Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua

Abstract

Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss‐of‐function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad‐resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR‐associated protein 9 (Cas9) gene‐editing technology. Reverse transcriptase‐polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230‐fold) and spinetoram (792‐fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene‐editing technology to confirm the role played by candidate target site mutations in insecticide resistance.

Effects of nicotinic acetylcholine receptor subunit deletion mutants on insecticide susceptibility and fitness in Drosophila melanogaster

“…Therefore, if the presence of a particular nAChR subunit mutant can lead to decreased susceptibility to insecticides, such a mutant in the field population can potentially lead to an increased risk of cross‐resistance to these insecticides. For example, the G275E mutation of α6 increased the resistance of spinosyns in S. exigua , and the R81T mutation of β1 increased the resistance of imidacloprid and thiamethoxam in A. gossypii 26, 56 . Therefore, from the perspective of insecticide resistance management, it is important to avoid the rotation of insecticides that attack the same target site.…”

“…For example, the G275E mutation of ⊍6 increased the resistance of spinosyns in S. exigua, and the R81T mutation of ⊎1 increased the resistance of imidacloprid and thiamethoxam in A. gossypii. 26,56 Therefore, from the perspective of insecticide resistance management, it is important to avoid the rotation of insecticides that attack the same target site. It is important to highlight, however, that a degree of caution should be applied when extrapolating experimental findings, probably point mutants are more powerful tools in analyzing neonicotinoid mode of action than deletion mutants.…”

Nicotinic modulation insecticides act on diverse receptor subtypes with distinct subunit compositions

“…The spinosyns including spinosad and spinoteram have been shown to act a population of nAChR that are not targeted by neonicotinoids, the binding site is also distinct to the orthosteric site [4]. The 6 subunit has been proposed as the main target of spinosyns since the resistance to spinosad in many insects is associated with loss-of-function mutations in the 6 gene [24], however, whether other subunits are involved is still unknown. We used spinoteram in bioassays and the results strongly indicated that spinosyns may specifically act on the 6 homomeric nAChR but not any other subtypes (Figure 4), which is consistent with a recent report using spinosad [25].…”

Drosophila nicotinic acetylcholine receptor subunits and their native interactions with insecticidal peptide toxins

“…An essential pre-requisite for effective design of new insecticides targeting these receptors is an understanding of their distinctive binding properties. For many reasons, including low expression in endogenous tissues or difficulties in expressing insect receptors in heterologous systems, the characterisation of functional insect receptors has been challenging ( Perry et al, 2021 ; Zuo et al, 2022 ; Salgado, 2021 ). Even in the tractable D. melanogaster insect model, there has been no systematic isolation of mutations in nAChR subunit genes, until recently, when Perry and colleagues described the generation of a new set of null mutations in nine out of the ten D. melanogaster subunit genes ( Perry et al, 2021 ).…”