IR20a-GAL4 labels a population of gustatory receptor neurons (GRNs) sensitive to specific amino acids and salts.

(A - B) IR20a-GAL4 expression is observed in approximately 20 GRNs in the labellum (A) and 2 GRNs in the fifth tarsal segment (B); arrows indicate the cell bodies labeled by IR20a-GAL4. (C) IR20a-GAL4 does not label cell bodies within the brain or ventral nerve cord (VNC); however, neuronal projections from the labellum, legs, and wings are visible in these central tissues. (D - G) Double-labeling experiments in the labellum, tarsi, and pharynx show that IR20a-GAL4 is co-expressed with the co-receptors IR25a (D) and IR76b (E). No overlap is observed with markers for bitter-sensing (GR66a-LexA) (F) or sweet-sensing (GR64f-LexA) (G) GRNs. Arrows indicate co-expression. Note that pseudocoloring was used for IR20a and IR76b in (E) for consistent visualization. (H) Single-cell calcium imaging of IR20a-GAL4 tarsal GRNs reveals robust responses to 100 mM L-arginine and L-proline, but not to other basic amino acids. Note that black squares under the bars indicate essential amino acids for flies. (I - K) Dose-response analysis of IR20a-GAL4 tarsal GRNs stimulated with 10 - 100 mM L-arginine (I - J) and 10 - 100 mM L-proline (K). (L) IR20a-GAL4 tarsal GRNs respond to 50 mM NaCl and KCl, but show no significant response to 50 mM LiCl. (M - N) IR20a-GAL4 tarsal GRNs exhibit plateaued calcium responses to NaCl at concentrations of 50 mM and higher. Data represent mean ± SEM (n = 8 - 18). Mann-Whitney test comparing ligands to H2O control, *p < 0.05, **p < 0.01, ***p < 0.001.

IR20a is required for low NaCl attraction and contributes to arginine-driven feeding behavior.

(A - C) Single-cell calcium imaging reveals that the response of IR20a-GAL4 tarsal GRNs to 50 mM NaCl is abolished in IR20a mutants, while responses to 100 mM L-arginine, L-proline, and 50 mM KCl remain intact. (n = 8 -16). (D - E) Behavioral attraction to 100 mM Arg (D) and 50 mM NaCl (E) is restored upon genetic rescue of IR20a. (n = 16 - 31). (F - G) IR20a is dispensable for preference toward 100 mM proline (Pro) (F) and 50 mM KCl (G). (n = 18 - 39). (H - I) Tarsal stimulation with arginine does not induce PER (H), but labellar stimulation with 100 mM arginine induces PER in an IR20a-dependent manner (I). (n = 17 – 34). (J - K) IR20a is required for PER induced by 50 mM NaCl, with stimulation in the tarsal or labellum (K). (n = 18 - 49). Data represent mean ± SEM. Mann-Whitney test (H), Kruskal-Wallis with Dunn’s post hoc test (C - G and I - K). Different letters denote differences between designated groups (p < 0.05)

IR20a functions with IR25a to detect arginine, while IR76b is required for low-salt sensitivity.

(A - C) IR25a is essential for both arginine and low-salt responses in IR20a tarsal GRNs. (D - F) IR76b is dispensable for arginine responses but is required for low-salt responses in IR20a tarsal GRNs. (G) Immunostaining for IR25a shows co-expression with Ppk23-GAL4 in f5a sensilla; arrows indicate co-expression. (H) Co-localization of Ppk23-GAL4 and IR76b-QF is restricted to a single cell body within the f5a sensilla; arrows indicate co-expression. (I - J) Ectopic expression of IR20a in Ppk23-GAL4 tarsal 5a GRNs is sufficient to confer sensitivity to NaCl and arginine. (K) IR25a is necessary for the arginine and salt sensitivity conferred by IR20a ectopic expression in 5a GRNs. (L) IR76b is not required for the arginine sensitivity but is required for the salt sensitivity conferred by IR20a misexpression in 5a GRNs. Data represent mean ± SEM (n = 8 -18). In (C) and (F), Kruskal-Wallis with Dunn’s post hoc test was used, different letters denote differences between designated groups (p < 0.05). In (J - L), Mann-Whitney test was used, red asterisks indicate significant differences between indicated groups, *p < 0.05, **p < 0.01.

Co-expression of IR20a and IR25a confer arginine response, whereas adding IR76b provides low NaCl sensitivity in S2 cells.

(A) Heat map of calcium responses in S2 cells transfected with various IR combinations. Only specific combinations confer sensitivity to arginine and NaCl. (B - F) Expression of IR20a, IR25a, or IR76b alone, as well as co-expression of IR20a with IR76b or IR25a with IR76b, failed to elicit arginine responses. (G - H) Co-expression of IR20a with IR25a, or triple express of IR20a, IR25a, and IR76b, confer arginine sensitivity. (I - N) Neither IR20a nor IR25a alone, nor their co-expression, elicited responses to NaCl, whereas expression of IR76b alone conferred low NaCl sensitivity. However, co-expression of IR76b with either IR20a or IR25a abolished NaCl sensitivity. (O) Triple expression of IR20a, IR25a, and IR76b confers NaCl response. Data represent mean ± SEM (n = 8 -17). Mann-Whitney test, *p < 0.05, **p < 0.01, ***p < 0.001.

