Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature


Circadian clocks are endogenous timers adjusting behaviour and physiology with the solar day1. Synchronized circadian clocks improve fitness2 and are crucial for our physical and mental well-being3. Visual and non-visual photoreceptors are responsible for synchronizing circadian clocks to light4,5, but clock-resetting is also achieved by alternating day and night temperatures with only 2–4 °C difference6,7,8. This temperature sensitivity is remarkable considering that the circadian clock period (~24 h) is largely independent of surrounding ambient temperatures1,8. Here we show that Drosophila Ionotropic Receptor 25a (IR25a) is required for behavioural synchronization to low-amplitude temperature cycles. This channel is expressed in sensory neurons of internal stretch receptors previously implicated in temperature synchronization of the circadian clock9. IR25a is required for temperature-synchronized clock protein oscillations in subsets of central clock neurons. Extracellular leg nerve recordings reveal temperature- and IR25a-dependent sensory responses, and IR25a misexpression confers temperature-dependent firing of heterologous neurons. We propose that IR25a is part of an input pathway to the circadian clock that detects small temperature differences. This pathway operates in the absence of known ‘hot’ and ‘cold’ sensors in the Drosophila antenna10,11, revealing the existence of novel periphery-to-brain temperature signalling channels.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: IR25a is expressed in ChO neurons.
Figure 2: IR25a is required for temperature synchronization to low-amplitude temperature cycles.
Figure 3: IR25a is required for clock protein oscillations in central clock neurons.
Figure 4: IR25a is required for temperature-induced leg nerve responses and confers temperature sensitivity to l-LNv.


  1. Dunlap, J. C., Loros, J. J. & DeCoursey, P. J. Chronobiology: Biological Timekeeping (Sinauer Associates, 2004)

  2. Ouyang, Y., Andersson, C. R., Kondo, T., Golden, S. S. & Johnson, C. H. Resonating circadian clocks enhance fitness in cyanobacteria. Proc. Natl Acad. Sci. USA 95, 8660–8664 (1998)

    Article  CAS  ADS  Google Scholar 

  3. Bechtold, D. A., Gibbs, J. E. & Loudon, A. S. Circadian dysfunction in disease. Trends Pharmacol. Sci. 31, 191–198 (2010)

    Article  CAS  Google Scholar 

  4. Helfrich-Förster, C., Winter, C., Hofbauer, A., Hall, J. C. & Stanewsky, R. The circadian clock of fruit flies is blind after elimination of all known photoreceptors. Neuron 30, 249–261 (2001)

    Article  Google Scholar 

  5. Hughes, S., Jagannath, A., Hankins, M. W., Foster, R. G. & Peirson, S. N. Photic regulation of clock systems. Methods Enzymol. 552, 125–143 (2015)

    Article  CAS  Google Scholar 

  6. Brown, S. A., Zumbrunn, G., Fleury-Olela, F., Preitner, N. & Schibler, U. Rhythms of mammalian body temperature can sustain peripheral circadian clocks. Curr. Biol. 12, 1574–1583 (2002)

    Article  CAS  Google Scholar 

  7. Wheeler, D. A., Hamblen-Coyle, M. J., Dushay, M. S. & Hall, J. C. Behavior in light-dark cycles of Drosophila mutants that are arrhythmic, blind, or both. J. Biol. Rhythms 8, 67–94 (1993)

    Article  CAS  Google Scholar 

  8. Maguire, S. E. & Sehgal, A. Heating and cooling the Drosophila melanogaster clock. Curr. Opin . Insect Sci. 7, 71–75 (2015)

    Google Scholar 

  9. Sehadova, H. et al. Temperature entrainment of Drosophila’s circadian clock involves the gene nocte and signaling from peripheral sensory tissues to the brain. Neuron 64, 251–266 (2009)

    Article  CAS  Google Scholar 

  10. Florence, T. J. & Reiser, M. B. Neuroscience: hot on the trail of temperature processing. Nature 519, 296–297 (2015)

    Article  CAS  ADS  Google Scholar 

  11. Gallio, M., Ofstad, T. A., Macpherson, L. J., Wang, J. W. & Zuker, C. S. The coding of temperature in the Drosophila brain. Cell 144, 614–624 (2011)

    Article  CAS  Google Scholar 

  12. Glaser, F. T. & Stanewsky, R. Temperature synchronization of the Drosophila circadian clock. Curr. Biol. 15, 1352–1363 (2005)

