Letter | Published:

Distinct activity-gated pathways mediate attraction and aversion to CO2 in Drosophila

Naturevolume 564pages420424 (2018) | Download Citation

Abstract

Carbon dioxide is produced by many organic processes and is a convenient volatile cue for insects1 that are searching for blood hosts2, flowers3, communal nests4, fruit5 and wildfires6. Although Drosophila melanogaster feed on yeast that produce CO2 and ethanol during fermentation, laboratory experiments7,8,9,10,11,12 suggest that walking flies avoid CO2. Here we resolve this paradox by showing that both flying and walking Drosophila find CO2 attractive, but only when they are in an active state associated with foraging. Their aversion to CO2 at low-activity levels may be an adaptation to avoid parasites that seek CO2, or to avoid succumbing to respiratory acidosis in the presence of high concentrations of CO2 that exist in nature13,14. In contrast to CO2, flies are attracted to ethanol in all behavioural states, and invest twice the time searching near ethanol compared to CO2. These behavioural differences reflect the fact that ethanol is a unique signature of yeast fermentation, whereas CO2 is generated by many natural processes. Using genetic tools, we determined that the evolutionarily conserved ionotropic co-receptor IR25a is required for CO2 attraction, and that the receptors necessary for CO2 avoidance are not involved in this attraction. Our study lays the foundation for future research to determine the neural circuits that underlie both state- and odorant-dependent decision-making in Drosophila.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

Processed data are available in a Dryad repository at https://doi.org/10.5061/dryad.2s8422f. Raw data are available from the corresponding author upon request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Guerenstein, P. G. & Hildebrand, J. G. Roles and effects of environmental carbon dioxide in insect life. Annu. Rev. Entomol. 53, 161–178 (2008).

  2. 2.

    Dekker, T. & Cardé, R. T. Moment-to-moment flight manoeuvres of the female yellow fever mosquito (Aedes aegypti L.) in response to plumes of carbon dioxide and human skin odour. J. Exp. Biol. 214, 3480–3494 (2011).

  3. 3.

    Thom, C., Guerenstein, P. G., Mechaber, W. L. & Hildebrand, J. G. Floral CO2 reveals flower profitability to moths. J. Chem. Ecol. 30, 1285–1288 (2004).

  4. 4.

    Buehlmann, C., Hansson, B. S. & Knaden, M. Path integration controls nest-plume following in desert ants. Curr. Biol. 22, 645–649 (2012).

  5. 5.

    Stange, G. Carbon dioxide is a close-range oviposition attractant in the Queensland fruit fly Bactrocera tryoni. Naturwissenschaften 86, 190–192 (1999).

  6. 6.

    Klocke, D., Schmitz, A. & Schmitz, H. Native Flies Attracted to Bushfires (Department of Environment and Conservation, The Government of Western Australia, 2009).

  7. 7.

    Suh, G. S. B. et al. A single population of olfactory sensory neurons mediates an innate avoidance behaviour in Drosophila. Nature 431, 854–859 (2004).

  8. 8.

    Faucher, C., Forstreuter, M., Hilker, M. & de Bruyne, M. Behavioral responses of Drosophila to biogenic levels of carbon dioxide depend on life-stage, sex and olfactory context. J. Exp. Biol. 209, 2739–2748 (2006).

  9. 9.

    Jones, W. D., Cayirlioglu, P., Kadow, I. G. & Vosshall, L. B. Two chemosensory receptors together mediate carbon dioxide detection in Drosophila. Nature 445, 86–90 (2007).

  10. 10.

    Ai, M. et al. Acid sensing by the Drosophila olfactory system. Nature 468, 691–695 (2010).

  11. 11.

    Faucher, C. P., Hilker, M. & de Bruyne, M. Interactions of carbon dioxide and food odours in Drosophila: olfactory hedonics and sensory neuron properties. PLoS ONE 8, e56361 (2013).

