| Summary: | Sex determination is an important and traditional biological process. In Lepidoptera, <i>Masculinizer</i> (<i>Masc</i>) and <i>doublesex</i> (<i>dsx</i>) are the essential genes for sex determination and play critical roles in sexual differentiation and development. The functions of <i>Masc</i> and <i>dsx</i> have been characterized in several model insect species. However, the molecular mechanism and sex determination functions of <i>Masc</i> and <i>dsx</i> in <i>Ostrinia furnacalis</i>, an agricultural pest, are still unknown. Here, we successfully used the CRISPR/Cas9 genome editing system to knock out <i>OfMasc</i> and <i>Ofdsx</i>. Mutation of <i>OfMasc</i> induced male external genital defects and sterility. Disruptions of the <i>Ofdsx</i> common region caused sex-specific defects in the external genitals and adult sterility. In addition, we found that <i>OfMasc</i> and <i>Ofdsx</i> can regulate the pigmentation genes that control wing pigmentation patterns. These results demonstrate that <i>OfMasc</i> and <i>Ofdsx</i> play key roles in the sex determination of <i>O. furnacalis</i>, and suggest novel genetic control approaches for the management of pests, including <i>O. furnacalis</i>.
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