Short communicationA knockout strain of Drosophila melanogaster confers a high level of resistance to spinosad
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, -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, -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, . Loss of function mutations in 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, and Df(2L)Exel6025/CyO which were sourced from the Bloomington Stock Centre, USA. They were maintained at 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 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 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 ( and ) 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.
References (30)
- et al.
A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX
Neuron
(1990) Studies on the mode of action of spinosad: insect symptoms and physiological correlates
Pestic. Biochem. Physiol.
(1998)- et al.
Desensitizing and non-desensitizing subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in cockroach neurons
J. Insect. Physiol.
(2004) - et al.
Spinosad resistance in the housefly, Musca domestica, is due to a recessive factor on autosome 1
Pestic. Biochem. Physiol.
(2003) - et al.
Selection and characterization of spinosad resistance in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)
Pestic. Biochem. Physiol.
(2006) Actions of insecticidal spinosyns on gamma-aminobutyric acid responses from small-diameter cockroach neurons
Pestic. Biochem. Physiol.
(2001)- et al.
Biology and genetics of a laboratory strain of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae), highly resistant to spinosad
Crop Prot.
(2003) - et al.
Biological properties of spinosad
Down Earth
(1997) - et al.
Loss of the membrane anchor of the target receptor is a mechanism of bioinsecticide resistance
Proc. Natl. Acad. Sci.
(2002) - et al.
A point mutation in a Drosophila GABA receptor confers insecticide resistance
Nature
(1993)
Identification of a gene associated with Bt resistance in Heliothis virescens
Science
Novel putative nicotinic acetylcholine receptor subunit genes, Dalpha5, Dalpha6 and Dalpha7, in Drosophila melanogaster identify a new and highly conserved target of adenosine deaminase acting on RNA-mediated A-to-I pre-mRNA editing
Genetics
Molecular characterization of Dalpha6 and Dalpha7 nicotinic acetylcholine receptor subunits from Drosophila: formation of a high-affinity alpha-bungarotoxin binding site revealed by expression of subunit chimeras
J. Neurochem.
Ion channels and synaptic organization: analysis of the Drosophila genome
Neuron
A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper)
Proc. Natl. Acad. Sci. USA
Cited by (164)
Uridine diphosphate glucosyltransferases are involved in spinosad resistance in western flower thrips Frankliniella occidentalis (Pergande)
2024, Journal of Hazardous MaterialsAberrant splicing of a nicotinic acetylcholine receptor alpha 6 subunit is associated with spinosad tolerance in the thrips predator Orius laevigatus
2024, Pesticide Biochemistry and PhysiologySelection and characterization of spinetoram resistance in field collected Drosophila melanogaster
2023, Pesticide Biochemistry and PhysiologyMicroRNA-263b confers imidacloprid resistance in Sitobion miscanthi (Takahashi) by regulating the expression of the nAChRβ1 subunit
2022, Pesticide Biochemistry and PhysiologyCRISPR-mediated knockout of nicotinic acetylcholine receptor (nAChR) α6 subunit confers high levels of resistance to spinosyns in Spodoptera frugiperda
2022, Pesticide Biochemistry and PhysiologyNACHO permits functional heterologous expression of an insect homomeric α6 nicotinic acetylcholine receptor
2022, Pesticide Biochemistry and Physiology