In vivo functional analysis of the Drosophila melanogaster nicotinic acetylcholine receptor Dα6 using the insecticide spinosad

Somers, Jason; Nguyen, Joseph; Lumb, Chris; Batterham, Philip; Perry, Trent

doi:10.1016/j.ibmb.2015.01.018

Cited by 59 publications

(75 citation statements)

References 49 publications

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“…Validating the significance of the Dmα6 subunit for insecticidal toxicity, introduction of an EMS-isolated mutation into a controlled genetic background via the CRISPR/ Cas9 system was itself sufficient to generate resistance to spinosad in D. melanogaster. 147) These findings from the model organism Drosophila, are in line with investigations of spinosyn resistance in agricultural pests. High-level resistance in the diamond back moth Plutella xylostella was widespread in Hawaii within 2 years of first use 148) and is associated with a mutation within an intron region of Pxα6, leading to gene mis-splicing and a predicted truncated Pxα6 nAChR subunit.…”

Section: Spinosynssupporting

confidence: 73%

“…151) Given these observations it has been suggested that any mutation that causes a loss of α6 will confer resistance to spinosad. 147) In all cases so far though, spinosyn resistance has been incompletely dominant. However such alterations in α6 invertebrate nAChRs are without any serious or even no observable fitness penalty associated.…”

Section: Spinosynsmentioning

confidence: 99%

“…However such alterations in α6 invertebrate nAChRs are without any serious or even no observable fitness penalty associated. 139,147) This may be a result of receptor redundancy, as the Dmα6 subunit shares a high degree of homology to the Dmα5 and Dmα7 subunits, which are known to be required for the formation of α-BGTX-sensitive but IMI insensitive nAChRs. 152) Biochemical, electrophysiological and resistance based-approaches tie in to collectively imply that the spinosyns act as allosteric modulators, at a binding site located within the TMD of α-BGTX-sensitive α6 containing invertebrate nAChRs.…”

Section: Spinosynsmentioning

confidence: 99%

“…152) Biochemical, electrophysiological and resistance based-approaches tie in to collectively imply that the spinosyns act as allosteric modulators, at a binding site located within the TMD of α-BGTX-sensitive α6 containing invertebrate nAChRs. 147) The vertebrate α7 is the closest vertebrate orthologue of Dmα6 and there are now many synthetic small molecule allosteric modulators identified. Thus it is reasonable to expect identification of fully synthetic low molecular weight allosteric modulators of invertebrate nAChRs is feasible.…”

Section: Spinosynsmentioning

confidence: 99%

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The invertebrate pharmacology of insecticides acting at nicotinic acetylcholine receptors

Crossthwaite

Bigot

Camblin

et al. 2017

Journal of Pesticide Science

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The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel composed of 5 protein subunits arranged around a central cation selective pore. Several classes of natural and synthetic insecticides mediate their effect through interacting at nAChRs. This review examines the basic pharmacology of the neonicotinoids and related chemistry, with an emphasis on sapfeeding insects from the order Hemiptera, the principle pest target for such insecticides. Although the receptor subunit stoichiometry for endogenous invertebrate nAChRs is unknown, there is clear evidence for the existence of distinct neonicotinoid binding sites in native insect preparations, which reflects the predicted wide repertoire of nAChRs and differing pharmacology within this insecticide class. The spinosyns are principally used to control chewing pests such as Lepidoptera, whilst nereistoxin analogues are used on pests of rice and vegetables through contact and systemic action, the pharmacology of both these insecticides is unique and different to that of the neonicotinoids.

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Section: Spinosynssupporting

confidence: 73%

Section: Spinosynsmentioning

confidence: 99%

Section: Spinosynsmentioning

confidence: 99%

Section: Spinosynsmentioning

confidence: 99%

See 2 more Smart Citations

The invertebrate pharmacology of insecticides acting at nicotinic acetylcholine receptors

Crossthwaite

Bigot

Camblin

et al. 2017

Journal of Pesticide Science

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“…Our results show that a reverse genetics strategy is exceptionally suitable for the elucidation of the MoA of insecticides and/or functional validation of mutations associated with insecticide resistance in a wide array of targets that are otherwise difficult to study. CRISPR/Cas9 has already been used in Drosophila for resistance research before (25,26). However, in this study, we have generated lines bearing homozygous recessive gain-of-function mutations in a single-copy gene, thus enabling comprehensive investigation-that is, comparative bioassays for these particular mutations against several insecticide classes.…”

Section: Discussionmentioning

confidence: 99%

Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis

Douris

Steinbach

Panteleri

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

157

166

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Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin-an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro

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Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella

Wang

et al. 2019

Pest Management Science

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BACKGROUND The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach. RESULTS Here, we successfully constructed a homozygous strain (Pxα6‐KO) with a 2‐nt deletion mutation of nAChRα6 by CRISPR/Cas9 coupled with non‐homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3–TM4 loop region. When compared to the background strain IPP‐S, the knockout strain Pxα6‐KO exhibited 229‐ and 1462‐fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3‐fold) effects on the toxicities of imidacloprid, abamectin, β‐cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2‐nt deletion mutation of nAChRα6 in the Pxα6‐KO strain. CONCLUSION In vivo functional investigation demonstrates the causality of the Pxα6 truncating mutation with high levels of resistance to spinosyns in P. xylostella. Our results suggest the Pxα6‐KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. © 2019 Society of Chemical Industry

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In vivo functional analysis of the Drosophila melanogaster nicotinic acetylcholine receptor Dα6 using the insecticide spinosad

Cited by 59 publications

References 49 publications

The invertebrate pharmacology of insecticides acting at nicotinic acetylcholine receptors

The invertebrate pharmacology of insecticides acting at nicotinic acetylcholine receptors

Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis

Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella

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