Use of Viscous medium to study anthelmintic drug action in Caenorhabditis elegans

Abstract

Caenorhabditis elegans is an appealing tool for experimental evolution and for working with antiparasitic drugs, from understanding the molecular mechanisms of drug action and resistance to uncover new drug targets. We present a new methodology for studying the impact of antiparasitic drugs in C. elegans. Viscous medium was initially designed for C. elegans maintenance during long-term evolution experiments. Viscous medium provides a less structured environment than the standard nematode growth media agar, yet the bacteria food source remains suspended. Further, the Viscous medium offers the worm population enough support to move freely, mate, and reproduce at a rate comparable to standard agar cultures. Here, the Viscous medium was adapted for use in antiparasitic research. We observed a similar sensitivity of C. elegans to anthelmintic drugs as in standard liquid media and statistical difference to the standard agar media through a larval development assay. Using Viscous medium in C. elegans studies will considerably improve antiparasitic resistance research, and this medium could be used in studies aimed at understanding long-term multigenerational drug activity.

Keywords: Caenorhabditis elegans; Anthelmintics; Larval development; Viscous medium.

Figure 1

Setup to wash worms on a large scale between generations. Worms were pooled into a 50 ml conical centrifugation tube and centrifuged at 5134 × g for 5 min at 20 °C. The supernatant was removed, and the worm pellet was resuspended. The resuspended worm pellet was washed with approximately 100 ml of S basal in the above system. Worms are placed on a wet filter, and 100 ml of S basal were added periodically. When about 10–15 ml of the solution remained in the filter, a Pasteur pipette was used to remove the worms, and the density of the worms was counted.

Figure 2

Caenorhabditis elegans tm830 lengths (A) and mean width (B) after 72 h incubation of L1 in either S or V medium or on NGM agar. Length, width, and volume were obtained by Wormsizer (v 1.2.5). Graphs show individual values for approximately 100 worms for each condition, means and SD are shown. P value obtained by 1way ANOVA with post test Tukey's multiple comparison test. ns indicates P value above 0.05, * indicates a P value below 0.05, and *** indicates a P value lower than 0.0001.

Figure 3

Concentration–response-curves of C. elegans tm830 worms in either S or V medium when exposed to (A) ivermectin, (B) moxidectin, (C) albendazole, and (D) levamisole. Worms were exposed to drug concentrations in triplicate and four independent biological replicates were included. Worms that developed into either the L4 or adult stage were counted as fully developed, while L1, L2, and L3 were considered not developed.

Figure 4

Development of C. elegans tm830 worms in S and V medium. Worms were measured for development after a 48 h incubation at 20 °C. Mean and standard deviation are reported. *** indicates a P value < 0.0001.