Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

Biology Centre of the Czech Academy of Sciences Institute of Entomology České Budějovice Czech Republic

Faculty of Science University of South Bohemia České Budějovice Czech Republic

See more in PubMed

Allada R, Emery P, Takahashi JS, Rosbash M.. 2001. Stopping time: the genetics of fly and mouse circadian clocks. Annu Rev Neurosci. 24:1091–1119. PubMed

Allada R, White NE, So WV, Hall JC, Rosbash M.. 1998. A mutant Drosophila homolog of mammalian clock disrupts circadian rhythms and transcription of period and timeless. Cell 93(5):791–804. PubMed

Bajgar A, Jindra M, Dolezel D.. 2013. Autonomous regulation of the insect gut by circadian genes acting downstream of juvenile hormone signaling. Proc Natl Acad Sci U S A. 110(11):4416–4421. PubMed PMC

Bargiello TA, Jackson FR, Young MW.. 1984. Restoration of circadian behavioral rhythms by gene-transfer in Drosophila. Nature 312(5996):752–754. PubMed

Barnes JW, Tischkau SA, Barnes JA, Mitchell JW, Burgoon PW, Hickok JR, Gillette MU.. 2003. Requirement of mammalian timeless for circadian rhythmicity. Science 302(5644):439–442. PubMed

Bazalova O, Kvicalova M, Valkova T, Slaby P, Bartos P, Netusil R, Tomanova K, Braeunig P, Lee HJ, Sauman I, et al.2016. Cryptochrome 2 mediates directional magnetoreception in cockroaches. Proc Natl Acad Sci U S A. 113(6):1660–1665. PubMed PMC

Benna C, Bonaccorsi S, Wulbeck C, Helfrich-Forster C, Gatti M, Kyriacou CP, Costa R, Sandrelli F.. 2010. Drosophila timeless2 is required for chromosome stability and circadian photoreception. Curr Biol. 20(4):346–352. PubMed

Benna C, Scannapieco P, Piccin A, Sandrelli F, Zordan M, Rosato E, Kyriacou CP, Valle G, Costa R.. 2000. A second timeless gene in Drosophila shares greater sequence similarity with mammalian tim. Curr Biol. 10(14):R512–513. PubMed

Ceriani MF, Darlington TK, Staknis D, Mas P, Petti AA, Weitz CJ, Kay SA.. 1999. Light-dependent sequestration of TIMELESS by CRYPTOCHROME. Science 285(5427):553–556. PubMed

Chiu JC, Ko HW, Edery I.. 2011. NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed. Cell 145(3):357–370. PubMed PMC

Cyran SA, Buchsbaum AM, Reddy KL, Lin MC, Glossop NRJ, Hardin PE, Young MW, Storti RV, Blau J.. 2003. vrille, Pdp1, and dClock form a second feedback loop in the Drosophila circadian clock. Cell 112(3):329–341. PubMed

Danbara Y, Sakamoto T, Uryu O, Tomioka K.. 2010. RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus. J Insect Physiol. 56(12):1738–1745. PubMed

Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TDL, Weitz CJ, Takahashi JS, Kay SA.. 1998. Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 280(5369):1599–1603. PubMed

Emery P, Stanewsky R, Helfrich-Forster C, Emery-Le M, Hall JC, Rosbash M.. 2000. Drosophila CRY is a deep brain circadian photoreceptor. Neuron 26(2):493–504. PubMed

Fedele G, Edwards MD, Bhutani S, Hares JM, Murbach M, Green EW, Dissel S, Hastings MH, Rosato E, Kyriacou CP.. 2014. Genetic analysis of circadian responses to low frequency electromagnetic fields in Drosophila melanogaster. PLoS Genet. 10(12):e1004804. PubMed PMC

Godinho SI, Maywood ES, Shaw L, Tucci V, Barnard AR, Busino L, Pagano M, Kendall R, Quwailid MM, Romero MR, et al.2007. The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period. Science 316(5826):897–900. PubMed

Gotter AL, Manganaro T, Weaver DR, Kolakowski LF, Possidente B, Sriram S, MacLaughlin DT, Reppert SM.. 2000. A time-less function for mouse timeless. Nat Neurosci. 3(8):755–756. PubMed

Grima B, Lamouroux A, Chelot E, Papin C, Limbourg-Bouchon B, Rouyer F.. 2002. The F-box protein slimb controls the levels of clock proteins period and timeless. Nature 420(6912):178–182. PubMed

Hara T, Koh K, Combs DJ, Sehgal A.. 2011. Post-translational regulation and nuclear entry of TIMELESS and PERIOD are affected in new timeless mutant. J Neurosci. 31(27):9982–9990. PubMed PMC

Hardin PE, Hall JC, Rosbash M.. 1990. Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels. Nature 343(6258):536–540. PubMed

Helfrich-Forster C. 2001. The locomotor activity rhythm of Drosophila melanogaster is controlled by a dual oscillator system. J Insect Physiol. 47:877–887.

