Investigating the <i>fruitless</i> -directed molecular and circuit architecture of courtship behavior in Drosophila <i>melanogaster</i> , <i>Drosophila simulans</i> and their hybrids

Understanding the mechanisms driving behavioral evolution enhances our knowledge of speciation and how behavioral potentials are encoded. Drosophila male courtship behaviors, which evolve rapidly, are directed by fruitless-expressing neurons. Here we investigate how species differences arise from shared molecular-genetic and circuit architecture. We examined courtship in pure species Drosophila melanogaster, Drosophila simulans, and hybrids. This design allowed us to assess how hybrid males integrate behavioral repertoires from both parental species. To identify the neural correlates for hybrid courtship behaviors, we examined fruitless-expressing neurons in hybrids. Their projection patterns resembled those in D. melanogaster, indicating largely conserved circuit architecture. To identify molecular differences that drive behavioral differences between species, we identified male-specific Fruitless target genes in both species, revealing conserved core and species-specific targets. Focusing on chemosensory receptors with D. melanogaster-specific Fruitless binding, we conducted a genetic screen in D. melanogaster, silencing neurons co-expressing fruitless and specific receptor genes. Courtship preference assays identified three additional olfactory receptor neuron subtypes that modulate behavior. Using a transsynaptic mapping approach, we mapped second-order projection neurons of these subtypes, revealing their targets in higher-order brain centers. This study uncovers new molecular and neural mechanisms underlying the specification and evolution of courtship behavior, highlighting how genetic and sensory inputs shape species-specific behavioral outcomes.