Abstract
The preference for a particular thermal range is a key determinant of the distribution of animal species. However, we know little on how temperature preference behaviour evolves during the colonization of new environments. Here we show that at least two distinct neurobiological mechanisms drive the evolution of temperature preference in flies of the genus Drosophila. Fly species from mild climates (D. melanogaster and D. persimilis) avoid both innocuous and noxious heat, and we show that the thermal activation threshold of the molecular heat receptor Gr28b.d precisely matches species-specific thresholds of behavioural heat avoidance. We find that desert-dwelling D. mojavensis are instead actively attracted to innocuous heat. Notably, heat attraction is also mediated by Gr28b.d (and by the antennal neurons that express it) and matches its threshold of heat activation. Rather, the switch in valence from heat aversion to attraction correlates with specific changes in thermosensory input to the lateral horn, the main target of central thermosensory pathways and a region of the fly brain implicated in the processing of innate valence1,2,3,4,5. Together, our results demonstrate that, in Drosophila, the adaptation to different thermal niches involves changes in thermal preference behaviour, and that this can be accomplished using distinct neurobiological solutions, ranging from shifts in the activation threshold of peripheral thermosensory receptor proteins to a substantial change in the way temperature valence is processed in the brain.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
27,99 € / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
199,00 € per year
only 3,90 € per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
All data are available in the main paper and Extended Data Figs. 1–7. Maps were created in MATLAB and traced in Adobe Illustrator; all other illustrations were created in Adobe Illustrator. Gr28b.d-coding sequences from D. persimilis (GenBank OQ624956), D. mojavensis (GenBank OQ621410) and D. mettleri (GenBank PP885916) are available via GenBank. Raw read sequences from RNA-seq of samples from D. mojavensis aristae are available via the Sequence Read Archive of the National Center for Biotechnology Information with the BioProject ID PRJNA948454. The following database was also used: EM Hemibrain:v1.2.1 (https://neuprint.janelia.org/). Source data are provided with this paper.
Code availability
No custom algorithms or computer code was produced as part of this work.
References
Das Chakraborty, S., Chang, H., Hansson, B. S. & Sachse, S. Higher-order olfactory neurons in the lateral horn support odor valence and odor identity coding in Drosophila. eLife 11, e74637 (2022).
Frechter, S. et al. Functional and anatomical specificity in a higher olfactory centre. eLife 8, e44590 (2019).
Knaden, M., Strutz, A., Ahsan, J., Sachse, S. & Hansson, B. S. Spatial representation of odorant valence in an insect brain. Cell Rep. 1, 392–399 (2012).
Lerner, H., Rozenfeld, E., Rozenman, B., Huetteroth, W. & Parnas, M. Differential role for a defined lateral horn neuron subset in naive odor valence in Drosophila. Sci. Rep. 10, 6147 (2020).
Varela, N., Gaspar, M., Dias, S. & Vasconcelos, M. L. Avoidance response to CO2 in the lateral horn. PLoS Biol. 17, e2006749 (2019).
Sinervo, B. et al. Erosion of lizard diversity by climate change and altered thermal niches. Science 328, 894–899 (2010).
Warren, M. S. et al. The decline of butterflies in Europe: problems, significance, and possible solutions. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2002551117 (2021).
Contributions to the genetics, taxonomy, and ecology of Drosophila pseudoobscura and its relatives. Ann. Entomol. Soc. Am. 39, 151 (1946).
Ito, F. & Awasaki, T. Comparative analysis of temperature preference behavior and effects of temperature on daily behavior in 11 Drosophila species. Sci. Rep. 12, 12692 (2022).
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).
Sayeed, O. & Benzer, S. Behavioral genetics of thermosensation and hygrosensation in Drosophila. Proc. Natl Acad. Sci. USA 93, 6079–6084 (1996).
Gibbs, A. G., Perkins, M. C. & Markow, T. A. No place to hide: microclimates of Sonoran Desert Drosophila. J. Therm. Biol 28, 353–362 (2003).
Kellermann, V. et al. Upper thermal limits of Drosophila are linked to species distributions and strongly constrained phylogenetically. Proc. Natl Acad. Sci. USA 109, 16228–16233 (2012).
Huda, A., Omelchenko, A. A., Vaden, T. J., Castaneda, A. N. & Ni, L. Responses of different Drosophila species to temperature changes. J. Exp. Biol. https://doi.org/10.1242/jeb.243708 (2022).
