Role of nicotinic acetylcholine receptor subunits in the mode of action of neonicotinoid, sulfoximine and spinosyn insecticides in Drosophila melanogaster

https://doi.org/10.1016/j.ibmb.2021.103547Get rights and content

Highlights

  • CRISPR/Cas9 used to systematically analyse the insect nAChR gene family.

  • Eight of the nine nAChR subunit mutants created could be maintained as homozygotes.

  • Receptor subunit combinations targeted within and between insecticide classes vary.

  • Insecticide targets are highly conserved across insect species.

Abstract

Insecticides remain valuable tools for the control of insect pests that significantly impact human health and agriculture. A deeper understanding of insecticide targets is important in maintaining this control over pests. Our study systematically investigates the nicotinic acetylcholine receptor (nAChR) gene family, in order to identify the receptor subunits critical to the insect response to insecticides from three distinct chemical classes (neonicotinoids, spinosyns and sulfoximines). Applying the CRISPR/Cas9 gene editing technology in D. melanogaster, we were able to generate and maintain homozygous mutants for eight nAChR subunit genes. A ninth gene (Dβ1) was investigated using somatic CRISPR in neural cells to overcome the low viability of the homozygous germline knockout mutant. These findings highlight the specificity of the spinosyn class insecticide, spinosad, to receptors containing the Dα6 subunit. By way of contrast, neonicotinoids are likely to target multiple receptor subtypes, beyond those receptor subunit combinations previously identified. Significant differences in the impacts of specific nAChR subunit deletions on the resistance level of flies to neonicotinoids imidacloprid and nitenpyram indicate that the receptor subtypes they target do not completely overlap. While an R81T mutation in β1 subunits has revealed residues co-ordinating binding of sulfoximines and neonicotinoids differ, the resistance profiles of a deletion of Dβ1 examined here provide new insights into the mode of action of sulfoxaflor (sulfoximine) and identify Dβ1 as a key component of nAChRs targeted by both these insecticide classes. A comparison of resistance phenotypes found in this study to resistance reported in insect pests reveals a strong conservation of subunit targets across many different insect species and that mutations have been identified in most of the receptor subunits that our findings would predict to have the potential to confer resistance.

Introduction

For more than a century insecticide resistance has reduced the capacity of successive generations of insecticides to control pest insects that damage crops and threaten human and animal health (Ffrench-Constant, 2013; McKenzie and Batterham, 1998; Nauen and Denholm, 2005; Whalon et al., 2008). Insecticides are still pivotal for the control of many pest insects, although a wide range of other practices are routinely implemented. Efforts to prevent or stall the evolution and spread of resistance to current and future insecticidal compounds are facilitated by a detailed understanding of their mode of action (Nauen et al., 2019; Sparks and Nauen, 2015). This knowledge is increasingly valuable in light of losses caused by ineffective pest control and the increasing technological and regulatory costs of bringing new insecticides to market (Sparks and Lorsbach, 2017).

The insect nervous system provides targets for a majority of the most commercially successful insecticides, including acetylcholinesterases (organophosphates and carbamates), voltage gated sodium channels (pyrethroids and DDT) and γ-aminobutyric acid (GABA) and Glutamate-gated chloride channels (cyclodienes, avermectins, and phenylpyrazoles) (Ffrench-Constant et al., 2016; Sparks and Nauen, 2015; Sparks et al., 2019b). The nicotinic acetylcholine receptor family (nAChRs) has, over time, been targeted by a number of insecticides of diverse chemical structures including nicotine, nereistoxin analogs, the neonicotinoids, spinosyns, sulfoximines, butenolids, and most recently, the mesoionics (Sparks and Nauen, 2015; Casida, 2018; Nauen et al., 2019; Matsuda et al., 2020). At present insecticides acting at the nAChR account for approximately 29% of the global insecticide market, rendering the nAChR the most used target site for insect pest control (Sparks et al., 2020).

