Requirements for four Irs for preferred high salt-containing food and chemogenetic and optogenetic control of Ir60b-positive GRNs

(A) Binary food choice assay comparing 30 Ir-mutants to the control strain (w1118) for high salt avoidance, n=8–12.

(B) Preference of indicated flies observed at various concentrations of NaCl, n=8–12.

(C) The gustatory response to the activation of Gr64f GRNs or Ir60b GRNs by feeding capsaicin to trpV1-expressing flies was tested. Binary food choice assays were performed with the indicated flies. The presence or absence of the transgene is indicated by "+" and "-", respectively. n=8.

(D) Optogenetics was employed to measure PER in the indicated flies using red light and sucrose stimulation at the same time. n=6.

Multiple sets of data were compared using single-factor ANOVA coupled with Scheffe’s post hoc test. Statistical significance compared with the control was denoted by asterisks (**p < 0.01). All error bars represent the standard error of the mean (SEM).

The requirement of pharyngeal Ir60b-GRNs in high salt avoidance

(A) Schematic representation illustrating the gustatory sensilla arrangement on the fly labellum, following Tanimura’s nomenclature.

(B) Tip recording analyses conducted on S4, S8, and L4 sensilla using control, Ir7cGAL4, Ir25a2, Ir60b3, and Ir76b1 strains at 300 mM NaCl, n=10–16.

(C) Representative sample traces obtained from S4 sensillum in (D).

(D) Binary food choice assays comparing specific GRN-ablated flies to the control strain, with each genotype indicated by different colors. n=12.

(E) Tip recording analyses conducted on S4 and S8 sensilla using specific GRN-ablated flies and the control strain at 300 mM NaCl, with each genotype indicated by different colors. n=16–20

(F-I) Proboscis extension reflex (PER) assay was performed using Ir25a2, Ir60b3, Ir76b1, and control strain, n=8–10.

(F) PER percentages induced by first 2% sucrose.

(G) PER percentages induced by first 2% sucrose with 300 mM NaCl.

(H) PER percentages induced by secondary 2% sucrose.

(I) PER percentages is induced by secondary 2% sucrose with 300 mM NaCl.

(J) Binary food choice assays for 300 mM salt avoidance were conducted with Poxn mutant (Poxn70–28/PoxnΔM22-B5), Ir25a2, Ir60b3, Ir76b1, double mutant (Poxn70- 28/PoxnΔM22-B5;Ir60b3), and control strain, n=9–12.

All error bars represent SEM. Multiple sets of data were compared using single-factor ANOVA coupled with Scheffe’s post hoc test. Statistical significance compared with the controls or the Poxn70–28/PoxnΔM22-B5 is denoted by black or red asterisks, respectively (**p < 0.01).

Measurement of food intake utilizing the DrosoX syste

(A-P) The figures present the ingestion amounts (A, C, E, G, I, K, M and O) and the ingestion index (B, D, F, H, I, J, L, N and P)

(A-N) The ingestion of (a)100 mM sucrose alone and (b) in combination with 300 mM NaCl was determined using the Droso-X assay

(A and B) Measurement of food intake with control, Ir25a2, Ir60b3, and Ir76b1, n=12. (C and D) Genetically recovered flies of Ir60b3driven by Ir60b-GAL4, n=12

(E and F) Genetically recovered flies of Ir25a2 driven by Ir25a-GAL4, n=12. (G and H) Genetically recovered flies of Ir76b1 driven by Ir76b-GAL4, n=12. (I and J) Genetically recovered flies of Ir25a2 driven by Ir60b-GAL4, n=12. (K and L) Genetically recovered flies of Ir76b1 driven by Ir60b-GAL4, n=12

(M and N) Genetically recovered flies of Ir60b3 driven by Ir25a-GAL4 and Ir76b-GAL4 respectively, n=12

(O and P) Measurement of food intake with control, Ir60b3, and Gr66aex83 at 100 mM sucrose versus 100 mM sucrose with 10 mM caffeine, n=12

All error bars represent SEM. Multiple sets of data were compared using single-factor ANOVA coupled with Scheffe’s post hoc test. Statistical significance compared with the controls (A and P) or each UAS only in the mutant condition (D, F, H, J, L and N), is denoted by color asterisks (**p < 0.01).

Immunohistochemistry and calcium imaging experiments in the IR60b GRNs

(A) Immunohistochemistry was performed using anti-GFP and anti-RFP to visualize the LSO in the pharynx of UAS-mCD8::GFP/Ir76b-QF2;Ir60b-GAL4/QUAS-tdTomato.

(B) Immunohistochemistry was conducted using anti-GFP and anti-RFP to visualize the LSO in the pharynx of Ir25a-GAL4/Ir76b-QF2;UAS-mCD8::GFP/QUAS-tdTomato.

(C) Heat map images illustrate changes in GCaMP6f fluorescence before and after stimulation with 300 mM NaCl using the indicated flies.

(D) Sample traces depict the responses of UAS-GCaMP6f;Ir60b-GAL4 to 300 mM NaCl with Ir25a2, Ir60b3, Ir76b1, and control strains. n=10–14.

(E) Quantification of UAS-GCaMP6f;Ir60b-GAL4 responses to various concentrations of NaCl on control, Ir25a2, Ir60b3, and Ir76b1, respectively, n=10–14.

(F) Quantification of GCaMP6f responses to 300 mM NaCl on the indicated mutants and rescued flies, n=8–10. The presence or absence of the transgene is indicated by "+" and "-", respectively.

(G) Quantification of UAS-GCaMP6f;Ir60b-GAL4 responses to 50 mM, 300 mM, and 500 mM of CaCl2, MgCl2, and KCl on control, n=10–14.

(H) Quantification of UAS-GCaMP6f;Ir60b-GAL4 responses to various concentrations of NaBr on control, Ir25a2, Ir60b3, and Ir76b1, respectively, n=10–14.

(I) Quantification of UAS-GCaMP6f;Ir60b-GAL4 responses to 5 mM and 50 mM concentrations of bitter compounds (quinine, caffeine, strychnine, lobeline, denatonium, and coumarin), n=8–10.

(J) Quantification of UAS-GCaMP6f;Ir60b-GAL4 responses to various concentrations of sucrose on control, Ir25a2, Ir60b3, and Ir76b1, respectively, n=10–14.All error bars represent the SEM. Multiple sets of data were compared using single-factor ANOVA coupled with Scheffe’s post hoc test. Statistical significance compared with the controls is indicated by asterisks (**p < 0.01).