Skip to main content

Thank you for visiting nature.com. 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.

  • Letter
  • Published:

A gut microbial factor modulates locomotor behaviour in Drosophila

Nature volume 563, pages 402–406 (2018)Cite this article

Subjects

Abstract

While research into the biology of animal behaviour has primarily focused on the central nervous system, cues from peripheral tissues and the environment have been implicated in brain development and function1. There is emerging evidence that bidirectional communication between the gut and the brain affects behaviours including anxiety, cognition, nociception and social interaction1,2,3,4,5,6,7,8,9. Coordinated locomotor behaviour is critical for the survival and propagation of animals, and is regulated by internal and external sensory inputs10,11. However, little is known about how the gut microbiome influences host locomotion, or the molecular and cellular mechanisms involved. Here we report that germ-free status or antibiotic treatment results in hyperactive locomotor behaviour in the fruit fly Drosophila melanogaster. Increased walking speed and daily activity in the absence of a gut microbiome are rescued by mono-colonization with specific bacteria, including the fly commensal Lactobacillus brevis. The bacterial enzyme xylose isomerase from L. brevis recapitulates the locomotor effects of microbial colonization by modulating sugar metabolism in flies. Notably, thermogenetic activation of octopaminergic neurons or exogenous administration of octopamine, the invertebrate counterpart of noradrenaline, abrogates the effects of xylose isomerase on Drosophila locomotion. These findings reveal a previously unappreciated role for the gut microbiome in modulating locomotion, and identify octopaminergic neurons as mediators of peripheral microbial cues that regulate motor behaviour in animals.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Fig. 1: Select gut bacteria modulate locomotor behaviour in flies.
Fig. 2: Xylose isomerase from L. brevis alters host locomotion.
Fig. 3: Octopamine mediates Xi-induced changes in locomotion.

Similar content being viewed by others

Data availability

All datasets generated are available from the corresponding authors upon request.

References

  1. Diaz Heijtz, R. et al. Normal gut microbiota modulates brain development and behavior. Proc. Natl Acad. Sci. USA 108, 3047–3052 (2011).

    Article  ADS  Google Scholar 

  2. Bravo, J. A. et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc. Natl Acad. Sci. USA 108, 16050–16055 (2011).

    Article  ADS  CAS  Google Scholar 

  3. Luczynski, P. et al. Microbiota regulates visceral pain in the mouse. eLife 6, e25887 (2017).

    Article  Google Scholar 

  4. Gacias, M. et al. Microbiota-driven transcriptional changes in prefrontal cortex override genetic differences in social behavior. eLife 5, e13442 (2016).

    Article  Google Scholar 

  5. Fischer, C. N. et al. Metabolite exchange between microbiome members produces compounds that influence Drosophila behavior. eLife 6, 1–25 (2017).

    Google Scholar 

  6. Leitão-Gonçalves, R. et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. PLoS Biol. 15, e2000862 (2017).

    Article  Google Scholar 

  7. Wong, A. C. N. et al. Gut microbiota modifies olfactory-guided microbial preferences and foraging decisions in Drosophila. Curr. Biol. 27, 2397–2404.e4 (2017).

    Article  CAS  Google Scholar 

  8. Liu, W. et al. Enterococci mediate the oviposition preference of Drosophila melanogaster through sucrose catabolism. Sci. Rep. 7, 13420 (2017).

    Article  ADS  Google Scholar 

  9. Sharon, G. et al. Commensal bacteria play a role in mating preference of Drosophila melanogaster. Proc. Natl Acad. Sci. USA 107, 20051–20056 (2010).

    Article  ADS  CAS  Google Scholar 

  10. Huston, S. J. & Jayaraman, V. Studying sensorimotor integration in insects. Curr. Opin. Neurobiol. 21, 527–534 (2011).

    Article  CAS  Google Scholar 

  11. Dickinson, M. H. et al. How animals move: an integrative view. Science 288, 100–106 (2000).

    Article  ADS  CAS  Google Scholar 

  12. Pearson, K. G. Common principles of motor control in vertebrates and invertebrates. Annu. Rev. Neurosci. 16, 265–297 (1993).

    Article  CAS  Google Scholar 

  13. Strausfeld, N. J. & Hirth, F. Deep homology of arthropod central complex and vertebrate basal ganglia. Science 340, 157–161 (2013).

    Article  ADS  CAS  Google Scholar 

  14. Martin, J. R., Ernst, R. & Heisenberg, M. Temporal pattern of locomotor activity in Drosophila melanogaster. J. Comp. Physiol. 184, 73–84 (1999).

    Article  CAS  Google Scholar 

  15. Erkosar, B., Storelli, G., Defaye, A. & Leulier, F. Host-intestinal microbiota mutualism: “learning on the fly”. Cell Host Microbe 13, 8–14 (2013).

    Article  CAS  Google Scholar 

  16. Wong, C. N., Ng, P. & Douglas, A. E. Low-diversity bacterial community in the gut of the fruitfly Drosophila melanogaster. Environ. Microbiol. 13, 1889–1900 (2011).