Insect nAChRs are members of the cys-loop ligand-gated ion-channel superfamily. In the model insect Drosophila melanogaster there are 10 receptor subunit genes (seven α-subunits and three β-subunits) (Sattelle et al., 2005). The insect nAChR is a pentameric assembly of these subunits, expressed mainly in the central nervous systems of insects (Sattelle and Breer, 1990). Thus, many possible combinations of these subunits could exist producing an array of nAChR subtypes, each potentially having different physiological properties. The endogenous ligand, acetylcholine (ACh), binds at the interface between two receptor subunits. The principal face of the binding pocket for ACh is formed by loops A-C of an α-subunit, while the complementary face is formed from loops D-F of the adjacent α or β subunit (Corringer et al., 2000). This subunit interface is also believed to be the primary site of binding for the neonicotinoid and sulfoximine insecticide classes (Ihara et al., 2015; Matsuda et al., 2005; Wang et al., 2016). In contrast other nAChR acting insecticides, the spinosyns, appear to act at an allosteric site (Geng et al., 2013; Puinean et al., 2013; Silva et al., 2016; Somers et al., 2015; Watson et al., 2010).

The Insecticide Resistance Action Committee (IRAC) (Nauen et al., 2019; Sparks and Nauen, 2015) classifies insecticides according to their mode of action. Compounds are also allocated to distinct subgroups based on their chemical structure and information demonstrating differential metabolism and reduced resistance from testing of resistant insect strains. Whereas the insecticides imidacloprid (IMI), nitenpyram (NIT) and sulfoxaflor (SFX) are categorized as nAChR competitive modulators; the neonicotinoids (including IMI & NIT) are both in the same subgroup (4A) (Nauen et al., 2019; Sparks and Nauen, 2015), while the sulfoximine, SFX, is a different subgroup (4C) (Sparks and Nauen 2015) due to differences in chemistry and metabolic resistance (Sparks et al., 2013; Watson et al., 2011, 2017). The spinosyns, (including spinosad (SPIN)) are nAChR allosteric modulator – site I compounds and are thus in an entirely different group (Group 5) (Sparks and Nauen, 2015).

Given the contributions of two different subunits to a single ligand binding pocket and that five interfaces are present in a pentameric receptor, it is important to determine which particular nAChR subunits are present and their arrangement. Heterologous expression studies have provided some insights into the interaction of insecticides at specific residues/possible receptor interfaces when insect nAChR subunits are co-expressed with vertebrate β2 or β4 subunits (Lansdell et al., 2008; Lansdell and Millar, 2000a, b; Matsuda et al., 1998; Matsuda et al., 2005) or with an accessory protein from Caenorhabditis elegans, RIC3 (Lansdell et al., 2012; Watson et al., 2010). Until recently, difficulties expressing insect nAChRs limited investigation via these methods (Lansdell et al., 2012; Watson et al., 2010); however co-expression of additional cofactors has led to robust functional expression of several combinations of nAChR subunits from D. melanogaster, Apis mellifera and Bombus terrestris in Xenopus laevis oocytes (Ihara et al., 2020).

Genetic studies offer another line of investigation into the mode of action of insecticides to improve our understanding of the roles and functions of the different nAChR subunits. For example, in D. melanogaster, loss of function mutants were used to show that the Dα1 and Dβ2 subunits are involved in binding of neonicotinoid insecticides including IMI and NIT (Perry et al., 2008). The modest levels of resistance measured for these mutants, singly and in combination, suggested that receptors comprised of additional subunits are targeted by neonicotinoids (Perry et al., 2008). Because the Dα1 and Dβ2 mutants were not highly resistant to SFX or SPIN insecticides (Perry et al., 2012), this suggested that the neonicotinoids, sulfoximines and spinosyns target different nAChR subtypes. In contrast to the neonicotinoids, the Dα6 subunit has been established as the main target of the spinosyns in Drosophila (Perry et al., 2007; Watson et al., 2010). Mutations in Dα6 or its orthologues are associated with high levels of resistance in Drosophila (Crouse et al., 2018; Perry et al., 2007; Sparks et al., 2019a; Watson et al., 2010) and a wide range of pest insects (Bao and Xu, 2011; Baxter et al., 2010; Geng et al., 2013; Puinean et al., 2013; Silva et al., 2016). One caveat to the findings of previous studies is that only a limited set of receptor mutations known to confer resistance were identified and tested, making it unclear as to whether other subunits play a role, either major or minor. The advent of the capability to specifically manipulate genomes at will provides a new avenue to understand the target site of insecticides and their respective resistance mechanisms (Perry and Batterham, 2018; Somers et al., 2015; Zimmer et al., 2016), and hence options for insecticide resistance management (IRM) programs.

