BACKGROUND: The beet armyworm, Spodoptera exigua, is a serious agricultural pest that is primarily controlled using chemical insecticides. Recently, resistance to the insecticide spinosad has been described in S. exigua field populations. To date, there has been no functional evidence proving the involvement of the nicotinic acetylcholine receptor (nAChR) ⊍6 mutation in spinosad resistance in S. exigua.RESULTS: In this study, using the CRISPR/Cas9 genome-editing system, a homozygous strain (Se⊍6-KO) with approximately 1760-bp deletion within Se⊍6 in S. exigua causing a premature truncation of Se⊍6 was successfully constructed. Insecticide bioassays showed that Se⊍6-KO exhibited 373-fold higher resistance to spinosad and 850-fold higher resistance to spinetoram compared to WH-S strain with the same genetic background but showed no significant change in susceptibility to emamectin benzoate and chlorantraniliprole. Genetic analysis revealed that Se⊍6-KO is inherited as an incompletely recessive trait. CONCLUSION:The results clearly demonstrated the functional role of Se⊍6 in resistance to spinosyn insecticides and provide an example of using genome editing to verify a target premature truncation associated with resistance.

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.

BACKGROUND: Insect cytochrome P450 monooxygenases play important roles in the detoxification metabolism of endogenous and exogenous compounds. Haedoxan A (HA) from Phryma leptostachya L. is a highly efficient natural pesticide used to control houseflies and mosquitos. CYP4C21 and CYP304A1 were previously demonstrated to be transcriptionally increased in Aedes albopictus in response to HA exposure, but their involvement in HA metabolism is unknown. RESULTS: Our data showed that CYP304A1 expression levels in A. albopictus were highest in third-instar larvae, and the expression level of CYP4C21 decreased significantly with the growth of instars, with the lowest occurring in the pupal stage. Compared with the control, the silencing of CYP304A1 and CYP4C21 genes by chitosan nanoparticle-mediated RNA interference could deplete 58.2% and 54.0% of the expression of corresponding genes, respectively. The bioassay data showed that knocking down the expression of CYP304A1 increased the mortality of A. albopictus when exposed to HA at LC 30 and LC 50 doses, but did not significantly increase mortality after silencing CYP4C21. Our data demonstrated that CYP304A1, but not CYP4C21, may be involved in HA detoxification. Moreover, the resistance ratio of CYP304A1 overexpressing flies was approximately 2-fold higher than that of the control line. The metabolized product of HA by CYP304A1 needs to be further confirmed by in vitro expression. CONCLUSION: This finding showed that inducibility was not always linked to detoxifying capabilities, and enhanced our understanding of the molecular basis of HA metabolic detoxification in A. albopictus.