    Article  CAS  Google Scholar 

  17. Schwarzer, M. et al. Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition. Science 351, 854–857 (2016).

    Article  ADS  CAS  Google Scholar 

  18. Lee, K.-A. et al. Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila. Cell 153, 797–811 (2013).

    Article  CAS  Google Scholar 

  19. Lemaitre, B. & Miguel-Aliaga, I. The digestive tract of Drosophila melanogaster. Annu. Rev. Genet. 47, 377–404 (2013).

    Article  CAS  Google Scholar 

  20. Kimura, K. I. & Truman, J. W. Postmetamorphic cell death in the nervous and muscular systems of Drosophila melanogaster. J. Neurosci. 10, 403–411 (1990).

    Article  CAS  Google Scholar 

  21. Tissot, M. & Stocker, R. F. Metamorphosis in Drosophila and other insects: the fate of neurons throughout the stages. Prog. Neurobiol. 62, 89–111 (2000).

    Article  CAS  Google Scholar 

  22. Blacher, E., Levy, M., Tatirovsky, E. & Elinav, E. Microbiome-modulated metabolites at the interface of host immunity. J. Immunol. 198, 572–580 (2017).

    Article  CAS  Google Scholar 

  23. Breton, J. et al. Gut commensal E. coli proteins activate host satiety pathways following nutrient-induced bacterial growth. Cell Metab. 23, 324–334 (2016).

    Article  CAS  Google Scholar 

  24. Mann, K., Gordon, M. D. & Scott, K. A pair of interneurons influences the choice between feeding and locomotion in Drosophila. Neuron 79, 754–765 (2013).

    Article  CAS  Google Scholar 

  25. Wong, A. C.-N., Dobson, A. J. & Douglas, A. E. Gut microbiota dictates the metabolic response of Drosophila to diet. J. Exp. Biol. 217, 1894–1901 (2014).

    Article  Google Scholar 

  26. Kim, E.-K., Park, Y. M., Lee, O. Y. & Lee, W.-J. Draft genome sequence of Lactobacillus brevis strain EW, a Drosophila gut pathobiont. Genome Announc. 1, e00938-13 (2013).

    Article  Google Scholar 

  27. Martino, M. E. et al. Resequencing of the Lactobacillus plantarum strain WJL genome. Genome Announc. 3, e01382-15 (2015).

    Article  Google Scholar 

  28. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.008 (2006).

    Article  Google Scholar 

  29. Yamanaka, K. Purification, crystallization and properties of the d-xylose isomerase from Lactobacillus brevis. Biochim. Biophys. Acta 151, 670–680 (1968).

    Article  CAS  Google Scholar 

  30. Ridley, E. V., Wong, A. C. N., Westmiller, S. & Douglas, A. E. Impact of the resident microbiota on the nutritional phenotype of Drosophila melanogaster. PLoS ONE 7, e36765 (2012).

    Article  ADS  CAS  Google Scholar 

  31. Yang, Z. et al. Octopamine mediates starvation-induced hyperactivity in adult Drosophila. Proc. Natl Acad. Sci. USA 112, 5219–5224 (2015).

    Article  ADS  CAS  Google Scholar 

  32. Chen, A. et al. Dispensable, redundant, complementary, and cooperative roles of dopamine, octopamine, and serotonin in Drosophila melanogaster. Genetics 193, 159–176 (2013).

    Article  CAS  Google Scholar 

  33. Riemensperger, T. et al. Behavioral consequences of dopamine deficiency in the Drosophila central nervous system. Proc. Natl Acad. Sci. USA 108, 834–839 (2011).

    Article  ADS  CAS  Google Scholar 

  34. Mithieux, G. et al. Portal sensing of intestinal gluconeogenesis is a mechanistic link in the diminution of food intake induced by diet protein. Cell Metab. 2, 321–329 (2005).

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  36. Roeder, T. Tyramine and octopamine: ruling behavior and metabolism. Annu. Rev. Entomol. 50, 447–477 (2005).

    Article  CAS  Google Scholar 

  37. Crocker, A. & Sehgal, A. Octopamine regulates sleep in Drosophila through protein kinase A-dependent mechanisms. J. Neurosci. 28, 9377–9385 (2008).

    Article  CAS  Google Scholar 

  38. Crocker, A., Shahidullah, M., Levitan, I. B. & Sehgal, A. Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior. Neuron 65, 670–681 (2010).

    Article  CAS  Google Scholar 

  39. Selcho, M., Pauls, D., El Jundi, B., Stocker, R. F. & Thum, A. S. The role of octopamine and tyramine in Drosophila larval locomotion. J. Comp. Neurol. 520, 3764–3785 (2012).

    Article  CAS  Google Scholar 

  40. Saraswati, S., Fox, L. E., Soll, D. R. & Wu, C. F. Tyramine and octopamine have opposite effects on the locomotion of Drosophila larvae. J. Neurobiol. 58, 425–441 (2004).

    Article  CAS  Google Scholar 

  41. Klaassen, L. W. & Kammer, A. E. Octopamine enhances neuromuscular transmission in developing and adult moths, Manduca sexta. J. Neurobiol. 16, 227–243 (1985).

    Article  CAS  Google Scholar 

  42. Weisel-Eichler, A. & Libersat, F. Neuromodulation of flight initiation by octopamine in the cockroach Periplaneta americana. J. Comp. Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 179, 103–112 (1996).

    Article  CAS  Google Scholar 

  43. Brembs, B., Christiansen, F., Pflüger, H. J. & Duch, C. Flight initiation and maintenance deficits in flies with genetically altered biogenic amine levels. J. Neurosci. 27, 11122–11131 (2007).

