Mutations in Dα1 or Dβ2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster
Introduction
The neonicotinic insecticides are widely used in the control of sucking insects (Mehlhorn et al., 1999; Tomizawa and Casida, 2003). Neonicotinoids act as agonists at the nicotinic acetylcholine receptor (nAChR), opening the channel and causing continuous depolarisation and firing of postsynaptic neurons resulting in paralysis and death (Bai et al., 1991). They act selectively on insect nAChRs, with only low-binding affinity and activity on vertebrate nAChRs (Tomizawa et al., 2000). The nAChRs belong to the ligand-gated ion-channel superfamily (LGIC) which also includes the excitatory, cationic selective 5-hydroxytryptamine receptor 3A (5HT3A) channel and the inhibitory, anionic selective γ-aminobutyric acid (GABA) receptors and glycine channels (Devillers-Thiery et al., 1993).
Discovery and crystallisation of the glial cell acetylcholine-binding protein of the snail Lymnaea stagnalis provides a useful model of the predicted tertiary and quaternary structure of the ligand-binding N-terminus (Brejc et al., 2001). Mutagenesis and in vitro expression studies have identified components of the large N-terminal domain involved in ligand binding (Arias, 2000). In the nAChR pentamer, the second transmembrane domains (M2) enclose the lumen of the ion channel (Dani, 1989), while several regions of M3, M4 and the intervening cytoplasmic domain have been postulated to have allosteric interactions. Several sites for phosphorylation and other modifications have been identified in both vertebrate and insect nAChRs which are thought to modulate responses to ligands and desensitisation (Charpantier et al., 2005; Jones et al., 2005; Marszalec et al., 2005; Thany et al., 2005). For example, the V285I mutation in M3 of the human muscle acetylcholine receptor α-subunit results in slower opening and faster closing of the channel (Wang et al., 1999).
Heterologous expression of Drosophila melanogaster nAChRs has so far been unable to directly examine the interactions between α- and β-subunits that must occur in vivo. No reports of in vitro expression of a fully functional receptor have been published for D. melanogaster. Most information to date about the functionality of Drosophila subunits has come from studies in which α-subunits were co-expressed with a vertebrate β-subunit (Bertrand et al., 1994; Lansdell and Millar, 2000), or N-terminal regions were fused with C-terminal regions of 5HT3 genes (Lansdell and Millar, 2004).
Resistance to neonicotinoids has appeared in field populations of insects, in some species prior to commercial introduction (Tomizawa and Casida, 2003). Neonicotinoid resistance by metabolic detoxification of the insecticide, arising initially due to selection by DDT, has been found in D. melanogaster (Daborn et al., 2002). Long-term selection in Bemisia tabaci produced strains up to 82-fold resistant (Prabhaker et al., 1997). A mutation in two nAChR α-subunits of Nilaparvata lugens reduced radiolabelled insecticide binding in assays with vertebrate β-subunits (Liu et al., 2005). This mutation, a replacement of a tyrosine with serine in a position homologous to Y151 of the Torpedo α1 subunit, occurred in N. lugens subunits Nlα1 and Nlα3, in a strain reported to have up to 250-fold resistance (Liu and Han, 2006). If this were to become established in field populations the high target site resistance could greatly reduce the efficacy of pest control with neonicotinoids. This study provides valuable insights into the nAChR subunits targeted by neonicotinoids. Furthermore, the mutations available for selection also provide a valuable complement to the domain of predictability currently attainable by heterologous expression systems.
Previous studies utilising EMS mutagenesis and selection in laboratory populations of insects have reproduced insecticide-resistance alleles previously detected in the field (McKenzie and Batterham, 1998). We chose a similar approach to investigate resistance to neonicotinoids in D. melanogaster. Due to the nature of the nAChR and its excitatory role in the nervous system, it was highly likely that target site resistance would be recessive. Support for this was provided by a recent study into a spinosad resistance mechanism in D. melanogaster where a knockout of the Dα6 nAChR subunit confers high levels of incompletely recessive resistance to spinosad (Perry et al., 2007).
To circumvent the difficulties with genome-wide screens for recessive mutations, we focused on specific chromosomal regions for which genes encoding nAChR subunits were deleted. Screening mutagenised chromosomes over such deletions permitted us to discern recessive, resistance-conferring mutations that were not masked by the wild-type susceptible allele. We found four highly resistant, recessive nAChR mutants by employing this approach. Mutations occurred in both α- and β-subunit encoding genes.
Section snippets
Chemicals
Analytical grade nitenpyram, imidacloprid, and thiamethoxam were provided by Novartis Animal Health Australasia P/L. Nitenpyram was dissolved in distilled water while imidacloprid and thiamethoxam were dissolved in acetone. All stock solutions were made to 0.1% (w/v).
Strains
Armenia (Armenia60) was sourced from the European Drosophila Stock Centre (Umeå, Sweden). 2363 (Df(3R)crb87-5, st1e1/TM3, Ser1) (Df2363) was sourced from the Bloomington Stock Centre (Indiana, USA). Deficiency stocks used in the
Isolation of resistant mutants
A preliminary screen of several deficiency strains uncovering various nAChR subunit genes (Dα1, Dα2, Dα5, Dα6, Dα7, Dβ1, Dβ2, or Dβ3; Supplementary Fig. 5) showed that Df2363 exhibited an elevated tolerance to nitenpyram. This strain is deficient across the cytological region 96A on chromosome 3 uncovering a cluster of three nAChR genes, Dα1, Dα2, and Dβ2. EMS mutagenesis (Section 2.4) generated four strains (Ems1, Ems2, Ems3, and Ems4) with high levels of survival (up to and above 45 ppm),
Plasticity of the CNS
The D. melanogaster genome encodes 10 putative nAChR subunits (Sattelle et al., 2005). Despite this relatively low number compared to other species with fully sequenced genomes, it still has the potential to express a diverse array of receptors by combining different subunits and their alternatively spliced isoforms as well as RNA editing (Sattelle et al., 2005). The actual combinations employed in vivo, their tissue distributions and developmental profiles and their differential responses to
Acknowledgements
This work was funded with financial support provided by Novartis Animal Health Australasia P/L and an Australian Research Council SPIRT grant as well as continuing support from an Australian Wool Innovation Postdoctoral Fellowship. Novartis also provided the neonicotinoid chemicals. The authors also thank John Damiano and Ayscha Hill-Williams for technical assistance with EMS treatment of flies and Phillip Daborn and Len Kelly for helpful comments on the manuscript.
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