    Article  CAS  Google Scholar 

  13. Wolfgang, W., Simoni, A., Gentile, C. & Stanewsky, R. The Pyrexia transient receptor potential channel mediates circadian clock synchronization to low temperature cycles in Drosophila melanogaster . Proc. R. Soc. Lond. B 280, 20130959 (2013)

    Article  Google Scholar 

  14. Rees, J. S. et al. In vivo analysis of proteomes and interactomes using parallel affinity capture (iPAC) coupled to mass spectrometry. Mol. Cell Proteomics 10, M110.002386 (2011)

    Article  Google Scholar 

  15. Abuin, L. et al. Functional architecture of olfactory ionotropic glutamate receptors. Neuron 69, 44–60 (2011)

    Article  CAS  Google Scholar 

  16. Benton, R., Vannice, K. S., Gomez-Diaz, C. & Vosshall, L. B. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila . Cell 136, 149–162 (2009)

    Article  CAS  Google Scholar 

  17. Rytz, R., Croset, V. & Benton, R. Ionotropic receptors (IRs): chemosensory ionotropic glutamate receptors in Drosophila and beyond. Insect Biochem. Mol. Biol. 43, 888–897 (2013)

    Article  CAS  Google Scholar 

  18. Petersen, L. K. & Stowers, R. S. A Gateway MultiSite recombination cloning toolkit. PLoS ONE 6, e24531 (2011)

    Article  CAS  ADS  Google Scholar 

  19. Sayeed, O. & Benzer, S. Behavioral genetics of thermosensation and hygrosensation in Drosophila . Proc. Natl Acad. Sci. USA 93, 6079–6084 (1996)

    Article  CAS  ADS  Google Scholar 

  20. Kaneko, H. et al. Circadian rhythm of temperature preference and its neural control in Drosophila . Curr. Biol. 22, 1851–1857 (2012)

    Article  CAS  Google Scholar 

  21. Hamada, F. N. et al. An internal thermal sensor controlling temperature preference in Drosophila . Nature 454, 217–220 (2008)

    Article  CAS  ADS  Google Scholar 

  22. Ni, L. et al. A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila . Nature 500, 580–584 (2013)

    Article  CAS  ADS  Google Scholar 

  23. Busza, A., Murad, A. & Emery, P. Interactions between circadian neurons control temperature synchronization of Drosophila behavior. J. Neurosci. 27, 10722–10733 (2007)

    Article  CAS  Google Scholar 

  24. Venken, K. J. et al. Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster . Nature Methods 6, 431–434 (2009)

    Article  CAS  Google Scholar 

  25. Kaneko, M. & Hall, J. C. Neuroanatomy of cells expressing clock genes in Drosophila: transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections. J. Comp. Neurol. 422, 66–94 (2000)

    Article  CAS  Google Scholar 

  26. Gummadova, J. O., Coutts, G. A. & Glossop, N. R. Analysis of the Drosophila Clock promoter reveals heterogeneity in expression between subgroups of central oscillator cells and identifies a novel enhancer region. J. Biol. Rhythms 24, 353–367 (2009)

    Article  CAS  Google Scholar 

  27. Kim, J. et al. A TRPV family ion channel required for hearing in Drosophila . Nature 424, 81–84 (2003)

    Article  CAS  ADS  Google Scholar 

  28. Park, J. H. & Hall, J. C. Isolation and chronobiological analysis of a neuropeptide pigment-dispersing factor gene in Drosophila melanogaster . J. Biol. Rhythms 13, 219–228 (1998)

    Article  CAS  Google Scholar 

  29. Liu, L. et al. Drosophila hygrosensation requires the TRP channels water witch and nanchung. Nature 450, 294–298 (2007)

    Article  CAS  ADS  Google Scholar 

  30. Chung, Y. D., Zhu, J., Han, Y. & Kernan, M. J. nompA encodes a PNS-specific, ZP domain protein required to connect mechanosensory dendrites to sensory structures. Neuron 29, 415–428 (2001)

    Article  CAS  Google Scholar 

  31. Ruben, M., Drapeau, M. D., Mizrak, D. & Blau, J. A mechanism for circadian control of pacemaker neuron excitability. J. Biol. Rhythms 27, 353–364 (2012)

    Article  CAS  Google Scholar 

  32. Konopka, R. J. & Benzer, S. Clock mutants of Drosophila melanogaster . Proc. Natl Acad. Sci. USA 68, 2112–2116 (1971)