Hirano A, Yumimoto K, Tsunematsu R, Matsumoto M, Oyama M, Kozuka-Hata H, Nakagawa T, Lanjakornsiripan D, Nakayama KI, Fukada Y.. 2013. FBXL21 Regulates oscillation of the circadian clock through ubiquitination and stabilization of cryptochromes. Cell 152(5):1106–1118. PubMed

Huang N, Chelliah Y, Shan Y, Taylor CA, Yoo SH, Partch C, Green CB, Zhang H, Takahashi JS.. 2012. Crystal structure of the heterodimeric CLOCK: BMAL1 transcriptional activator complex. Science 337(6091):189–194. PubMed PMC

Ikeno T, Katagiri C, Numata H, Goto SG.. 2011. Causal involvement of mammalian-type cryptochrome in the circadian cuticle deposition rhythm in the bean bug Riptortus pedestris. Insect Mol Biol. 20(3):409–415. PubMed

Ikeno T, Numata H, Goto SG.. 2011. Photoperiodic response requires mammalian-type cryptochrome in the bean bug Riptortus pedestris. Biochem Biophys Res Commun. 410(3):394–397. PubMed

Johnson KP, Dietrich CH, Friedrich F, Beutel RG, Wipfler B, Peters RS, Allen JM, Petersen M, Donath A, Walden KKO, et al.2018. Phylogenomics and the evolution of hemipteroid insects. Proc Natl Acad Sci U S A. 115(50):12775–12780. PubMed PMC

Kamae Y, Tomioka K.. 2012. timeless is an essential component of the circadian clock in a primitive insect, the firebrat Thermobia domestica. J Biol Rhythms. 27(2):126–134. PubMed

Kaniewska MM, Vaněčková H, Doležel D, Kotwica-Rolinska J.. 2020. Light and temperature synchronizes locomotor activity in the Linden bug, Pyrrhocoris apterus. Front Physiol. 11:242. PubMed PMC

Kawahara AY, Plotkin D, Espeland M, Meusemann K, Toussaint EFA, Donath A, Gimnich F, Frandsen PB, Zwick A, Dos Reis M, et al.2019. Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths. Proc Natl Acad Sci U S A. 116(45):22657–22663. PubMed PMC

Koh K, Zheng X, Sehgal A.. 2006. JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS. Science 312(5781):1809–1812. PubMed PMC

Konopka RJ, Benzer S.. 1971. Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci U S A. 68(9):2112–2116. PubMed PMC

Kotwica-Rolinska J, Chodakova L, Chvalova D, Kristofova L, Fenclova I, Provaznik J, Bertolutti M, Wu BC, Dolezel D.. 2019. CRISPR/Cas9 genome editing introduction and optimization in the non-model insect Pyrrhocoris apterus. Front Physiol. 10:891. PubMed PMC

Kotwica-Rolinska J, Pivarciova L, Vaneckova H, Dolezel D.. 2017. The role of circadian clock genes in the photoperiodic timer of the linden bug Pyrrhocoris apterus during the nymphal stage. Physiol Entomol. 42(3):266–273.

Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin XW, Maywood ES, Hastings MH, Reppert SM.. 1999. mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98(2):193–205. PubMed

Kurien P, Hsu PK, Leon J, Wu D, McMahon T, Shi G, Xu Y, Lipzen A, Pennacchio LA, Jones CR, et al.2019. TIMELESS mutation alters phase responsiveness and causes advanced sleep phase. Proc Natl Acad Sci U S A. 116(24):12045–12053. PubMed PMC

Martinek S, Inonog S, Manoukian AS, Young MW.. 2001. A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock. Cell 105(6):769–779. PubMed

Matsumoto A, Ukai-Tadenuma M, Yamada RG, Houl J, Uno KD, Kasukawa T, Dauwalder B, Itoh TQ, Takahashi K, Ueda R, et al.2007. A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock. Genes Dev. 21(13):1687–1700. PubMed PMC