IR20a works with IR25a to mediate synergistic responses to arginine and NaCl.

(A - B) PER assays reveal that a combination of arginine and NaCl elicits a synergistic response that is dependent on IR20a. (C - D) Calcium imaging shows that ectopic expression of IR20a in Ppk23-GAL4 5a GRNs confers the synergistic response to arginine-NaCl mixture. (E - F) Calcium imaging shows that IR25a, but not IR76b, is required for the synergistic response conferred by ectopic expression of IR20a in Ppk23-GAL4 5a GRNs. (G - K) Calcium imaging in S2 cells shows that co-expression of IR20a and IR25a confers a synergistic response to the arginine-NaCl mixture, whereas triple expression of IR20a, IR25a, and IR76b does not show a synergistic response to the arginine-NaCl mixture. Data represent mean ± SEM (n = 35 - 60 for PER assays, while n = 8 - 18 for calcium imaging). Kruskal-Wallis with Dunn’s post hoc test used in (A - C, H, and J), Different letters denote differences between designated groups (p < 0.05), while Mann-Whitney test used in (E - F), **p < 0.01.

IR20a provides tonic sensitivity that cooperates with the state-modulated IR56b pathway.

(A) Two-choice feeding assays show that amino acid deprivation increases preference for arginine, independent of IR20a. (B) Two-choice feeding assays show that salt deprivation increases preference for low NaCl, independent of IR20a. (C - D) Calcium imaging confirms that IR20a-GAL4 tarsal GRN responses to NaCl are comparable in salt-deprived and salt-fed flies. (E) Two-choice feeding assays show that the state-dependent change in salt preference is dependent on IR56b. (F - G) Calcium imaging reveals that the state-dependent response of IR56b GRNs to NaCl requires IR56b. Data represent mean ± SEM (n = 11 - 31 for two-choice feeding assays, while n = 8 -12 for calcium imaging). Mann-Whitney test, *p < 0.05, **p < 0.01, ***p < 0.001.

IR20a is specifically required for arginine and NaCl attraction.

(A - B) IR20a is not required for 25 mM lysine preference in PER assays. (n = 18 – 35). (C - D) IR20a is not required for preference in amino acid mixture in PER assays, n = 16 - 40. Group A: 25 mM each of Ser, Thr, and Phe. Group B:100 mM Met, 75 mM Ile, 50 mM Val and Leu, 25 mM Trp. Group C:100 mM Gly, 75 mM Ala, 50 mM His, 25 mM Cys. Group D: 25 mM Glu, 50 mM Gln, 50 mM Asn, 25 mM Asp, 0.5 mM Tyr. (E) Ablation of IR20a-GAL4 by UAS-hid reduces PER response to both arginine and NaCl. Data represent mean ± SEM (n = 38 - 49). Kruskal-Wallis with Dunn’s post hoc test, different letters denote differences between designated groups (p < 0.05).

Neither co-expression of IR20a and IR25a nor triple expression of IR20a, IR25a, and IR76b in S2 cells confers broad amino acid sensitivity.

(A - B) Calcium imaging shows that heterologous expression of IR20a and IR25a, or triple expression of IR20a, IR25a, and IR76b in S2 cells does not elicit responses to various amino acid mixtures. Group A: 25 mM each of Ser, Thr, and Phe. Group B:100 mM Met, 75 mM Ile, 50 mM Val and Leu, 25 mM Trp. Group C:100 mM Gly, 75 mM Ala, 50 mM His, 25 mM Cys. Group D: 25 mM Glu, 50 mM Gln, 50 mM Asn, 25 mM Asp、0.5 mM Tyr. Data represent mean ± SEM (n = 8 -14).

Synergistic responses to an arginine and NaCl mixture in S2 cells.

(A–D) Calcium imaging shows that heterologous expression of IR20a and IR25a, or triple expression of IR20a, IR25a, and IR76b in S2 cells does not elicit synergistic responses to 10 or 25 mM of arginine-NaCl mixture. Data represent mean ± SEM (n = 8 -10).

IR56b is required for state-dependent low NaCl response.

(A) The state-dependent increase in salt preference is abolished in IR56b mutants, and this phenotype is rescued by reintroducing IR56b. (B - C) Calcium respond to H2O in IR56b controls, mutants and rescue. (D - E) Calcium responses to sucrose are independent on salt-fed condition in wild-type control, IR56b mutants and rescue. Data represent mean ± SEM (n = 8 - 13). Mann-Whitney test, *p < 0.05.