Suvorov, A. et al. Widespread introgression across a phylogeny of 155 Drosophila genomes. Curr. Biol. 32, 111–123 (2022).
Simoes, J. M. et al. Robustness and plasticity in Drosophila heat avoidance. Nat. Commun. 12, 2044 (2021).
Ni, L. et al. A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila. Nature 500, 580–584 (2013).
Thorne, N. & Amrein, H. Atypical expression of Drosophila gustatory receptor genes in sensory and central neurons. J. Comp. Neurol. 506, 548–568 (2008).
Mishra, A. et al. The Drosophila Gr28bD product is a non-specific cation channel that can be used as a novel thermogenetic tool. Sci. Rep. 8, 901 (2018).
Arenas, O. M. et al. Activation of planarian TRPA1 by reactive oxygen species reveals a conserved mechanism for animal nociception. Nat. Neurosci. 20, 1686–1693 (2017).
Hamada, F. N. et al. An internal thermal sensor controlling temperature preference in Drosophila. Nature 454, 217–220 (2008).
Frank, D. D., Jouandet, G. C., Kearney, P. J., Macpherson, L. J. & Gallio, M. Temperature representation in the Drosophila brain. Nature 519, 358–361 (2015).
Liu, W. W., Mazor, O. & Wilson, R. I. Thermosensory processing in the Drosophila brain. Nature 519, 353–357 (2015).
Marin, E. C. et al. Connectomics analysis reveals first-, second-, and third-order thermosensory and hygrosensory neurons in the adult Drosophila brain. Curr. Biol. 30, 3167–3182 (2020).
Schlegel, P. et al. Whole-brain annotation and multi-connectome cell typing of Drosophila. Nature 634, 139–152 (2024).
Alpert, M. H., Gil, H., Para, A. & Gallio, M. A thermometer circuit for hot temperature adjusts Drosophila behavior to persistent heat. Curr. Biol. 32, 4079–4087 (2022).
Alpert, M. H. et al. A circuit encoding absolute cold temperature in Drosophila. Curr. Biol. 30, 2275–2288 (2020).
Jouandet, G. C. et al. Rapid threat assessment in the Drosophila thermosensory system. Nat. Commun. 14, 7067 (2023).
Stratman, R. & Markow, T. A. Resistance to thermal stress in desert Drosophila. Funct. Ecol. 12, 965–970 (1998).
Govek, K. W. et al. CAJAL enables analysis and integration of single-cell morphological data using metric geometry. Nat. Commun. 14, 3672 (2023).
Auer, T. O. et al. Olfactory receptor and circuit evolution promote host specialization. Nature 579, 402–408 (2020).
Stensmyr, M. C., Dekker, T. & Hansson, B. S. Evolution of the olfactory code in the Drosophila melanogaster subgroup. Proc. Biol. Sci. 270, 2333–2340 (2003).
Toda, Y. et al. Early origin of sweet perception in the songbird radiation. Science 373, 226–231 (2021).
Laursen, W. J., Schneider, E. R., Merriman, D. K., Bagriantsev, S. N. & Gracheva, E. O. Low-cost functional plasticity of TRPV1 supports heat tolerance in squirrels and camels. Proc. Natl Acad. Sci. USA 113, 11342–11347 (2016).
Yang, S. et al. A paradigm of thermal adaptation in penguins and elephants by tuning cold activation in TRPM8. Proc. Natl Acad. Sci. USA 117, 8633–8638 (2020).
Sprengelmeyer, Q. D. et al. Recurrent collection of Drosophila melanogaster from wild African environments and genomic insights into species history. Mol. Biol. Evol. 37, 627–638 (2020).
Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).
Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
Gratz, S. J. et al. Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196, 961–971 (2014).
Masumoto, M., Ohde, T., Shiomi, K., Yaginuma, T. & Niimi, T. A baculovirus immediate-early gene, ie1, promoter drives efficient expression of a transgene in both Drosophila melanogaster and Bombyx mori. PLoS ONE 7, e49323 (2012).
Caron, S. J., Ruta, V., Abbott, L. F. & Axel, R. Random convergence of olfactory inputs in the Drosophila mushroom body. Nature 497, 113–117 (2013).
Hayashi, T. T. et al. Mushroom body input connections form independently of sensory activity in Drosophila melanogaster. Curr. Biol. 32, 4000–4012.e5 (2022).
