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Neuropeptides play pivotal roles in modulating circadian rhythms. Pigment-dispersing factor (PDF) is critical to the circadian rhythms in  Drosophila  locomotor activity. Here, we demonstrate that diuretic hormone 31 (DH31) complements PDF function in regulating free-running rhythmicity using male flies. We determined that  Dh31 loss-of-function  mutants ( Dh31   #51  ) showed normal rhythmicity, whereas  Dh31   #51   ;Pdf   01   double mutants exhibited a severe arrhythmic phenotype compared to  Pdf-null  mutants ( Pdf   01  ). The expression of tethered-PDF or tethered-DH31 in clock cells, posterior dorsal neurons 1 (DN1ps), overcomes the severe arrhythmicity of  Dh31   #51   ;Pdf   01   double mutants, suggesting that DH31 and PDF may act on DN1ps to regulate free-running rhythmicity in a hierarchical manner. Unexpectedly, the molecular oscillations in  Dh31   #51   ;Pdf   01   mutants were similar to those in  Pdf   01   mutants in DN1ps, indicating that DH31 does not contribute to molecular oscillations. Furthermore, a reduction in  Dh31 receptor (Dh31r)  expression resulted in normal locomotor activity and did not enhance the arrhythmic phenotype caused by the  Pdf receptor (Pdfr)  mutation, suggesting that PDFR, but not DH31R, in DN1ps mainly regulates free-running rhythmicity. Taken together, we identify a novel role of DH31, in which DH31 and PDF hierarchically regulate free-running rhythmicity through DN1ps.

Neuropeptides PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor activity