The neuronal response of the adult flies to cholesterol.

(A) Schematic diagram of the fly labellum. (B) Average frequencies of action potential generated from S7, I8, and L6 sensilla upon the application of different concentrations of cholesterol (n = 10−12). (C) Representative sample traces of S7, I8, and L6 from (B). (D) Electrophysiological responses of control flies produced from all the labellum sensilla in response to 0.1% cholesterol (n = 10−12). (E) Electrophysiological analysis of the neuron-ablated flies by the inwardly rectifying potassium channel Kir2.1 in presence of 0.1% cholesterol (n = 10−12). (F) Representative sample traces of the S7 sensilla from (E). All error bars represent SEM. Single-factor ANOVA was combined with Scheffe’s post hoc analysis to compare multiple datasets. Asterisks indicate statistical significance compared to the control group (**P < 0.01).

Ionotropic receptors (IRs) are responsible for sensing cholesterol.

(A) Tip recording analysis of 0.1% cholesterol with control and 32 Ir mutants on S7 sensilla (n = 10−16). (B) Tip recording analysis of the Ir7g2, Ir25a Df/Ir25a2, Ir51b2, Ir56d2, and Ir76b2 (n = 10−16). (C) Electrophysiology of the RNAi lines of the Ir7g, Ir25a, Ir51b, Ir56d, and Ir76b driven by the Gr33a-GAL4 and ppk23-GAL4 in S7 sensilla (n = 10−16). (D) Representative sample traces of (F) for control, mutants, and rescue lines. (E) Heatmap representing the dose responses of the control and the candidate mutants Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1 analyzed using electrophysiology (n = 10−16). (F) Tip recordings of control, Ir7g1, Ir25a2, Ir51b1, Ir56d1, Ir76b1, and genetically recovered flies driven by their own GAL4 and Gr33a-GAL4 on S7 sensilla with 0.1% cholesterol (n = 10−14). All error bars represent SEM. Single-factor ANOVA was combined with Scheffe’s post hoc analysis to compare multiple datasets. Asterisks indicate statistical significance compared to the control group; the red asterisks indicate statistical significance between the control and the rescued flies (**P < 0.01).

Ir7g, Ir25a, Ir51b, Ir56d, and Ir76b are required for the perception of cholesterol.

(A) Behavioral analysis of the w1118 adult flies toward different doses of cholesterol. Sucrose (2 mM) was included on both sides (n = 6). (B) Sex-wise feeding assay analysis toward 0.1% cholesterol (n = 6). (C) Binary food choice assay of specific gustatory receptor neuron (GRN)-ablated flies toward 0.1% cholesterol; +/-indicates the presence or absence of the transgene, respectively (n = 6). (D) Two-way choice assay of the control, Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1 toward 0.1% cholesterol (n = 6). (E) Feeding assay analysis of Ir7g2, Ir25a Df, Ir51b2, Ir56d2, and Ir76b2 (n = 6). (F) Dose response of control, Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1 toward different concentrations of cholesterol (10-5%, 10-4%, 10-3%, 10-2%, and 10-1%) via binary food choice assay (n = 6). (G) Rescue of Ir7g1, Ir25a2, Ir51b1, Ir56d1, and Ir76b1 defects by expressing the wild-type cDNA under the control of the respective GAL4 drivers (n = 6). All error bars represent SEM. Single-factor ANOVA was combined with Scheffe’s post hoc analysis to compare multiple datasets. Asterisks indicate statistical significance compared to the control group; the red asterisks indicate statistical significance between the control and the rescued flies (**P < 0.01).

Recapitulation of Ir7g, Ir25a, Ir51b, Ir56d, and Ir76b on L– and I-type sensilla.

(A) Schematic representation of heteromultimeric association of Ir7g, Ir51b, and Ir56d in I-type sensilla for cholesterol taste processing using Gr33a-GAL4. (B) Tip recordings were conducted from I9 sensilla after activation with 0.1% cholesterol by overexpression of UAS-Ir7g, UAS-Ir25a, UAS-Ir51b, UAS-Ir56d, and UAS-Ir76b in bitter-sensing gustatory receptor neurons (GRNs) via Gr33a-GAL4 (n = 10−16). (C) Schematic presentation of misexpression of Ir7g, Ir51b, and Ir56d in L-type sensilla using Gr5a-GAL4. (D) Tip recording of indicated flies from L6 sensilla Gr5a-GAL4 (n = 10−16). (E) Behavioral ectopic analysis for attraction or aversion to 0.1% cholesterol in flies misexpressing Ir7g, Ir51b, and Ir56d in sweet-sensing GRNs (Gr5a-GAL4). The IRs were ectopically expressed in an Ir56d1 and Ir7g1mutant background (n = 6). The red asterisks indicate the comparison of the combination of two UAS lines (Ir7g, Ir56d and Ir51b, Ir56d) driven by Gr5a-GAL4 with all the single UAS line including combination of Ir7g and Ir51b. All error bars represent the SEM. Single-factor ANOVA was combined with Scheffe’s post hoc analysis to compare multiple datasets. Asterisks indicate statistical significance compared with the control (**P < 0.01).

Key resources table

Electrophysiological analysis of different doses of MβCD.

Electrophysiological analysis of different bitter GRs and TRP lines in the presence of 10-1% CHL, with subset expression of Ir56d in bitter GRNs.

Binary food choice assay with CHL and MβCD.