The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.

Vegetative insecticidal proteins (Vips) are widely used in pest management, but Vip resistance is a big threat. DNA methylation plays important roles in regulating the response of biological organisms to environmental stress. In this study, DNA methylation map was developed for fall armyworm (FAW, Spodoptera frugiperda), and its function in regulating FAW Vip3Aa resistance was explored. FAW was screened by Vip3Aa for 10 generations, and bioassays indicated that Vip3Aa resistance increased trans-generationally. Based on the comparison of DNA methylation maps between Vip3Aa-resistant and -susceptible strains showed that gene body methylation was positively correlated with its expression. Moreover, the study demonstrated that a reduction in the methylation density within the gene body of a 3’5’-cyclic nucleotide phosphodiesterase gene resulted in decreased expression and increased resistance of FAW to Vip3Aa, which was validated through RNAi experiments. The mechanism of Vip3Aa resistance will improve the understanding of DNA methylation and its function in lepidoptera and provide a new perspective for making strategies to pest management.

The sex of Lepidoptera species can typically be identified at the pupal or adult stage by their morphological characteristics. However, for most species, no obvious sex-specific traits can be used at the larval or embryonic stage. The growing interest in studying sex determination and differentiation, along with the potential application of research findings in genetic regulation techniques, is promoting the advancement of new sexing methods at early developmental stages. In this study, the sex of individual eggs and larvae was successfully identified by means of W chromosome molecular markers, which were identified through analysing the previously published W chromosome sequence data of Spodoptera frugiperda (Lepidoptera: Noctuidae). Additionally, we present methods to extract DNA and RNA from individual eggs and larval hemolymph. These techniques provide a simple and dependable method to identify sex at pre-pupal stages. This approach could potentially be extended to other Lepidoptera species of which the W chromosome information is available.

Bacillus thuringiensis ( Bt ) crops expressing Vip3Aa are highly efficacious in controlling major lepidopteran pests and delaying evolution of pest resistance. Although practical resistance to Vip3Aa in the field has not been reported, to proactively manage the pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa. This is particularly important for the fall armyworm ( Spodoptera frugiperda ), one of the most destructive pests around the world, which has evolved practical resistance to Bt crystal (Cry) toxins. Here, a highly Vip3Aa-resistant (resistance ratio: 5,562-fold) strain of S. frugiperda was selected in the laboratory. Results from bulked segregant analysis, fine-scale mapping, and genetic linkage analysis indicate that a mutation in the midgut-specific chitin synthase gene, SfCHS2 , is strongly associated with high-level resistance to Vip3Aa. The resistance is ascribed to the transcriptional variation caused by retrotransposon insertion. The same variation of SfCHS2 was also detected in a field population. Importantly, knockout of SfCHS2 via CRISPR/Cas9 in susceptible S. frugiperda confers its complete resistance (>10,000-fold) to Vip3Aa. Also, we demonstrate that knockout of CHS2 can result in complete resistance to Vip3Aa in additional lepidopteran species, suggesting a general role of this gene in Vip3Aa resistance among lepidopteran pests. These results reported here would contribute to monitor and management of pest resistance to Vip3Aa. ### Competing Interest Statement The authors have declared no competing interest.

Introduction: The development of insecticide resistance in Spodoptera frugiperda populations is a serious threat to the crop industry. Given the spread of invasive resistant populations, prospective monitoring should be accelerated, and the development of diagnostic tools for rapid and accurate assessments of insecticide resistance is essential.Methods: First, the discriminating dose and diagnostic time of the kit were determined by the glass vial method based on a susceptible strain. Then, pests that were collected from field populations were used to determine their susceptibility to seven insecticides by using the diagnostic kit. Finally, the accuracy of the kit was verified based on correlation analyses and the likelihood of insecticide control failure was assessed.Results: Here, we describe a diagnostic kit that enables the rapid detection of resistance to chlorpyrifos, bifenthrin, deltamethrin, lambda-cyhalothrin, phoxim, chlorantraniliprole and chlorfenapyr within 1-2 h in S. frugiperda at diagnostic doses of 0.98, 0.84, 0.38, 1.64, 0.0082, 1.75 and 0.65 μg/cm2, respectively. The linear equation between mortalities under diagnostic doses and actual resistance ratios measured by the diet-overlay bioassay was determined. The high correlation indicates that the insecticide resistance levels diagnosed by the kit were consistent with the results of the diet-overlay bioassay. Moreover, we found a significant negative correlation between diagnostic mortality and the likelihood of control failure for bifenthrin (r = −0.899, p = 0.001), deltamethrin (r = −0.737, p = 0.024) and lambda-cyhalothrin (r = −0.871, p = 0.002).Discussion: The insecticide resistance diagnostic kit for S. frugiperda is a user-friendly tool (portable, short detection time). Its excellent performance qualifies the kit as a reliable screening tool for identifying effective insecticides in sustainable resistance management.

The fall armyworm, Spodoptera frugiperda (J. E. Smith), has become one of the most damaging pests worldwide since its invasion of Africa, Asia and Oceania from 2016, threatening plants in 76 families including important crops. Genetics-based methods have proved to be an efficient way to control pests, especially invasive species, but many difficulties must be overcome to develop a transgenic insect strain, especially for a non-model species. Here we thus sought to identify a visible marker that would facilitate the distinction between genetically modified (GM) and non-transgenic insects, thereby simplifying mutation identification and facilitating the broader application of genome editing tools in non-model insects. Five genes (sfyellow-y, sfebony, sflaccase2, sfscarlet, and sfok) that are orthologs of well-studied genes in pigment metabolism were knocked out using the CRISPR/Cas9 system to identify candidate gene markers. Two genes, Sfebony and Sfscarlet, were identified responsible for body and compound eye coloration, respectively, in S. frugiperda, and could be potential visual markers for genetics-based pest management strategies.

ATP-binding cassette transporter B1 (ABCB1, or P-glycoprotein) is known to be an important participant in multidrug resistance in mammals, and it also has been proved as a transporter for some insecticides in several lepidopteran insects, yet the precise function of this transporter in Spodoptera frugiperda is unknown. Here, we generated a SfABCB1 knockout strain of the S. frugiperda using the CRISPR/Cas9 system to explore its potential roles in determining susceptibility to chemical insecticides or Bt toxins. Bioassay results showed that the susceptibility of SfABCB1 knockout strain to beta-cypermethrin, chlorantraniliprole and emamectin benzoate were significantly increased compared with the wild-type strain DH19, whereas there were no changes to Bt toxins for Cry1Ab, Cry1Fa and Vip3Aa. Our results revealed that SfABCB1 plays important roles in the susceptibility of S. frugiperda to beta-cypermethrin, chlorantraniliprole and emamectin benzoate, and imply that overexpression of ABCB1 may contribute to beta-cypermethrin, chlorantraniliprole and emamectin benzoate resistance in S. frugiperda.