Letter | Published:

A gut microbial factor modulates locomotor behaviour in Drosophila

Naturevolume 563pages402406 (2018) | Download Citation

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.

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

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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

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

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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.

Competing interests

The authors declare no competing interests.

Corresponding authors

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

Extended data figures and tables

  1. 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 (bd, fh), 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.

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

    a, Experimental design (be) 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. bd, 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 (il) 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. ik, 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 (bf, il) 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.

  3. 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. be, 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.

  4. 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.

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

    ac, 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.ctyrA, n = 18; E.ctrpC, n = 17; E.cmanX, n = 45; E.ctreA, n = 46; E.cxylA, 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 (ai) or log-rank (i) post hoc tests were used for statistical analysis. Data are representative of at least two independent trials for each experiment.

  6. 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.

  7. 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 (ac, e, f, hk) 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.

  8. 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). bh, 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.

  9. 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.

  10. 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.

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

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