| Summary: | <i>Spodoptera frugiperda</i> (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 <i>S. frugiperda</i> is largely unknown. In this study, we cloned and functionally characterized <i>Doublesex</i> (<i>dsx</i>), a “molecular switch” modulating sexual dimorphism in <i>S. frugiperda</i> using male- and female-specific isoforms. Given that Lepidoptera is recalcitrant to RNAi, CRISPR/Cas9-mediated mutagenesis was employed to construct <i>S. frugiperda</i> mutants. Specifically, we designed target sites on exons 2, 4, and 5 to eliminate the common, female-specific, and male-specific regions of <i>S. frugiperda dsx</i> (<i>Sfdsx</i>), 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 <i>dsx</i> variants in <i>S. frugiperda</i> led to significantly reduced fecundity and fertility in adults of corresponding sex. Our combined results not only confirm the conserved function of <i>dsx</i> in <i>S. frugiperda</i> sex differentiation but also provide empirical evidence for <i>dsx</i> as a potential target for the Sterile Insect Technique (SIT) to combat this globally invasive pest in a sustainable and environmentally friendly way.
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