An insertion in intron 3 of nAChR α6 subunit is associated with spinosad resistance in the western flower thrips Frankliniella occidentalis

Derived from natural products of a soil bacteria, spinosad is a highly effective insecticide with a broad spectrum and a sound environmental profile. The mode of action of spinosad is the disruption of a neural mechanism, targeting primarily at the α6 subunit of nicotinic acetylcholine receptors (nAChRs) of the insect nervous system. Insect pests, including the western flower thrips, Frankliniella occidentalis (Pergande), however, have developed resistance to spinosad by producing truncated α6 subunits. Here, we cloned and compared the splicing variants and genomic DNAs of α6 subunit using a pair of spinosad susceptible (Ivf03) and resistant (NIL-R) near-isogenic F. occidentalis lines with the same genetic back-ground. The resultant structural differences in α6 subunit between Ivf03 and NIL-R were further validated in Sf9 cells using a mini-gene construct. Results showed 100% truncation of the α6 subunit transcripts in the NIL-R strain, and the presence of seven INDELs (insertions and deletions) within α6 subunit between the two strains, where a 230 bp insertion in intron 3 of NIL-R had inverted repeat at both ends. In Sf9 cells, this insertion sequence was able to mediate 92.7% truncation of the mini-gene transcript. More importantly, the ratio of insertion is positively correlated with spinosad resistance levels in both lab (4) and field (8) F. occidentalis populations (y=53.28x-4.054, R²=0.9527, N=12). This study not only advances our mechanistic understanding of spinosad resistance in F. occidentalis, but also provides a diagnostic tool for monitoring the development of resistance in the field.