A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis.

Alin M Puinean,Toby Collins,Stuart J Lansdell,Pablo Bielza,Neil S Millar

doi:10.1111/jnc.12029

Alin M Puinean, Toby Collins + Show 3 more

Open Access

https://doi.org/10.1111/jnc.12029

Copy DOI

Abstract

High levels of resistance to spinosad, a macrocyclic lactone insecticide, have been reported previously in western flower thrips, Frankliniella occidentalis, an economically important insect pest of vegetables, fruit and ornamental crops. We have cloned the nicotinic acetylcholine receptor (nAChR) α6 subunit from F. occidentalis (Foα6) and compared the nucleotide sequence of Foα6 from susceptible and spinosad-resistant insect populations (MLFOM and R1S respectively). A single nucleotide change has been identified in Foα6, resulting in the replacement of a glycine (G) residue in susceptible insects with a glutamic acid (E) in resistant insects. The resistance-associated mutation (G275E) is predicted to lie at the top of the third α-helical transmembrane domain of Foα6. Although there is no direct evidence identifying the location of the spinosad binding site, the analogous amino acid in the C. elegans glutamate-gated chloride channel lies in close proximity (4.4 Å) to the known binding site of ivermectin, another macrocyclic lactone pesticide. The functional consequences of the resistance-associated mutation have been examined in the human nAChR α7 subunit. Introduction of an analogous (A272E) mutation in α7 abolishes the modulatory effects of spinosad whilst having no significant effect upon activation by acetylcholine, consistent with spinosad having an allosteric mechanism of action.

Highlights

Spinosad is a macrocyclic lactone, isolated from the microorganism Saccharopolyspora spinosa (Sparks et al 1998; Thompson et al 2000)
It is only relatively recently that target-site resistance has been reported for insecticides acting on nicotinic acetylcholine receptor (nAChR) (Millar and Denholm 2007; Wolstenholme and Kaplan 2012)
Point mutations altering single amino acids in nAChR a or b subunits have been described that are associated with resistance to neonicotinoid insecticides (Liu et al 2005; Bass et al 2011)

Summary

Introduction

Spinosad is a macrocyclic lactone, isolated from the microorganism Saccharopolyspora spinosa (Sparks et al 1998; Thompson et al 2000). The inhibitory glutamate-gated chloride channel (GluCl), found in several invertebrate species, has close structural similarity to nAChRs and is the target site for ivermectin (Fig. 1), another macrocyclic lactone pesticide (Wolstenholme 2010). In common with all Cys-loop receptors, nAChRs and GluCls are transmembrane proteins in which five subunits are arranged around a central ion channel pore. The conventional orthosteric agonist binding site is located within the extracellular domain of Cys-loop receptors at the interface between two adjacent subunits (Sine, 2002 #1539). In the case of ivermectin, there is clear evidence that it interacts with an allosteric site in the transmembrane domain of GluCls (Hibbs and Gouaux 2011). The binding site of spinosad on nAChRs is less well defined, but there is evidence that it acts an allosteric ligand (Salgado and Saar 2004) at a site that is distinct from the conventional extracellular agonist binding site (Orr et al 2009)

Methods

Results

Conclusion