Comparative single-cell transcriptomic atlases reveal conserved and divergent features of drosophilid central brains

To explore how brains change upon species evolution, we generated the first whole central brain comparative single-cell transcriptomic atlases of three closely-related but ecologically-distinct drosophilids: D. melanogaster, D. simulans and D. sechellia. D. melanogaster and D. simulans are cosmopolitan generalists, while the island endemic D. sechellia exhibits extreme niche specialism on the ripe noni fruit of the Morinda citrifolia shrub. The global cellular composition of central brains is well-conserved in the three Drosophila species, but we predicted a few cell types (perineurial glia, sNPF and Dh44 neurons) with divergent frequencies. Gene expression analysis revealed that distinct cell types within the central brain evolve at different rates and patterns; notably, several glial cell types exhibit the greatest divergence between species. Compared to D. melanogaster, the cellular composition and gene expression patterns of the central brain in D. sechellia displays greater deviation than those of D. simulans, indicating that the distinctive ecological specialization of D. sechellia is reflected in the structure and function of its brain. Gene expression changes in D. sechellia encompass metabolic and ecdysone signaling genes, indicative of adaptations to its novel ecological demands. Additional single-cell transcriptomic analysis on D. sechellia revealed genes and cell types responsive to noni juice supplementation, showing glial cells as key sites for both physiological and genetic adaptation to novel conditions. Our comparative transcriptomic atlases of drosophilid brains will provide an entry point to more broadly study the evolvability of nervous systems across and beyond the Drosophila genus. Nuclei of Drosophila central brains were isolated using homogenization and FACS sorting and were analyzed by single-nucleus RNA-sequencing.