An expression atlas of chemosensory ionotropic glutamate receptors identifies a molecular basis of carbonation detection
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(91) . ! . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Supplementary Figure 1. Projections of Ir-Gal4 expressing sensory neurons in the larval central nervous system Immunofluorescence with anti-GFP (green) and nc82 (magenta) on wholemount brains of third instar larvae, revealing the projection patterns of Ir-Gal4expressing neuron populations in the brain and ventral nerve cord (as schematised in the cartoon at the top left). Images for Ir7a, Ir7b, Ir7g, Ir94e and Ir94h drivers have been adapted from Croset et al., 201628. Genotypes are of the form: w;UAS-mCD8:GFP;IrX-Gal4. SEZ: Subesophageal Zone; VNC: Ventral Nerve Cord. Scale bars: 20 μm.. 4.
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(95) . Supplementary Figure 2. Projections of Ir-Gal4 expressing sensory neurons in the adult central nervous system Immunofluorescence with anti-GFP (green) and nc82 (magenta) on wholemount adult brains and ventral nerve cords (schematised top left), revealing the projection patterns of Ir-Gal4-expressing neuron populations. Grey panels indicate no expression was detected for that Gal4 driver. Genotypes are of the form: UAS-mCD8:GFP;IrX-Gal4. Scale bars: 50 μm (brain), 100 μm (ventral nerve cord).. 5.
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(103) . !" !" !. Supplementary Figure 3. An Ir56dGal4 allele shows a similar expression pattern to Ir56d-Gal4 (a) Schematic representing the generation of the Ir56dGal4 allele. The entire Ir56d exon was substituted with the Gal4 sequence. In an additional step, the DsRed marker used for screening of positive recombination events was removed with Cre recombinase. HA: Homology Arm. Unfilled arrowheads point to the sgRNA targets. (b) Immunofluorescence with anti-GFP (green), overlaid on a bright-field image, of a whole mount proboscis of a w;Ir56dGal4;UAS-mCD8:GFP animal. Scale bar: 25 μm. (c) Immunofluorescence with anti-GFP (green) and anti-nc82 (blue) on a whole mount brain of a w;Ir56dGal4;UAS-mCD8:GFP animal. Scale bar: 50 μm (right).. 6.
(104) Supplementary Figure 4. Analysis of carbonation-evoked feeding by Expresso (a) Feeding success, latency to first bout, total consumption per fly, number of meal bouts and average bout volume for male w1118 flies (n=30 per tastant) feeding from water, 100 mM NaHCO3 pH 8.5 or 100 mM NaHCO3 pH 6.5 solutions. (b) Feeding success, latency to first bout, total consumption per fly, number of meal bouts and average bout volume for male w1118 flies (n=30 per tastant) feeding from solutions containing 5 mM sucrose, 5 mM sucrose + 100 mM NaHCO3 pH 8.5 or 5 mM sucrose + 100 mM NaHCO3 pH 6.5 solutions. ns: non-significant (pairwise comparisons using Tukey and Kramer (Nemenyi) test with Tukey-Dist approximation for independent samples).. 7.
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(107) . . )!" '$"% . Supplementary Figure 5. IR56d is required for physiological and behavioural responses to hexanoic acid (a) Colour-coded images (reflecting the maximal GCaMP3 fluorescence intensity changes) in w;Bl/+;UAS-GCaMP3,Ir56d-Gal4/+ animals stimulated with distilled water, 1 M sucrose and 1% (v/v) hexanoic acid. (b) Quantification of changes in GCaMP3 fluorescence (∆F/F) upon stimulation with the indicated chemicals (as in (a)) to the proboscis of the flies. Genotypes: Control: w;Bl/+;UAS-GCaMP3,Ir56d-Gal4/+ (n=7); Mutant: w;Ir56d1/Ir56d1;UAS-GCaMP3,Ir56d-Gal4/+ (n=8); Rescue: w;Ir56d1,UASIr56d/Ir56d1;UAS-GCaMP3,Ir56d-Gal4/+ (n=11). *P<0.05, **P<0.01, ***P<0.001 (Wilcoxon rank sum test with Bonferroni correction for multiple comparisons). (c) Fraction of w1118 (n=36) or Ir56d1 mutant (n=33) flies showing proboscis extension reflex (PER) to 1% (v/v) hexanoic acid and 100 mM sucrose. Error bars represent the ±95% binomial confidence intervals ***P<0.001 (Fisher exact test).. 8.
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