  12. 12.

    Lin, H., Chu, L., Fu, T., Dickson, B. J. & Chiang, A. Parallel neural pathways mediate CO2 avoidance responses in Drosophila. Science 340, 1338–1341 (2013).

  13. 13.

    Sorey, M. L. et al. Invisible CO 2 Gas Killing Trees at Mammoth Mountain, California US Geological Survey Fact Sheet 172-96 (USGS, 2000).

  14. 14.

    Hubbard, H. G. Insect life in the hot springs of Yellowstone National Park. Can. Entomol. 23, 226–235 (1891).

  15. 15.

    van Breugel, F., Riffell, J., Fairhall, A. & Dickinson, M. H. Mosquitoes use vision to associate odor plumes with thermal targets. Curr. Biol. 25, 2123–2129 (2015).

  16. 16.

    van Breugel, F. & Dickinson, M. H. Plume-tracking behavior of flying Drosophila emerges from a set of distinct sensory-motor reflexes. Curr. Biol. 24, 274–286 (2014).

  17. 17.

    Kim, I. S. & Dickinson, M. H. Idiothetic path integration in the fruit fly Drosophila melanogaster. Curr. Biol. 27, 2227–2238 (2017).

  18. 18.

    Wasserman, S., Salomon, A. & Frye, M. A. Drosophila tracks carbon dioxide in flight. Curr. Biol. 23, 301–306 (2013).

  19. 19.

    Yorozu, S. et al. Distinct sensory representations of wind and near-field sound in the Drosophila brain. Nature 458, 201–205 (2009).

  20. 20.

    Gaudry, Q., Nagel, K. I. & Wilson, R. I. Smelling on the fly: sensory cues and strategies for olfactory navigation in Drosophila. Curr. Opin. Neurobiol. 22, 216–222 (2012).

  21. 21.

    Kwon, J. Y., Dahanukar, A., Weiss, L. A. & Carlson, J. R. The molecular basis of CO2 reception in Drosophila. Proc. Natl Acad. Sci. USA 104, 3574–3578 (2007).

  22. 22.

    Ni, L. et al. The ionotropic receptors IR21a and IR25a mediate cool sensing in Drosophila. eLife 5, e13254 (2016).

  23. 23.

    Enjin, A. et al. Humidity sensing in Drosophila. Curr. Biol. 26, 1352–1358 (2016).

  24. 24.

    Silbering, A. F. et al. Complementary function and integrated wiring of the evolutionarily distinct Drosophila olfactory subsystems. J. Neurosci. 31, 13357–13375 (2011).

  25. 25.

    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).

  26. 26.

    Robertson, H. M. & Kent, L. B. Evolution of the gene lineage encoding the carbon dioxide receptor in insects. J. Insect Sci. 9, 19 (2009).

  27. 27.

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

  28. 28.

    Lighton, J. R. B. Discontinuous gas exchange in insects. Annu. Rev. Entomol. 41, 309–324 (1996).

  29. 29.

    Hetz, S. K. & Bradley, T. J. Insects breathe discontinuously to avoid oxygen toxicity. Nature 433, 516–519 (2005).

  30. 30.

    Ramdya, P. et al. Mechanosensory interactions drive collective behaviour in Drosophila. Nature 519, 233–236 (2015).

  31. 31.

    Ai, M. et al. Ionotropic glutamate receptors IR64a and IR8a form a functional odorant receptor complex in vivo in Drosophila. J. Neurosci. 33, 10741–10749 (2013).

  32. 32.

    Hall, M. L. Brew by the numbers: add up what’s in your beer. Zymurgy 1995-01-01, 54–61 (1995).

  33. 33.

    Straw, A. D., Branson, K., Neumann, T. R. & Dickinson, M. H. Multi-camera real-time three-dimensional tracking of multiple flying animals. J. R. Soc. Interface 8, 395–409 (2011).

  34. 34.