    Article  CAS  ADS  Google Scholar 

  33. Sweeney, S. T., Broadie, K., Keane, J., Niemann, H. & O’Kane, C. J. Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14, 341–351 (1995)

    Article  CAS  Google Scholar 

  34. Zhang, Y., Liu, Y., Bilodeau-Wentworth, D., Hardin, P. E. & Emery, P. Light and temperature control the contribution of specific DN1 neurons to Drosophila circadian behavior. Curr. Biol. 20, 600–605 (2010)

    Article  CAS  Google Scholar 

  35. Saina, M. & Benton, R. Visualizing olfactory receptor expression and localization in Drosophila . Methods Mol. Biol. 1003, 211–228 (2013)

    Article  CAS  Google Scholar 

  36. Yoshii, T., Todo, T., Wülbeck, C., Stanewsky, R. & Helfrich-Förster, C. Cryptochrome is present in the compound eyes and a subset of Drosophila’s clock neurons. J. Comp. Neurol. 508, 952–966 (2008)

    Article  CAS  Google Scholar 

  37. Rush, B. L., Murad, A., Emery, P. & Giebultowicz, J. M. Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophila ovary. J. Biol. Rhythms 21, 272–278 (2006)

    Article  CAS  Google Scholar 

  38. Gentile, C., Sehadova, H., Simoni, A., Chen, C & Stanewsky, R . Cryptochrome antagonizes synchronization of Drosophila’s circadian clock to temperature cycles. Curr. Biol. 23, 185–195 (2013)

    Article  CAS  Google Scholar 

  39. Croset, V. et al. Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genet. 6, e1001064 (2010)

    Article  Google Scholar 

  40. Levine, J. D., Funes, P., Dowse, H. B. & Hall, J. C. Signal analysis of behavioral and molecular cycles. BMC Neurosci. 3, 1 (2002)

    Article  Google Scholar 

  41. Simoni, A. et al. A mechanosensory pathway to the Drosophila circadian clock. Science 343, 525–528 (2014)

    Article  CAS  ADS  Google Scholar 

  42. Wilson, R. I. & Corey, D. P. The force be with you: a mechanoreceptor channel in proprioception and touch. Neuron 67, 349–351 (2010)

    Article  CAS  Google Scholar 

  43. Stanewsky, R. et al. Temporal and spatial expression patterns of transgenes containing increasing amounts of the Drosophila clock gene period and a lacZ reporter: mapping elements of the PER protein involved in circadian cycling. J. Neurosci. 17, 676–696 (1997)

    Article  CAS  Google Scholar 

Download references


We thank P. Emery, J. Albert, J. Jepson, P. Garrity, and A. Samuel for discussions and sharing of unpublished results, J. Giebultowicz for anti-TIM antibodies, J. Albert and J. Jepson for fly stocks, C. Tardieu and R. Kavlie for help with qPCR, D. Carr for assistance with the temperature recording setup, and M. Ogueta-Gutierrez for help with figure preparations. The drawing for Fig. 4a was generated by Polygonal Tree ( This work was supported by BBSRC grants BB/H001204 to R.S., BB/J0-18589/-17221 to R.S. and J.J.L.H., and a CSC PhD fellowship to C.C. V.C. was supported by a Boehringer Ingelheim Foundation Fellowship. Research in R.B.’s laboratory was supported by European Research Council Starting Independent Researcher and Consolidator Grants (205202 and 615094). Mass spectrometry analysis was supported by Wellcome Trust grant 099135/Z/12/Z.

Author information

Authors and Affiliations



C.C., E.B., R.S., J.J.L.H., R.B. and K.S.L. conceived, designed, and supervised the project. C.C., E.B., M.X., V.C., and J.S.R. performed experiments. C.C., E.B., and J.S.R. analysed data, and R.S. wrote the paper, with feedback from all authors.