Li, J., Mahoney, B. D., Jacob, M. S. & Caron, S. J. C. Visual input into the Drosophila melanogaster mushroom body. Cell Rep. 32, 108138 (2020).
Scheffer, L. K. et al. A connectome and analysis of the adult Drosophila central brain. eLife https://doi.org/10.7554/eLife.57443 (2020).
Feng, L., Zhao, T. & Kim, J. neuTube 1.0: A new design for efficient neuron reconstruction software based on the SWC format. eNeuro https://doi.org/10.1523/ENEURO.0049-14.2014 (2015).
Arshadi, C., Gunther, U., Eddison, M., Harrington, K. I. S. & Ferreira, T. A. SNT: a unifying toolbox for quantification of neuronal anatomy. Nat. Methods 18, 374–377 (2021).
Huang, H., Liu, Y., Yuan, M. & Marron, J. S. Statistical significance of clustering using soft thresholding. J. Comput. Graph. Stat. 24, 975–993 (2015).
Acknowledgements
We thank M. Stensmyr, T. Bozza, H. Lee and members of the Gallio Lab for comments; D. Bennett, A. Park, P. Seng, I. Baker and especially A. Kuang, J. Hua and P. Laminette for technical assistance in the early stages of the work; R. Suhendra for help with data analysis; and M. Stensmyr for the gift of fly drawings in Figs. 1, 3 and 5. This work was supported by National Institutes of Health, National Institute of Neurological Disorders and Stroke grants R01NS086859, R21NS130554 (to M.G.) and 1F31NS129270 (to M.C.), the PEW Scholars Program (M.G.) and a grant from the Trienens Institute for Sustainability and Energy at Northwestern University (A.P.).
Author information
Authors and Affiliations
Contributions
Conceptualization: M.G., A.P. and M.C.; behavioural experiments and analysis: M.C., A.A., E.E.Z., I.D.M.-G. and J.M.S.; electrophysiology and analysis: O.M.A., M.C. and Y.S.; CRISPR mutants and transgenesis: M.C. and A.P.; RNA sequencing and analysis: E.E.Z., A.P. and M.C.; in vivo electroporation and analysis: M.C. and M.H.A.; microscopy: H.G., M.C. and M.H.A.; visualization: M.G. and M.C.; funding acquisition: M.G.; supervision: M.G. and A.P.; writing: M.G. and M.C.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature thanks Sophie Caron and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Alignment of Gr28b.d protein sequences across Drosophila species.
(a) Multiple alignment of Gr28b.d protein sequences from 25 Drosophila species (names abbreviated to first three letters, e.g. D. mel = D. melanogaster), (b) and of the 4 species D. melanogaster, D. persimilis, D. mojavensis, and D. mettleri. (c) Location of amino acid differences between the indicated species (red=different). (histogram: bars = mean pairwise amino acid sequence identity across species, green = 100%, yellow = <100% and ≥30%, orange = <30%; highlighted residues below denote divergence from the consensus sequence and are colored by amino acid identity using the default Geneious color scheme).
Extended Data Fig. 2 The D. mojavensis Gr28b.d null mutant displays normal avoidance of noxious heat.
For each panel, the data plotted on the left is the preference index for wild-type D. mojavensis (WT), and the data plotted on the right is the preference index of D. mojavensis Gr28b.dΔ1 mutants. In all cases, flies were given a choice between 25 °C and a TT of 40° or 45 °C, respectively. The transparent box on the left represents wild type D. mojavensis data re-drawn from Fig. 1 to allow for direct comparison. Ø indicates the wild type and mutant preference distributions are not statistically different (two-tail two sample t-test, *p < 0.05; p = [0.678,0.720] for WT vs. mutant for 40 °C and 45 °C, respectively; red lines=mean, grey boxes=one standard deviation, empty boxes=95% confidence interval for the mean, empty circles=datapoints; each datapoint is one group of 15 flies; N = 11 datapoints /condition).
Extended Data Fig. 3 Labelling, reconstructing, and analyzing thermosensory projection neurons in Drosophila brains.
