Yidong Wu

Background: The cotton bollworm Helicoverpa armigera is a major global pest controlled by genetically engineered crops expressing Bacillus thuringiensis (Bt) toxins, including Vip3Aa. While Vip3Aa is widely deployed, the genetic basis of resistance remains poorly understood. Previous work identified disruption of a thyroglobulin-like gene (HaVipR1) as one mechanism of resistance, suggesting additional loci may be involved. Results: Using linkage analysis, transcriptomics, long-read sequencing, and CRISPR-Cas9 gene editing, we identify a second thyroglobulin-like gene, HaVipR2, as a novel mediator of Vip3Aa resistance. Resistance in a field-derived H. armigera line was shown to be monogenic, recessive, and autosomal, mapping to chromosome 29. Long-read sequencing revealed a ∼16 kb transposable element insertion disrupting HaVipR2, which was undetectable using standard short-read approaches. CRISPR-Cas9 knockout of HaVipR2 conferred >900-fold resistance, confirming its causal role. Comparative analyses show that HaVipR1 and HaVipR2 share conserved domain architecture, indicating that thyroglobulin-domain proteins represent a recurrent target of resistance evolution. Conclusions: Our findings establish thyroglobulin-domain proteins as a new class of Bt resistance genes in Lepidoptera and demonstrate that transposable element insertions can drive adaptive resistance while evading detection by conventional methods. These results highlight the importance of long-read sequencing and accurate genome annotation for resistance monitoring and provide new insights into the molecular basis and evolution of Vip3Aa resistance.

BACKGROUND: The beet armyworm, Spodoptera exigua , is a polyphagous pest with long‐distance migration capabilities. It has been a major crop pest in southeastern China since the 1980s, but only emerged as a significant pest in northwestern China (Xinjiang) over the past decade. As a consequence of intensive insecticide use, this pest has developed serious resistance. Although insecticide resistance has been documented in southeastern populations, data on the current resistance status, particularly in Xinjiang populations, remain limited. RESULTS: We evaluated resistance in 13 southeastern populations collected in 2023 and five Xinjiang populations collected in 2024 to four insecticides by comparing their median lethal concentrations (LC ₅₀ s) with those of the reference WH‐S strain. All 18 field populations exhibited high‐level resistance to emamectin benzoate (EB; 711‐ to 8790‐fold) and chlorantraniliprole (103‐ to 1422‐fold) relative to a susceptible laboratory strain, whereas resistance to broflanilide and chlorfenapyr was absent or limited. Genotyping identified high frequencies of the CYP9A186 F116V (0.55–1.00) and RyR I4743M/K (0.92–1.00) mutations, which are associated with resistance to EB and chlorantraniliprole, respectively. Resistance magnitudes and resistance‐allele frequencies were similar in Xinjiang and southeastern China. Furthermore, resistance to EB and chlorantraniliprole has markedly increased over time (from 2009 to 2024) in southeastern populations. CONCLUSION: Our results demonstrate widespread, high‐level resistance to EB and chlorantraniliprole in S. exigua across China and near‐fixation of two major resistance alleles. Although broflanilide and chlorfenapyr currently remain effective alternatives, they should be used judiciously given the ≈10‐fold existing variation in susceptibility to both insecticides. Coordinated geographical resistance monitoring and management are needed for S. exigua in China and elsewhere, given its strong migratory and dispersal capabilities. © 2026 Society of Chemical Industry.

The beet armyworm, Spodoptera exigua, threatens agriculture due to its rapid evolution of insecticide resistance. CYP9A9a and CYP9A9b are close paralogous P450s, and mutant CYP9A9a (V40L/Q160H/T208A) was previously linked to cyhalothrin resistance but unvalidated in vitro. Here, we studied esfenvalerate resistance mediated by mutant CYP9A9a in the field-derived strain FX21 (960-fold resistance to esfenvalerate) using in vitro and genetic approaches. Both CYP9A9a (1500-fold) and CYP9A9b (670-fold) are overexpressed in the larval midgut. Kinetic analyses showed that CYP9A9a has a similar substrate affinity but a 2.3-fold higher catalytic turnover, yielding 1.9-fold greater metabolic efficiency than CYP9A9b. CRISPR knockout of CYP9A9b alone did not alter susceptibility, whereas dual knockout reduced resistance by 294-fold, underscoring CYP9A9a's essential role. Thus, CYP9A9a is the principal resistance determinant via qualitative mutations and quantitative overexpression, making it a key marker for monitoring pyrethroid resistance in S. exigua. We also address why CYP9A9b exhibits in vitro activity but does not confer resistance in vivo.

BACKGROUND: Bacillus thuringiensis (Bt) insecticidal proteins, including crystalline (Cry) proteins and vegetative insecticidal proteins (Vips), are extensively used in transgenic crops due to their efficacy, low environmental impact, and safety. The fall armyworm, Spodoptera frugiperda , has evolved practical resistance to Cry1Fa, yet no practical resistance to Vip3Aa has been documented. However, both laboratory selection and field screen studies indicate a high potential for this pest to evolve resistance to Vip3Aa, making it crucial to evaluate potential resistance genes. HaVipR1 has recently been identified as a key determinant of Vip3Aa resistance in the cotton bollworm, Helicoverpa armigera . This study investigated whether the HaVipR1 ‐homologous gene in S. frugiperda ( SfVipR1 ) is similarly involved in Vip3Aa resistance. RESULTS: We employed CRISPR/Cas9 technology to generate a homozygous knockout strain of SfVipR1 . In comparison with the parent susceptible YJ‐19 strain, the knockout strain (Sfru‐KO) exhibited high‐level resistance to Vip3Aa (>1850‐fold) but showed no cross resistance to Cry1Fa. Resistance to Vip3Aa in Sfru‐KO is autosomal, recessive, and genetically linked with SfVipR1 . CONCLUSION: Disruption of SfVipR1 results in high‐level resistance to Vip3Aa, highlighting SfVipR1 has a critical role in Vip3Aa toxicity in S. frugiperda , despite the exact mechanism remaining unclear. Early detection of SfVipR1 mutant alleles in the field is essential for developing adaptive resistance management strategies against S. frugiperda . © 2025 Society of Chemical Industry.