    Article  CAS  Google Scholar 

  44. van Breugel, F., Suver, M. P. & Dickinson, M. H. Octopaminergic modulation of the visual flight speed regulator of Drosophila. J. Exp. Biol. 217, 1737–1744 (2014).

    Article  Google Scholar 

  45. Han, D. D., Stein, D. & Stevens, L. M. Investigating the function of follicular subpopulations during Drosophila oogenesis through hormone-dependent enhancer-targeted cell ablation. Development 127, 573–583 (2000).

    CAS  PubMed  Google Scholar 

  46. Selkrig, J. et al. The Drosophila microbiome has a limited influence on sleep, activity, and courtship behaviors. Sci. Rep. 8, 10646 (2018).

    Article  ADS  Google Scholar 

  47. Nishino, R. et al. Commensal microbiota modulate murine behaviors in a strictly contamination-free environment confirmed by culture-based methods. Neurogastroenterol. Motil. 25, 521–528 (2013).

    Article  CAS  Google Scholar 

  48. Lendrum, J. E., Seebach, B., Klein, B. & Liu, S. Sleep and the gut microbiome: antibiotic-induced depletion of the gut microbiota reduces nocturnal sleep in mice. Preprint at https://www.biorxiv.org/content/early/2017/10/05/199075 (2017).

  49. Berridge, C. W. Noradrenergic modulation of arousal. Brain Res. Rev. 58, 1–17 (2008).

    Article  CAS  Google Scholar 

  50. Monastirioti, M., Linn, C. E. Jr & White, K. Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine. J. Neurosci. 16, 3900–3911 (1996).

    Article  CAS  Google Scholar 

  51. Clyne, J. D. & Miesenböck, G. Sex-specific control and tuning of the pattern generator for courtship song in Drosophila. Cell 133, 354–363 (2008).

    Article  CAS  Google Scholar 

  52. Shiga, Y., Tanaka-Matakatsu, M. & Hayashi, S. A nuclear GFP/β-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Dev. Growth Differ. 38, 99–106 (1996).

    Article  CAS  Google Scholar 

  53. Lee, W. C. & Micchelli, C. A. Development and characterization of a chemically defined food for Drosophila. PLoS ONE 8, e67308 (2013).

    Article  ADS  CAS  Google Scholar 

  54. Brummel, T., Ching, A., Seroude, L., Simon, A. F. & Benzer, S. Drosophila lifespan enhancement by exogenous bacteria. Proc. Natl Acad. Sci. USA 101, 12974–12979 (2004).

    Article  ADS  CAS  Google Scholar 

  55. Ren, C., Webster, P., Finkel, S. E. & Tower, J. Increased internal and external bacterial load during Drosophila aging without life-span trade-off. Cell Metab. 6, 144–152 (2007).

    Article  CAS  Google Scholar 

  56. Ryu, J.-H. et al. Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science 319, 777–782 (2008).

    Article  ADS  CAS  Google Scholar 

  57. Storelli, G. et al. Lactobacillus plantarum promotes Drosophila systemic growth by modulating hormonal signals through TOR-dependent nutrient sensing. Cell Metab. 14, 403–414 (2011).

    Article  CAS  Google Scholar 

  58. Shin, S. C. et al. Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling. Science 334, 670–674 (2011).

    Article  ADS  CAS  Google Scholar 

  59. Chiu, J. C., Low, K. H., Pike, D. H., Yildirim, E. & Edery, I. Assaying locomotor activity to study circadian rhythms and sleep parameters in Drosophila. J. Vis. Exp. 43, 2157 (2010).

    Google Scholar 

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

    Article  Google Scholar 

  61. Wolf, F. W., Rodan, A. R., Tsai, L. T.-Y. & Heberlein, U. High-resolution analysis of ethanol-induced locomotor stimulation in Drosophila. J. Neurosci. 22, 11035–11044 (2002).

    Article  CAS  Google Scholar 

  62. Simon, J. C. & Dickinson, M. H. A new chamber for studying the behavior of Drosophila. PLoS ONE 5, e8793 (2010).

    Article  ADS  Google Scholar 

  63. White, K. E., Humphrey, D. M. & Hirth, F. The dopaminergic system in the aging brain of Drosophila. Front. Neurosci. 4, 205 (2010).

    Article  Google Scholar 

  64. Mendes, C. S., Bartos, I., Akay, T., Márka, S. & Mann, R. S. Quantification of gait parameters in freely walking wild type and sensory deprived Drosophila melanogaster. eLife 2, e00231 (2013).

    Article  Google Scholar 

  65. Shaw, P. J., Cirelli, C., Greenspan, R. J. & Tononi, G. Correlates of sleep and waking in Drosophila melanogaster. Science 287, 1834–1837 (2000).

    Article  ADS  CAS  Google Scholar 

  66. Yu, Y. et al. Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila. eLife 5, e15693 (2016).

    Article  Google Scholar 

  67. Qi, W. et al. A quantitative feeding assay in adult Drosophila reveals rapid modulation of food ingestion by its nutritional value. Mol. Brain 8, 87 (2015).

