A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella

Jing Wang,Xingliang Wang,Stuart J Lansdell,Jianheng Zhang,Neil S Millar,Yidong Wu

doi:10.1016/j.ibmb.2016.02.001

Abstract

Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella.

Highlights

The spinosyns are a family of secondary metabolites from the aerobic fermentation of the soil bacterium Saccharopolyspora spinosa on nutrient media
We have identified high levels of resistance to spinosad in a laboratory-selected strain of the diamondback moth, P. xylostella
We have demonstrated that resistance is associated with a 3-aa deletion in the fourth transmembrane domain of the nicotinic acetylcholine receptors (nAChRs) a6 subunit (Pxa6)

Summary

Introduction

The spinosyns are a family of secondary metabolites from the aerobic fermentation of the soil bacterium Saccharopolyspora spinosa on nutrient media. They show potent insecticidal activities against a broad spectrum of pest insects (Thompson et al, 2000). The first commercial product of spinosyns, is a mixture of spinosyn A (the major component) and spinosyn D (the minor component). Spinetoram is a semi-synthetic spinosyn, which is developed by making two synthetic modifications to fermentationderived spinosyns (Kirst, 2010; Dripps et al, 2011). Spinetoram has improved insecticidal efficacy and a broader pest insect spectrum (Geng et al, 2013). The mode of action of spinosyns involves a binding site on the nicotinic acetylcholine receptors (nAChRs) that is distinct from that targeted by neonicotinoids (Millar and Denholm, 2007; Watson et al, 2010; Sparks et al, 2012; Geng et al, 2013)

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