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

Chris Bass,William T. Garrood,Manuela Eckel-Zimmer,Christoph T. Zimmer,T.G. Emyr Davies,Martin S. Williamson,A. Mirel Puinean

doi:10.1016/j.ibmb.2016.04.007

Chris Bass, William T. Garrood + Show 5 more

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https://doi.org/10.1016/j.ibmb.2016.04.007

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Abstract

Spinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad, and in the process identified this particular subunit as the specific target site. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. The causal role of this mutation has been called into question as, to date, functional evidence proving its involvement in resistance has been limited to the study of vertebrate receptors. Here we use the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. Reverse transcriptase-PCR and sequencing confirmed the presence of the mutation in Dα6 transcripts of mutant flies and verified that it does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in flies with the mutation compared to flies of the same genetic background minus the mutation, clearly demonstrating the functional role of this amino acid substitution in resistance to spinosad. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target-site mutation and provides an example of how recent advances in genome editing can be harnessed to study insecticide resistance.

Highlights

Insecticide resistance is an exceptional example of rapid adaptive evolution and has provided a range of insights into the diversity of genetic alterations that occur in response to novel selective pressures
All offspring tested by PCR and sequencing exhibited mutations around the clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 target site and the ratio of INDEL mutations mediated by NEJR to homology-directed repair (HDR) mediated insertion of the oligo template was 40:60
China and the USA have failed to detect any nicotinic acetylcholine receptor (nAChR) a6 sequence or expression differences between the susceptible and resistant strains prompting the authors to question the role of nAChR a6 subunit in spinosad resistance (Hou et al, 2014)

Summary

Introduction

Insecticide resistance is an exceptional example of rapid adaptive evolution and has provided a range of insights into the diversity of genetic alterations that occur in response to novel selective pressures. A common mechanism of insect resistance to insecticides, termed ‘target-site resistance’ involves alterations (mutations) in the insecticide target protein that reduce its sensitivity to insecticide. Target-site resistance most frequently involves point mutations at select positions in the target receptor as small changes to proteins are least likely to disrupt their, usually important, native function (ffrench-Constant et al, 1998). The nicotinic acetylcholine receptor (nAChR) a6 subunit is a rare example of an insecticide target-site that can tolerate more radical alterations as it appears to be a redundant target (Perry et al, 2007). Several lines of research indicate that spinosad binds at a site distinct from the neonicotinoid insecticides one exerting its effect through an allosteric mechanism (Orr et al, 2009; Puinean et al, 2013; Salgado and Saar, 2004)

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