    Article  Google Scholar 

  68. Chakrabarti, S., Poidevin, M. & Lemaitre, B. The Drosophila MAPK p38c regulates oxidative stress and lipid homeostasis in the intestine. PLoS Genet. 10, e1004659 (2014).

    Article  Google Scholar 

Download references

Acknowledgements

We thank H. Chu, G. Sharon, W.-L. Wu, J. K. Scarpa, E. D. Hoopfer and the Mazmanian laboratory for critiques; A. A. Aravin and K. Fejes Tόth for use of their laboratory space; D. J. Anderson, H. A. Lester, V. Gradinaru and M.-F. Chesselet for discussions; A. R. Sandoval, M. Meyerowitz and M. Smalley for technical support; Y. Garcia-Flores for administrative support; D. C. Hall for creating custom Python scripts; W.-J. Lee for the L. brevisEW, L. plantarumWJL and Acetobacter pomorum bacterial strains; the Yale Coli Genetic Stock Center for wild-type and mutant E. coli strains; M. H. Dickinson, D. J. Anderson, A. A. Aravin, and K. Fejes Tόth for fly lines; the GlycoAnalytics Core for help with carbohydrate analysis; and M. Fischbach and M. Funabashi for the pGID023 vector and advice. Imaging was performed in the Biological Imaging Facility, with the support of the Caltech Beckman Institute and the Arnold and Mabel Beckman Foundation. C.E.S. was partially supported by the Center for Environmental Microbial Interactions at Caltech. This project was funded by grants from the NIH (NS085910) and the Heritage Medical Research Institute to S.K.M.

Reviewer information

Nature thanks P. Bercik, C.-F. Wu and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Authors and Affiliations

  1. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Catherine E. Schretter & Sarkis K. Mazmanian

  2. Protein Expression Center, Beckman Institute, California Institute of Technology, Pasadena, CA, USA

    Jost Vielmetter

  3. Department of Physics, Columbia University, New York, NY, USA

    Imre Bartos, Zsuzsa Marka & Szabolcs Marka

  4. GlycoAnalytics Core, University of California, San Diego, CA, USA

    Sulabha Argade

Authors
  1. Catherine E. Schretter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jost Vielmetter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Imre Bartos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zsuzsa Marka
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Szabolcs Marka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sulabha Argade
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sarkis K. Mazmanian
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.E.S. designed, performed and analysed most of the experiments. J.V. assisted with experimental design for biochemical analysis. I.B., Z.M., and S.M. assisted with gait analysis experiments. S.A. performed carbohydrate quantification. C.E.S. and S.K.M supervised the project. C.E.S. and S.K.M. wrote the manuscript with assistance from all authors.

Corresponding authors

Correspondence to Catherine E. Schretter or Sarkis K. Mazmanian.

Ethics declarations

Competing interests

The authors declare no competing interests.

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 Effects of colonization level, bacterial strain, and host diet on L. brevis modulation of locomotion.

a, Colony-forming units (CFU) per individual fly (mean ± s.e.m.) for L.p or L.b mono-associated flies. L.p, n = 15; L.b, n = 18. b, Average speed of Conv, Ax and L.b mono-associated female or male flies. Females: Conv, n = 90; Ax, n = 92; L.b, n = 89; Males: Conv, n = 100; Ax, n = 100; L.b, n = 95. c, d, Average speed of Ax flies or flies mono-associated with L.b strains EW, Bb14 or P-2. c, Ax, n = 58; L.b EW, n = 57; L.b Bb14, n = 57. d, Ax, n = 45; L.b EW, n = 28; L.b P-2, n = 42. e, Average speed of Ax or L.b mono-associated flies raised on different diet compositions from eclosion until day 7. Diet 1 (left): Ax, n = 20; L.b, n = 21; diet 2 (middle): Ax, n = 18; L.b, n = 16; diet 3 (right): Ax, n = 6; L.b, n = 6. f, Average speed during walking bouts for Conv, Ax, L.p and L.b groups. Conv, n = 23; Ax, n = 35; L.p, n = 22; L.b, n = 22. g, Tripod index for Conv, Ax, L.p and L.b groups. Conv, n = 6; Ax, n = 7; L.p, n = 5; L.b, n = 5. h, Average speed of Ax flies or flies mono-associated with L.p or L.b alone or in combination (1:1). Ax, n = 18; L.p, n = 24; L.b, n = 24; L.p + L.b, n = 24. Box-and-whisker plots show median and IQR; whiskers show either 1.5 × IQR of the lower and upper quartiles or range. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Specific P values are in the Supplementary Information. Unpaired Student’s t-test (a), Kruskal–Wallis and Dunn’s (b–d, f–h), or Mann–Whitney U (e) post hoc tests were used for statistical analysis. Data are representative of at least three independent trials for each experiment.

