Functional validation of target-site resistance mutations against sodium channel blocker insecticides (SCBIs) via molecular modeling and genome engineering in Drosophila

Abstract

Voltage-gated sodium channels are the target of several insecticides including DTT, pyrethroids, and SCBIs like indoxacarb and metaflumizone. SCBIs are an alternative insecticide resistance management (IRM) strategy against several pests resistant to other compounds. However, resistance to SCBIs has been reported in several pests, in most cases implicating metabolic resistance mechanisms, although in certain indoxacarb resistant populations of Plutella xylostella and Tuta absoluta, two mutations in the domain IV S6 segment of the voltage-gated sodium channel, F1845Y and V1848I have been identified, and have been postulated through in vitro electrophysiological studies to contribute to target-site resistance.In order to functionally validate in vivo each mutation in the absence of confounding resistance mechanisms, we have employed a CRISPR/Cas9 strategy to generate strains bearing homozygous F1845Y or V1848I mutations in the para (voltage-gated sodium channel) gene of Drosophila melanogaster. We performed toxicity bioassays of these strains compared to wild-type controls of the same genetic background. Our results indicate both mutations confer moderate resistance to indoxacarb (RR: 6 -10.2), and V1848I to metaflumizone (RR: 8.4). However, F1845Y confers very strong resistance to metaflumizone (RR: >3400), a finding that may be related to the specific binding of each insecticide to its target.

A first, naturally occurring substitution at the second pyrethroid receptor of voltage‐gated sodium channel of Aedes aegypti

“…At present, however, whether the V253F + M374I + G923S + F1534C haplotype actually confers the same level of resistance as V410L + S723T + V1016I + F1534C is unclear because genetic studies alone cannot exclude the presence of another resistance factor hitchhiking to the VGSC haplotype. Further studies, especially electrophysiological and genome editing analyses, 35 are required to fully understand the contribution of the V253F substitution to the resistance phenotype.…”

Significance and interpretation of molecular diagnostics for insecticide resistance management of agricultural pests

“…In addition, the dominance and fitness cost of certain resistance mutations was determined [31,36,38,39], which further increases the value of a certain molecular marker for IRM [38,[40][41][42]. As introgression of a marker is not feasible for most insect species (however, see [43,44] for exceptions), gene editing in Drosophila and/or pest species have also been a very useful tool for validating and measuring the role and effect of certain mutations in resistance against insecticides [30,[45][46][47][48][49][50][51][52][53].…”

‘What I cannot create, I do not understand’: functionally validated synergism of metabolic and target site insecticide resistance

“…Deltamethrin (99.6%) of technical grade was purchased from Sigma Aldrich (Darmstadt, Germany) and used in contact assays and topical applications. Contact assays were performed as described previously [28]. More specifically, 20 adult female flies (1-3 days old, as per [20,28]) were used for each toxicity assay.…”

“…Contact assays were performed as described previously [28]. More specifically, 20 adult female flies (1-3 days old, as per [20,28]) were used for each toxicity assay. Flies were collected in plastic vials and transferred in scintillation vials coated with insecticide.…”