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Background: Vegetative insecticidal proteins (Vips) are widely used in pest management, but Vip tolerance poses a significant threat. DNA methylation plays important roles in regulating the response of biological organisms to environmental stress, and it may also regulate fall armyworm (FAW, Spodoptera frugiperda) Vip3Aa tolerance., Results: In this study, a DNA methylation map was developed for FAW, and its function in regulating FAW Vip3Aa tolerance was explored. The FAW genome-wide DNA methylation map showed that exons were preferred regions for DNA methylation and housekeeping genes were highly methylated. FAW was screened using Vip3Aa for ten generations, and bioassays indicated that Vip3Aa tolerance increased trans-generationally. A comparison of DNA methylation maps between Vip3Aa-tolerant and -susceptible strains showed that gene body methylation was positively correlated with gene expression level. FAW exhibits significant variation in DNA methylation among individuals, and Vip3Aa screening induces epigenetic variation based on DNA methylation. Moreover, the study demonstrated that a reduction in methylation density within the gene body of a 3'5'-cyclic nucleotide phosphodiesterase gene resulted in decreased expression and increased tolerance of FAW to Vip3Aa, which was validated through RNA interference experiments., Conclusion: The DNA methylation map and mechanism of Vip3Aa tolerance improve our understanding of DNA methylation and its function in Lepidoptera and provide a new perspective for developing pest management strategies. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

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., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized control of some major pests. However, more than 25 cases of field-evolved practical resistance have reduced the efficacy of transgenic crops producing crystalline (Cry) Bt proteins, spurring adoption of alternatives including crops producing the Bt vegetative insecticidal protein Vip3Aa. Although practical resistance to Vip3Aa has not been reported yet, better understanding of the genetic basis of resistance to Vip3Aa is urgently needed to proactively monitor, delay, and counter pest resistance. This is especially important for fall armyworm ( Spodoptera frugiperda ), which has evolved practical resistance to Cry proteins and is one of the world's most damaging pests. Here, we report the identification of an association between downregulation of the transcription factor gene SfMyb and resistance to Vip3Aa in S. frugiperda . Results from a genome-wide association study, fine-scale mapping, and RNA-Seq identified this gene as a compelling candidate for contributing to the 206-fold resistance to Vip3Aa in a laboratory-selected strain. Experimental reduction of SfMyb expression in a susceptible strain using RNA interference (RNAi) or CRISPR/Cas9 gene editing decreased susceptibility to Vip3Aa, confirming that reduced expression of this gene can cause resistance to Vip3Aa. Relative to the wild-type promoter for SfMyb , the promoter in the resistant strain has deletions and lower activity. Data from yeast one-hybrid assays, genomics, RNA-Seq, RNAi, and proteomics identified genes that are strong candidates for mediating the effects of SfMyb on Vip3Aa resistance. The results reported here may facilitate progress in understanding and managing pest resistance to Vip3Aa.

The rapid evolution of resistance in agricultural pest poses a serious threat to global food security. However, the mechanisms of resistance through metabolic regulation are largely unknown. Here, we found that a GST gene cluster was strongly selected in North China (NTC) population, and it was significantly genetically-linked to lambda-cyhalothrin resistance. Knockout of the GST cluster using CRISPR/Cas9 significantly increased the sensitivity of the knockout strain to lambda-cyhalothrin. Haplotype analysis revealed no non-synonymous mutations or structural variations in the GST cluster, whereas GST_119 and GST_121 were significantly overexpressed in the NTC population. Silencing of GST_119 or co-silencing of GST_119 and GST_121 with RNAi significantly increased larval sensitivity to lambda-cyhalothrin. We also identified additional GATAe transcription factor binding sites in the promoter of NTC_GST_119. Transient expression of GATAe in Hi5 cells activated NTC_GST_119 and Xinjiang (XJ)_GST_119 transcription, but the transcriptional activity of NTC_GST_119 was significantly higher than that of XJ_GST_119. These results demonstrate that variations in the regulatory region result in complex expression changes in the GST cluster, which enhances lambda-cyhalothrin resistance in field-populations. This study deepens our knowledge of the evolutionary mechanism of pest adaptation under environmental stress and provides potential targets for monitoring pest resistance and integrated management., (© 2023. Springer Nature Limited.)

