Abstract
Understanding the mechanisms driving behavioral evolution enhances our knowledge of speciation and how behavioral potentials are genetically encoded. Male courtship behaviors, which evolve rapidly, are critical for pre-mating isolation. To investigate the genetic basis of species-specific courtship, we generated hybrid males by crossing two Drosophila melanogaster strains with D. simulans Lhr males. This design allowed us to assess how genetic background and female species identity influence male courtship behavior. In both single-pair and mate-choice assays, hybrid males displayed behavioral plasticity, adjusting their courtship strategies based on the female species. The maternal D. melanogaster strain significantly shaped hybrid behavioral repertoires. To identify the neural correlates, we examined fruitless ( fru )-expressing neurons in hybrids. Their projection patterns resembled those in D. melanogaster , indicating conserved circuit architecture. Using CUT&Tag, we identified Fru (M) target genes in both species, revealing conserved core and species-specific Fru (M) targets. Focusing on chemosensory receptors with D. melanogaster -specific Fru (M) binding, we conducted a genetic screen in D. melanogaster , silencing neurons co-expressing fru P1 and specific receptor genes. Courtship preference assays identified three additional olfactory receptor neuron subtypes that modulate species-specific behavior. Using trans-Tango , 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.