Figure 6. A subset of bitter GRNs responds to hexanoic acids in an IR25a/IR76b independent manner.

(A) Representative still images of the fifth tarsal segment of the prothoracic leg show maximum Ca²⁺ responses in the bitter GRNs associated with 5b and 5 s sensilla upon stimulation by indicated ligands. ΔF indicates the changes in fluorescence light intensity of the cell body before/after ligand application. (B) Representative fluorescence traces (top) and corresponding Ca²⁺ responses (bottom) of the 5b- and 5s-associated bitter GRNs upon stimulation by indicated ligands. The gray line underneath the fluorescence traces indicates time of ligand application. Hexanoic acid elicits highly significant Ca²⁺ responses in the 5b- associated bitter GRNs (compared to carrier). 1 mM denatonium was used as a positive ligand control. 1% ethanol was used as a carrier to facilitate solubilization of high concentrations of hexanoic acid (2.5%). Each bar represents the mean ± SEM of Ca²⁺ imaging with 10–30 female prothoracic legs. Two-tailed, Mann-Whitney U test versus carrier (1% ethanol), ***p<0.001, ns: not significant. Fly genotype is Gr33a^GAL4 UAS-GCaMP6m/+. (C and D) Cellular responses to fatty acid in bitter GRNs do not require IR25a and IR76b. Ca²⁺ responses of the 5b-assoicated bitter GRNs of IR25a (C) or IR76b (D) mutant flies to indicated ligands. Ca²⁺ responses of GRNs of mutants to hexanoic acid were not significantly reduced when compared to control flies. Each bar represents the mean ± SEM of Ca²⁺ imaging with 4–42 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 C: Gr33a^GAL4/+; UAS-GCaMP6m/+ (Control, black), IR25a² Gr33a^GAL4/IR25a²; UAS-GCaMP6m/+ (IR25a^-/-, white), and IR25a² Gr33a^GAL4/IR25a² UAS-IR25a; UAS-GCaMP6m/+ (Rescue, grey); Fly Genotypes D: Gr33a^GAL4 UAS-GCaMP6m/+ (Control, black), Gr33a^GAL4 UAS-GCaMP6m/+; IR76b²/IR76b² (Ir76b^-/-, white) and Gr33a^GAL4 UAS-GCaMP6m/UAS-IR76b; IR76b²/IR76b² (Rescue, grey). See Figure 6—figure supplement 1—source data 1 for hexanoic acid responses of 5s-associated bitter GRNs from IR25a or IR76b mutant flies. Source data for summary graphs are provided in Figure 6—source data 1.

Figure 6—figure supplement 1. Hexanoic acid responses of 5s -associated bitter GRNs of IR25a (A) or IR76b (B) mutant flies.

Because the overall responses are so low, the relevance of differences between some genotypes in the IR25a group is unclear. Each bar represents the mean ± SEM of Ca²⁺ imaging with 4–39 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 6C and D. Source data for summary graphs are provided in Figure 6—figure supplement 1—source data 1.