Drug-resistant Drosophila indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin

Abstract

The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid. Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media. The resistant strain, glc(1), is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity. Binding assays using glc(1) head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin. A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamate-gated chloride channel subunit DmGluClalpha. To examine the effect of this mutation on the biophysical properties of DmGluClalpha channels, it was introduced into a recombinant DmGluClalpha, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes. Nodulisporic acid directly activated wild-type and mutant DmGluClalpha channels. However, mutant channels were approximately 10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluClalpha channels.

Figure 1

Nodulisporic acid and ivermectin binding to wild-type and glc¹ Drosophila head membranes. (A) Specific binding of [³⁵S]nodulisporic acid to wild-type (filled symbols) and glc¹ (open symbols, two separate experiments) Drosophila head membranes. Specific binding was defined as total binding, measured in triplicate, minus binding in the presence of 100 nM unlabeled nodulisporic acid. Data were fit to the binding isotherm of the form: B = (B_max⋅L)/(K_d + L), where B is the concentration of specifically bound ligand, B_max is the maximal number of binding sites, L is the free ligand concentration, and K_d is the equilibrium dissociation constant. (B) Specific binding of [³⁵S]ivermectin to wild-type (filled symbols) and glc¹ (open symbols, two separate experiments) Drosophila head membranes.

Figure 2

In situ hybridization of DmGluClα probe to cytological position 92A-B on Or-R polytene chromosome 3 (A). The DmGluClα probe hybridizes to heterozygous deficiency chromosome DfB16 (B) but not Df12 (C).

Figure 3

Spontaneous Locomotion. Twenty to thirty-three flies of specified genotypes were tested in spontaneous locomotion assays. Data are presented as medians and quantiles, where the center lines indicate medians, boxes represent the 25% and 75% quantiles, bars the 10% and 90% quantiles, and black squares are the individual data points.

Figure 4

Nodulisporic acid activates DmGluClα. An oocyte expressing DmGluClα exhibited a rapidly activating, reversible current in response to application of 100 μM glutamate. In the same oocyte, 1 μM nodulisporic acid activated a current that was essentially irreversible on wash-out of nodulisporic acid and measured 60% of the glutamate-activated current.

Figure 5

Agonist sensitivity of DmGluClα P299S. Dose-response curves for nodulisporic acid (A), ivermectin (B), and glutamate (C) activation of DmGluClα wild-type (filled circles) and P299S (open squares) channels. Oocytes injected with DmGluClα wild-type or P299S RNA were superfused with varying drug concentrations, and currents were normalized to the current activated by saturating concentrations of glutamate. Data were fit to the Hill equation with n_h = 2.