Extended Data Fig. 2 Post-eclosion microbial signals decrease host locomotion.

a, Experimental design (b–e) in which Ax flies were associated with L.b either directly after (day 0, dark green arrows) or 3–5 days after (light green arrows) eclosion. b–d, Average speed (b), average bout length (c) and average speed during walking bouts (d) of Ax flies and flies mono-associated with L.b at either day 0 or day 3–5. b, Ax, n = 46; L.b 0 d, n = 47; L.b 3–5 d, n = 43. c, Ax, n = 18; L.b 0 d, n = 18; L.b 3–5 d, n = 6. d, Ax, n = 36; L.b 0 d, n = 36; L.b 3–5 d, n = 12. e, Average speed of Conv flies, Ax flies and flies mono-associated with L.b at either day 0 or day 3–5. Conv, n = 11; Ax, n = 53; L.b 0 d, n = 53; L.b 3–5 d, n = 52. f, Average speed of Conv, Ax and Conv flies treated with antibiotics for 3 days after eclosion (ABX). Conv, n = 32; Ax, n = 36; ABX, n = 36. g, Average speed of OregonR (OR) and Canton-S (CS) Conv flies and Conv flies treated with antibiotics for 3 days after eclosion (ABX). OR: Conv, n = 20; ABX, n = 22; CS: Conv, n = 12; ABX, n = 17. h, Experimental design (i–l) in which conventionally reared flies were treated with antibiotics (ABX, black arrow) for 3 days following eclosion. All flies were subsequently placed on irradiated medium either without supplementation (ABX) or associated with L.p (blue arrows) or L.b (green arrows) for the 3 days before testing. i–k, Average speed (i), average bout length (j) and average speed during walking bouts (k) calculated for ABX, L.p- and L.b-associated flies. i, ABX, n = 29; L.p, n = 24; L.b, n = 35. j, ABX, n = 36; L.p, n = 30; L.b, n = 35. k, ABX, n = 42; L.p, n = 30; L.b, n = 35. l, Daily activity of ABX, L.p and, L.b groups (virgin female OregonR flies) over a 2-day 12 h light:12 h dark cycle period, starting at time 0. White boxes represent lights on and grey boxes represent lights off. n = 6 per condition. Box-and-whisker plots show median and IQR; whiskers show either 1.5 × IQR of the lower and upper quartiles or range. *P < 0.05, **P < 0.01. Specific P values are in the Supplementary Information. Kruskal–Wallis and Dunn’s (b–f, i–l) or Mann–Whitney U (g) post hoc tests were used for statistical analysis. Data are representative of at least two independent trials for each experiment.

Extended Data Fig. 3 Bacterial-derived products from L. brevis alter locomotion.

a, Average speed of Ax flies, L.p or L.b mono-associated flies, and Ax flies treated with CFS from L.p or L.b. Ax, n = 45; L.p, n = 17; L.b, n = 42; L.p CFS, n = 17; L.b CFS, n = 16. b–e, Average speed (b), average bout length (c), average speed during walking bouts (d) and daily activity (e) of Ax flies and Ax virgin female OregonR flies treated with CFS from L.p or L.b. White boxes represent lights on and grey boxes represent lights off. b, Ax, n = 23; L.p CFS, n = 20; L.b CFS, n = 20. c, Ax, n = 23; L.p CFS, n = 20; L.b CFS, n = 17. d, Ax, n = 22; L.p CFS, n = 21; L.b CFS, n = 17. e, Ax, n = 8; L.p CFS, n = 8; L.b CFS, n = 4. f, Average speed of Ax, L.b mono-associated and Ax uracil-treated flies. Ax, n = 96; L.b, n = 88; 0.1 nM uracil, n = 41; 10 nM uracil, n = 18. Box-and-whisker plots show median and IQR ; whiskers show either 1.5 × IQR of the lower and upper quartiles or range. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Specific P values are in the Supplementary Information. Kruskal–Wallis and Dunn’s post hoc tests were used for statistical analysis. Data are representative of at least two independent trials for each experiment.

Extended Data Fig. 4 Locomotor phenotypes are independent of food intake, anti-microbial peptides, and the immune deficiency (IMD) and Toll pathways.

a, Average speed of wild-type background (OregonR, Wt) and Imd−/− flies placed on either medium alone or medium supplemented with antibiotics (ABX) following eclosion. Wt: Conv, n = 16; ABX, n = 17; IMD−/−: Conv, n = 24; ABX, n = 25. b, Average speed of wild-type background (Canton-S, Wt) and Ti−/− flies placed on either medium alone or medium supplemented with antibiotics (ABX) following eclosion. Wt: Conv, n = 15; ABX, n = 17; Ti−/−: Conv, n = 10; ABX, n = 11. c, qRT–PCR of immune-related transcripts (mean ± s.e.m.) in Ax and Ax L.p or L.b CFS-treated flies. Dpt (also known as DptA): Ax, n = 8; L.p CFS, n = 10; L.b CFS, n = 10; Drs: Ax, n = 10; L.p CFS, n = 10; L.b CFS, n = 10; Cec (also known as CecA1): Ax, n = 8; L.p CFS, n = 10; L.b CFS, n = 10; AttA: Ax, n = 5; L.p CFS, n = 5; L.b CFS, n = 5; Duox: Ax, n = 3; L.p CFS, n = 5; L.b CFS, n = 5. d, Amount ingested by Ax and Ax L.p or L.b CFS-treated flies over 10 trials during MAFE assay. Ax, n = 6; L.p CFS, n = 5; L.b CFS, n = 6. e, Intestinal content measured through supplementing the diet of Conv, Ax, and L.p- or L.b-CFS-treated Ax flies with blue food dye. Conv, n = 7; Ax, n = 13; L.p CFS, n = 7; L.b CFS, n = 10. Box-and-whisker plots show median and IQR; whiskers show either 1.5 × IQR of the lower and upper quartiles or range. *P < 0.05, **P < 0.01, ****P < 0.0001. Specific P values are in the Supplementary Information. Mann–Whitney U (a, b), one-way ANOVA and Bonferroni (c), and Kruskal–Wallis and Dunn’s (d, e) post hoc tests were used for statistical analysis. Data are representative of at least two independent trials for each experiment. Dpt, diptericin; Drs, drosomycin; Cec, cecropin; AttA, attacin-A; Duox, dual oxidase.

