1. Volume 27, Issue 18, Pages 2741-2750.e4 (September 2017)
Ionotropic Receptors Mediate Drosophila Oviposition Preference through Sour Gustatory Receptor Neurons 
Yan Chen, Hubert Amrein 
Current Biology 
Volume 27, Issue 18, Pages e4 (September 2017)
DOI: /j.cub
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2. Current Biology 2017 27, 2741-2750.e4DOI: (10.1016/j.cub.2017.08.003)
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3. Figure 1
IR25a-Gal4 and IR76b-Gal4 Are Co-expressed in a GRN Not Expressing Markers for Bitter and Sweet Neurons
(A) Immunostaining of the fifth tarsal segment of a w1118/w1118; IR25a-GAL4/IR76b-QF UAS-mCD8GFP; QUAS-mtdTomato-3XHA/+ fly. IR25a and IR76b are broadly co-expressed in a large number of tarsal GRNs. Inset: schematic drawing of chemosensory sensilla in the fifth tarsal segment.
(B) Immunostaining of the fifth tarsal segment of a w1118/w1118; IR76b-QF UAS-mCD8GFP/Gr33a-GAL4 Gr64f-GAL4; QUAS-mtdTomato-3XHA/+ fly. Bitter and sweet GRNs express IR76b, but each sensillum harbors one (or two) additional GRN(s) that is not labeled by bitter and sweet Gr-GAL4 drivers.
(A and B) Numbers indicate the average number of labeled GRNs of the three taste sensilla: 5b and 5s+5v (mean ± SEM; 6 ≤ n ≤ 8). Neurons of the 5a sensillum (dotted oval) respond to pheromones and were excluded in the count. Arrows indicate non-sweet and non-bitter neurons.
(C) Endogenous IR25a, visualized by anti-IR25a antibody, and IR76b-GAL4 are co-expressed in IR76bonly GRNs of the foreleg. Genotype: w1118/w1118; IR76b-GAL4 Gr66a-LexA/UAS-GCaMP6m; Gr64f-LexA/LexAop-GAL80. Note that the bitter GRN of the 5b sensillum (∗) does not express Gr66-LexA.
(D) Immunostaining of the ventral nerve cord in the thorax of a w1118/w1118; IR76b-QF UAS-mCD8GFP/Gr33a-GAL4 Gr64f-GAL4; QUAS-mtdTomato-3XHA/+ fly. Tarsal projections of GRNs expressing IR76b (red) terminate predominantly in the thoracic ganglion, whereas most tarsal projections of GRNs expressing bitter Gr genes and a subset of GRNs expressing sweet Gr genes (green) bypass the ganglion to terminate in the subesophageal zone (SEZ). f, foreleg; h, hindleg; m, midleg; w, wing.
(E) Projections of tarsal and wing IR76bonly GRNs predominantly terminate in the thoracic ganglion. Genotype: w1118/w1118; IR76b-GAL4 Gr66a-LexA/UAS-GCaMP6m; Gr64f-LexA/LexAop-GAL80.
GRNs of the 5a sensillum (dotted oval) were not counted in (A) and (B), as these neurons are thought to sense pheromones [13, 15]. All scale bars represent 10 μm. See also Figure S1.
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4. Figure 2
IR76bonly GRNs Associated with 5b and 5s Sensilla Specifically Respond to Many Carboxylic and Mineral Acids
Ca2+ imaging was performed on the fifth segment of forelegs of females with the genotype w1118/w1118; IR76b-GAL4, Gr66a-LexA/UAS-GCaMP6m; Gr64f-LexA/lexAop-GAL80 (IR76bonly).
(A) Schematic image of chemosensory sensilla in the fifth segment of tarsi (left), and a live image of an IR76bonly neuron after stimulation with 100 mM acetic acid (middle). Relative fluorescence change (ΔF) is visualized (right). Scale bars represent 10 μm. Asterisks indicate bitter GRNs in 5b sensilla, which do not express Gr66a-LexA.
