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A Dα6 knockout strain of Drosophila melanogaster confers a high level of resistance to spinosad

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Abstract

A null mutation of the nicotinic acetylcholine receptor (nAChR) subunit Dα6, in Drosophila melanogaster, confers 1181-fold resistance to a new and increasingly important biopesticide, spinosad. This study's molecular characterisation of a spinosad resistance mechanism identifies Dα6 as a major spinosad target in D. melanogaster. Although D. melanogaster is not a major field pest, target site resistances found in this species are often conserved in pest species. This, combined with the high degree of evolutionary conservation of nAChR subunits, suggests that mutations in Dα6 orthologues may underpin the spinosad resistance identified in several economically important field pests.

Introduction

The evolution of resistance to different insecticides has made effective control of pests increasingly dependent on the use of new classes of chemicals. One such group, the spinosyns, derive from fermentation products of Saccharopolyspora spinosa which have varying degrees of insecticidal activity (Sparks et al., 1995, Bret et al., 1997). S. spinosa produces a large number of these secondary metabolites. Two of these form the commercialised insecticide formula, spinosad (85% spinosyn A :15% spinosyn D) (marketed as Success1). Efficacy has been reported against Diptera, Lepidoptera and Thysanoptera as well as many other insects (Bret et al., 1997, Thompson et al., 1995).

The targets of spinosyn A and D are thought to be the nicotinic acetylcholine receptors (nAChRs) with some evidence to suggest additional targeting to the GABA receptor (Salgado, 1997, Watson, 2001). Involuntary contractions and neuron over-excitation are initial responses to spinosad with paralysis and death following prolonged exposure. This is due to exhaustion of the firing capability of the neurons and not through damage to the nervous system (Salgado, 1998). While both spinosyns and the neonicotinoid class insecticides target nAChRs, spinosyns do not displace a neonicotinoid (imidacloprid) from receptors. This suggests that these insecticides bind to non-overlapping sites (Salgado, 1997) and/or may bind to separate classes of nAChR's (Salgado and Saar, 2004).

High-level resistance to spinosad has been documented in field populations of several pest species including Spodoptera exigua (Beet armyworm, >345-fold) (Wang et al., 2006), Plutella xylostella (Diamondback moth, 20,600-fold) (Sayyed et al., 2004, Zhao et al., 2002), Heliothis virescens (Tobacco budworm, 669-fold) (Wyss et al., 2003), and Musca domestica (Housefly, >150-fold) (Shono and Scott, 2003). This study characterises a recessive spinosad resistance (1181-fold) in a strain of Drosophila melanogaster. This resistance is caused by a loss of function mutation in an nAChR subunit gene, Dα6. Loss of function mutations in Dα6 orthologues may lead to spinosad resistance in field populations of insect pests.

Section snippets

Materials and methods

D. melanogaster strains used in this study were: the control strain, y; cn bw sp, deficiencies, {w;Df(2L)s1402,P{w+mC=lacW}s1402/CyO} and Df(2L)Exel6025/CyO which were sourced from the Bloomington Stock Centre, USA. They were maintained at 25C and reared on standard semolina media.

The insecticide formulation, spinosad (120 g/l) was added to media after appropriate dilutions in water for screening. Survival to adult emergence was recorded for a range of spinosad concentrations from 0 to 100 ppm.

Results

Deficiency strains hemizygous for known nAChR subunit genes in D. melanogaster were screened with spinosad (not shown). The Df(2L)s1402/CyO strain was identified as being resistant. Dosage mortality analysis showed that the Df(2L)s1402/CyO strain was 1181-fold resistant to spinosad compared to a susceptible strain, y;cn bw sp (Table 1). The Dα6 nAChR subunit gene is located in the region deleted on the deficiency chromosome. Investigation of resistance status of heterozygotes revealed that the

Discussion

This paper demonstrates that a loss of function mutation in the Dα6 nAChR subunit gene is responsible for spinosad resistance in D. melanogaster. nAChRs are also involved in mechanisms of resistance to neonicotinoids, with amino acid substitutions in the ligand binding region of two nAChR subunit genes (Nlα1 and Nlα3) conferring resistance to imidacloprid in Nilaparvata lugens (Liu et al., 2005). Loss of function has previously only been associated with resistance for targets outside the

Acknowledgements

This project was supported by a stipend provided through an Australian Research Council SPIRT Grant awarded to John McKenzie and Philip Batterham.

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