Project Summary A central question in the field of Evo-Devo is how genes controlling embryonic development change during evolution. Many recent advances in Evo-Devo have identified genetic changes that are associated with the acquisition of, or changes in, external body features, such as alterations in pigmentation patterns or development of body armor. In contrast, our studies are novel in this field as they have revealed unexpected genetic variation underlying a highly conserved trait: the shared segmented body plan of insects. The genes controlling segmentation encode transcription factors that are required for embryonic development and viability. The cohort of genes responsible for segment formation include pair-rule genes (PRGs) identified in the model insect Drosophila. Mutations in Drosophila PRGs result in lethality accompanied by loss of alternate segmental regions. Thus, it was surprising to find differences in the presence, expression, or function of PRGs in different insect taxa. The work proposed here is designed to understand the mechanistic basis for this genetic variation, which we have observed in different insect lineages. To carry out functional studies, we have developed molecular genetic approaches in diverse insect species in our lab. The establishment of multiple non-model systems simultaneously within one lab has synergistic effects due to sharing of protocols and troubleshooting strategies, allowing us to develop new techniques more effectively in different species. With these tools in hand, we will examine the underlying bases of specific scenarios of genetic variation: In Aim 1, we will ask how PR-patterning is achieved without canonical PRGs in the milkweed bug Oncopeltus where we have found novel utilization of PRGs compared to Drosophila. Aim 2 will explore the evolutionary trajectory of the Oncopeltus PRG network across insect groups. Aim 3 will decipher mechanisms underlying loss of an essential PRG in both mosquitoes and butterflies. These studies will contribute to our understanding of fundamental mechanisms regulating embryonic development and how these mechanisms have changed during the radiation of insects. This project will train postdoctoral fellows, graduate students, and at least five undergraduate students in molecular biology, genetics, and molecular evolution. Establishment of molecular techniques in non-model and emerging model insect species, including expression analysis, RNA interference, CRISPR/Cas9, FAIRE-seq, and transgenesis, not only allows us to answer fundamental questions about embryonic development, but also provides molecular tools for translational studies of insects that pose a risk to human health.