[
2005]
RNA interference (RNAi) is a recently discovered phenomenon in which doublestranded RNA (dsRNA) silences endogenous gene expression in a sequencespecific manner (Fire et al., 1998). Since its discovery, the use of RNAi has become widely employed in many organisms to specifically knock down gene function. RNAi shares a remarkable degree of similarity with silencing phenomena in other organisms (Cogoni et al., 1999a; Sharp, 1999). For instance, RNAi, posttranscriptional gene silencing in plants and cosuppression in fungi can all be activated by the presence of aberrant RNAs (Maine, 2000; Tijsterman et al., 2002a). Additionally, plant, worm, and fly cells or extracts undergoing RNA-mediated interference all contain small dsRNAs, around 25 nucleotides in length, identical to the sequences present in the silenced gene (Baulcombe, 1996; Hammond et al., 2000; Zamore et al., 2000; Catalanotto et al., 2000). The high degree of similarity between these RNA-mediated silencing phenomena supports the notion that they were derived from an ancient and conserved pathway used to regulate gene expression, presumably to eliminate defective RNAs and to defend against viral infections and transposons. (Zamore, 2002). Components of RNAi have also been implicated in developmental processes, suggesting that RNAi may play a broader role in regulating gene expression (Smardon et al., 2000; Knight et al., 2001; et al., Ketting et al., 2001). Although we have learned much about the general mechanisms underlying RNAi, a detailed understanding of how RNAi works remains to be elucidated. In this chapter we will discuss first the biology of RNAi, then the genes required for its function, and we will end with a discussion on recent findings that have implicated chromatin silencing in the mechanism of RNAi.
[
Adv Exp Med Biol,
2010]
Nematode neuropeptide systems comprise an exceptionally complex array of approximately 250 peptidic signaling molecules that operate within a structurally simple nervous system of approximately 300 neurons. A relatively complete picture of the neuropeptide complement is available for Caenorhabditis elegans, with 30 flp, 38 ins and 43 nlp genes having been documented; accumulating evidence indicates similar complexity in parasitic nematodes from clades I, III, IV and V. In contrast, the picture for parasitic platyhelminths is less clear, with the limited peptide sequence data available providing concrete evidence for only FMRFamide-like peptide (FLP) and neuropeptide F (NPF) signaling systems, each of which only comprises one or two peptides. With the completion of the Schmidtea meditteranea and Schistosoma mansoni genome projects and expressed sequence tag datasets for other flatworm parasites becoming available, the time is ripe for a detailed reanalysis ofneuropeptide signalingin flatworms. Although the actual neuropeptides provide limited obvious value as targets for chemotherapeutic-based control strategies, they do highlight the signaling systems present in these helminths and provide tools for the discovery of more amenable targets such as neuropeptide receptors or neuropeptide processing enzymes. Also, they offer opportunities to evaluate the potential of their associated signaling pathways as targets through RNA interference (RNAi)-based, target validation strategies. Currently, within both helminth phyla, theflp signaling systems appear to merit further investigation as they are intrinsically linked with motor function, a proven target for successful anti-parasitics; it is clear that some nematode NLPs also play a role in motor function and could have similar appeal. At this time, it is unclear if flatworm NPF and nematode INS peptides operate in pathways that have utility for parasite control. Clearly, RNAi-based validation could be a starting point for scoring potential target pathways within neuropeptide signaling for parasiticide discovery programs. Also, recent successes in the application of in planta-based RNAi control strategies for plant parasitic nematodes reveal a strategy whereby neuropeptide encoding genes could become targets for parasite control. The possibility of developing these approaches for the control of animal and human parasites is intriguing, but will require significant advances in the delivery of RNAi-triggers.
[
1994]
Nematodes have been cultured continuously in the laboratory since 1944 when Margaret Briggs Gochnauer isolated and cultured the free-living hermaphroditic species Caenorhabditis briggsae. Work with C. briggsae and other rhabditid nematodes, C. elegans, Rhabditis anomala, and R. pellio, demonstrated the relative ease with which they could be cultured. The culturing techniques described here were developed for C. elegans, but are generally suitable (to varying degrees) for other free-living nematodes. Whereas much of the early work involved axenic culturing, most of these techniques are no longer in common use and are not included here. In the 1970s C. elegans became the predominant research model due to work by Brenner and co-workers on the genetics and development of this species. An adult C. elegans is about 1.5 mm long, and under optimal laboratory conditions has a life cycle of approximately 3 days. There are two sexes, males and self-fertile hermaphrodites, that are readily distinguishable as adults. The animals are transparent throughout the life cycle, permitting observation of cell divisions in living animals using differential interference microscopy. The complete cell lineage and neural circuitry have been determined and a large collection of behavioral and anatomical mutants have been isolated. C. elegans has six developmental stages: egg, four larval stages (L1-L4), and adult. Under starvation conditions or specific manipulations of the culture conditions a developmentally arrested dispersal stage, the dauer larva, can be formed as an alternative third larval stage. Many of the protocols included here and other experimental protocols have been summarized in "The Nematode Caenorhabditis elegans". We also include a previously unpublished method for long-term chemostat cultures of C. elegans. General laboratory culture conditions for nematode parasites of animals have been described, but none of these nematodes can be cultured in the laboratory through more than one life cycle. Marine nematodes and some plant parasites have been cultured xenically or with fungi. Laboratory cultivation of several plant parasites on Arabidopsis thaliana seedlings in agar petri plates has also been reported.