Herein we report our findings from the first systematic investigation of the potential involvement of Drosophila melanogaster nAChR subunits in the mode of action of these three insecticide classes (neonicotinoids, sulfoximines, spinosyns). We took advantage of gene editing techniques using Clustered regularly Interspaced Palindromic Repeats (CRISPR) and the CRISPR Associated Protein 9 (Cas9) nuclease to disrupt the function of nine of the ten individual nAChR subunit genes in D. melanogaster. Our findings help to refine the list of subunits that are involved in the insecticidal activity of the tested nAChR-acting compounds and the level of resistance conferred from loss of function. They also provide insights into the potential combinations of subunits that may co-assemble into native insect nAChR subtypes, an area highly relevant to the understanding of insecticide mode of action and resistance as well as insect neurophysiology. A detailed understanding of an insecticide's mode of action is an important component for the development of effective IRM strategies (Nauen et al., 2019; Sparks and Nauen, 2015), especially those involving the rotation of groups possessing a different mode of action, in an effort to reduce selection for resistance and cross-resistance (Roush, 1989).

Section snippets

Compounds

Spinosad (Success®; Dow AgroSciences), imidacloprid 99% (Pestanal®; Sigma-Aldrich), nitenpyram 99% (Pestanal®; Sigma-Aldrich) were purchased commercially. Sulfoxaflor (99%, racemic mixture) was synthesised and provided by Dow AgroSciences (now Corteva Agriscience).

Toxicology bioassays

D. melanogaster larval bioassays were performed as described previously (Perry et al., 2012). Briefly, 1st instar larvae were collected and reared on semolina-based fly media containing selected doses of the insecticides. Control

nAChR subunit deletions created using CRISPR/Cas9

Deletions of varying lengths were isolated for nine nAChR subunit genes using a variety of CRISPR/Cas9 methods (Fig. 1). Flies homozygous for a deletion of Dα2, Dα3 or Dβ3 were viable. Consistent with published data, the Dα1, Dβ2, Dα4, Dα6 and Dα7 mutants were also viable (Fayyazuddin et al., 2006; Perry et al., 2007, 2008; Shi et al., 2014). With the exception of Dβ1, the mean 1st instar to adult viability of these mutants on untreated media ranged from 84% to 112.8% when compared to the

Germline and somatic CRISPR/Cas9 gene editing can be used to examine insecticide targets

The capacity of CRISPR/Cas9 methods to specifically target and manipulate genes to validate their role in insecticide resistance generated in the lab (Somers et al., 2015), or identified in the field (Zimmer et al., 2016), is clear. Here, we used CRISPR/Cas9 to systematically create mutations in nAChR subunit genes to examine the contribution of individual gene family members to resistance phenotypes. Microinjection of the CRISPR/Cas9 components in combination with transgenic approaches (Gratz

Declarations of competing interest

T. Perry and P. Batterham received the sulfoxaflor compound and funding assistance towards sulfoxaflor bioassays from Dow AgroSciences. T. C. Sparks currently acts as a consultant for the agrochemical industry including Corteva Agriscience.

Acknowledgements

We thank Mr. Gerald Watson and Dr. Melissa Siebert (Corteva Agriscience) for useful discussions and feedback during the preparation of this article. Dow AgroSciences synthesised sulfoxaflor and provided funding assistance for sulfoxaflor studies. Fly strains were sourced from the Bloomington Drosophila Stock Centre and the Australian Drosophila Biomedical Research Support Facility provided quarantine facilities for imported fly strains. Funding for this research was provided through an

References (76)

  • T. Perry et al.

    The biology of insecticidal activity and resistance

    Insect Biochem. Mol. Biol.

    (2011)
  • T. Perry et al.

    Effects of mutations in Drosophila nicotinic acetylcholine receptor subunits on sensitivity to insecticides targeting nicotinic acetylcholine receptors

    Pestic. Biochem. Physiol.

    (2012)
  • T. Perry et al.

    Mutations in Dalpha1 or Dbeta2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster

    Insect Biochem. Mol. Biol.

    (2008)
  • T. Perry et al.

    A Dalpha6 knockout strain of Drosophila melanogaster confers a high level of resistance to spinosad

    Insect Biochem. Mol. Biol.

    (2007)
  • T. Perry et al.

