title: Calmodulin disruption impacts growth and motility in juvenile liver fluke.
abstract: BACKGROUND
  Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.
  
  
  METHODS
  In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.
  
  
  RESULTS
  Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.
  
  
  CONCLUSIONS
  These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.
authors.affiliation: Microbes & Pathogen Biology: Institute for Global Food Security, School of Biological Sciences, Queen's, University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK. e.mccammick@qub.ac.uk.
authors.forename: Erin M
authors.initials: EM
authors.lastname: McCammick
authors.affiliation: Microbes & Pathogen Biology: Institute for Global Food Security, School of Biological Sciences, Queen's, University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK. paul.mcveigh@qub.ac.uk.
authors.forename: Paul
authors.initials: P
authors.lastname: McVeigh
authors.affiliation: Microbes & Pathogen Biology: Institute for Global Food Security, School of Biological Sciences, Queen's, University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK. p.mccusker@qub.ac.uk.
authors.forename: Paul
authors.initials: P
authors.lastname: McCusker
authors.affiliation: Microbes & Pathogen Biology: Institute for Global Food Security, School of Biological Sciences, Queen's, University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK. D.Timson@brighton.ac.uk.
authors.forename: David J
authors.initials: DJ
authors.lastname: Timson
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Penglais, Aberystwyth, Ceredigion, SY23 3FL, UK. rom@aber.ac.uk.
authors.forename: Russell M
authors.initials: RM
authors.lastname: Morphew
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Penglais, Aberystwyth, Ceredigion, SY23 3FL, UK. pmb@aber.ac.uk.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Brophy
authors.affiliation: Microbes & Pathogen Biology: Institute for Global Food Security, School of Biological Sciences, Queen's, University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK. n.marks@qub.ac.uk.
authors.forename: Nikki J
authors.initials: NJ
authors.lastname: Marks
authors.affiliation: Microbes & Pathogen Biology: Institute for Global Food Security, School of Biological Sciences, Queen's, University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK. a.mousley@qub.ac.uk.
authors.forename: Angela
authors.initials: A
authors.lastname: Mousley
authors.affiliation: Microbes & Pathogen Biology: Institute for Global Food Security, School of Biological Sciences, Queen's, University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK. a.maule@qub.ac.uk.
authors.forename: Aaron G
authors.initials: AG
authors.lastname: Maule
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D005210:Fasciola hepatica / Q000235:genetics / Q000254:growth & development*; D006801:Humans; D007150:Immunohistochemistry; D010641:Phenotype; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics; D018510:RNA, Helminth / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: D002147:Calmodulin; D012330:RNA, Double-Stranded; D018510:RNA, Helminth; D034741:RNA, Small Interfering
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