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The free-living nematode Caenorhabditis elegans is a formidable experimentally tractable model organism that offers key advantages in accelerating nematicide discovery. We report the screening of drug-like libraries using an high-throughput C. elegans assay, based on an automated infrared motility reader. As a proof of concept, we screened the “Pathogen Box” library, and identical results to a previous screen using Haemonchus contortus were obtained. We then screened an in-house library containing a diversity of compound families. Our results identified novel nematicidal scaffolds and illustrate the value of C. elegans in accelerating nematicide discovery using a non labor-intensive automated assay that provides a simple overnight readout.


Synchronized L4 C.elegans worms were removed from culture plates and washed three times with K saline (NaCl 51 mM, KCl 32 mM) by centrifugation at 1000 g. Worms were plated in 96-well flat microtiter plates (Costar). Approximately 60 worms per well were seeded in 80 uL of K saline containing 0.015% bovine serum albumin (BSA), and their basal movement was measured for 30 min to normalize the 100% movement activity for each well at the beginning of the assay. Then, the drugs were added to a final volume of 100 uL per well. A dose-dependent assay for ivermectin was carried out in the 0.01–10 um range in 1% DMSO. The levamisole dose–response assay concentration range was 1–1000 um. No DMSO was used in the levamisole assays. Motility, using WMicrotracker One, was measured for 90 min. Following the protocol described above, Pathogen Box compounds were added at a 50 um final concentration in 1% DMSO in a final volume of 100 uL per well. Control wells with vehicle only were assayed by octuplicate. Motility (using WMicrotracker ONE) was measured for 17,5 h. Putative positives were rescreened using the same method. The dose-dependent motility response for tolfenpyrad was carried out in the 2–30 um range in 1% DMSO.


Initially, we tested two commercially available nematicides (ivermectin and levamisole) known to kill worms by paralysis through different mechanisms. The dose–response curves obtained for both compounds (Figure 3) clearly showed that the assay was suitable for screening drugs that affect motility. The EC50 was 0.19±0.01 um for ivermectin and 6.4±0.3 um for levamisole. Tolfenpyrad compound was identified when this library was screened using H. contortus L3 and L4 larval stages. The dose–response curve showed that tolfenpyrad was fully active at a concentration as low as 10 um (Figure 4b), with an EC50 of 3.6±0.2 um. The worms treated with tolfenpyrad were then examined under a stereoscopic microscope, and a clear lethal phenotype was observed.

Vet Sci. 2019 Mar 17;6(1). pii: E29. doi: 10.3390/vetsci6010029

Risi G, Aguilera E, Ladós E, Suárez G, Carrera I, Álvarez G, Salinas G