PROJECT SUMMARY/ABSTRACT Circadian rhythms are the backing track to the symphony of life. Circadian rhythms not only allow organisms to coordinate their behavior and physiology with daily and seasonal changes but also temporally coordinate internal processes across organ systems. However, genetic, molecular, circuit, metabolic, and behavioral studies of chronobiology have been conducted primarily in male animals. Although the overall architecture of circadian systems is conserved across phylogeny and between sexes, circadian systems are not identical between sexes. This proposal exploits the Drosophila melanogaster genetic model to identify sex differences in circadian output signaling from the brain clock to peripheral metabolic tissues. We have identified two major knowledge gaps: (1) Sex differences in circuitry and signaling from the brain clock to clock output regions that communicate time of day to peripheral tissues; and (2) Sex differences in peripheral responses to circadian entrainment at baseline and when exposed to clock desynchronizing stressors. Our published and preliminary data has identified sex differences in the role of specific clock output neuropeptides in regulating circadian behavior and shown that age and high-fat diet have separable effects on dampening circadian rhythms of locomotor behavior and peripheral gene expression. In Project 1, we will use in vivo and in silico circuit mapping techniques to identify how sex-specific circuitry interacts with circadian clock outputs, identify sex specific transcriptional programs that alter morphology, cell number, connections, and physiology of clock output neurons, and use CRISPR knockout screening to identify specific signaling molecules and cognate receptors that contribute to sexually dimorphic circadian output signaling. In Project 2, we will use a novel long-term circadian luminescence imaging approach to identify how circadian gene transcription rhythms break down in peripheral tissues in res