McKenna DD, Shin S, Ahrens D, Balke M, Beza-Beza C, Clarke DJ, Donath A, Escalona HE, Friedrich F, Letsch H, et al.2019. The evolution and genomic basis of beetle diversity. Proc Natl Acad Sci U S A. 116(49):24729–24737. PubMed PMC

Menet JS, Pescatore S, Rosbash M.. 2014. CLOCK: BMAL1 is a pioneer-like transcription factor. Genes Dev. 28(1):8–13. PubMed PMC

Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB, Ware J, Flouri T, Beutel RG, et al.2014. Phylogenomics resolves the timing and pattern of insect evolution. Science 346(6210):763–767. PubMed

Netusil R, Tomanova K, Chodakova L, Chvalova D, Doleze D, Ritz T, Vacha M.. 2021. Cryptochrome-dependent magnetoreception in a heteropteran insect continues even after 24 h in darkness. J Exp Biol. 224(19): jeb243000. PubMed

Nose M, Tokuoka A, Bando T, Tomioka K.. 2017. timeless2 plays an important role in reproduction and circadian rhythms in the cricket Gryllus bimaculatus. J Insect Physiol. 105:9–17. PubMed

Ozkaya O, Rosato E.. 2012. The circadian clock of the fly: a neurogenetics journey through time. Adv Genet. 77:79–123. PubMed

Peres R, Amaral FG, Marques AC, Neto JC.. 2014. Melatonin production in the sea star Echinaster brasiliensis (Echinodermata). Biol Bull. 226(2):146–151. PubMed

Peschel N, Chen KF, Szabo G, Stanewsky R.. 2009. Light-dependent interactions between the Drosophila circadian clock factors cryptochrome, Jetlag, and Timeless. Curr Biol. 19(3):241–247. PubMed

Peschel N, Veleri S, Stanewsky R.. 2006. Veela defines a molecular link between Cryptochrome and Timeless in the light-input pathway to Drosophila's circadian clock. Proc Natl Acad Sci USA. 103(46):17313–17318. PubMed PMC

Pivarciova L, Vaneckova H, Provaznik J, Wu BC, Pivarci M, Peckova O, Bazalova O, Cada S, Kment P, Kotwica-Rolinska J, et al.2016. Unexpected geographic variability of the free running period in the Linden bug Pyrrhocoris apterus. J Biol Rhythms. 31(6):568–576. PubMed

Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW.. 1998. Double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation. Cell 94(1):83–95. PubMed

Putker M, Wong DCS, Seinkmane E, Rzechorzek NM, Zeng A, Hoyle NP, Chesham JE, Edwards MD, Feeney KA, Fischer R, et al.2021. CRYPTOCHROMES confer robustness, not rhythmicity, to circadian timekeeping. EMBO J. 40(7):e106745. PubMed PMC

Rothenfluh A, Abodeely M, Price JL, Young MW.. 2000. Isolation and analysis of six timeless alleles that cause short- or long-period circadian rhythms in Drosophila. Genetics 156(2):665–675. PubMed PMC

Rubin EB, Shemesh Y, Cohen M, Elgavish S, Robertson HM, Bloch G.. 2006. Molecular and phylogenetic analyses reveal mammalian-like clockwork in the honey bee (Apis mellifera) and shed new light on the molecular evolution of the circadian clock. Genome Res. 16(11):1352–1365. PubMed PMC

Rutila JE, Suri V, Le M, So WV, Rosbash M, Hall JC.. 1998. CYCLE is a second bHLH-PAS clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell 93(5):805–814. PubMed

Saez L, Young MW.. 1996. Regulation of nuclear entry of the Drosophila clock proteins period and timeless. Neuron 17(5):911–920. PubMed

Schmid B, Helfrich-Forster C, Yoshii T.. 2011. A new ImageJ plug-in “ActogramJ” for chronobiological analyses. J Biol Rhythms. 26(5):464–467. PubMed

Sehgal A, Price JL, Man B, Young MW.. 1994. Loss of circadian behavioral rhythms and per RNA oscillations in the Drosophila mutant timeless. Science 263(5153):1603–1606. PubMed

Siepka SM, Yoo SH, Park J, Song WM, Kumar V, Hu YN, Lee C, Takahashi JS.. 2007. Circadian mutant overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression. Cell 129(5):1011–1023. PubMed PMC

