encodes one of the most abundant neuropeptides in flatworms and a homologue of vertebrate neuropeptide Y (NPY). Double stranded (ds)RNAs, delivered by electroporation and soaking (4-8h), failed to trigger consistent Me-npf-1 transcript knock-down in adult worms; small interfering RNAs (siRNAs) were also ineffective. Identical approaches resulted in significant and consistent transcript knock-down of actin transcript (71+/-4%) following soaking in Me-act-1 dsRNA. Similar successes were seen with hydrophobic lipid-binding protein (Me-lbp-1), with a dsRNA inducing significant target transcript reduction (72+/-5%). To confirm the validity of the observed transcript knock-downs we further investigated Me-act-1 RNAi worms for associated changes in protein levels, morphology and phenotype. Me-act-1 RNAi worms displayed significant reductions in both filamentous actin immunostaining (62+/-3%) and the amount of actin detected in Western blots (54+/-13%). Morphologically, Me-act-1 RNAi worms displayed profound tegumental disruption/blebbing. Further, muscle tension recordings from Me-act-1 RNAi worms revealed a significant reduction in both the number of worms contracting in response to praziquantel (20+/-12%) and in their contractile ability. These data demonstrate, to our knowledge for the first time, a functional RNAi pathway in a cestode and show that the robust knock-down of abundant gene transcripts is achievable using long dsRNAs following short exposure times.
authors.affiliation: Molecular Biosciences-Parasitology, School of Biological Sciences, Queen's University Belfast, Belfast, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Pierson
authors.forename: Angela
authors.initials: A
authors.lastname: Mousley
authors.forename: Lynda
authors.initials: L
authors.lastname: Devine
authors.forename: Nikki J
authors.initials: NJ
authors.lastname: Marks
authors.forename: Tim A
authors.initials: TA
authors.lastname: Day
authors.forename: Aaron G
authors.initials: AG
authors.lastname: Maule
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002589:Cestoda / Q000033:anatomy & histology / Q000235:genetics* / Q000302:isolation & purification; D041981:Gastrointestinal Tract; D055785:Gene Knockdown Techniques / Q000379:methods*; D015801:Helminth Proteins / Q000235:genetics; D007150:Immunohistochemistry; D009129:Muscle Tonus; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000235:genetics; D034622:RNA Interference; D012756:Sheep / Q000469:parasitology
chemical_list: D000199:Actins; D015801:Helminth Proteins; D009479:Neuropeptides; C076101:neuropeptide F