    Stowers, J. R. et al. Virtual reality for freely moving animals. Nat. Methods 14, 995–1002 (2017).

Download references

Acknowledgements

We thank A. Straw for the 3D tracking software. Several colleagues provided mutants: R. Benton (quadruple mutant), R. Stanewsky (IR25a and rescue); G. Suh (IR8a); and M. Gallio and M. Stensmyr (IR40a). R. Benton, E. Hong and J. Riffell contributed helpful comments. This work was funded by grants from NIH (NIH1RO1DCO13693-01, U01NS090514) and the Simons Foundation.

Reviewer information

Nature thanks S. Combes, M. Frye, L. Vosshall and R. Wilson for their contribution to the peer review of this work.

Author information

Author notes

    • Floris van Breugel

    Present address: University of Nevada, Reno, NV, USA

Affiliations

  1. California Institute of Technology, Pasadena, CA, USA

    • Floris van Breugel
    • , Ainul Huda
    •  & Michael H. Dickinson

Authors

  1. Search for Floris van Breugel in:

  2. Search for Ainul Huda in:

  3. Search for Michael H. Dickinson in:

Contributions

F.v.B. and M.H.D. conceived the experiments. A.H. made genetic recombinants. F.v.B. and A.H. performed experiments. F.v.B. analysed data. F.v.B. and M.H.D. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Michael H. Dickinson.

Extended data figures and tables

  1. Extended Data Fig. 1 Drosophila prefer early fermentations, at peak CO2 production.

    a, Alcohol by volume for apple juice and sugar fermented with champagne yeast over the course of two weeks, measured with a hydrometer. CO2 production was calculated from the stoichiometry of fermentation (1 sugar molecule yields 2 ethanol and 2 CO2 molecules), corresponding to the derivative of alcohol by volume. n = 4 independent ferments; the results were very consistent. b, Trap assay. c, Preference index exhibited by flies in three two-choice assays, using traps shown in b. Flies were presented with two traps: one was a completed 14-day-old ferment that had been stored in the refrigerator, the second was a fresh ferment aged 2, 7 or 12 days old. The positive preference index indicates a preference for the fresh ferment. The red line shows the linear regression (P < 0.001, r2 = 0.28). n = 12 trials per condition. The mean and standard deviation of the total captured flies for each trial was 105 ± 59. d, CO2 concentration in 500-ml fly-rearing bottles under common laboratory conditions. n = 6 trials per condition. e, Measurement setup for the data shown in d. f, Time course of CO2 concentration measurement for three bottles filled with different concentrations of CO2. n = 3 per calibration gas. g, Peak measured CO2 concentration versus actual CO2 concentration for the calibration gases (black). Coloured lines show the measured peak concentrations for the actual fly-food bottles, and the resulting CO2 concentrations shown in d. In all panels, shading indicates the bootstrapped 95% confidence intervals around the mean.

