An eye on RNAi in nematode parasites Aaron G. Maule, Paul McVeigh, Johnathan J. Dalzell, Louise Atkinson, Angela Mousley and Nikki J. Marks Molecular Bioscience–Parasitology, Institute of Agri-Food and Land Use, School of Biological Sciences, Queen’s University Belfast, Belfast BT9 7BL, UK RNA interference (RNAi) has revolutionised approaches to gene function determination. From a parasitology perspective, gene function studies have the added dimen- sion of providing validation data, increasingly deemed essential to the initial phases of drug target selection, pre-screen development. Notionally advantageous to those working on nematode parasites is the fact that Caenorhabditis elegans research spawned RNAi discov- ery and continues to seed our understanding of its fundamentals. Unfortunately, RNAi data for nematode parasites illustrate variable and inconsistent susceptibili- ties which undermine confidence and exploitation. Now well-ensconced in an era of nematode parasite genomics, we can begin to unscramble this variation. RNAi in nematodes – early optimism The discovery of RNAi-based gene silencing in Caenorhabditis elegans [1] and the subsequent realisa- tion that RNAi was a widespread phenomenon occurring in animals and plants appeared to provide the first real opportunity to apply a reverse genetics approach to gene function determinations in parasitic nematodes [2]. Within the context of parasite-based research, gene function studies generate target-validation data in that RNAi-induced phenotypic aberration can endorse drug target candidature [3,4]. This marked a significant step forward for parasitologists in that RNAi could provide an alternative to using C. elegans as a surrogate target validation tool, a process demanding the assumption of functional conservation. The earliest demonstrations of RNAi-based knockdown of gene transcripts in nematode parasites [5,6] served to seed the early optimism and spawned the accumulation of reports on RNAi successes in parasitic nematodes across a variety of life-stages, species and clades. However, sprin- kled among these were studies reporting difficulties in the induction of RNAi or inconsistencies in RNAi datasets for parasitic nematodes. These difficulties were further highlighted in a number of reports and reviews that voiced concerns on the applicability of RNAi in animal parasitic nematodes (APNs) [7–10]. RNAi in plant parasitic nematodes In plant parasitic nematodes (PPNs), RNAi has been dem- onstrated in both sedentary (Glossary) endoparasites (Meloidogyne incognita infective J2 s and eggs [11–18], Meloidogyne artiellia eggs [19], Globodera rostochiensis J2 s [20,21], Globodera pallida J2 s [6,16,22], Heterodera glycines J2 s [6,23–26], Meloidogyne javanica J2 s [27,28]) and in migratory endoparasites (Bursaphelenchus xylophi- lus L2, L3, L4 and adults [29–31] and Radopholus similis mixed stage larvae [32]) (Box 1). Indeed, published RNAi data for PPNs are overwhelmingly positive with almost unanimous success in the knockdown of genes using dsRNAs or siRNAs. Review Glossary Amphidial canals: these take the form of two channels that are contiguous with the external environment and encase the amphidial (sensory) nerves and accessory cells as they track posteriorly from the head. Carbamoylcholine chloride: a cholinergic agonist that stimulates both muscarinic and nicotinic subtypes of acetylcholine receptors and has been shown to stimulate the activity of the feeding tube or pharynx (pump) that facilitates nematode feeding. Chemical ligaturing: in this context it designates the use of a chemical compound that paralyses the feeding tube or pharynx (pump) and so prevents the nematode from feeding. Dicer complex: initiates RNAi by processing long double-stranded RNA (dsRNA) into small interfering RNA (siRNA). Endocytic/phagocytic processes: the cellular based uptake of extracellular molecules by engulfing them through processes involving invagination of the outer plasma membrane; these processes have been implicated in the movement of double stranded RNA in some species. Excretory/secretory system: a term that refers to nematode excretory systems that encompass ducts, pores and gland cells; a key role includes excess water removal. Exsheathed: the removal, escape from or shedding of the residual outer- surface/cuticle retained from a previous moult by some species of nematode. Hypodermis: a syncytial layer that encases the nematode body and secretes the outer cuticle. Migratory endoparasite: commonly, nematode parasites that migrate through host plant root tissue to access multiple distinct feeding sites. Nonsense-mediated decay: a cellular mRNA quality control mechanism which facilitates the detection and degradation of premature termination (nonsense) mutations thus preventing the expression of erroneous proteins. Octopamine: a biogenic amine which acts as a neurotransmitter in some invertebrates; in plant parasitic nematodes it stimulates pumping of the feeding tube/pharynx. Pharyngeal or intestinal lumen: the central space through which the tubular pharynx (muscular feeding pump) or intestine conduct food posteriorly. Pharyngeal pumping: rhythmical contractions (pumps) of the feeding tube (pharynx) that pass substances from the mouth towards the intestine. Pharyngeal stimulant: a drug or similar substance that increases activity (i.e. pumping) of the nematode feeding tube (pharynx) and so triggers uptake through the mouth. Resorcinol: a dihydroxy benzene that can be used to stimulate pumping of the feeding tube/pharynx in plant nematodes. Secondary amplification response: primary short interfering RNAs (siRNAs) hybridise with complementary mRNA and can facilitate the generation of secondary siRNAs through the actions of RNA-dependent RNA polymerases thereby amplifying the impact of the original double stranded RNA and/or primary siRNAs. Sedentary endoparasite: commonly, plant parasitic nematodes that, as adults, are stationary and reside in a fixed feeding location within the host. Serotonin: a monoamine neurotransmitter in multicellular animals that stimulates pumping of the feeding tube/pharynx in some nematodes; also known as 5-hydroxytryptamine (5-HT). Corresponding author: Maule, A.G. (a.maule@qub.ac.uk) 1471-4922/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2011.07.004 Trends in Parasitology, November 2011, Vol. 27, No. 11 505