| Summary: | (1) Background: <i>Abdominal-A</i> (<i>Abd-A</i>) and <i>Ultrabithorax</i> (<i>Ubx</i>) are homeotic genes that determine the identity and morphology of the thorax and abdomen in insects. The Asian corn borer, <i>Ostrinia furnacalis</i> (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 <i>Abd-A</i> and <i>Ubx</i> would disrupt the abdominal body planning in <i>O. furnacalis</i>. (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 <i>OfAbd-A</i> 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 <i>Lab</i>, <i>Pd</i>, <i>Dfd</i>, <i>Antp</i>, and <i>Abd-B</i>. The mutagenesis of <i>OfUbx</i> 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 <i>Dfd</i>, <i>Abd-B</i>, and <i>Wnt1</i>. (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.
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