Drosophila-type timeless (dTIM) is a key clock protein in fruit flies, regulating rhythmicity and light-mediated entrainment. However, functional experiments indicate that its contribution to the clock differs in various insects. Therefore, we conducted a comprehensive phylogenetic analysis of dTIM across animals and dated its origin, gene duplications, and losses. We identified variable and conserved protein domains and pinpointed animal lineages that underwent the biggest changes in dTIM. While dTIM modifications are only mildly affected by changes in the PER protein, even the complete loss of PER in echinoderms had no impact on dTIM. However, changes in dTIM always co-occur with the loss of CRYPTOCHROMES or JETLAG. This is exemplified by the remarkably accelerated evolution of dTIM in phylloxera and aphids. Finally, alternative d-tim splicing, characteristic of Drosophila melanogaster temperature-dependent function, is conserved to some extent in Diptera, albeit with unique alterations. Altogether, this study pinpoints major changes that shaped dTIM evolution.

• Keywords
• Evolutionary biology, Genetics, Molecular biology, Neuroscience,
• Publication type
• Journal Article MeSH

Daily and annually cycling conditions manifested on the Earth have forced organisms to develop time-measuring devices. Circadian clocks are responsible for adjusting physiology to the daily cycles in the environment, while the anticipation of seasonal changes is governed by the photoperiodic clock. Circadian clocks are cell-autonomous and depend on the transcriptional/translational feedback loops of the conserved clock genes. The synchronization among clock centers in the brain is achieved by the modulatory function of the clock-dependent neuropeptides. In insects, the most prominent clock neuropeptide is Pigment Dispersing Factor (PDF). Photoperiodic clock measures and computes the day and/or night length and adjusts physiology accordingly to the upcoming season. The exact mechanism of the photoperiodic clock and its direct signaling molecules are unknown but, in many insects, circadian clock genes are involved in the seasonal responses. While in Drosophila, PDF signaling participates both in the circadian clock output and in diapause regulation, the weak photoperiodic response curve of D. melanogaster is a major limitation in revealing the full role of PDF in the photoperiodic clock. Here we provide the first description of PDF in the linden bug, Pyrrhocoris apterus, an organism with a robust photoperiodic response. We characterize in detail the circadian and photoperiodic phenotype of several CRISPR/Cas9-generated pdf mutants, including three null mutants and two mutants with modified PDF. Our results show that PDF acts downstream of CRY and plays a key role as a circadian clock output. Surprisingly, in contrast to the diurnal activity of wild-type bugs, pdf null mutants show predominantly nocturnal activity, which is caused by the clock-independent direct response to the light/dark switch. Moreover, we show that together with CRY, PDF is involved in the photoperiod-dependent diapause induction, however, its lack does not disrupt the photoperiodic response completely, suggesting the presence of additional clock-regulated factors. Taken together our data provide new insight into the role of PDF in the insect's circadian and photoperiodic systems.

• Keywords
• CRISPR/Cas9, Pyrrhocoris apterus, circadian clock, cryptochrome-m, diapause, photoperiodic clock, pigment dispersing factor,
• Publication type
• Journal Article MeSH