Natural variation in a chloride channel subunit confers avermectin resistance in C. elegans

Abstract

Resistance of nematodes to anthelmintics such as avermectins has emerged as a major global health and agricultural problem, but genes conferring natural resistance to avermectins are unknown. We show that a naturally occurring four-amino-acid deletion in the ligand-binding domain of GLC-1, the alpha-subunit of a glutamate-gated chloride channel, confers resistance to avermectins in the model nematode Caenorhabditis elegans. We also find that the same variant confers resistance to the avermectin-producing bacterium Streptomyces avermitilis. Population-genetic analyses identified two highly divergent haplotypes at the glc-1 locus that have been maintained at intermediate frequencies by long-term balancing selection. These results implicate variation in glutamate-gated chloride channels in avermectin resistance and provide a mechanism by which such resistance can be maintained.

Figure 1. Sensitivity to abamectin differs between N2 and CB4856

(A) Time course of frequency of body bends (n≥20 for each strain). * indicates a significant (P<0.01) difference from CB4856. The legend depicts the strains and their respective colors on graphs A and B. (B) Frequency of body bends at 30 minutes plotted against different doses of abamectin for N2 (orange, n≥12 at each dose), CB4856 (blue, n≥12 at each dose), F1 progeny from crosses between N2 males and CB4856 hermaphrodites (red, n≥18 at each dose) and F1 progeny from crosses between CB4856 males and N2 hermaphrodites (green, n≥28 at each dose). Heterozygotes were scored only at 0.375 μg/ml, 0.4 μg/ml and 0.5 μg/ml abamectin. Error bars are standard errors of the mean for each strain. ns: non-significant difference.* indicates P<0.01. Pvalues are from Mann-Whitney tests for concentrations 0–0.3 μg/ml. For the higher concentrations, where more than two groups were involved, a Kruskal-Wallis test followed by Dunn’s multiple comparison test was performed.

Figure 2. Linkage analysis and fine mapping of the chromosome V locus

In all figures CB4856 and N2 are color coded in blue and orange respectively. (A) LOD scores plotted against marker positions. Red line: 5% genome-wide significance threshold obtained after 10,000 permutations of the data. (B) The QTL identified on Chromosome V, with blue dashed lines showing the 1.5 LOD-drop interval. (C) Distribution of trait values of RIAILs grouped by genotypes at the marker corresponding to the maximum LOD score on chromosome V. (D) Genotypes of RIAILs with breakpoints within the QTL interval. Vertical bars with dots are genotyped markers. (E) Boxplots of body bend frequency of RIAILs. Each dot represents a single individual. (F) Top: 107 kb genomic interval within the QTL on chromosome V. Middle: Horizontal gray bars depict region of the genome (upper panel) covered by each fosmid. The bottom pull out shows the 4.5 kb N2-genomic fragment. (G) Boxplots of the frequency of body bends in abamectin of parental and transgenic strains in CB4856 background harboring indicated N2 fosmids and a genomic clone. Pooled data of rescue lines for each fosmid or genomic fragment are plotted (individual lines are shown in Fig. S3). ns: non-significant difference *: (P<0.001) from CB4856.

Figure 3. Common naturally-occurring deletion in glc-1 confers abamectin resistance

In all figures CB4856 and N2 are color coded in blue and orange respectively and ns: non-significant difference. (A) Boxplots of frequency of body bends for the indicated strains in abamectin. N2/glc-1(pk54) and CB4856/glc-1(pk54) are F1 heterozygotes. Bottom panel: Introgression strains and their schematic genotypes. The red vertical line depicts glc-1(pk54). * significantly different (P<0.001) from CB4856. (B) Boxplots of frequency of body bends for the parent strains (as in 3A) and transgenic strains in the CB4856 background. GFP, genomic DNA clone of glc-1, and the glc-1 cDNA with and without mutations, were expressed under a 1.1 kb glc-1 promoter and an unc-54 3′UTR. Boxplots show pooled data of individual lines shown in Fig. S3. CB4856 was transformed with glc-1 cDNA harboring the following mutations: glc-1(A20T), glc-1(indel) and glc-1(T346A). * P<0.001 significant difference from GFP. (C) Genome-wide association mapping for fraction of worms paralyzed in abamectin. Significantly associated markers (P<3×10⁻⁷) are indicated by red lines. (D) Boxplots of body bends per ten seconds in 0.375 μg/ml abamectin are plotted for each wild isolate and the indicated F1 heterozygotes. * significantly different from the corresponding wild isolate (P<0.01).

Figure 4. glc-1 shows signatures of balancing selection and confers resistance to S. avermitilis

(A) Elevated divergence in the glc-1 region between N2 and CB4856. Upper panel: Sequence alignment of ~23 kb between N2 and CB4856 by mvista (6). Percent identity is plotted against physical distance. Gray bars: regions of the CB4856 genome that could not be reliably sequenced. Red triangles indicate indels. Black line: glc-1, zoomed in the lower panel. Lower panel: The gene structure of glc-1 is shown above the alignment. Exons, introns and UTR’s are colored in blue, pink and cyan, respectively. Numerals indicate the number of SNPs in each exon. Gray triangles represent indels. (B) Neighbor-joining tree constructed from 1604 base pairs of glc-1 sequence. Blue and orange clades: CB4856-like and N2-like sequences, respectively. Red: strains resistant to abamectin relative to N2. (C) glc-1(pk54) confers resistance to Streptomyces avermitilis. Box plots of brood size without S. avermitilis (upper panel) and with 100 μl S. avermitilis (lower panel) for the parental and indicated introgression strains. * represent significant differences (P<0.05), ns non-significant differences.