(a) TPN labeling strategy using electroporation of dextran-conjugated dyes, followed by imaging in Front view (on anteroposterior axis) and Top view (on dorsoventral axis) of the brain. (b) Following the acquisition of 2-photon z stacks, 2D outlines of brain regions are segmented to produce a 3D model of the CA and LH which is used for image registration (to align images to a representative brain for each species). Neurons are traced in the registered volumes using neuTube and visualized alongside the 3D CA and LH volumes. (c) Maximum projections and corresponding 3D neuronal reconstructions of five labelled TPN-V in D. melanogaster and D. mojavensis brains. (d) Neuron reconstructions are exported in SWC format, and intrinsic neuronal morphology is quantified with the CAJAL package. UMAP embedding of pairwise distances from CAJAL analysis alone shows neuronal morphologies sort out by species in the Lateral Horn (LH) but not as much for the Calyx (CA). (e) Center of Mass (COM) of each projection pattern is measured in FIJI/ImageJ using a z-projection for each z-stack cropped around either the CA or LH (see methods for details); XY plots represent average ± STD of 5 COM measurements for each species, the COM of the D. melanogaster EM reconstruction is shown for comparison. Next, a matrix of pairwise taxicab distances is computed between the COMs. (f) Pairwise distance matrices from CAJAL and COM analysis are normalized to their max values, summed, and used as input for hierarchical clustering analysis and UMAP embedding. Abbreviations: Thermosensory Receptor Neurons, TRNs; Thermosensory Projection Neurons, TPNs; Posterior Antennal Lobe, PAL; LH, Lateral Horn; CA, Calyx. Scale bars in a = 10 μm and in c = 25 μm.
Extended Data Fig. 4 Frequency of Thermosensory Projection Neuron cell types labelled in dye electroporation experiments across Drosophila species.
(a-d) Counts (tables) and frequencies (visualized as radial bar graphs) of Thermosensory Projection Neuron (TPN) cell types labeled in dye-filling experiments, arranged by species. Included are all experiments that successfully labelled a small number of TPNs (up to 3-4) allowing unambiguous identification of each cell type. We excluded experiments in which only local interneurons of the PAL were labeled, where too many TPNs were labeled, or where labeling of olfactory projections prevented us from classifying TPNs. (Note that individual percents for cell types add up to more than 100% due to double- and triple- labeling of TPNs in the same brains).
Extended Data Fig. 5 Thermosensory Lateral Horn Output Neuron II (TLHON-II) is the primary output of TPN-V and is critical for heat avoidance behavior in D. melanogaster.
(a) Connectomic diagram illustrating the pathways that relay temperature information from the hot thermosensory receptor neurons (TRNs) to the Thermosensory Lateral Horn Output Neuron II (TLHON-II). TPN-V is the major output of the aristal hot TRNs (comprising 52.67% of hot TRN output synapses to TPNs) and the major direct path of connectivity from hot TRNs to Thermosensory Lateral Horn Output Neuron II (TLHON-II), which is in turn the major target of TPN-V (black arrows represent alternative direct pathways of connectivity between hot TRNs and TLHON-II, Ns=number of synapses). (b) D. melanogaster EM reconstruction of TPN-V (red) and TLHON-II (light blue). (c) TPN-V::TLHON-I synapses (yellow dots) and TPN-V::TLHON-II synapses within the LH (blue dots) tile the TPN-V axonal arbor within the LH (synapse ___location was reconstructed from EM data, see methods for details). (d) A split-Gal4 driver exclusively expressed in TLHON-II (maximum projections of 2-photon z-stacks of the brain and VNC of TLHON-II-Gal4>GFP animal). Scale bars = 25 μm. (e) Genetic silencing using TLHON-II:Gal4>Kir2.1 impacts heat avoidance (*p < 0.05, 2-way ANOVA, p = [0.748, 1.95e-3, 4.15e-5, 4.91e-7] for test temperatures 25, 30, 35, and 40, respectively) (f) Optogenetic activation via TLHON-II:Gal4>CsChrimson is sufficient to induce avoidance of red light (*p < 0.05 indicates significant difference from zero in two-way one sample t-test, p = [0.440, 0.983, 2.54e-5] for Gal4/+, UAS/+, and Gal4>UAS, respectively). (in e-f red lines=mean, grey boxes=one standard deviation, empty boxes=95% confidence interval for the mean, circles=groups of 15 and 25 flies each for e and f, respectively; N = 8 and 9 groups/condition for e and f, respectively). Abbreviations: Lateral Horn, LH; Calyx, CA; Posterior Antennal Lobe, PAL; Mushroom Body, MB; Superior Medial Protocerebrum, SMP.