(B) Ca2+ response (ΔF/F [%]) of IR76bonly GRNs after stimulation with the indicated ligands. Response profiles are similar for the 5b- (top) and 5s- (middle) associated sour GRNs (i.e., IR76bonly). 5v sensilla (bottom) harbor two similarly tuned IR76bonly GRNs that respond only to malic and glycolic acid (at high concentration), as well as to 500 mM NaCl. Representative traces to 100 mM citric acid are shown to the right of each graph. An amino acid (aa) mix (40 mM) contains all 20 amino acids (each at 2 mM). Acetic acid concentrations: 10, 100, and 500 mM; citric and tartaric acid concentrations: 1, 10, and 100 mM; malic and glycolic acid concentrations: 10 mM; HCl concentrations: 1, 10, 25, and 50 mM. Data are shown as the mean, and error bars indicate SEM; Student’s t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; 6 ≤ n ≤ 11; den, denatonium benzoate, lob, lobeline, quin, quinine, suc, sucrose.
(C) Ca2+ response of IR76bonly GRNs in 5b and 5s sensilla stimulated with 100 mM acetate and citrate buffered solutions. Data are shown as means, and error bars indicate the SEM. 6 ≤ n ≤ 10.
See also Figures S2 and S3.
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5. Figure 3
Activation of Sour GRNs Is Dependent on Both IR76b and IR25a Function
Ca2+ response (ΔF/F [%]) upon stimulation with the indicated acids in control (A and B), homozygous IR76b mutant (A), homozygous IR25a mutant (B), and the respective rescue flies (A and B).
(A) Absence of IR76b leads to a complete loss of sour GRN responses, which is rescued in the presence of an IR76b transgene, expressed under the control of IR76b-GAL4. Genotypes: w1118/w1118; IR76b-GAL4 Gr66a-LexA/UAS-GCaMP6m; Gr64f-LexA/LexAop-Gal80 (IR76b control); w1118/w1118; IR76b-GAL4 Gr66a-LexA/UAS-GCaMP6m; Gr64f-LexA IR76b2/IR76b1 LexAop-GAL80 (IR76b mutant); and w1118/w1118; IR76b-GAL4 Gr66a-LexA/UAS-IR76b UAS-GCaMP6m; Gr64f-LexA IR76b2/IR76b1 LexAop-GAL80 (IR76b rescue).
(B) Absence of IR25a leads to a complete loss of sour GRN responses, which is rescued in the presence of an IR25a transgene, expressed under the control of IR25a-GAL4. Note that because the IR25a-Gal4 and Gr66a-LexA transgenes and IR25a2 are all located on the second chromosome, rescue experiments could not be carried out with the GAL80 suppressor transgenes. Consequently, only the 5b sensillum was analyzed, because the neurons in the 5v and 5s sensilla are in too close a proximity for unequivocal identification. Genotypes: w1118/w1118; IR25a-GAL4/+; UAS-GCaMP6m/+ (IR25a control); w1118/w1118; IR25a-GAL4 IR25a2/IR25a2; UAS-GCaMP6m/+ (IR25a mutant); and w1118/w1118; IR25a-Gal4 IR25a2/IR25a2 UAS-IR25a; UAS-GCaMP6m/+ (IR25a rescue).
Concentrations used: 100 mM for acetic acid, citric acid, and tartaric acid; 10 mM for glycolic acid and malic acid; 50 mM for HCl; 50 mM and 500 mM for NaCl; and 100 and 500 mM for KCl. All data are shown as the mean, and error bars indicate SEM; one-way ANOVA with post hoc Bonferroni correction, different letters indicate a significant difference with p < 0.05; 6 ≤ n ≤ 12.
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6. Figure 4 Oviposition Preference Is Mainly Mediated by Tarsal Sour GRNs
(A) Representative example of female oviposition preference on acid-containing sugar-agar: the majority of eggs are laid on the acid-containing half of the plate (left). w1118 females have a strong, dose-dependent preference for oviposition on acid-containing sugar-agar (right) (see also Figure S4).
