Testing requirements for Irs for avoiding high salt-containing food, and chemogenetic and optogenetic control of Ir60b GRNs. (A) Binary food choice assays (1 mM sucrose versus 5 mM sucrose and 300 mM NaCl) comparing 30 Ir mutants to the control strain (w1118) for high salt avoidance, n=8–12. (B) Preferences of the indicated flies for 1 mM sucrose versus 5 mM sucrose and 0—1000 mM NaCl. n=8– 12. (C) Testing the effects of 100 μM capsaicin after expressing the rat TRPV1 channel (UAS-trpV1) either in Class A GRNs or Ir60b GRNs under the control of the Gr64f-GAL4 or the Ir60b-GAL4, respectively. The flies were given a choice between 2 mM sucrose or 2 mM sucrose plus 100 μM capsaicin. The presence or absence of the various transgenes is indicated by “+” and “-”, respectively. n=8. (D) Testing the effects of light activation of various classes of GRNs. UAS-CsChrimson was expressed in Class A GRNs (driven by the Gr5a-GAL4), Class B GRNs (driven by the Gr66a-GAL4) or in Ir60b GRNs (driven by the Ir60b-GAL4). The flies were then simultaneously exposed to red lights (650 nm; WP-5700, 3M, USA) for 5 sec while 2% sucrose was applied to labellum and the percent PER was recorded. n=6. Data were compared using single-factor ANOVA coupled with Scheffe’s post hoc test. Statistical significance compared with the control. Means ± SEMs. **p < 0.01.

Contributions of different classes of GRNs to high salt avoidance. (A) Schematic showing the names of sensilla bristles on the labellum 65. (B) Tip recordings conducted on S3, S7, and L3 sensilla using 300 mM NaCl and the indicated flies. n=10–16. (C) Representative traces obtained from S3 sensilla. (D) Binary food choice assays (1 mM sucrose versus 5 mM sucrose and 300 mM NaCl) after inactivating different classes of GRNs with UAS-Kir2.1, driven by the indicated GAL4 drivers: Class A (Gr64f; blue), Class B (Gr66a; red), Class C (ppk28; green) and Class D (ppk23; purple). Significances were determined by comparing to the UAS-Kir2.1 only control (black). n=12. (E) Tip recordings conducted by stimulating S3 and S7 sensilla with 300 mM NaCl from flies with different classes of GRNs inactivated with UAS-Kir2.1. See panel D for legend. n=16–20. (F—I) PER assays performed using the control strain (w1118; black) and Ir25a2 (red), Ir60b3 (blue), Ir76b1 (green). n=8–10. (F) PER percentages induced by 2% sucrose (1st offering). (G) PER percentages induced by 2% sucrose (2nd offering). (H) PER percentages induced by 2% sucrose with 300 mM NaCl (1st offering). (I) PER percentages induced by 2% sucrose with 300 mM NaCl (2nd offering). (J) Binary food choice assays for 300 mM salt avoidance were conducted with the control strain, Poxn (Poxn70–28/PoxnΔM22-B5), Ir25a2, Ir60b3, Ir76b1, and Poxn;Ir60b3. n=9–12. Data were compared using single-factor ANOVA coupled with Scheffe’s post hoc test. Statistical significances compared with the control flies or the Poxn mutant are denoted by black and red asterisks, respectively. Means ± SEMs. **p < 0.01.

Measuring volume of food intake in Ir mutants using the DrosoX system. (A—N) Each fly was exposed to two capillaries, one of which contained 100 mM sucrose (a), and the other contained 100 mM sucrose and 300 mM NaCl (b). (O and P) Each fly was exposed to two capillaries, one of which contained 100 mM sucrose (c), and the other contained 100 mM sucrose and 10 mM caffeine (d). (A, C, E, G, I, K, M and O) Volumes of the two food options consumed by the indicated flies over the course of 6 hrs. (B, D, F, H, I, J, L, N and P) Ingestion indexes to indicate the relative consumption of the two foods. Ingestion indexes were calculated in each time point using the following equation: [(Ingestion volume of 100 mM sucrose and 300 mM NaCl or 10 mM caffeine)-(Ingestion volume of 100 mM sucrose)]/[(Ingestion volume of 100 mM sucrose and 300 mM NaCl or 10 mM caffeine)+(Ingestion volume of 100 mM sucrose)] n=12. Multiple sets of data were compared using single-factor ANOVA coupled with Scheffe’s post hoc test. Statistical significances were relative to the control and determined for the Ingestion indexes only. In all panels, the controls were w1118. The colors of the asterisks match the colors of the genotypes in the corresponding panels. Means ± SEMs. **p < 0.01.

GCaMP6f responses of Ir60b GRNs to NaCl and other chemicals. (A) Relative staining of the Ir60b reporter (green, anti-GFP) and the Ir76b reporter (red; anti-dsRed) in the LSO of UAS-mCD8::GFP/Ir76b-QF2;Ir60b-GAL4/QUAS-tdTomato flies. Merge is to the right. (B) Relative staining of the Ir25a reporter (green, anti-GFP) and the Ir76b reporter (red; anti-dsRed) in the LSO of Ir25a-GAL4/Ir76b-QF2;UAS-mCD8::GFP/QUAS-tdTomato. Merge is to the right. (C—J) Peak GCaMP6f responses (ΔF/F) of Ir60b GRNs in flies expressing UAS-GCaMP6f under control of the indicated GAL4 driver. (C) Heat map images illustrating changes in GCaMP6f fluorescence before and after stimulation with 300 mM NaCl using the indicated flies. (D) Sample traces depicting GCaMP6f responses to 300 mM NaCl. The traces are from the indicated flies expressing UAS-GCaMP6f driven by the Ir60b-GAL4. n=10– 14. (E) GCaMP6f responses to various concentrations of NaCl in the indicated flies. UAS-GCaMP6f was driven by the Ir60b-GAL4. n=10–14. (F) GCaMP6f responses to 300 mM NaCl in the indicated mutants and in the absence or presence of the corresponding rescue transgene indicated by “-” and “+”, respectively. n=8–10. (G) GCaMP6f responses to 50 mM, 300 mM, and 500 mM of CaCl2, MgCl2, and KCl in control flies. n=10–14. (H) GCaMP6f responses of Ir60b GRNs from the indicated flies to various concentrations of NaBr. n=10–14. (I) GCaMP6f responses to 5 mM and 50 mM concentrations of bitter compounds (quinine, caffeine, strychnine, lobeline, denatonium, and coumarin). n=8–10. (J) GCaMP6f responses to various concentrations of sucrose in Ir60b GRNs from the indicated flies. n=10–14. Multiple sets of data were compared using single-factor ANOVA coupled with Scheffe’s post hoc test. Statistical significance compared with the controls. Means ± SEMs. **p < 0.01.