Corresponding author

Correspondence to Ralf Stanewsky.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 IR25a and Nocte physically interact in vivo and are expressed in femur and antennal ChO neurons.

a, In vivo co-immunoprecipitation experiments using protein extracts from fly heads. Head lysates were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were examined by western blotting using anti-Flag and anti-IR25a antibody. Input represents 30% of cell lysates used in the pull-down experiment. The genotypes of the flies used were: tim > IR25a: UAS-GFP/UAS-IR25a; tim-gal4:67/+. tim > IR25a+FLAG-NOCTE: UAS-FSNH/UAS-IR25a; tim-gal4:67/+. The bracket indicates that NOCTE–Flag runs as a double band on western blots. For uncropped gel images, see Supplementary Fig. 1. b, Overview of the antennal and femur ChO adapted from refs 13, 42. c, d, Labelling of the JO neurons by IR25a-gal4 (c) and F-gal4 (d) driven membrane bound mCD8–GFP and nuclear-localized DsRed expression. Note that IR25a is expressed in only a subset of JO neurons. e, f, Same flies as in c, d analysed for IR25a expression in the femoral ChO. Again, only subsets of the ChO neurons express IR25a. Arrows in c, e point to ChO neuron nuclei. g, Labelling of the ChO neurons in JO and femur by nocte-gal4-driven membrane-bound GFP and nuclear DsRed expression9. Scale bar, 20 μm.

Extended Data Figure 2 Spatial and quantitative IR25a mRNA and protein expression in CNS and PNS tissues and efficiency of RNAi-mediated knockdown.

a, Analysis of IR25a-gal4 and IR25a in the third segment of the antenna reveals expression in coeloconic sensilla16. Schematic adapted from15. b, c, Determination of IR25a and nocte mRNA levels in femur and retinal tissues by semiquantitative RT–PCR; rp49 was used as control. For uncropped gel data, see Supplementary Fig. 1. d, e, qPCR analysis of IR25a mRNA levels in whole heads (d), or dissected body parts (as indicated) (e) from flies of the genotypes indicated. Pan-neuronal elav-gal4 knockdown (d) decreased IR25a mRNA >75% or >90%, using one or two different RNAi lines combined, respectively. ****P < 0.0001, ***P < 0.001, *P < 0.05, one-way ANOVA followed by Bonferroni correction. f, IR25a is not expressed in the central brain and clock neurons. Left, IR25a immunolabelling of a Canton S brain reveals no signals. Middle, same brain labelled with anti-TIM reveals expression in clock neurons. Right, merge. Brains were dissected in LD at ZT20. Scale bar, 10 μm. g, IR25a-gal4 is not expressed in clock neurons and largely absent from the brain. Left, nuclear DsRed driven by IR25a-gal4. Second from left, anti-PDF staining showing LNv and their projections. Middle, anti-PER (diluted 1:5,000)43 showing all clock neurons. Second from right, merge, showing two IR25a-gal4 positive cell in the antennal lobe, not co-localized with any of the clock neurons. These cells were observed in 4/8 hemispheres and always on the same side of the brain. Right, magnified view of circled area in the merged image. Scale bar, 30 μm.

Extended Data Figure 3 IR25a is required for temperature synchronization to low-amplitude temperature cycles but not for high-amplitude temperature cycles.

a, Canton S, IR25a−/−, and nocte1 flies were exposed to LD at 20 °C for 5 days (left) or LL at 25 °C for 2 days (right), followed by exposure to a 12 h:12 h 20 °C:29 °C (left) or 16 °C:25 °C (right) temperature cycles in LL, which after 6–7 days was delayed or advanced by 6 h, respectively. Warmer temperature indicated by red and orange shading, respectively. b, Actograms and daily averages of Canton S and IR25a−/−, and IR25a−/− flies containing a genomic IR25a rescue construct (rescue) exposed to 18 °C:20 °C temperature cycles in LL (left) and 21 °C:23 °C temperature cycles in LL (right). Warm phase in actograms indicated by orange shading. Histogram colour coding as in Fig. 2. For quantification see Fig. 2e.

Extended Data Figure 4 IR25a is not required for temperature synchronization to high- but to low-amplitude temperature cycles and IR25a−/− flies show normal LD and DD behaviour.

a, Canton S and IR25a−/− flies were exposed to LL at 25 °C for 2–3 days, followed by exposure to high-amplitude 12 h:12 h temperature cycles in LL, which after 5–6 days were delayed by 6 h. Double plotted average actograms depicting the daily activity levels and environmental conditions during the entire experiment are shown. Actual temperatures are colour coded and indicated below the entrainment index calculations. Numbers (n) indicated in the bars. b, As in a but flies were initially kept in LD and DD for 2 days each (left) or LD (right), before being exposed to two phase delayed (left) or advanced (right) temperature cycles in DD at the temperatures indicated. n.s., not significant. c, Behaviour of IR25a−/− and rescue flies during DD and 25 °C:27 °C temperature cycles with 8 h delay and during DD and 21 °C:23 °C temperature cycles with a 8 h advance compared to the previous LD cycle (at 25 °C). Warm phase is indicated by orange shading. d, Canton S and IR25a−/− flies during LD and DD conditions at 25 °C (see Extended Data Table 2 for period calculations).

