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.

It is well known that insect larval midgut cadherin protein serves as a receptor of Bacillus thuringiensis (Bt) crystal Cry1Ac or Cry1Ab toxins, since structural mutations and downregulation of cad gene expression are linked with resistance to Cry1Ac toxin in several lepidopteran insects. However, the role of Spodoptera frugiperda cadherin protein (SfCad) in the mode of action of Bt toxins remains elusive. Here, we investigated whether SfCad is involved in susceptibility to Cry1Ab or Cry1Fa toxins. In vivo, knockout of the SfCad gene by CRISPR/Cas 9 did not increase tolerance to either of these toxins in S. frugiperda larvae. In vitro cytotoxicity assays demonstrated that cultured insect TnHi5 cells expressing GFP-tagged SfCad did not increase susceptibility to activated Cry1Ab or Cry1Fa toxins. In contrast, expression of another well recognized Cry1A receptor in this cell line, the ABCC2 transporter, increased the toxicity of both Cry1Ab and Cry1Fa toxins, suggesting that SfABCC2 functions as a receptor of these toxins. Finally, we showed that the toxin-binding region of SfCad did not bind to activated Cry1Ab, Cry1Ac, nor Cry1Fa. All these results support that SfCad is not involved in the mode of action of Cry1Ab or Cry1Fa toxins in S. frugiperda.

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.

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.

Gene editing technologies are playing an increasingly important role in the study of insect gene functions. Appropriate incubation humidity helps improve the hatching rate of insect eggs. However, to date, no study has evaluated the impact of humidity on the hatching rate of gene-edited eggs or the subsequent development of the larvae. Using the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) as a model, we investigated the weight and hatching rate of microinjected eggs under different humidity conditions. We determined the larval development time and pupal weight and calculated the larval gene editing efficiency. The results showed that post-microinjection incubation under high humidity (RH = 95%) had a significant influence on CRISPR/Cas9 gene-edited eggs. The key research results indicate that, compared with the incubation environment with RH ≤ 80%, after 48 h of incubation under 95% RH, the hatching rate of H. armigera eggs increased by more than 27.5%, with higher egg saturation and greater egg weight; meanwhile, the larval developmental duration was shorter and the pupal weight was also greater. These results establish a humidity-regulated developmental recovery protocol for gene-edited insects, which provides theoretical and practical support for optimizing post-microinjection recovery processes in gene-edited insects.