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. 2018 Oct 12;9:4252. doi: 10.1038/s41467-018-06453-1

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — Carbonation promotes IR56d-dependent feeding behaviours: a Schematic of the two-choice positional preference arena assay. Flies can choose to feed from two substrates comprising stacks of blotting paper soaked in different tastant solutions on opposite sides of a 94 mm diameter Petri dish. Fly position was quantified automatically and used to calculate a Preference Index (PI) as indicated below the scheme. b Two-choice assay showing the preference of the indicated genotypes for a carbonated solution (100 mM NaHCO₃ pH 6.5) over a control non-carbonated solution (100 mM NaHCO₃ pH 8.5). Genotypes: w¹¹¹⁸ (n = 21 arenas; 70–80 flies per arena); Ir56d mutant: w;Ir56d¹/Ir56d² (n = 21 arenas); Rescue: w;Ir56d¹,UAS-Ir56d/Ir56d²;Ir56d-Gal4/+ (n = 14 arenas). ns non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Wilcoxon signed rank test (H₀ = 0)). c Two-choice assay showing the preference of w¹¹¹⁸ flies (n = 24 arenas) for a PBS pH 6.5 solution vs. a PBS pH 8.5 solution. d Two-choice assay showing the preference of w¹¹¹⁸ flies (n = 10 arenas) for a carbonated solution (100 mM NaHCO₃ pH 6.5) over a non-carbonated solution (100 mM NaHCO₃ pH 8.5) that was supplemented with NaCl to achieve a final [Na⁺] of 150 mM. **P < 0.01 (Wilcoxon signed rank test (H₀ = 0)). e Fraction of w¹¹¹⁸ flies (n = 36) showing the proboscis extension reflex (PER) to the tastants indicated (100 mM NaHCO₃ at pH 6.5 or pH 8.5, 100 mM sucrose). Error bars represent the ±95% binomial confidence intervals; *P < 0.05 (Fisher exact test). f Fraction of w¹¹¹⁸ flies (n = 36) showing PER to commercial carbonated water, non-carbonated water and 100 mM sucrose. Error bars represent the ±95% binomial confidence intervals; ns non-significant (Fisher exact test). g Fraction of flies of the indicated genotypes (n = 14 for all) showing PER upon optogenetic stimulation using red light. Genotypes (left-to-right along the x-axis): (i) w;Bl/CyO;Gr5a-Gal4/TM6B (ii) w;Bl/CyO;Ir56d-Gal4/TM6B (iii) w;Bl/CyO;E409-Gal4/TM6B (iv) w;UAS-CsChrimson/CyO;TM2/TM6B (v) w;UAS-CsChrimson/CyO;Gr5a-Gal4/TM6B (vi) w;UAS-CsChrimson/CyO;Ir56d-Gal4/TM6B (vii) w;UAS-CsChrimson/CyO;E409-Gal4/TM6B. Error bars represent the ±95% binomial confidence intervals; ns non-significant, ***P < 0.001 (Fisher exact test)