  2. Extended Data Fig. 2 Responses of flies to odours at different concentrations.

    a, CO2 concentration on the landing platform (green), and at two distances downwind from the downwind edge of the platform (red and purple). Measurements (shown with points) were made for low flow rates (shown in the inset), and values at larger flowrates were extrapolated based on a linear model for measurements made at the 2-cm distance. This was necessary because the CO2 sensor could not accurately report concentrations higher than 0.5% CO2. b, Diagram that illustrates the theoretical boundary layer used to confirm our measurements (see Methods). c, The responses of flies to odours is consistent across a wide range of concentrations. Data plotted as in Fig. 2e, for additional flow rates. Points indicate individual data points (each trajectory contributes a single point). For each odour, we recorded the following n = number of trajectories for each of the concentrations (listed left to right). H2O, 128, 183 and 79; CO2, 195, 106, 125 and 48; ethanol, 173, 171 and 47; and vinegar, 219, 193 and 248. In all panels, shading indicates the bootstrapped 95% confidence intervals around the median. dg, Comparison of the results from experiments with the landing platform from c and the constrained walking arena used in Fig. 3. Scattergram is repeated from c, 60 ml min−1 CO2. To compare the data from the wind tunnel experiment to the walking arena from Fig. 3, we calculated a bootstrapped time trace.The time trace is the bootstrapped mean and 95% confidence intervals for the normalized number of flies that would have been on the platform, had all the flies landed simultaneously. The green shading is only provided for reference; the odour was never turned off in these wind tunnel experiments. e, Time trace from d overlaid on the normalized number of non-starved flies near the 5% CO2 source during the dusk time period in the walking arena, copied from Fig. 3d. f, Same as d, but for ethanol, 60 ml min−1. g, Time trace from f overlaid on the normalized number of non-starved flies near the 5% ethanol source during the dusk time period in the walking arena. Data are not shown, but are very similar to Fig. 3d ethanol case with starved flies. We chose non-starved flies for the comparisons because wind tunnel experiments were done with non-starved flies. We chose the 60 ml min−1 case because the CO2 concentration in the wind tunnel matches the 5% CO2 stimulus in the walking experiments.

  3. Extended Data Fig. 3 Walking arena geometry and odour stimulus.

    a, Photograph of walking arena, with the lid removed. b, Annotated photograph of the walking arena as seen from above, taken with the machine vision camera that is used for tracking. c, Odour control for the three delivery architectures, with odour off. d, Odour control for the three delivery architectures, with odour on. In our experiments, the port through which odour is delivered was alternated.

  4. Extended Data Fig. 4 Responses to CO2 are strongest at 5% concentration and are unaffected by social dynamics.

    a, Control and 5% CO2 responses for individual flies. For these experiments, we starved a single two-day old wild-type (HCS) female fly for either 24 h or 3 h before starting the experiment. In every other way, the data are plotted as in Fig. 4. The data shown were collected from n = 29 individual flies, in which each fly was subject to a 20-h long experiment with n= 14 5% CO2 stimuli and n = 10 control stimuli. b, CO2 responses exhibited by flies to three concentrations of CO2. For these experiments, we starved groups of 10 flies for 24 h before starting the experiment. Flies were presented with 0%, 1.7% or 5% CO2 in one set of experiments, and 0% or 15% in another set. Data are plotted as in Fig. 4. n = 20–170 trials per condition. To explain the complex dynamics of the approach behaviour under the different CO2 concentrations, we made a very simple agent-based model with the pseudocode shown in c; see Supplementary Information for additional discussion. d, Dynamics of the CO2 attraction of flies can be explained by the simple agent-based model described in c. Preference indices are shown for the results of n = 100 iterations of the model, under three different CO2 concentrations. The data are plotted in the same manner as b. The key insight offered by this model is that although our agents were programmed to exhibit the same behaviour towards 1.7% and 5% CO2, the decreased likelihood of them detecting the lower concentration CO2 in conjunction with the long-term aversion results in an apparent indifference towards low concentrations of CO2. e, To show that flies are indeed attracted to the low (1.7%) concentration of CO2, we used a different analysis that calculated the number of times that flies approached the CO2 source during the course of each 10-min stimulus. Pairwise statistics were determined with the two-sample Kolmogorov–Smirnov test (test statistics were 0.57, 0.83 and 0.41 for comparisons between 0% and 1.7%, 5%, and 15%). f, Time course of the number of times that flies approach the CO2 source, in 5-min intervals. In each panel, the shading shows the bootstrapped 95% confidence intervals around the mean.

  5. Extended Data Fig. 5 Temperature measurements in the walking arena show no correlation with CO2 or clean air stimuli.

    a, Temperature over the course of 16 h (see Methods). As in our experiments, every 40 min a 10-min CO2 stimulus identical to that used in Fig. 4 was applied either to the side of the arena with the temperature probe (green shading) or to the opposite side of the arena (blue shading). b, c, Data from a time-aligned and baseline-subtracted for CO2 and control trials, respectively.