(B) Silencing of all IR76b-GAL4 GRNs or sour (i.e., IR76bonly) GRNs abolishes acid preference, whereas silencing only the sweet and bitter GRNs has no effect. Genotypes: w1118/w1118 (WT); w1118/w1118; UAS-Kir2.1/+ (UAS control); IR76b-GAL4/UAS-Kir2.1 (silencing IR76b GRNs); w1118/w1118; IR76b-GAL4 Gr66a-LexA/UAS-Kir2.1; Gr64f-LexA/LexAop-GAL8o (silencing sour GRNs); and w1118/w1118; Gr33a-GAL4 Gr64f-GAL4/UAS-Kir2.1 (silencing bitter/sweet GRNs).
(C) All major taste organs contribute to oviposition preference for acetic acid (left), but removal of the forelegs has a more severe effect than removal of any other taste organ. The loss of oviposition preference for citric acid after removal of any specific taste organ is less pronounced, and significant reduction is only observed when the forelegs are removed. Genotype: w1118/w1118 (see also Figure S5).
Data are shown as the mean, and error bars indicate SEM; Student’s t test for (A), ∗∗∗p < 0.001; 20 ≤ n ≤ 30. One-way ANOVA with post hoc Bonferroni correction for (B) and (C), different letters indicate significant difference with p < 0.05; 23 ≤ n ≤ 39. O.I., oviposition index.
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7. Figure 5
Female Oviposition Preference for Acid-Containing Food Is Mediated by IR76b and IR25a
(A) Females lacking IR76b lost preference for oviposition on acid-containing sugar-agar, a phenotype that can be rescued by expressing IR76b under the control of IR76b-GAL4. Note that oviposition preference is also restored when IR76b is only expressed in sour GRNs (lane 7). Genotypes: IR76b+ control (w1118/w1118) (lane 1); w1118/w1118; IR76b1/IR76b1 (lane 2); w1118/w1118; IR76b-GAL4/+; IR76b1/IR76b1 (lane 3); w1118/w1118; UAS-IR76b/+; IR76b1/IR76b1 (lane 4); w1118/w1118; IR76b-GAL4/UAS-IR76b; IR76b1/IR76b1 (lane 5); w1118/w1118; IR76b-GAL4 Gr66a-LexA/+; Gr64f-LexA IR76b2/ R76b1 LexAop-GAL8o (lane 6); and w1118/w1118; IR76b-GAL4 Gr66a-LexA/UAS-IR76b; Gr64f-LexA IR76b2/ R76b1 LexAop-GAL8o (lane 7).
(B) Females lacking IR25a lost preference for oviposition on acid-containing sugar-agar. This phenotype was not rescued by expression of a UAS-IR25a gene under the control of the IR25a-GAL4 driver (lane 5), but it was rescued when expressed under the control of IR76b-GAL4 (lanes 8 and 9; see text). Genotypes: IR25a+ control (w1118/w1118) (lane 1); w1118/w1118; IR25a2/IR25a2 (lane 2); w1118/w1118; IR25a-GAL4/+; IR25a2/IR25a2 (lane 3); w1118/w1118; UAS-IR25a/+; IR25a2/IR25a2 (lane 4); w1118/w1118; IR25a-GAL4 IR25a2/IR25a2 UAS-IR25a (lane 5); w1118/w1118; IR76b-GAL4 IR25a2/IR25a2 (lane 6); w1118/w1118; IR76b-GAL4 IR25a2/IR25a2 antenna removed (lane 7); w1118/w1118; IR76b-GAL4 IR25a2/IR25a2 UAS-IR25a (lane 8); and w1118/w1118; IR76b-GAL4 IR25a2/IR25a2 UAS-IR25a antenna removed (lane 9).
All data are shown as the mean, and error bars indicate SEM; one-way ANOVA with post hoc Bonferroni correction, different letters indicate significant difference with p < 0.05; 20 ≤ n ≤ 39.
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