Extended Data Fig. 5 Modulation of locomotion by the bacterial enzyme, xylose isomerase.

a–c, Average speed of Ax flies or Ax flies treated with unaltered, protease-treated (Typ, trypsin; PK, proteinase-K) or heat-treated (100 °C) L.b CFS. a, Ax, n = 18; L.b CFS, n = 18; +Typ, n = 17; –Typ, n = 17. b, Ax, n = 23; L.b CFS, n = 18; +PK, n = 23; –PK, n = 23. c, n = 18. d, Average speed of Ax flies treated with amylase-treated PBS (Ax), amylase-treated L.b CFS (+ amyl L.b CFS) or unaltered L.b CFS (–amyl L.b CFS). Ax, n = 30; +amyl, n = 17; –amyl, n = 30. e, Average speed of Ax flies or flies mono-associated with L.b, L.p, A. pomorum (A.p), or E. coli (E.c). Below shows the presence (+) or absence (–) of Xi based on NCBI Blastn (xylA locus) and Blastp (Xi) results. Ax, n = 30; L.b, n = 30; L.p, n = 29; A.p, n = 30; E.c, n = 18. f, Average speed of Ax flies and flies mono-associated with either WT E.c or single gene knockout strains of E.c (∆tyrA, ∆trpC, ∆manX, ∆treA, ∆xylA). Ax, n = 65; E.c; n = 52; E.c∆tyrA, n = 18; E.c∆trpC, n = 17; E.c∆manX, n = 45; E.c∆treA, n = 46; E.c∆xylA, n = 20. g, Daily activity of Conv, Ax and Ax virgin female OregonR flies treated with L.b CFS, L.b∆xylA CFS or Xi* over a two-day 12 h light:12 h dark cycle period, starting at time 0. White boxes represent lights on and grey boxes represent lights off. Conv, n = 16; Ax, n = 24; L.b CFS, n = 19; L.b∆xylA CFS, n = 20; Xi*, n = 8. h, Average speed of Ax flies and Ax flies treated with L.b CFS or Xi*. Ax, n = 16; L.b CFS, n = 11; 10 µg/ml Xi*, n = 12; 100 µg/ml Xi*, n = 14. i, Lifespan measurements for Ax flies and Ax flies treated with L.p CFS, L.b CFS, or Xi*. Asterisks represent significance at the time point measured by Kruskal–Wallis and Dunn’s post hoc test. Inset image shows survival at day 7 (mean ± s.e.m.). Ax, n = 4 groups; L.p CFS, n = 5 groups; L.b CFS, n = 5 groups; Xi*, n = 4 groups. j, Percentage of apoptotic cells (mean ± s.e.m.) in the intestines of Conv flies, Ax flies and Ax flies treated with L.p CFS, L.b CFS or Xi*. Conv, n = 7; Ax, n = 5; L.p CFS, n = 4; L.b CFS, n = 6; Xi*, n = 6. Box-and-whisker plots show median and IQR; whiskers show either 1.5 × IQR of the lower and upper quartiles or range. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Specific P values are in the Supplementary Information. Kruskal–Wallis and Dunn’s (a–i) or log-rank (i) post hoc tests were used for statistical analysis. Data are representative of at least two independent trials for each experiment.

Extended Data Fig. 6 Sleep analysis for mono-colonized flies and flies treated with bacterial factors.

a, Twenty-four-hour sleep profiles (mean ± s.e.m.) of Conv, Ax, L.p- and L.b-colonized virgin female OregonR flies with the number of sleep bouts in 60-min time windows and total sleep in the light or dark phase. n = 8 flies per condition. b, Twenty-four-hour sleep profiles (mean ± s.e.m.) of Conv, Ax, L.b CFS, L.b∆xylA CFS and Xi* treated Ax virgin female OregonR flies with the number of sleep bouts in 60-min time windows and total sleep in the light or dark phase. Conv, n = 17; Ax, n = 25; L.b CFS, n = 19; L.b∆xylA CFS, n = 21; Xi*, n = 8. Box-and-whisker plots show median and IQR; whiskers show range. *P < 0.05. Specific P values are in the Supplementary Information. Kruskal–Wallis and Dunn’s post hoc tests were used for statistical analysis. Data are representative of at least two independent trials for each experiment.