Extended Data Fig. 6 Labelling olfactory projections in the D. mojavensis brain reveals largely conserved Lateral Horn organization.
(a) Schematic of the Drosophila olfactory system. Olfactory Receptor Neurons (ORNs) send axons to the Antennal Lobe (AL) where they connect with specific second-order Olfactory Projection Neurons (OPNs) that relay information to higher brain centers such as the CA and LH. The ORN/OPN circuit of glomerulus DM1 is schematized next to an EM reconstruction of the D. melanogaster DM1 OPN. (b) Loading of dextran-conjugated dyes on the antennal nerve allows visualization of stochastic subsets of ORN axons that can be recognized due to the stereotype position of glomeruli (a D. mojavensis DM1 is shown) and used as a target for electroporation. (c) Innervation of the CA and LH by DM1-OPN is identical in D. mojavensis (in neuronal reconstructions and 2-photon z-stacks) as compared to D. melanogaster (EM reconstructions). Stacks from three individual D. mojavensis flies are shown to highlight stereotypy of innervation (right). (d) To label both olfactory and thermosensory projections in the same brain, a red dye was first used to load the aristal nerve while a green dye was later used to load the antennal nerve (see methods for details). This allows independent visualization of PAL and AL glomeruli as targets for 2-color dye-labelling experiments. (e) 2-photon imaging demonstrates that DM1-PN innervation (white, labelled with AlexaFluorTM 594 dye) of the LH is essentially identical in D. mojavensis as compared to D. melanogaster (green, EM). In contrast, D. mojavensis TPN-V innervates only the posterior LH (red, labelled with AlexaFluorTM 488 dye), while the D. melanogaster TPN-V counterpart innervates more broadly (red, EM). Abbreviations: Lateral Horn, LH; Calyx, CA; Antennal Lobe, AL; Posterior Antennal Lobe, PAL; Olfactory Receptor Neurons, ORNs; Olfactory Projection Neurons, OPNs. Scale bars = 25 μm.
Extended Data Fig. 7 Temperature preference behavior and TPN-V innervation are species-specific and consistent across populations.
(a-c) Preference indexes measured from additional strains of D. persimilis, D. melanogaster, and D. mojavensis, demonstrate consistent behavior within populations of the same species (compare to data shown in Fig. 1b–d, based on different strains -see methods). (a) A D. persimilis strain from Santa Rosa, California (strain 24) (N = 8,8,8,7,7,7 groups of 15 flies for test temperatures 15, 20, 25, 30, 35, and 40 °C, respectively). (b) A wild-derived D. melanogaster strain from Egypt (EG16; N = 13,11,12,12 groups of 15 flies for test temperatures 15, 20, 25, 30, and 35 °C, respectively). (c) A D. mojavensis wrigleyi strain from Catalina Island (D. m. wrigleyi is a subspecies of D. mojavensis; N = 20,19,18,20 groups of 15 flies for 25, 30, 35, and 40 °C, respectively) (d-f) 2-photon z-stacks of dye-labelled TPN-V in each of the three strains shows consistent anatomy within species (compare to data shown in main Figs. 4 and 5, based on different strains of the same species -see methods). (g) A distance matrix of reconstructions (see methods) shows additional strains cluster by species (highlighted branches/datapoints represent additional strains generated from 2P images in d-f, whereas greyed out ones are re-plotted from Fig. 5). Abbreviations: Lateral Horn, LH; Calyx, CA. Scale bars = 25 μm. Asterisks: lateral Accessory Calyx (lACA). Arrowheads = ventral anterior LH, the dotted line is for reference. (In a-c: red lines=mean, filled boxes=one standard deviation, empty boxes=95% CI of the mean, empty circles=groups of flies, grey shading = approx. favorite thermal range, and Ø denotes preference indexes not significantly different from zero in two-tail one sample t-test; p < 0.05, p = [6.30e-3, 3.33e-4, 0.747, 2.47e-5, 7.67e-6, 1.55e-7] for a, p = [2.22e-9, 9.38e-6, 0.488, 1.34e-5, 1.76e-10] for b, and p = [0.888, 3.66e-8, 1.58e-7, 1.13e-5] for c).
Supplementary information
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Capek, M., Arenas, O.M., Alpert, M.H. et al. Evolution of temperature preference in flies of the genus Drosophila. Nature 641, 447–455 (2025). https://doi.org/10.1038/s41586-025-08682-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-025-08682-z