    Expression of insect alpha6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction

    Insect Biochem. Mol. Biol.

    (2015)
  • W.M. Silva et al.

    Mutation (G275E) of the nicotinic acetylcholine receptor alpha6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: gelechiidae)

    Pestic. Biochem. Physiol.

    (2016)
  • J. Somers et al.

    In vivo functional analysis of the Drosophila melanogaster nicotinic acetylcholine receptor Dalpha6 using the insecticide spinosad

    Insect Biochem. Mol. Biol.

    (2015)
  • T.C. Sparks et al.

    Insecticides, biologics and nematicides: updates to IRAC's mode of action classification - a tool for resistance management

    Pestic. Biochem. Physiol.

    (2020)
  • T.C. Sparks et al.

    IRAC: mode of action classification and insecticide resistance management

    Pestic. Biochem. Physiol.

    (2015)
  • T.C. Sparks et al.

    Sulfoxaflor and the sulfoximine insecticides: chemistry, mode of action and basis for efficacy on resistant insects

    Pestic. Biochem. Physiol.

    (2013)
  • T.C. Sparks et al.

    The new age of insecticide discovery-the crop protection industry and the impact of natural products

    Pestic. Biochem. Physiol.

    (2019)
  • G.B. Watson et al.

    A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression

    Insect Biochem. Mol. Biol.

    (2010)
  • G.B. Watson et al.

    Novel nicotinic action of the sulfoximine insecticide sulfoxaflor

    Insect Biochem. Mol. Biol.

    (2011)
  • G.B. Watson et al.

    Characterization of a nicotinic acetylcholine receptor binding site for sulfoxaflor, a new sulfoximine insecticide for the control of sap-feeding insect pests

    Pestic. Biochem. Physiol.

    (2017)
  • C.T. Zimmer et al.

    A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster

    Insect Biochem. Mol. Biol.

    (2016)
  • W.S. Abbott

    A method of computing the effectiveness of an insecticide

    J. Econ. Entomol.

    (1925)
  • S. Anders et al.

    Differential expression analysis for sequence count data

    Genome Biol.

    (2010)
  • S. Anders et al.

    HTSeq--a Python framework to work with high-throughput sequencing data

    Bioinformatics

    (2015)
  • B. Bao et al.

    Identification of gene expression changes associated with the initiation of diapause in the brain of the cotton bollworm, Helicoverpa armigera

    BMC Genom.

    (2011)
  • C. Bass et al.

    Mutation of a nicotinic acetylcholine receptor beta subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae

    BMC Neurosci.

    (2011)
  • S.W. Baxter et al.

    Mis-spliced transcripts of nicotinic acetylcholine receptor alpha6 are associated with field evolved spinosad resistance in Plutella xylostella (L.)

    PLoS Genet.

    (2010)
  • M.E. Beck et al.

    Insight into the binding mode of agonists of the nicotinic acetylcholine receptor from calculated electron densities

    ChemPhysChem

    (2015)
  • J.E. Casida

    Neonicotinoids and other insect nicotinic receptor competitive modulators: progress and prospects

    Annu. Rev. Entomol.

    (2018)
  • P.J. Corringer et al.

    Nicotinic receptors at the amino acid level

    Annu. Rev. Pharmacol. Toxicol.

    (2000)
  • V. Croset et al.

    Cellular diversity in the Drosophila midbrain revealed by single-cell transcriptomics

    Elife

    (2018)
  • G.D. Crouse et al.

    De novo design of potent, insecticidal synthetic mimics of the spinosyn macrolide natural products

    Sci. Rep.

    (2018)
  • J. Crow

    Genetics of insect resistance to chemicals

    Annu. Rev. Entomol.

    (1957)
  • P. Cutler et al.

    Investigating the mode of action of sulfoxaflor: a fourth-generation neonicotinoid

    Pest Manag. Sci.

    (2013)
  • Cited by (0)

    1

    Present address, School of Biological Sciences, Monash University, 18 Innovation Walk, Clayton, Victoria, 3800, Australia.

    2

    Present address, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece.

    3

    Present address, School of Science, STEM College, RMIT University, Melbourne, Victoria, Australia.

    4

    Present address Agrilucent LLC, 1547 N State St. PMB 207, Greenfield, IN, 46140, USA.

    View full text