Fig 1.
PCA plots (A. males, B. females) with regularised log transformed data, based on the 500 most variable genes, and heatmaps (C. males, D. females) showing Poisson distance between samples. MHco3 = drug-susceptible parent, MHco18 = drug-resistant parent, F2IVM = ivermectin-treated genetic cross F2, F2BZ = benzimidazole-treated F2, F2CTL = untreated control F2. The datapoint symbol in the PCA and the sample number in heatmap relate to the order that the samples were processed (circle = 1st, triangle = 2nd, square = 3rd).
Fig 2.
Scatter plot showing differentially expressed genes in comparisons of resistant and susceptible populations (adj P < 0.01), with log2 fold change of males shown on the x-axis and females on the y-axis. Grey points represent genes that are differentially expressed only in parental (MHco18 vs MHco3) comparisons, i.e. likely to represent between-strain differences not associated with ivermectin resistance. If blue they are also differentially expressed in the F2IVM vs MHco3 and F2IVM vs F2CTL comparisons, i.e. likely to be specifically associated with ivermectin resistance. The most highly upregulated gene in resistant isolates is the H. contortus homologue of C. elegans cky-1 (HCON_00155390). Equivalent scatterplots for all pairwise comparisons are shown in S5 Fig.
Fig 3.
Chromosome V karyoplots showing genomic position of genes with significant upregulation (yellow) or downregulation (blue). Each point represents a differentially expressed gene (adj P < 0.01) and point size corresponds to significance. Panels A-D compare ivermectin-resistant versus ivermectin-susceptible populations (A. MHco18 vs MHco3 males, B. MHco18 vs MHco3 females, C. F2IVM vs MHco3 males, D. F2IVM vs MHco3 females). Panel E compares an ivermectin-resistant versus a mixed (resistant and susceptible) population (F2IVM vs F2CTL males). Panel F shows genetic differentiation (FST) between the F3 generation of the genetic cross pre- and post- ivermectin selection to highlight the major locus of ivermectin selection [18]. Panel G shows relative absence of differential expression at the ivermectin QTL in a comparison with a benzimidazole selected population (F2BZ vs F2CTL males). Upregulation of the H. contortus homologue of C. elegans cky-1 (HCON_00155390) is identified in ivermectin resistant populations only.
Fig 4.
Volcano plots showing differentially expressed genes in male (A) and female (B) samples (adj P < 0.01). Blue points represent genes that are only differentially expressed in the parental (MHco18 vs MHco3) comparisons. If purple, they are also differentially expressed in the F2IVM vs MHco3 comparisons. If yellow (males only), they are also differentially expressed in the F2IVM vs F2CTL comparison.
Fig 5.
Expression analysis of HCON_00162780:pgp-11.
Normalised read counts for male (A) and female (B) RNA-seq samples highlighting higher constitutive expression in all male populations relative to female populations. C. Comparison of RNA-seq and RT-qPCR data (mean of three biological replicates) for MHco18 males relative to MHco3 males, showing constitutive upregulation in the ivermectin resistant isolate. For RNA-seq data, error bars show the log fold change standard error (lfcSE) and for RT-qPCRs, error bars show the standard deviation.