  6. Extended Data Fig. 6 Flies do not respond to a stimulus of clean air (without CO2).

    Data plotted as in Fig. 4, but for a 0% CO2 stimulus. n = 17–81 trials per condition. Shading indicates the bootstrapped 95% confidence intervals around the mean.

  7. Extended Data Fig. 7 IR25a is required for CO2 attraction and IR40a is not.

    As in Fig. 4, the data from each experimental group are sorted according to the mean speed during the reference period of 5 min before the odour stimulus. In addition, for each mutant we show two sets of panels corresponding to: (1) flies that were starved for 24 h or 3 h before experiments conducted at 23 °C, and (2) flies that were starved for 3 h before experiments done at 32 °C. This arrangement is in contrast to Fig. 4, in which data from the two temperature groups are combined. a, b, Responses of two IR25a mutants and a bacterial artificial chromosome rescue to a 5% CO2 stimulus (a) and a 0% CO2 stimulus (b). c, d, Responses of an IR40a mutant to a 5% CO2 stimulus (c) and a 0% CO2 stimulus (d). n = 4–78 trials per condition. Shading indicates the bootstrapped 95% confidence intervals around the mean.

  8. Extended Data Fig. 8 IR25a is required for ethanol attraction but not vinegar attraction.

    Data plotted as in Fig. 4. Experiments were done with 24-h-starved flies only. a, b, Responses to 3 ml min−1 air passed through a bottle of pure ethanol added to 20 ml min−1 clean air. c, Control responses with 3 ml min−1 of clean air added to 20 ml min−1 of clean air. d, e, Responses to 3 ml min−1 air passed through a bottle of pure vinegar added to 20 ml min−1 clean air. f, Control responses with 3 ml min−1 of clean air added to 20 ml min−1 of clean air. n = 14−70 trials per condition. Shading indicates the bootstrapped 95% confidence intervals around the mean.

  9. Extended Data Fig. 9 Drosophila are attracted to fatal levels of CO2.

    Top, Photograph of two flies that were fatally attracted to a 200 ml min−1 CO2 stimulus. Bottom, trajectories for these two flies before they became anaesthetized and died. Colour encodes time, starting at purple and ending at green or yellow.

  10. Extended Data Fig. 10 Flies and mosquitoes both increase CO2 production when shaken.

    Red shading indicates the time during which the vial was shaken. We tested four groups of 10–20 animals for flies (black) and mosquitoes (blue). CO2 was measured with a LiCorr-6262. See Methods for details.

Supplementary information

  1. Supplementary Information

    This file contains statistics and reproducibility data and a discussion of the effects of CO2 concentration.

  2. Reporting Summary

  3. Video 1

    Drosophila find CO2 aversive during periods of low activity. Flies’ responses to the onset of CO2 (red) during an afternoon trial in our walking arena, corresponding to data shown in Fig. 3d (5% CO2, 24 hrs starved). Playback is approximately 10x real time. This video is a representative example of the 42 trials.

  4. Video 2

    Drosophila find CO2 attractive during periods of high activity. Flies’ responses to the onset of CO2 (red) during a dusk trial in our walking arena, corresponding to data shown in Fig. 3d (5% CO2, 24 hrs starved). Same group of flies as video 1. Playback is approximately 10x real time. This video is a representative example of the 30 trials.

  5. Video 3

    Drosophila’s response to CO2 during high activity is qualitatively similar to their response towards ethanol. Flies’ responses to the onset of ethanol (red) during a dusk trial in our walking arena, corresponding to data shown in Fig. 3d (5% Ethanol, 24 hrs starved). Playback is approximately 10x real time. This video is a representative example of the 30 trials.

About this article

Publication history

Received

Accepted

Published

Issue Date

DOI

https://doi.org/10.1038/s41586-018-0732-8

Comments

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.