Abstract
Hox genes play critical roles in specifying the regionalization of the body axis across metazoa, with the exception of the anterior dorsal head of bilaterian animals, which instead is instructed by a deeply conserved set of non-Hox regulators. The anterior dorsal head is also a hot spot of evolutionary diversification, raising the question as to the developmental-genetic underpinnings of such innovation. Onthophagine dung beetles develop evolutionarily novel and highly diversified horns on the dorsal head used as weapons during intrasexual conflicts. Preliminary RNAseq data unexpectedly documented Hox gene expression in the dorsal head of premetamorphic onthophagine larvae. Motivated by this observation, we aimed to (i) investigate the genomic content and arrangement of the Hox cluster across three onthophagine species, and (ii) assess expression patterns and (iii) potential functions of the anterior Hox genes labial, proboscipedia, and Deformed in patterning the adult beetle head and cephalic horns. We document an unexpected derived Hox cluster configuration in the Onthophagus sagittarius genome, a species with apomorphic cephalic horn morphology. Yet despite this genomic rearrangement, embryonic expression patterns of labial and proboscipedia as well as the adult segment patterning functions of proboscipedia and Deformed were found to be conserved. In contrast, labial RNAi revealed an adult head patterning function outside horn-forming regions previously undescribed for any insect. Lastly, we show that electrosurgical ablation of the presumptive larval labial-expressing head region phenocopies this conspicuous adult labial RNAi defect. We discuss the implications of these data for current models of insect head development and diversification.