Fig 1.
The high-throughput phenotyping assay allows for rapid dose-response assessments across genetically diverse C. elegans strains.
(A) Strains were passaged for three generations to reduce generational effects of starvation. (B) Strains were bleach synchronized to collect embryos and then hatched and arrested at the L1 larval stage. (C) After 24 hours, a serial dilution containing eight concentrations for each anthelmintic was created. The serial dilution was added to an aliquot of E. coli. This mixture was fed to the nematodes. (D) After 48 hours of growth, animals were imaged to collect phenotypic measurements. (E) Data were cleaned, and dose-response analysis was performed. Detailed descriptions of all steps can be found in Methods. Created with BioRender.com. Modified from a previous version [23].
Table 1.
Drug class, subclass, and drugs used in this study.
Drug classes with defined Mode of Action (MoA) are listed.
Fig 2.
Dose-response curves for benzimidazoles (BZs).
Normalized animal lengths (y-axis) are plotted for each strain as a function of the dose of benzimidazole supplied in the high-throughput phenotyping assay (x-axis); Albendazole, Benomyl, Fenbendazole, Mebendazole, and Thiabendazole. Strains are denoted by color. Lines extending vertically from points represent the standard deviation from the mean response. Statistical normalization of animal lengths is described in Methods.
Fig 3.
Variation in benzimidazole (BZ) EC10 dose-response and slope estimates can be explained by genetic variation across strains.
(A) Strain-specific EC10 estimates (e) for each benzimidazole are displayed for each strain. Standard errors for each strain- and anthelmintic-specific EC10 estimates are shown. (B) Strain-specific slope estimates (b) for each benzimidazole are displayed for each strain. Standard errors for each strain- and anthelmintic-specific slope estimate are indicated by the line extending vertically from each point. (C) The broad-sense (x-axis) and narrow-sense heritability (y-axis) of normalized animal length measurements were calculated for each concentration of each benzimidazole (Methods; Broad-sense and narrow-sense heritability calculations). The color of each cross corresponds to the log-transformed dose for which those calculations were performed. The horizontal line of the cross corresponds to the confidence interval of the broad-sense heritability estimate obtained by bootstrapping, and the vertical line of the cross corresponds to the standard error of the narrow-sense heritability estimate.
Fig 4.
Dose-response curves for macrocyclic lactones (MLs).
Normalized animal lengths (y-axis) are plotted for each strain as a function of the dose of macrocyclic lactone supplied in the high-throughput phenotyping assay (x-axis). Macrocyclic lactones are organized by (A) Avermectins: Abamectin, Doramectin, Eprinomectin, Ivermectin, Selamectin; and (B) Milbemycins: Milbemycin and Moxidectin. Strains are denoted by color. Lines extending vertically from points represent the standard deviation from the mean response. Statistical normalization of animal lengths is described in Methods.
Fig 5.
Variation in macrocyclic lactone (ML) EC10 dose-response and slope estimates can be explained by genetic variation across strains.
(A) Strain-specific EC10 estimates (e) for each macrocyclic lactone are displayed for each strain. Standard errors for each strain- and anthelmintic-specific EC10 estimates are indicated by the line extending vertically from each point. (B) Strain-specific slope estimates (b) for each macrocyclic lactone are displayed for each strain. Standard errors for each strain- and anthelmintic-specific slope estimate are indicated by the line extending vertically from each point. (C) The broad-sense (x-axis) and narrow-sense heritability (y-axis) of normalized animal length measurements were calculated for each concentration of each macrocyclic lactone (Methods; Broad-sense and narrow-sense heritability calculations). The color of each cross corresponds to the log-transformed dose for which those calculations were performed. The horizontal line of the cross corresponds to the confidence interval of the broad-sense heritability estimate obtained by bootstrapping, and the vertical line of the cross corresponds to the standard error of the narrow-sense heritability estimate. EC10, estimated slope, and heritability could not be calculated for moxidectin and therefore, not plotted.
Fig 6.
Dose-response curves for nicotinic acetylcholine receptor (nAChR) agonists.
Normalized animal lengths (y-axis) are plotted for each strain as a function of the dose of anthelmintic supplied in the high-throughput phenotyping assay (x-axis); Levamisole, Morantel, and Pyrantel. Strains are denoted by color. Lines extending vertically from points represent the standard deviation from the mean response. Statistical normalization of animal lengths is described in Methods.
Fig 7.
Variation in nicotinic acetylcholine receptor agonists (nAChR agonists) EC10 dose-response and slope estimates can be explained by genetic variation across strains.
(A) Strain-specific EC10 estimates (e) for each nicotinic acetylcholine receptor agonist are displayed for each strain. Standard errors for each strain- and anthelmintic-specific EC10 estimates are indicated by the line extending vertically from each point. (B) Strain-specific slope estimates (b) for each nicotinic acetylcholine receptor agonist are displayed for each strain. Standard errors for each strain- and anthelmintic-specific slope estimate are indicated by the line extending vertically from each point. (C) The broad-sense (x-axis) and narrow-sense heritability (y-axis) of normalized animal length measurements were calculated for each concentration of each nicotinic acetylcholine receptor agonist (Methods; Broad-sense and narrow-sense heritability calculations). The color of each cross corresponds to the log-transformed dose for which those calculations were performed. The horizontal line of the cross corresponds to the confidence interval of the broad-sense heritability estimate obtained by bootstrapping, and the vertical line of the cross corresponds to the standard error of the narrow-sense heritability estimates.
Fig 8.
Variation in EC10 and dose-response slope estimates can be explained by genetic variation across strains.
(A) The relative potency of each anthelmintic for each strain compared to the N2 strain is shown. Solid points denote strains with significantly different relative resistance to that anthelmintic compared to the N2 strain (Student’s t-test and subsequent Bonferroni correction with a padj < 0.05). Faded points denote strains not significantly different than the N2 strain. Asterisks denote strains with normalized estimates greater than ten compared to the N2 strain. See S14 Fig for the relative potency of all strains in each anthelmintic. Anthelmintic drugs with undefined EC10 estimates (estimates greater than the maximum dose to which animals were exposed) are not shown. (B) For each anthelmintic, the relative steepness of the dose-response slope inferred for that strain compared to the N2 strain is shown. Solid points denote strains with significantly different dose-response slopes for that anthelmintic compared to the N2 strain (Student’s t-test and subsequent Bonferroni correction with a padj < 0.05). Faded points denote strains without significantly different slopes than the N2 strain. Asterisks represent strains with slope estimates greater than 20 compared to the N2 strain. See S15 Fig for the slope estimates of all strains in each anthelmintic. Anthelmintic drugs with undefined slope estimates are not shown. The broad class to which each anthelmintic belongs is denoted by the strip label for each facet.