Spodoptera frugiperda (J. E. Smith), an emerging invasive pest worldwide, has posed a serious agricultural threat to the newly invaded areas. Although somatic sex differentiation is fundamentally conserved among insects, the sex determination cascade in S. frugiperda is largely unknown. In this study, we cloned and functionally characterized Doublesex (dsx), a “molecular switch” modulating sexual dimorphism in S. frugiperda using male- and female-specific isoforms. Given that Lepidoptera is recalcitrant to RNAi, CRISPR/Cas9-mediated mutagenesis was employed to construct S. frugiperda mutants. Specifically, we designed target sites on exons 2, 4, and 5 to eliminate the common, female-specific, and male-specific regions of S. frugiperda dsx (Sfdsx), respectively. As expected, abnormal development of both the external and internal genitalia was observed during the pupal and adult stages. Interestingly, knocking out sex-specific dsx variants in S. frugiperda led to significantly reduced fecundity and fertility in adults of corresponding sex. Our combined results not only confirm the conserved function of dsx in S. frugiperda sex differentiation but also provide empirical evidence for dsx as a potential target for the Sterile Insect Technique (SIT) to combat this globally invasive pest in a sustainable and environmentally friendly way.

(1) Background: Abdominal-A (Abd-A) and Ultrabithorax (Ubx) are homeotic genes that determine the identity and morphology of the thorax and abdomen in insects. The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Pyralidae), is a devastating maize pest throughout Asia, the Western Pacific, and Australia. Building on previous knowledge, we hypothesized that the knockout of Abd-A and Ubx would disrupt the abdominal body planning in O. furnacalis. (2) Methods: CRISPR/Cas9-targeted mutagenesis was employed to decipher the functions of these homeotic genes. (3) Results: Knockout insects demonstrated classical homeotic transformations. Specifically, the mutagenesis of OfAbd-A resulted in: (1) Fused segments and segmental twist during the larval stage; (2) Embryonic lethality; and (3) The pleiotropic upregulation of other homeotic genes, including Lab, Pd, Dfd, Antp, and Abd-B. The mutagenesis of OfUbx led to: (1) Severe defects in the wing pads, which limited the ability of the adults to fly and mate; (2) Female sterility; and (3) The pleiotropic upregulation of other homeotic genes, including Dfd, Abd-B, and Wnt1. (4) Conclusions: These combined results not only support our hypothesis, but they also strengthen the potential of using homeotic genes as molecular targets for the genetic control of this global insect pest.

Cyetpyrafen is a compound that lacks inherent uptake and systemic translocation activity. If mites do not come into direct contact with the pesticide solution on leaves, the efficacy cannot be achieved. Controlling the particle size can potentially play a crucial role in the manifestation of efficacy. In this study, high-throughput formulation technology was used to systematically screen a large number of adjuvants to obtain cyetpyrafen formulations. The particle size of the active ingredient in the formulation was measured. By examining the dynamic light scattering and contact angle, we simulated the actual process of the efficacy transmission of cyetpyrafen formulations against Tetranychus cinnabarinus. Our results showed that the activity of cyetpyrafen increases as the particle size decreases, suggesting that reducing the particle size can enhance the coverage and deposition on crop leaves, and further improve the dispersion efficiency and enhance spreading capabilities. Furthermore, controlling the particle size at 160 nm resulted in an LC₅₀ value of 0.2026, which is approximately double than that of the commercial product. As a novel pesticide for mites, our study presents the most effective cyetpyrafen formulation in practice. Our findings provide valuable insights into controlling other mite species that pose a threat to agricultural products.

Oogenesis in insects is a carefully orchestrated process, facilitating the formation of female gametes, which is regulated by multiple extrinsic and intrinsic factors, including ovary serine protease (Osp). As a member of the serine protease family, Osp is a homolog of Nudel, a maternally required protease defining embryonic dorsoventral polarity in Drosophila. In this study, we used CRISPR/Cas9-mediated mutagenesis to functionally characterize Osp in the Asian corn borer, Ostrinia furnacalis, a devastating maize pest throughout Asia and Australia. Building on previous knowledge, we hypothesized that knockout of Osp would disrupt embryonic development in O. furnacalis females. To examine this overarching hypothesis, we (1) cloned and characterized Osp from O. furnacalis, (2) designed target sites on exons 1 and 4 to construct a CRISPR/Cas9 mutagenesis system, and (3) documented phenotypic impacts among O. furnacalis Osp mutants. As a result, we (1) examined the temporal-spatial expression profiles of OfOsp, which has an open reading frame of 5648 bp in length and encodes a protein of 1873 amino acids; (2) established O. furnacalis Osp mutants; and (3) documented recessive, female-specific sterility among OfOsp^F mutants, including absent or deformed oviducts and reduced fertility in female but not male mutants. Overall, the combined results support our initial hypothesis that Osp is required for embryonic development, specifically ovarian maturation, in O. furnacalis females. Given its substantial impacts on female sterility, Osp provides a potential target for the Sterile Insect Technique (SIT) to manage Lepidoptera pests in general and the species complex Ostrinia in particular.

Lysinibacillus sphaericus harboring Binary (BinA and BinB) toxins is highly toxic against Anopheles and Culex mosquito larvae. The Anopheles Ag55 cell line is a suitable model for investigating the mode of Bin toxin action. Based on the low-levels of α-glycosidase Agm3 mRNA in Ag55 cells and the absence of detectable Agm3 proteins, we hypothesized that a scavenger receptor could be mediating Bin cytotoxicity. Preliminary RNA interference knockdown of the expressed scavenger receptors, combined with Bin cytotoxicity assays, was conducted. The scavenger Receptor C1 (SCRC1) became the focus of this study, as a putative receptor for Bin toxins in Ag55 cells, and SCRBQ2 was selected as a negative control. Open reading frames encoding SCRC1 and SCRBQ2 were cloned and expressed in vitro, and polyclonal antibodies were prepared for immunological analyses. The RNAi silencing of SCRC1 and SCRBQ2 resulted in the successful knockdown of both SCRC1 and SCRBQ2 transcripts and protein levels. The cytolytic toxicity of Bin against Ag55 cells was severely reduced after the SCRC1-RNAi treatment. The phagocytic receptor protein SCRC1 mediates endocytosis of the Bin toxin into Ag55 cells, thereby facilitating its internal cytological activity. The results support a mechanism of the Bin toxin entering Ag55 cells, possibly via SCRC1-mediated endocytosis, and encourage investigations into how Bin is transferred from its bound form on the midgut epithelial cells into the epithelial endocytic system.