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

Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.

• Keywords
• bride of doubletime, casein kinase 1, circadian clock, doubletime, evolution, temperature compensation,
• Publication type
• Journal Article MeSH

Circadian clocks are synchronized with the external environment by light and temperature. The effect of these cues on behavior is well-characterized in Drosophila, however, little is known about synchronization in non-model insect species. Therefore, we explored entrainment of locomotor activity by light and temperature in the linden bug Pyrrhocoris apterus (Heteroptera), an insect species with a strong seasonal response (reproductive diapause), which is triggered by both photoperiod and thermoperiod. Our results show that either light or temperature cycles are strong factors entraining P. apterus locomotor activity. Pyrrhocoris is able to be partially synchronized by cycles with temperature amplitude as small as 3°C and more than 50% of bugs is synchronized by 5°C steps. If conflicting zeitgebers are provided, light is the stronger signal. Linden bugs lack light-sensitive (Drosophila-like) cryptochrome. Notably, a high percentage of bugs is rhythmic even in constant light (LL) at intensity ∼400 lux, a condition which induces 100% arrhythmicity in Drosophila. However, the rhythmicity of bugs is still reduced in LL conditions, whereas rhythmicity remains unaffected in constant dark (DD). Interestingly, a similar phenomenon is observed after temperature cycles entrainment. Bugs released to constant thermophase and DD display weak rhythmicity, whereas strong rhythmicity is observed in bugs released to constant cryophase and DD. Our study describes the daily and circadian behavior of the linden bug as a response to photoperiodic and thermoperiodic entraining cues. Although the molecular mechanism of the circadian clock entrainment in the linden bug is virtually unknown, our study contributes to the knowledge of the insect circadian clock features beyond Drosophila research.

• Keywords
• Pyrrhocoris apterus, circadian clock, constant light, entrainment, photoperiod, synchronization, temperature compensation, thermoperiod,
• Publication type
• Journal Article MeSH