Extended Data Figure 5 Antennal IR25a expression is not necessary for synchronization of locomotor activity rhythms to temperature cycles.

Ablation of antennae as indicated. a, IR25a−/− and rescue flies were exposed to the same condition used in Fig. 2. Actograms and daily averages as described before. b, Quantification of behaviour as described in Fig. 2. The data of IR25a−/− with normal antennae was taken from Fig. 2a. n.s., not significant.

Extended Data Figure 6 Knocking down IR25a expression via RNA interference disrupts synchronization of locomotor activity rhythms to temperature cycles (25 °C:27 °C in LL).

a, b, Behaviour of flies with spatially restricted IR25a knockdown mediated by IR25a-gal4 (a), ChO specific F-gal4, and nompC-gal4 (b) driven IR25-RNAi expression, respectively. Control flies are UAS-dicer2/Y; IR25-gal4/+ (a), and UAS-dicer2/Y;+/+; F-gal4/+ or UAS-dicer2/Y;+/+; nompC-gal4/+ (b). Test flies carry the same transgenes, but in addition one or two copies of the IR25a-RNAi line indicated. Actograms and daily averages as described for Fig. 2a. c, Progeny of the respective UAS-IR25a-RNAi lines crossed to y w (left three columns) and flies from (a, b) and the other gal4 drivers indicated, were exposed to the same LL and temperature cycle conditions used in Fig. 2a. As controls, UAS-dicer, gal4 driver lines were crossed to y w and F1 males containing UAS-dicer/Y and the respective gal4/+ were tested. Numbers of analysed individuals (n) are indicated above each column. Entrainment was quantified as in Fig. 2c. ****P < 0.0001, one-way ANOVA followed by Bonferroni correction.

Extended Data Figure 7 Rescue of TIM oscillations in clock neurons during low-amplitude temperature cycles and normal TIM oscillations during LD and high-amplitude temperature cycles.

a, b, TIM levels in clock neurons during LL 25 °C:27 °C temperature cycles at the indicated time points (ZT) in the genotypes indicated. At least 8 brain hemispheres per time point were analysed for each genotype. Scale bars, 10 µm. Data in b are mean ± s.e.m. c, Quantification of TIM levels in clock neurons during LD (25 °C) in Canton S and IR25a−/− mutant brains. d, TIM oscillations in different clock-neuronal groups in IR25a−/− are restored in 25 °C:29 °C temperature cycles in LL. At least 8 brain hemispheres per time point were analysed for each genotype and condition. Error bars indicate s.e.m.

Extended Data Figure 8 Ectopic expression and heat responses of TRPA1 and IR8a in l-LNv clock neurons.

a, Whole-cell current clamp recordings of Pdf-gal4/UAS-TrpA1; Pdf-RFP (top trace, red) and Pdf-gal4/UAS-IR8a-RFP (bottom trace, black) brains exposed to a temperature ramp from 18 °C to 30 °C and back to 18 °C. Note the additional depolarization of the TrpA1 neuron at higher temperatures. b, Compared to control (Fig. 4), recordings from TrpA1-expressing neurons show a large increase in firing rate with temperature which the IR8a expressing neurons do not. c, d, In comparison to control neurons (data taken from Fig. 4) the membrane potential of TrpA1 expressing neurons is more positive at 30 °C (open bars) and the input resistance is also significantly reduced in TrpA1 at 18 °C. e, f, The firing rate at 18 °C is higher for IR8a neurons but only the Q10 of TrpA1 is different to control. Bars are means and whiskers s.e.m., n indicated in bars, *P < 0.05, ***P < 0.001, ANOVA followed by Tukey test.

Extended Data Table 1 Mass spectrometry data from fly heads from three different genotypes
Extended Data Table 2 Rhythm analysis of control and IR25a mutant flies under free running (DD) conditions at different ambient temperatures

Supplementary information

Supplementary Figure

This file contains gels for Extended Data Figures 1 and 2. (PDF 141 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chen, C., Buhl, E., Xu, M. et al. Drosophila Ionotropic Receptor 25a mediates circadian clock resetting by temperature. Nature 527, 516–520 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing