Laboratory of Molecular Chronobiology
Biological clocks are found in most living organisms and their fundamental properties are highly conserved in vertebrates and invertebrates. Our laboratory is using various insect species to explore important questions of chronobiology and insect physiology. We combine research on established genetic models (Drosophila melanogaster) with studies on insects that have remarkably interesting biology, but only limited experimental tools are available (Pyrrhocoris apterus, Chymomyza costata, Periplaneta americana). We uniquely utilize the power of reverse genetics approaches (RNAi, genome editing, homologous recombination), “omic“-approaches (genomics, transcriptomics, peptidomics), in vitro techniques (tissue cultures, yeast two-hybrid assay), microsurgical interventions and behavioral experiments.
The biological processes of our interest include:
- Molecular, anatomical and genetic basis of insect photoperiodic timers (also known as photoperiodic clocks)
- Insect diapause and seasonality
- Evolution of circadian genes and circadian clocks
- Neuroendocrinology of Pyrrhocoris apterus
- Geographic variability in photoperiodic timers and circadian clocks
For details see “Current research projects“ (bottom of this webpage).
NEWS FROM OUR LAB:
- March 2021: A paper on insulin signaling in ticks from Petr Kopacek's group is online (Kozelkova et al., 2021)
- March 2020: Vlastik's paper on "Insulin Receptor Evolution" is accepted in prestigious Molecular Biology and Evolution (https://academic.oup.com/mbe/article/37/6/1775/5758269)
- Samarjeet defended his PhD, congratulations!
- Magda's first first-author paper is out (Kaniewska et al., 2020)
- Aska's paper describing a new insect neuropeptide EFLamide is accepted in IBMB: Kotwica-Rolinska et al.
- Lenka Chodáková defended her PhD, congratulations!
- Our joint effort with Vacha's group materialized in a paper by Premek Bartos et al.
- Aska's paper technical paper on CRISPR/CAS9 is online: Kotwica-Rolinska et al. 2019
- January 2016: Olina’s paper is accepted in PNAS
Current research projects
under construction: the linden bug:
Pyrrhocoris apterus as a new model species
The linden, Pyrrhocoris apterus, served as an excellent model of classical insect endocrinology for more than 50 years. However, neither functional genetics, nor genomic tools were available. Our goal is to introduce these techniques to P. apterus and establish this insect species as a model organism with full palette of experimental tools, especially:
Reverse genetics: Systemic RNA interference is routinely used for gene knock down in P. apterus (see Smykal et al. 2020, Bajgar et al., 2013a; Smykal et al., 2014; Buresova et al., 2016). To create either complete null mutants or specifically modify gene of interest, we established genome editing in P. apterus (Kotwica-Rolinska et al. 2019). Or first unique mutants are phenotypically characterized.
- Neuropeptide inventory: P. apterus served as a model for insect endocrinology, yet, its neuropeptide repertoire is only very poorly known. Therefore we prospected the genome and transcriptome for genes and transcripts coding for neuropeptides and their receptors. The physical existence of processed peptides is further confirmed by MALDI-TOF-MS/MS in collaboration with Dr. Christian Wegener (Wurzburg University), and anatomical localization is assessed with ICC experiments. See Aska's paper on a new insect neuropeptide (Kotwica-Rolinska et al.).
Microsurgery: The size and anatomy of P. apterus allows for fine microsurgical operations, such as removing Corpus Allatum (P. apterus has only one gland), extirpation of pars intercerebralis region (Bajgar et al., 2013b), or eye removal.
Photoperiodic timer and mechanisms regulating insect Diapause
Our major interest is diapause and its regulation, particularly seasonal switch depending on day-length measurement called photoperiodic timer (clock). Our goal is identifying architecture of photoperiodic timer at the genetic, molecular and anatomical levels. We mostly use two insect species: Pyrrhocoris apterus (Heteroptera) and Chymomyza costata (Diptera).
Pyrrhocoris apterus (Heteroptera) undergoes adult reproductive diapause, which is characterized by absence of Juvenile Hormone synthesis.
Using gene silencing in P. apterus, we confirmed the role of the circadian clock genes in photoperiodic timer (Kotwica-Rolinska et al., 2017).
- Using P. apterus, we described plasticity in Juvenile Hormone (JH) reception, where one universal receptor protein (MET) interacts with various partners in tissue specific manner (Smykal et al, 2014). One of responses requires interaction of MET with circadian proteins and output relies on novel, non-cyclical feedback between Par domain protein 1 and Cryptochrome (Bajgar et al., 2013a; Bajgar et al., 2013b).
- Surprisingly, JH not required for male mating behavior or fertility, whereas circadian genes are needed for the photoperiod-dependent switch from diapause to reproductive growth of MAGs and for mating (Urbanova et al., 2016)
Drosophilid fly Chymomyza costata undergoes photoperiodically regulated larval diapause.
- With robust photoperiodic diapause and adaptaion to temperate regions, Chymomyza costata is an interesting species to study circadian clock (Dolezel, 2019).
- Using RNAseq technology, we characterized transcription profiles associated with photoperiodic diapause induction. Short day photoperiod triggering diapause was associated to remarkable inhibition of 20-hydroxy ecdysone (20-HE) signalling during the photoperiod-sensitive stage of C. costata larval development (Poupardin et al., 2015).
- Using cell culture assays, we defined molecular mechanism of non-diapause (npd) mutant strain in C. costata. The 1855 bp deletion in npd strian removes crucial regulatory cis-elements as well as the minimal promoter, being subsequently responsible for the lack of tim mRNA expression (Kobelkova et al., 2010).
Smýkal V., Pivarči M., Provazník J., Bazalová O., Jedlička P., Lukšan O., Horák A., Vaněčková H., Beneš V., Fiala I., Hanus R., Doležel D. (2020) Complex evolution of insect insulin receptors and homologous decoy receptors, and functional significance of their multiplicity. Molecular Biology and Evolution 37 : 1775-1789. DOI: 10.1093/molbev/msaa048
Kotwica-Rolinska J., Pivarčiová L., Vaněčková H., Doležel D. (2017) The role of circadian clock genes in the photoperiodic timer of the linden bug, Pyrrhocoris apterus, during the nymphal stage. Physiological Entomology 42 : 266-273.
- Urbanová V., Bazalová O., Vaněčková H., Doležel D. (2016) Photoperiod regulates growth of male accessory glands through juvenile hormone signaling in the linden bug, Pyrrhocoris apterus. Insect Biochemistry and Molecular Biology 70: 184-190. DOI: 10.1016/j.ibmb.2016.01.003
- Doležel D. (2015) Photoperiodic time measurement in insects. Current Opinion in Insect Science 7: 98-103. DOI: 10.1016/j.cois.2014.12.002
- Poupardin R., Schöttner K., Korbelová J., Provazník J., Doležel D., Pavlinic D., Beneš V., Koštál V. (2015) Early transcriptional events linked to induction of diapause revealed by RNAseq in larvae of drosophilid fly, Chymomyza costata. BMC Genomics 16: 702. DOI: 10.1186/s12864-015-1907-4
- Smýkal V., Bajgar A., Provazník J., Fexová S., Buřičová M., Takaki K., Hodková M., Jindra M., Doležel D. (2014) Juvenile hormone signaling during reproduction and development of the linden bug, Pyrrhocoris apterus. Insect Biochemistry and Molecular Biology 45: 69-76.
- Bajgar A., Jindra M., Doležel D. (2013) Autonomous regulation of the insect gut by circadian genes acting downstream of juvenile hormone signaling. Proceedings of the National Academy of Sciences of the United States of America 110: 4416-21.
Ditrich T., Janda V., Vaněčková H., Doležel D. (2018) Climatic variation of supercooling point in the linden bug Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae). Insects 9 : article number 144.
- Pivarciova, Provaznik, Vaneckova, Pivarci, Peckova, Bazalova, Wu, Cada, Kment, Kotwica-Rolinska, Dolezel: Unexpected geographic variability of free running period in the linden bug, Pyrrhocoris apterus (Journal of Biological Rhythms, DOI: 10.1177/0748730416671213)
Publication connected to geographic variability, but on a different insect model:
- Paolucci S., Dalla Benetta E., Salis L., Doležel D., van de Zande L., Beukeboom L.W. (2019) Latitudinal variation in circadian rhythmicity in Nasonia vitripennis. Behavioral Sciences 9 : article number: 115. DOI: 10.3390/bs9110115
Geographic adaptations of the linden bug, Pyrrhocoris apterus, and its phylogeography
We use our favorite model organism, Linden bug (Pyrrhocoris apterus), to study diapause and particularly its photoperiodic regulation. Seasonal environmental changes are mostly influenced by latitude and altitude of particular locality. Indeed, we observe remarkable variability in photoperiodic and circadian clocks between geographical field-lines of P. apterus. Robust analysis of P. apterus phylogeography is needed to shed some light on origin of these adaptations. In collaboration with Prof. Roger Butlin (University of Sheffield) we are performing RAD sequencing and analysis of samples from approximately 150 localities.
Current collection of P. apterus (see the map) was only possible thanks to generous help of following colleagues: Adam Bajgar, Alejandro Cabezas-Cruz, Aleksandra Konjevic, Barbara Lis, Jerzy A. Lis, Carl-Cedric Coulianos, Rodolfe Costa, Dora Nagy, Eva Hola, Hanka Vaneckova, Iva Fukova, Jana Pavlová, Joanna Kotwica-Rolinska, Jula Lukes, Kai Schuette, Kajka Straznicka, Lucia Salis, Lukas Cizek, Lukas Drag, Manuel Baena, Marek Jindra, Marketa Ondrackova, Martin Kaltenpoth, Martin Vacha, Matilde Eizaguirre, Milan Stech, Milena Damulewicz, Olina Bazalova, Petr Kment, Petra Sekyrova, Plamen Kalushkov, Radka Zavodska, Ramon Albajes, Stanislav Rada, Teemu Rintala, Vlastimil Smykal, Xanti Pagola, Zejlko Popovic,
However, several geographical regions are either underrepresented or even absent completely (see red circles in the map below). Therefore, we would really welcome samples (either dead or alive) from following regions:
Europe – England, Sweden, Norway, Denmark, Mediterranean islands (Sicily, Corsica, Sardinia, islands in Aegean sea), Romania, Moldavia, Portugal, Belarus, Ukraine, Russia,
North Africa– Morocco, Tunisia, Algeria, Egypt
East – Turkey, Syria, Lebanon, Jordan, Iraq, Iran, Afghanistan, Pakistan, Oman, Saudi Arabia, Emirates, Georgia, Azerbaijan, Turkmenistan, Tajikistan, Uzbekistan, Kyrgyzstan, Kazakhstan, Russia, South West Mongolia, North West China
Sample collection: The optimal way for storing and transporting samples seems to be 96% ethanol (non-denatured). 2ml screw cup tubes can accommodate 5 - 8 bugs from the same locality. We are ready to send prepared tubes filled with EtOH, just send an e-mail request, please. Although we are happy even for one individual bug, 10-15 specimens from one locality are optimal. It is important to have the report of the locality (such as GPS position or locality name from the map, elevation is also very useful). Please, if possible, it would be also great to mention, if the locality was urbanized area (town, park and similar area modified by human) or if the locality is more similar to original nature-like type (forest, steppe, mountains). In ideal case we would like to have samples collected approximately 200 km apart in lowlands, while in mountains or complex terrain even higher density is helpful (down to tens of kilometers).
Living bugs – an alternative is to collect live specimens. Adults survive with wet piece of cotton or cellulose in small paper box for more than one week, as long as it is not too hot. We are really happy to obtain living specimens for establishing colonies, where circadian and photoperiodic phenotypes can be characterized.
Species identification - Pyrrhocoris apteruscan be quite easily identified thanks to its aposematic coloration. Both, larvae or adults can be collected and are welcome. We are also seeking for samples of closely related pyrrhocorids, such as P. sibiricus, P. marginalis, and genus Scantius (see the pictures below).
Contact: David Dolezel, firstname.lastname@example.org; Institute of Entomology; Biology center CAS; Branisovska 31; 370 05 Ceske Budejovice; Czech Republic
Circadian clock - evolution, comparative studies
Circadian biological clocks are found in most living organisms and their fundamental properties are highly conserved in vertebrates and invertebrates. The fruitfly, Drosophila melanogaster served as a premier insect species for the molecular analysis of the circadian rhythms, and we enjoy using its powerful genetic tools as well (Singh et al., 2019). Because of the functional similarities of the circadian clocks among all metazoans, it was suggested that the molecular mechanisms underlying the clock function would be conserved as well. Surprisingly, we can see remarkable differences between the molecular regulations of the circadian timing system even among holometabolous insects. The long-term goal of our laboratory is to gain a better understanding of the cellular and molecular mechanisms that underlie circadian rhythmicity and to shed some light on evolution of circadian and photoperiodic clocks. We also explore role of circadian genes in a non-clock processes, i.e., in magnetoreception (Bartos et al. 2019, Bazalova et al., 2016).
Singh S., Giesecke A., Damulewicz M., Fexová S., Mazzotta G.M., Stanewsky R., Doležel D. (2019) New Drosophila circadian clock mutants affecting temperature compensation induced by targeted mutagenesis of timeless. Frontiers in Physiology 10 : article number: 1442.
Bartoš P., Netušil R., Slabý P., Doležel D., Ritz T., Vácha M. (2019) Weak radiofrequency fields affect the insect circadian clock. Journal of the Royal Society Interface 16 : article number: 20190285. DOI: 10.1098/rsif.2019.0285
Bazalová O., Doležel D. (2017) Daily Activity of the Housefly, Musca domestica, Is Influenced by Temperature Independent of 3' UTR period Gene Splicing G3: Genes, Genomes, Genetics 7 : 2637-2649. DOI: 10.1534/g3.117.042374
- Bazalova O., Kvicalova M., Valkova T., Slaby P., Bartos P., Netusil R., Tomanova K., Braeunig P., Lee H.-J., Sauman I., Damulewicz M., Provaznik J., Pokorny R., Dolezel D., Vacha M. (2016) Cryptochrome 2 mediates directional magnetoreception in cockroaches.Proceedings of the National Academy of Sciences of the United States of America 113: 1660-1665. DOI:10.1073/pnas.1518622113