Helminth parasites have been largely refractory to genetic analysis, and as a result, functional studies of gene products have been limited by available technologies, guided largely by predictions from primary structure. This has posed a considerable problem to research efforts in this area thus far, and will clearly be a limiting factor in addressing questions raised by the large number of expressed sequence tags (ESTs) currently being generated from these organisms. This is particularly acute in the case of sequences for which no clear function can be ascribed. Moreover, even when a specific function appears evident from the primary structure, proteins may often perform unexpected roles, particularly in the context of parasitism. Development of genetic knockout techniques have not been possible due to the intrinsic difficulty in genetic crosses and the lack of methods for heritable transformation, although some progress has been made in transient expression of DNA and RNA by ballistic transfer [1, 2]. RNA interference (RNAi) offers an opportunity to address some of these problems. Initially performed on Caenorhabditis elegans by microinjection [3, 4], delivery of double stranded RNA (dsRNA) through the intestine was subsequently achieved via ingestion of transfected Escherichia coli [5], and by direct soaking of worms in dsRNA [6]. Dissemination from the nematode intestine to other somatic tissues and the germ line has been demonstrated, and somewhat surprisingly, the RNAi effect can be remarkably long-lived. Thus for many genes, interference may persist for several days postinjection and may in some cases be inherited in subsequent generations [6]. We therefore decided to assay whether RNAi could be applied to the parasitic nematode Nippostrongylus brasiliensis, utilising secreted acetylcholinesterases (AChEs) as targets. These enzymes are encoded by three separate genes expressed by fourth stage larvae (L4) and adult stages of this parasite. AChE B and AChE C are 90% identical in amino acid sequence, whereas AChE A is 63Á/64% identical to the others [7, 8]. In addition, N. brasiliensis expresses at least one other gene encoding a non-secreted (neuromuscular) AChE. Although these have yet to be cloned, the protein (s) can be discriminated from secreted variants by their characteristic slow migration in non-denaturing polyacrylamide gels stained for AChE activity [9]. Adult worms were recovered from the jejunum of rats at different times post-infection and soaked overnight in dsRNA. This was produced by T3 and T7 RNA polymerase following insertion of cDNA fragments into pBluescript, and in vitro transcription with T3 and T7 RNA polymerase (RiboMAX, Promega). Optimal results were obtained with dsRNA concentrations of 1 mg ml (1 or greater, and incubation was generally carried out in a volume of less than 200 ml of DEPC-treated phosphate buffered saline (PBS) at 37 8C. Following overnight (16 h) incubation, worms were washed 3)/in PBS and cultured in vitro for up to 6 days as previously described [10]. Culture medium was changed daily, passed through 0.2 mM filters and assayed for protein and AChE content [11]. Initial experiments were carried out with dsRNA derived from a full length cDNA for AChE B, ie 1799 bp [7]. Fig. 1A demonstrates that incubation in this dsRNA

Abbreviations: AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; ACh, acetylcholine; ASCh, acetylthiocholine; BuSCh, butyrylthiocholine; RNAi, RNA interference.