Extended Data Fig. 7 Xylose isomerase activity and key carbohydrates are involved in Xi-mediated changes in locomotion.

a, b, Average speed of Ax flies and Ax flies treated with Xi* or 100 µg/ml of d-fructose, d-glucose, d-xylose or d-xylulose. a, Ax, n = 16; Xi*, n = 13; d-fructose, n = 13; d-glucose, n = 15. b, Ax, n = 26; Xi*, n = 21; d-xylose, n = 22; d-xylulose, n = 18. c, Average speed of Ax flies and Ax flies treated with Xi* or Xi* inactivated by 5 mM EDTA. Ax, n = 21; Xi*, n = 16; Xi* + EDTA, n = 18. d, Carbohydrate levels (mean ± s.e.m.) in Ax and Xi*-treated fly medium. Each sample is from 0.1 g fly medium and represents a separate vial. n = 3 samples per condition. e, Carbohydrate levels (mean ± s.e.m.) in Ax, Xi*, and EDTA-treated Xi* flies. Each sample contains five flies. n = 5 samples per condition. f, Trehalose levels (mean ± s.e.m.) in Conv, Ax, and Xi*-treated flies. Conv, n = 9 samples; Ax, n = 6 samples; Xi*, n = 3 samples. g, Trehalose levels (mean ± s.e.m.) in Ax and L.b-colonized flies. n = 15 samples per condition. h, Average speed of Ax and Xi*-treated flies supplemented with either trehalose (Treh, 10 mg/ml) or arabinose (Ara, 10 mg/ml) for 3 days before testing. Ax, n = 40; Xi*, n = 40; Xi* + Treh, n = 39; Xi* + Ara, n = 18. i, Average speed of Ax flies and Xi*- or ribose (Ribo, 10 mg/ml)-treated flies. Ax, n = 29; Xi*, n = 25; Ribo, n = 12. j, Average speed of Conv and Ax flies supplemented with trehalose (Treh, 10 mg/ml) for 3 days before testing. Conv, n = 15; Ax, n = 22; Conv + Treh, n = 18; Ax + Treh, n = 15. k, Average speed of Ax and Xi* or EDTA-treated Xi* Ax flies subsequently left untreated or supplemented with trehalose (Treh, 10 mg/ml) for 3 days before testing. Ax, n = 27; Xi, n = 19; Xi + EDTA, n = 24; Xi + Treh, n = 19; Xi + EDTA + Treh, n = 25. Box-and-whisker plots show median and IQR; whiskers show 1.5 × IQR of the lower and upper quartiles. *P < 0.05, **P < 0.01, ***P < 0.001. Specific P values are in the Supplementary Information. Kruskal–Wallis and Dunn’s (a–c, e, f, h–k) or Mann–Whitney U (d, g) post hoc tests were used for statistical analysis. Data are representative of at least two independent trials for each experiment. Gluc, glucose; Fruc, fructose; Mann, mannose; Xylu, xylulose; Treh, trehalose; Ribo, ribose.

Extended Data Fig. 8 Thermogenetic activation of neuromodulator-GAL4 lines.

a, Experimental design in which Conv flies (Canton-S) were treated with antibiotics (ABX, black arrow) for 3 days following eclosion. All flies were subsequently placed on irradiated medium either without supplementation or treated with L.b CFS (green arrows) for 3 days. One hour before and during testing, flies were either exposed to 27 °C (red line) to facilitate thermogenetic activation or kept at 20 °C (blue line). b–h, Average speed of flies previously treated with antibiotics and subsequently left untreated (ABX) or treated with L.b CFS for 3 days before testing. b, UAS: ABX, n = 15; L.b CFS, n = 14; GAL4: ABX, n = 24; L.b CFS, n = 20; GAL4> UAS (27 °C): ABX, n = 14; L.b CFS, n = 9; GAL4> UAS (20 °C): ABX, n = 16; L.b CFS, n = 11. c, UAS: ABX, n = 24; L.b CFS, n = 24; GAL4: ABX, n = 24; L.b CFS, n = 23; GAL4> UAS (27 °C): ABX, n = 25; L.b CFS, n = 26; GAL4> UAS (20 °C): ABX, n = 19; L.b CFS, n = 19. d, UAS: ABX, n = 26; L.b CFS, n = 18; GAL4: ABX, n = 36; L.b CFS, n = 24; GAL4> UAS (27 °C): ABX, n = 53; L.b CFS, n = 23; GAL4> UAS (20 °C): ABX, n = 21; L.b CFS, n = 7. e, UAS: ABX, n = 34; L.b CFS, n = 26; GAL4: ABX, n = 34; L.b CFS, n = 28; GAL4> UAS (27 °C): ABX, n = 10; L.b CFS, n = 17; GAL4> UAS (20 °C): ABX, n = 17; L.b CFS, n = 13. f, UAS: ABX, n = 36; L.b CFS, n = 30; GAL4: ABX, n = 40; L.b CFS, n = 31; GAL4> UAS (27 °C): ABX, n = 19; L.b CFS, n = 17; GAL4> UAS (20 °C): ABX, n = 14; L.b CFS, n = 8. g, UAS: ABX, n = 21; L.b CFS, n = 12; GAL4: ABX, n = 28; L.b CFS, n = 24; GAL4> UAS (27 °C): ABX, n = 24; L.b CFS, n = 20; GAL4> UAS (20 °C): ABX, n = 16; L.b CFS, n = 15. h, UAS: ABX, n = 31; L.b CFS, n = 20; GAL4: ABX, n = 31; L.b CFS, n = 29; GAL4> UAS (27 °C): ABX, n = 16; L.b CFS, n = 17; GAL4> UAS (20 °C): ABX, n = 18; L.b CFS, n = 14. Box-and-whisker plots show median and IQR; whiskers show range. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Specific P values are in the Supplementary Information. Mann–Whitney U post hoc tests following a two-way ANOVA were used for statistical analysis. Data are representative of at least two independent trials for each experiment.

