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Animals need to integrate information across neuronal networks that direct reproductive behaviors and circadian rhythms. In Drosophila, the master regulatory transcription factors that direct courtship behaviors and circadian rhythms are co-expressed in a small set of neurons. In this study we investigate the role of these neurons in both males and females. We find sex-differences in the number of these fruitless and Clock -expressing neurons ( fru ∩ Clk neurons) that is regulated by male-specific Fru. We assign the fru ∩ Clk neurons to the electron microscopy connectome that provides high resolution structural information. We also discover sex-differences in the number of fru -expressing neurons that are post-synaptic targets of Clk -expressing neurons, with more post-synaptic targets in males. When fru ∩ Clk neurons are activated or silenced, males have a shorter period length. Activation of fru ∩ Clk neurons also changes the rate a courtship behavior is performed. We find that activation and silencing fru ∩ Clk neurons impacts the molecular clock in the sLNv master pacemaker neurons, in a cell-nonautonomous manner. These results reveal how neurons that subserve the two processes, reproduction and circadian rhythms, can impact behavioral outcomes in a sex-specific manner.

Drosophila melanogaster reproductive behaviors are orchestrated by fruitless neurons. We performed single-cell RNA-sequencing on pupal neurons that produce sex-specifically spliced fru transcripts, the fru P1-expressing neurons. Uniform Manifold Approximation and Projection (UMAP) with clustering generates an atlas containing 113 clusters. While the male and female neurons overlap in UMAP space, more than half the clusters have sex differences in neuron number, and nearly all clusters display sex-differential expression. Based on an examination of enriched marker genes, we annotate clusters as circadian clock neurons, mushroom body Kenyon cell neurons, neurotransmitter- and/or neuropeptide-producing, and those that express doublesex . Marker gene analyses also show that genes that encode members of the immunoglobulin superfamily of cell adhesion molecules, transcription factors, neuropeptides, neuropeptide receptors, and Wnts have unique patterns of enriched expression across the clusters. In vivo spatial gene expression links to the clusters are examined. A functional analysis of fru P1 circadian neurons shows they have dimorphic roles in activity and period length. Given that most clusters are comprised of male and female neurons indicates that the sexes have fru P1 neurons with common gene expression programs. Sex-specific expression is overlaid on this program, to build the potential for vastly different sex-specific behaviors., Competing Interests: CP, CA, SB, BF, AG, MA No competing interests declared, (© 2023, Palmateer et al.)