Circadian and photoperiodic control of melatonin in Drosophila ananassae

Melatonin is a widely conserved indoleamine with well-established roles in circadian timekeeping, sleep regulation, and physiological coordination across taxa. In Drosophila melanogaster, early studies reported daily melatonin rhythms, but little is known about melatonin dynamics in other Drosophila species. Here, we measured melatonin secretion in >14,000 Drosophila ananassae across 12 circadian timepoints under four simulated photoperiods: LD 8:16 (short day), LD 12:12 (equatorial), LD 16:8 (long day), and LD 20:4 (arctic summer). Using LC-MS/MS detection in pooled whole-fly samples, we identified robust, bimodal melatonin rhythms across all conditions, with conserved peaks at approximately ZT7 and ZT13 irrespective of daylength. Melatonin levels were significantly elevated under the short-day photoperiod compared to all others (p < 0.0005), indicating photoperiod-sensitive amplitude modulation. These findings confirm the presence of circadian melatonin rhythms in Drosophila, replicate a previously reported early-day peak observed in D. melanogaster, and reveal a second rhythm component not described in earlier studies. The data suggest that melatonin secretion in Drosophila is governed by an endogenous clock and phase-locked to lights-on, even in the absence of nighttime darkness. This work refines our understanding of melatonin rhythmicity in insects and underscores its potential role as a conserved circadian output across species.