Extended Data Fig. 9 Activation of octopaminergic neurons in flies carrying a null allele for Tβh (TβhM18).

Average speed of flies previously treated with antibiotics and subsequently left untreated (ABX) or treated with L.b CFS for 3 days before testing. UAS: ABX, n = 15; L.b CFS, n = 14; GAL4: ABX, n = 28; L.b CFS, n = 20; GAL4> UAS (27 °C): ABX, n = 11; L.b CFS, n = 13; GAL4> UAS (20 °C): ABX, n = 9; L.b CFS, n = 8. Box-and-whisker plots show median and IQR; whiskers show range. Specific P values are in the Supplementary Information. Mann–Whitney U post hoc tests following a two-way ANOVA were used for statistical analysis. Data are representative of at least two independent trials.

Extended Data Fig. 10 Octopamine mediates L. brevis- and Xi-induced changes in locomotion.

a, Average speed of Ax and L.b CFS-treated Ax flies left untreated or supplemented with octopamine (OA, 10 mg/ml) or l-dopa (1 mg/ml) for 3 days. Ax, n = 26; Ax + OA, n = 27; Ax + l-dopa, n = 6; L.b CFS, n = 35; L.b CFS + OA, n = 26; L.b CFS + l-dopa, n = 6. b, RT–qPCR (mean ± s.e.m.) for transcripts from heads of Ax and L.b CFS-treated Ax flies. Tdc2: n = 5; Tβh, n = 5; Ddc: Ax, n = 3; L.b CFS, n = 5; Tph: n = 7. c, qRT–PCR (mean ± s.e.m.) for transcripts from heads of Ax or Xi*-treated Ax flies. Ax, n = 5 samples; Xi*, n = 6 samples. d, Average speed of Ax and L.b CFS-treated Ax flies left untreated or supplemented with tyramine (TA, 10 mg/ml) for 3 days. Ax, n = 21; Ax + TA, n = 10; L.b CFS, n = 10; L.b CFS+TA, n = 9. e, Average speed of control lines and flies expressing DTI in octopaminergic and tyraminergic neurons outside the ventral nerve cord. All flies were previously treated with antibiotics and subsequently left untreated (ABX) or treated with Xi* for 3 days before testing. GAL4;GAL80: Ax, n = 25; Xi*, n = 18; UAS: Ax, n = 26; Xi*, n = 21; GAL4> UAS: Ax, n = 39; Xi*, n = 23. f, Average speed of control lines and flies expressing Tβh RNAi in all neurons. All flies were previously treated with antibiotics and subsequently left untreated (ABX) or treated with L.b CFS for 3 days before testing. UAS: n = 9; GAL4: Ax, n = 24; L.b CFS, n = 19; GAL4> UAS: Ax, n = 24; L.b CFS, n = 21. g, Tβh mRNA measured from heads of flies previously treated with antibiotics. Error bars represent range. n = 2 samples per condition. h, Average speed of Ax and Xi*-treated Ax flies left untreated or supplemented with mianserin (Mian; 2 mg/ml) for 3 days. Ax, n = 14; Xi*, n = 15; Xi* + Mian, n = 15. i, Average speed of Conv, Ax and Xi*-treated Ax flies left untreated or supplemented with mianserin (2 mg/ml) for 3 days. Conv, n = 13; Ax, n = 28; Xi*, n = 24; Conv + Mian, n = 27; Ax + Mian, n = 22; Xi* + Mian, n = 22. j, Average speed of wild-type background (w+, Wt) and Tdc2-null mutants (Tdc2RO54) either left untreated or after treatment with antibiotics for 3 days following eclosion. Wt Conv, n = 13; Wt ABX, n = 21; Tdc2RO54 Conv, n = 28; Tdc2RO54 ABX, n = 34. k, Average speed of wild-type background (Canton-S, Wt) and Tβh-null mutants (TβhM18) either left untreated or after treatment with antibiotics for 3 days following eclosion. Wt Conv, n = 38; Wt ABX, n = 42; TβhM18 Conv, n = 25; TβhM18 ABX, n = 33. l, Model of bacterial modulation of host locomotion. Box-and-whisker plots show median and IQR; whiskers show either 1.5 × IQR of the lower and upper quartiles or range. *P < 0.05, **P < 0.01, ***P < 0.001. Specific P values are in the Supplementary Information. Kruskal–Wallis and Dunn’s (a, d, h, i), unpaired Student’s t-test (b, c) or Mann–Whitney U (e, f, j, k) post hoc tests were used for statistical analysis. Data are representative of at least two independent trials for each experiment. Tdc, tyrosine decarboxylase; Tβh, tyramine beta-hydroxlyase; Ddc, DOPA decarboxylase; Tph, tryptophan hydroxylase.

Supplementary information

Supplementary Data

This file contains data on sample size and statistics

Reporting Summary

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schretter, C.E., Vielmetter, J., Bartos, I. et al. A gut microbial factor modulates locomotor behaviour in Drosophila. Nature 563, 402–406 (2018). https://doi.org/10.1038/s41586-018-0634-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-018-0634-9

Keywords

This article is cited by

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.

Search

Advanced search

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