RNA interference in Fasciola gigantica: Establishing and optimization of experimental RNAi in the newly excysted juveniles of the fluke.

Arun Anandanarayanan,Anju Varghese,Opinder Krishen Raina,Muthu Sankar,Hniang Lalrinkima,Ajayta Rialch,Mar Siles-Lucas

doi:10.1371/journal.pntd.0006109

Abstract

Fasciolosis caused by Fasciola gigantica is a neglected tropical disease but a constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries of Asia and Africa. Resistance to commonly used anthelmintics in Fasciola has increased the need to search for alternative therapeutic targets. RNA interference is the current tool of choice in the search for such targets in Fasciola. The susceptibility of juvenile Fasciola hepatica to double stranded (ds) RNA induced RNAi has been established but in F. gigantica a single preliminary report on RNAi induced mRNA transcript knockdown is available. Here we optimized conditions for RNAi in the liver fluke F.gigantica targeting six genes including superoxide dismutase (SOD), σ class of glutathione-s-transferase (GST), cathepsin (Cat) L1-D, Cat B1, Cat B2 and Cat B3 that showed robust transcriptional silencing of the targets following exposure of the newly excysted juveniles (NEJs) to long (170–223 nt) dsRNA. Knockdown was shown to be concentration dependent with significant mRNA transcript suppression occurring at 5 ng / μl that showed further suppression with the increase in the dsRNA concentration. The dsRNA induced persistent silencing of the mRNA transcript of SOD and σGST up to 15 days of observation. Delivery of the long dsRNA and siRNA to the newly excysted juveniles by soaking method was found to be efficient by tracking the uptake and diffusion of Cy3 labelled siRNA and long dsRNA in the flukes. Off-target effects of dsRNA trigger on some of the non-target genes were detected in the present investigation on RNAi in F. gigantica. The dsRNA induced superoxide dismutase protein suppression while impact of RNAi on other target proteins was not studied. There is no in vitro culture system for prolonged survival of the F. gigantica and in the present study in vitro maintenance of the NEJs is reported for a period of 3 weeks. The present study is the first attempt on optimization of RNAi protocols in F. gigantica where long dsRNA allowed for an efficient and persistent gene silencing, opening prospects for functional validation of putative vaccine and therapeutic targets in this neglected parasite.

Highlights

IntroductionFasciola gigantica (tropical liver fluke) and F. hepatica (temperate liver fluke) are the two causative agents of fasciolosis in livestock and are economically important veterinary parasites due to the substantial production and monetary losses that these parasites cause to the livestock industry
Fasciola gigantica and F. hepatica are the two causative agents of fasciolosis in livestock and are economically important veterinary parasites due to the substantial production and monetary losses that these parasites cause to the livestock industry
In parasitic flatworms most progress has been reported for schistosomes [22, 23, 24] that has demonstrated the utility of RNA interference (RNAi) in functional genomics in trematodes

Summary

Introduction

Fasciola gigantica (tropical liver fluke) and F. hepatica (temperate liver fluke) are the two causative agents of fasciolosis in livestock and are economically important veterinary parasites due to the substantial production and monetary losses that these parasites cause to the livestock industry. Fasciolosis caused by F. gigantica is a neglected tropical disease but a constraint on the growth and productivity of cattle, buffaloes and sheep in the tropical countries of Asia and Africa [1, 2]. In parasitic flatworms most progress has been reported for schistosomes [22, 23, 24] that has demonstrated the utility of RNAi in functional genomics in trematodes. These studies have indicated that genes are not affected; off-target effects can occur and developmental stages display different susceptibility to interference [25]. Successful gene silencing by RNAi reported in Opisthorchis viverrini, F. hepatica and Clonorchis sinensis [26, 27, 28] will provide framework for utilization of this

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