The fall armyworm, Spodoptera frugiperda, is a devastating pest in its native range Western Hemisphere and has become a major invasive pest around the globe. Transgenic crops producing Bt toxins have been widely used to control S. frugiperda. However, the evolution of resistance threatens the sustainability of Bt crops. Field-evolved S. frugiperda resistance to Bt crops was observed in America, whereas, no case of field-resistance was reported in its newly invaded East Hemisphere. Here we investigated the molecular mechanism of a Cry1Ab-resistant LZ-R strain of S. frugiperda, which selected 27-generations using Cry1Ab after being collected in corn fields from China. Complementation tests between LZ-R strain and SfABCC2-KO strain, which have been knockout SfABCC2 gene and confer 174-fold resistance to Cry1Ab, showed a similar level of resistance in the F 1 -progeny as their parent stains, indicating that a common locus of SfABCC2 mutation in LZ-R stain. Sequencing of the full length of SfABCC2 cDNA from LZ-R strain, we characterize a novel mutation allele of SfABCC2. Cross-resistance results showed that Cry1Ab-resistance strain also confers >260-fold resistance to Cry1F, with no cross-resistance to Vip3A. These results provided evidence of a novel SfABCC2 mutation allele in the newly invaded East Hemisphere of S. frugiperda., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Vip3Aa is a novel insecticidal protein secreted by Bacillus thuringiensis ( Bt ) during its vegetative growth stages. It has high insecticidal activity against lepidopteran pests such as Spodoptera frugiperda , and has no cross-resistance with Cry insecticidal proteins. As a new type of insecticide, it plays an important role in controlling agricultural pests. However, the insecticidal mechanism of the Vip3Aa toxin, especially its definite receptors, have not been fully revealed. In this study, the previously reported Vip3Aa receptor genes Sf-FGFR and Sf-SR-C were knocked out separately using the CRISPR/Cas9 system. Bioassay results showed that the sensitivity of these two knockout strains to Vip3Aa were not significantly changed compared to that of the normal strain. The current results are not consistent with the previously reports that Sf-SR-C and Sf-FGFR were the receptors of Vip3Aa in vitro. This suggests that the Sf-SR-C and Sf-FGFR genes we tested may not be critical in the mode of action of Vip3Aa in vivo in Spodoptera frugiperda .

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 .

The insecticidal Vip3 proteins, secreted by Bacillus thuringiensis ( Bt ) during its vegetative growth phase, are currently used in Bt crops to control insect pests, and are genetically distinct from known insecticidal Cry proteins. Compared with Cry toxins, the mechanisms of Vip3 toxins are still poorly understood. Here, the responses of Spodoptera frugiperda larvae after Vip3Aa challenge are characterized. Using an integrative analysis of transcriptomics and proteomics, we found that Vip3Aa has enormous implications for various pathways. The downregulated genes and proteins were mainly enriched in metabolic pathways, including the insect hormone synthesis pathway, whereas the upregulated genes and proteins were mainly involved in the caspase-mediated apoptosis pathway, along with the MAPK signaling and endocytosis pathways. Moreover, we also identified some important candidate genes involved in apoptosis and MAPKs. The present study shows that exposure of S. frugiperda larvae to Vip3Aa activates apoptosis pathways, leading to cell death. The results will promote our understanding of the host response process to the Vip3Aa, and help us to better understand the mode of action of Vip3A toxins.

Background: The fall armyworm Spodoptera frugiperda is a major agricultural pest that has invaded the East Hemisphere since 2016, generating a serious threat to food security worldwide including Africa and Asia. The Cry toxins produced by Bacillus thuringiensis (Bt) have been shown to be effective against this insect pest. In different insect ABC transporters (ABCC2 or ABCC3) have been shown to be involved as receptors of some Cry1 toxins. Here we analyzed the role of SfABCC2 and SfABCC3 in the toxicity of Cry1Fa and Cry1Ab toxins in this insect pest., Results: Two S. frugiperda SfABCC2 and SfABCC3 knockout strains, coding for potential functional Bt receptors, were created using CRISPR/Cas9 genome editing system. Both knockout strains showed resistance to both Cry1Fa and Cry1Ab toxins compared with the susceptible strain. SfABCC2 knockout strain showed higher resistance to both Cry toxins than SfABCC3 knockout strain, suggesting a major role of SfABCC2 in the mode of action of these Cry toxins. In addition, expression of SfABCC2 and SfABCC3 genes in Trichoplusia ni Hi5 cells also increased the susceptibility to Cry1Ab and Cry1Fa toxins, in agreement with the genome editing results. The double knockout of SfABCC2 and SfABCC3 strain was not viable in contrast to other lepidopteran species. Furthermore, we report here that SfABCC2 or SfABCC3 knockout strains increased their susceptibility to abamectin and spinosad insecticides., Conclusion: We provide functional evidence that in S. frugiperda these two ABCC transporters serve as receptors of Bt Cry1Fa and Cry1Ab toxins. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)