(A) Schematic of the feeding suppression assay. (B) Absorbance (at 625 nm) of extracts from homogenised UAS-VR1/+;IR60c-Gal4/+ larvae and controls (UAS-VR1/+ and IR60c-Gal4/+; “+” represents Canton S-derived chromosomes) after animals have fed on 50 mM sucrose containing 100 μM capsaicin and 0.4% Brilliant Blue for 30 minutes. (Kruskal-Wallis, ***p < 0.001; N = 11 groups of 30 larvae per genotype). (C,D) Absorbance of extracts from homogenised UAS-Shibirets1/+;;UAS-Shibirets1/IR60c-Gal4 larvae and controls (UAS-Shibirets1/+;;UAS-Shibirets1/+ and IR60c-Gal4/+; “+” represents Canton S-derived chromosomes) after animals have fed on 50 mM sucrose containing 0.4% Brilliant Blue for 30 minutes at the restrictive (C) or permissive (D) temperature. (Kruskal-Wallis, ***p < 0.001; N = 6 groups of 30 larvae per genotype). (E) Absorbance of extracts from homogenised larvae of the same genotypes as in (C) after animals have fed on 50 mM sucrose containing 0.4% Brilliant Blue for 30 minutes at the restrictive temperature. (Kruskal-Wallis, p > 0.05; N ≥ 12 groups of 30 larvae per genotype). (F–G) Absorbance of extracts from homogenised larvae of the same genotypes as in (C) after animals have fed on 50 mM sucrose containing 500 mM alanine and 0.4% Brilliant Blue for 30 minutes at the restrictive (F) or permissive (G) temperature. (Kruskal-Wallis, ***p < 0.001; N ≥ 12 groups of 30 larvae per genotype). (H–I) Absorbance of extracts from homogenised larvae of the same genotypes as in (C) after animals have fed on 50 mM sucrose containing 500 mM lysine (H) or 500 mM glycine (I) and 0.4% Brilliant Blue for 30 minutes at the restrictive temperature. (Kruskal-Wallis, ***p < 0.001; N ≥ 12 groups of 30 larvae per genotype). (J) Preference Indices of larvae in which IR60c-Gal4 neurons are silenced with Kir2.1 (UAS-Kir2.1:GFP/+;IR60c-Gal4/+), together with controls (UAS-Kir2.1:GFP/+ and IR60c-Gal4/+; “+” represents Canton S-derived chromosomes) for 50 mM sucrose versus 50 mM sucrose +500 mM alanine. (Wilcoxon Signed Rank Test, **p < 0.01, ***p < 0.001; N ≥ 69 groups of ~15 animals per genotype). (K) Absorbance of extracts from homogenised wild-type (w1118), homozygous IR76b1 mutant and heterozygous IR76b1/+ (“+” represents w1118 derived chromosomes) larvae after animals have fed on 50 mM sucrose containing 500 mM alanine and 0.4% Brilliant Blue for 30 minutes. (Kruskal-Wallis, ***p < 0.001; N = 16 groups of 30 larvae per genotype).