title: Non-nematode-derived double-stranded RNAs induce profound phenotypic changes in Meloidogyne incognita and Globodera pallida infective juveniles.
abstract: Nine non-nematode-derived double-stranded RNAs (dsRNAs), designed for use as controls in RNA interference (RNAi) screens of neuropeptide targets, were found to induce aberrant phenotypes and an unexpected inhibitory effect on motility of root knot nematode Meloidogyne incognita J2s following 24h soaks in 0.1 mg/ml dsRNA; a simple soaking procedure which we have found to elicit profound knockdown of neuronal targets in Globodera pallida J2s. We have established that this inhibitory phenomenon is both time- and concentration-dependent, as shorter 4h soaks in 0.1 mg/ml dsRNA had no negative impact on M. incognita J2 stage worms, yet a 10-fold increase in concentration to 1 mg/ml for the same 4h time period had an even greater qualitative and quantitative impact on worm phenotype and motility. Further, a 10-fold increase of J2s soaked in 0.1 mg/ml dsRNA did not significantly alter the observed phenotypic aberration, which suggests that dsRNA uptake of the soaked J2s is not saturated under these conditions. This phenomenon was not initially observed in potato cyst nematode G. pallida J2s, which displayed no aberrant phenotype, or diminution of migratory activity in response to the same 0.1 mg/ml dsRNA 24h soaks. However, a 10-fold increase in dsRNA to 1mg/ml was found to elicit comparable irregularity of phenotype and inhibition of motility in G. pallida, to that initially observed in M. incognita following a 24h soak in 0.1 mg/ml dsRNA. Again, a 10-fold increase in the number of G. pallida J2s soaked in the same volume of 1 mg/ml dsRNA preparation did not significantly affect the observed phenotypic deviation. We do not observe any global impact on transcript abundance in either M. incognita or G. pallida J2s following 0.1 mg/ml dsRNA soaks, as revealed by reverse transcriptase-PCR and quantitative PCR data. This study aims to raise awareness of a phenomenon which we observe consistently and which we believe signifies a more expansive deficiency in our knowledge and understanding of the variables inherent to RNAi-based investigation.
authors.affiliation: Molecular Biosciences-Parasitology, School of Biological Sciences, Queen's University Belfast, Belfast BT9 7BL, Northern Ireland, UK.
authors.forename: Johnathan J
authors.initials: JJ
authors.lastname: Dalzell
authors.forename: Steven
authors.initials: S
authors.lastname: McMaster
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Johnston
authors.forename: Rachel
authors.initials: R
authors.lastname: Kerr
authors.forename: Colin C
authors.initials: CC
authors.lastname: Fleming
authors.forename: Aaron G
authors.initials: AG
authors.lastname: Maule
mesh_terms: D000818:Animals; D018551:Solanum lycopersicum / Q000469:parasitology; D009348:Nematoda / Q000187:drug effects* / Q000254:growth & development; D010641:Phenotype; D010935:Plant Diseases / Q000469:parasitology; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000494:pharmacology*; D018510:RNA, Helminth / Q000235:genetics / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011198:Solanum tuberosum / Q000469:parasitology*; D013997:Time Factors; D014431:Tylenchoidea / Q000187:drug effects* / Q000254:growth & development
chemical_list: D012330:RNA, Double-Stranded; D018510:RNA, Helminth