A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation onCaenorhabditis elegansexposed to cysteine proteinases

Abstract

SUMMARYPlant cysteine proteinases (CPs) fromCarica papayakill parasitic and free-living nematodesin vitroby hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughputCaenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series ofC. elegans busmutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5–24 h, bothbusmutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.