The circadian clock conveys thermal and photoperiodic cues to modulate EYES ABSENT via the neuropeptide PDF to regulate seasonal physiology

Organisms adapt to seasonal changes in photoperiod and temperature to survive; however, the mechanisms by which these signals are integrated in the brain are poorly understood. We previously reported that EYES ABSENT (EYA) in Drosophila shows higher levels in cold temperature or short photoperiod, and genetic ablation of eya in the fly brain inhibits reproductive dormancy, suggesting that EYA promotes winter physiology. Nevertheless, the mechanisms by which EYA senses seasonal cues are unclear. Pigment-Dispersing Factor (PDF) is a neuropeptide important for photoentrainment and regulation of circadian output rhythms. Interestingly, PDF also regulates reproductive dormancy, suggesting that it may mediate the function of the circadian clock in modulating seasonal physiology. In this study, we investigated the role of PDF signaling in mediating the impact of EYA on seasonal biology. First, we subjected flies to different photoperiodic and temperature regimes and observed that PDF abundance is lower in cold and short days, compared to warm and long days. Interestingly, the response of PDF to seasonal cues is opposite of what was observed for EYA. We then determined the potential for PDF to convey seasonal cues and modulate EYA function in seasonality by assessing coexpression of EYA and PDF receptor. Our results indicated that PDF receptor (PDFR) is indeed coexpressed with EYA in the fly brain, including in the circadian clock neuronal network and neurons in the pars intercerebralis . We then manipulated PDF signaling in eya + cells to show that PDF modulates seasonal adaptations in daily activity rhythm and ovary development via EYA-dependent and independent mechanisms. At the molecular level, manipulating PDF signaling impacted EYA protein abundance. Specifically, we showed that protein kinase A (PKA), an effector of PDF signaling, phosphorylates EYA and promotes its degradation. This explains the opposite responses of PDF and EYA abundance to changes in seasonal cues. In summary, our results support a model in which PDF signaling negatively modulates EYA levels to regulate seasonal physiology, linking the circadian clock to the modulation of seasonal adaptations. ### Competing Interest Statement The authors have declared no competing interest.