Figure 7. Bitter GRNs inhibit acceptance of high concentration of hexanoic acid.

(A) Hexanoic acid dose response profiles of the sweet (left) and the bitter (right) GRNs associated with the 5b sensilla. sweet GRN reaches a maximal response already at 1%, while bitter GRNs responses further increases as ligand concentration increases. Each bar represents the mean ± SEM of Ca²⁺ imaging with 7–30 female prothoracic legs. Bars with different letters are significantly different (Kruskal-Wallis test by ranks with Dunn’s multiple comparison tests, p<0.05). Each y-axis delineates groups for Kruskal-Wallis test. Fly genotypes: Gr64f-GAL4 UAS-GCaMP6m/+ and Gr33a^GAL4/+;UAS-GCaMP6m/+. For hexanoic acid response profiles of the sweet GRNs, or the bitter GRN associated with 5 s/5v sensilla, see Figure 7—figure supplement 1. (B) PER responses of wild-type flies (w¹¹¹⁸) to different concentrations of hexanoic acid. At high concentration (2.5%) hexanoic acids induces a much lower PER response compared to more modest concentrations (0.5%–1%). Each symbol represents the mean ± SEM of PER responses (n = 61–142 flies). Symbols with different letters are significantly different (Kruskal-Wallis test by ranks with Dunn’s multiple comparison tests, p<0.01). Each y-axis delineates groups for Kruskal-Wallis test. (C) Inactivation of bitter GRN leads to concentration dependent PER responses to hexanoic acid, while control flies show a response profile similar to w¹¹¹⁸ flies (see B). Each symbol represents the mean ± SEM of PER responses (n = 23–121 flies). Symbols with different letters are significantly different (Kruskal-Wallis test by ranks with Dunn’s multiple comparison tests, p<0.05). Each square bracket delineates groups for Kruskal-Wallis test. Fly genotypes: Gr33a^GAL4/+ (Gr33a^GAL4 alone), UAS-TNT/+ (UAS-TNT alone) and Gr33a^GAL4/UAS-TNT. (D) Inactivation of bitter GRNs has no effect on cellular responses of sweet GRNs to hexanoic acid. Ca²⁺ responses of sweet GRNs to 2.5% hexanoic acid are similar regardless of whether a functional bitter GRN is present. Bitter GRNs was inactivated by expressing UAS-TNT under the control of Gr33a^GAL4. Each bar represents the mean ± SEM of 10–15 female GRNs from prothoracic legs. Two-tailed, Mann-Whitney U test between the flies lacking functional bitter GRNs and control flies, p<0.05, ns: not significant. Each y-axis delineates groups for Mann-Whitney U test. Fly genotypes: Gr64f^LexA/lexAop-GCaMP6m (control) and Gr33a^GAL4/UAS-TNT; Gr64f^LexA/lexAop-GCaMP6m (Gr33a > TNT). Source data for summary graphs are provided in Figure 7—source data 1.

Figure 7—figure supplement 1. Hexanoic acid responses of 5s/5v associated sweet (A and B) or bitter (C) GRNs.

Both the 5s- (A) and the 5v- (B) associated sweet GRN show a maximal Ca²⁺ response at the relative low concentration of 0.5% hexanoic acid. The 5s-associated bitter GRNs shows only weak Ca²⁺ response to hexanoic acid, but they are significantly higher than responses to carrier (C). Each bar represents the mean ± SEM of Ca²⁺ imaging with 13–55 female prothoracic legs. Bars with different letters are significantly different (Kruskal-Wallis test by ranks with Dunn’s multiple comparison tests, p<0.05). Each y-axis delineates groups for Kruskal-Wallis test. Genotypes are same as genotypes in Figure 7A. Source data for summary graphs are provided in Figure 7—figure supplement 1—source data 1.

Figure 7—figure supplement 2. Bitter GRNs suppress PER responses to high concentration of hexanoic acid.

Flies lacking functional bitter GRNs (Gr33a^GAL4/UAS-Kir2.1) show significantly higher PER responses to 2.5% hexanoic acid concentration, compared to control flies (Gr33a^GAL4 or UAS-Kir2.1 alone). Each symbol represents the mean ± SEM of PER responses (n = 33–121 flies). Symbols with different letters represent significant differences (Kruskal-Wallis test by ranks with Dunn’s multiple comparison tests, p<0.05). Each square bracket delineates groups for Kruskal-Wallis test. Fly genotypes: Gr33a^GAL4/+ (Gr33a^GAL4 alone), UAS-Kir2.1-GFP/+ (UAS-Kir2.1 alone) and Gr33a^GAL4/UAS-Kir2.1-GFP (Gr33a^GAL4/UAS-Kir2.1). Source data for summary graphs are provided in Figure 7—figure supplement 2—source data 1.