Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction

Abstract

Insecticide research has often relied on model species for elucidating the resistance mechanisms present in the targeted pests. The accuracy and applicability of extrapolations of these laboratory findings to field conditions varies but, for target site resistance, conserved mechanisms are generally the rule rather than the exception (Perry et al., 2011). The spinosyn class of insecticides appear to fit this paradigm and are a pest control option with many uses in both crop and animal protection. Resistance to spinosyns has been identified in both laboratory-selected and field-collected pest insects. Studies using the model insect, Drosophila melanogaster, have identified the nicotinic acetylcholine receptor subunit, Dα6 as an important target of the insecticide spinosad (Perry et al., 2007; Watson et al., 2010). Field-isolated resistant strains of several agricultural pest insects provide evidence that resistance cases are often associated with mutations in orthologues to Dα6 (Baxter et al., 2010; Puinean et al., 2013). The expression of these receptors is difficult in heterologous systems. In order to examine the biology of the Dα6 receptor subunit further, we used Drosophila as a model and developed an in vivo rescue system. This allowed us to express four different isoforms of Dα6 and show that each is able to rescue the response to spinosad. Regulatory sequences upstream of the Dα6 gene able to rescue the resistance phenotype were identified. Expression of other D. melanogaster subunits revealed that the rescue phenotype appears to be Dα6 specific. We also demonstrate that expression of pest insect orthologues of Dα6 from a variety of species are capable of rescuing the spinosad response phenotype, verifying the relevance of this receptor to resistance monitoring in the field. In the absence of a robust heterologous expression system, this study presents an in vivo model that will be useful in analysing many other aspects of these receptors and their biology.

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

“…In the model insect D. melanogaster , the Dα6 subunit was the first reported major target for spinosad in a strain with a null mutant Dα6 subunit . Using ethyl methanesulfonate (EMS) mutagenesis, premature truncations of Dα6 protein caused a loss of function, resulting in spinosad resistance . Using the in vivo GAL4‐UAS expression system, Dα6/Da7 chimeric subunit studies highlighted the importance of the Dα6 C‐terminal region for spinosad binding.…”

“…25 Using ethyl methanesulfonate (EMS) mutagenesis, premature truncations of D 6 protein caused a loss of function, resulting in spinosad resistance. 9,49 Using the in vivo GAL4-UAS expression system, D 6/Da7 chimeric subunit studies highlighted the importance of the D 6 C-terminal region for spinosad binding. Furthermore, employing the CRISPR/Cas9 editing system, P146S mutation in D 6 was confirmed to lead to spinosad resistance.…”

Invasion biology of spotted wing Drosophila (Drosophila suzukii): a global perspective and future priorities

“…This species has provided a wealth of information on the genetics and molecular basis of insecticide resistance in insects (Bogwitz et al 2005;Perry et al 2015), which will be valuable in understanding resistance in SWD, should it develop.…”

Analysis of persistent changes to γ-aminobutyric acid receptor gene expression in Plutella xylostella subjected to sublethal amounts of spinosad

“…The ubiquitous reduction of dADAR gene expression increased spinosad sensitivity, whereas reduction of dADAR expression in either cholinergic neurons or muscle led to a decrease in spinosad susceptibility (Rinkevich and Scott, 2012). Further, considerable evidence suggests that the expression of pest insect orthologues of Da6 in various species can rescue the spinosad response phenotype, indicating the relevance of this receptor for resistance monitoring in the field (Perry et al, 2015). Although whether GABAR is a direct target site of spinosad is not known, available evidence supports that the expression of the GABAR gene is related to the sub-lethal concentration selection generation; the differences in the expression levels of this gene were a kind of rescue response to spinosad.…”