Investigation of the molecular-genetic basis for courtship differences between Drosophila melanogaster, Drosophila simulans and their hybrids

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 FruM target genes in both species, revealing conserved core and species-specific FruM targets. Focusing on chemosensory receptors with D. melanogaster-specific FruM 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. Article SummaryWe examined how genes and circuits shape courtship behaviors in Drosophila melanogaster and Drosophila simulans. Hybrid males adjust their behavior based on the species of female, suggesting flexible courtship. We focused on the fruitless gene, which controls male courtship. fru neurons in hybrids closely resembled those in Drosophila melanogaster. We identified both shared and species-specific FruM target genes. A genetic screen of odorant receptor neurons revealed populations that help males distinguish females. Neuroanatomical mapping shows the odor information is relayed to different higher order brain regions. The results uncover molecular and neural circuit mechanisms underlying species differences in behaviors.