Smykal V, Bajgar A, Provaznik J, Fexova S, Buricova M, Takaki K, Hodkova M, Jindra M, Dolezel D.. 2014. Juvenile hormone signaling during reproduction and development of the linden bug, Pyrrhocoris apterus. Insect Biochem Mol Biol. 45:69–76. PubMed

Smýkal V, Pivarči M, Provazník J, Bazalová O, Jedlička P, Lukšan O, Horák A, Vaněčková H, Beneš V, Fiala I, et al.2020. Complex rvolution of insect insulin receptors and homologous decoy receptors, and functional significance of their multiplicity. Mol Biol Evol. 37(6):1775–1789. PubMed PMC

Stanewsky R, Kaneko M, Emery P, Beretta B, Wager-Smith K, Kay SA, Rosbash M, Hall JC.. 1998. The cry(b) mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95(5):681–692. PubMed

Urbanová V, Bazalová O, Vaněčková H, Dolezel D.. 2016. Photoperiod regulates growth of male accessory glands through juvenile hormone signaling in the linden bug, Pyrrhocoris apterus. Insect Biochem Mol Biol. 70:184–190. PubMed

Wan G, Hayden AN, Iiams SE, Merlin C.. 2021. Cryptochrome 1 mediates light-dependent inclination magnetosensing in monarch butterflies. Nat Commun. 12(1):771. PubMed PMC

Wipfler B, Letsch H, Frandsen PB, Kapli P, Mayer C, Bartel D, Buckley TR, Donath A, Edgerly-Rooks JS, Fujita M.. 2019. Evolutionary history of Polyneoptera and its implications for our understanding of early winged insects. Proc Natl Acad Sci USA. 116:3024–3029. PubMed PMC

Xia J, Guo Z, Yang Z, Han H, Wang S, Xu H, Yang X, Yang F, Wu Q, Xie W, et al.2021. Whitefly hijacks a plant detoxification gene that neutralizes plant toxins. Cell 184(7):1693–1705 e1617. PubMed

Xu J, Jarocha LE, Zollitsch T, Konowalczyk M, Henbest KB, Richert S, Golesworthy MJ, Schmidt J, Dejean V, Sowood DJC, et al.2021. Magnetic sensitivity of cryptochrome 4 from a migratory songbird. Nature 594(7864):535–540. PubMed

Yoshii T, Ahmad M, Helfrich-Forster C.. 2009. Cryptochrome mediates light-dependent magnetosensitivity of drosophila's circadian clock. PLoS Biol. 7(4):e1000086. PubMed PMC

Yu WJ, Houl JH, Hardin PE.. 2011. NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator. Curr Biol. 21(9):756–761. PubMed PMC

Yuan Q, Metterville D, Briscoe AD, Reppert SM.. 2007. Insect cryptochromes: gene duplication and loss define diverse ways to construct insect circadian clocks. Mol Biol Evol. 24(4):948–955. PubMed

Zehring WA, Wheeler DA, Reddy P, Konopka RJ, Kyriacou CP, Rosbash M, Hall JC.. 1984. P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic Drosophila melanogaster. Cell 39(2 Pt 1):369–376. PubMed

Zhang L, Jones CR, Ptacek LJ, Fu YH.. 2011. The genetics of the human circadian clock. Adv Genet. 74:231–247. PubMed

Zhang Y, Markert MJ, Groves SC, Hardin PE, Merlin C.. 2017. Vertebrate-like CRYPTOCHROME 2 from monarch regulates circadian transcription via independent repression of CLOCK and BMAL1 activity. Proc Natl Acad Sci USA. 114(36):E7516–E7525. PubMed PMC

Zylka MJ, Shearman LP, Levine JD, Jin XW, Weaver DR, Reppert SM.. 1998. Molecular analysis of mammalian timeless. Neuron 21(5):1115–1122. PubMed

Coevolution of Drosophila-type timeless with partner clock proteins

c-Jun N-terminal kinase signaling in aging

Circadian rhythms and circadian clock gene homologs of complex alga Chromera velia

Steroid receptor coactivator TAIMAN is a new modulator of insect circadian clock

Evolution of proteins involved in the final steps of juvenile hormone synthesis

Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila

Pigment Dispersing Factor Is a Circadian Clock Output and Regulates Photoperiodic Response in the Linden Bug, Pyrrhocoris apterus