The role of Hox genes in shaping embryonic external morphology of the primitive insect <i>Thermobia domestica</i> (Zygentoma)

Insects represent one of the most evolutionarily successful groups, with their diversity hypothesized to be related to the regulatory roles of Hox genes, a set of related genes encoding homeodomain transcription factors determining the identity of segments along the anterior-posterior axis of the embryo. However, functional insights into the roles of Hox genes in primitive ametabolous insects, which represent the critical transition from aquatic crustaceans to winged insects, have been limited. In this study, we identified complete protein-coding sequences of 10 Hox genes in the Zygentoma Thermobia domestica, and applied clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas 9) mediated gene knockout (KO) to decipher their functions. We found that the roles of pb, Dfd, and Scr are vital in specifying the appendages of the head in T. domestica, and these roles are relatively conserved in crustaceans and winged insects. Antp is essential for the development of the prothorax segment and the first pair of legs in T. domestica. Ubx and abd-A fully repress appendage development in the abdomen of T. domestica, which implies a functional switch from crustaceans to insects. Additionally, the role of ftz in segmenting the abdomen of T. domestica suggests it has acquired new functions in primitive insects, beyond its traditional Hox-like roles. Although KOs of lab, Hox3, and Abd-B did not result in obvious external phenotypic changes, they led to a significant decrease in hatching rates and substantial deviations in daily survival numbers compared to the negative control. These findings underscore the indispensable roles of all Hox genes during the embryonic development of T. domestica. Our study sheds new light on the functional evolution of Hox genes in ametabolous insects and enhances our understanding of the genetic underpinnings of insect development and diversification.