Variation in anthelmintic responses are driven by genetic differences among diverseC. eleganswild strains

Abstract

Treatment of parasitic nematode infections in humans and livestock relies on a small arsenal of anthelmintic drugs that have historically reduced parasite burdens. However, anthelmintic resistance (AR) is increasing, and little is known about the molecular and genetic causes of resistance for most drugs. The free-living roundworm Caenorhabditis elegans has proven to be a tractable model to understand AR, where studies have led to the identification of molecular targets of all major anthelmintic drug classes. Here, we used genetically diverse C. elegans strains to perform dose-response analyses across 26 anthelmintic drugs that represent the three major anthelmintic drug classes (benzimidazoles, macrocyclic lactones, and nicotinic acetylcholine receptor agonists) in addition to seven other anthelmintic classes. First, we found that C. elegans strains displayed significant variation in anthelmintic responses across drug classes. Dose-response trends within a drug class showed that the C. elegans strains elicited similar responses within the benzimidazoles but variable responses in the macrocyclic lactones and nicotinic acetylcholine receptor agonists. Next, we compared the effective concentration estimates to induce a 10% maximal response (EC10) and slope estimates of each dose-response curve of each strain to the reference strain, N2, which enabled the identification of anthelmintics with population-wide differences to understand how genetics contribute to AR. Because genetically diverse strains displayed differential susceptibilities within and across anthelmintics, we show that C. elegans is a useful model for screening potential nematicides. Third, we quantified the heritability of responses to each anthelmintic and observed a significant correlation between exposure closest to the EC10 and the exposure that exhibited the most heritable responses. Heritable genetic variation can be explained by strain-specific anthelmintic responses within and across drug classes. These results suggest drugs to prioritize in genome-wide association studies, which will enable the identification of AR genes.

Quantitative tests of albendazole resistance in beta-tubulin mutants

“…CRISPR-Cas9 genome editing was used to generate deletions of each beta-tubulin gene in the N2 laboratory strain genetic background (Figure 2). Edited strains with single deletions of each beta-tubulin gene were phenotyped in DMSO and ABZ using a previously described high-throughput assay (HTA) that quantitatively measures nematode development (Shaver et al, 2023; Widmayer et al, 2022). Briefly, strains were bleach synchronized and embryos were titered into 96-well plates.…”

“…Animals were grown for 48 hours with constant shaking at 180 rpm, after which, animals were treated with 50 mM sodium azide in M9 buffer to straighten and paralyze the animals for imaging. Following 10 minutes of exposure to sodium azide, each plate was imaged using a Molecular Devices ImageXpress Nano microscope (Molecular Devices, San Jose, CA) with a 2X objective (Shaver et al, 2023).…”

Screening of the Pandemic Response Box identifies anti-microsporidia compounds

“…Here we show that MMV1782387, a benzimidazole carbamate, has amongst the strongest inhibition of N. parisii and relatively low host toxicity in C. elegans. Benzimidazoles are known to inhibit C. elegans and natural resistance to these compounds has arisen through genetic variation in betatubulin [57,58]. Host toxicity has been shown to be mediated through inhibition of neuronal betatubulin [59].…”

Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure usingCaenorhabditis elegans

“…Data analysis steps have been modified from previous reports [37,92]. All analyses were performed using the R statistical environment (version 4.2.1) unless stated otherwise.…”

“… Objects with a Worm_Length > 30 pixels, 100 microns, were removed from the CellProfiler data to (A) retain L1 and MDHD-sized animals and (B) remove unwanted particles [93]. By using the Worm_Length > 30 pixels threshold to retain small sensitive animals, more small objects, such as debris, were also retained [37]. R/ easyXpress [89] was used to filter measurements from worm objects within individual wells with statistical outliers and to parse measurements from multiple worm models down to single measurements for single animals. The data were visualized by drug, drug concentration, assay, strain, and worm model for two purposes.…”