Article Figures & Data
Figures
- Figure 1.
Responses toward putative bitter tastants in larval ingestion and choice assays. A, Schematic drawing of the ingestion assay. Two agarose gel plates were used, with one containing only 1% indigo carmine dye (left, white) and one also containing a bitter substance (right, shaded). Thirty larvae were placed on each plate and allowed to forage and feed freely for 90 min. The ratio of dye ingested by the two groups was measured to calculate the I.I.. B, For the choice preference assay, larvae were placed onto a quadrant plate made with two types of agarose gel: agarose only (white quadrants) and tastant included (shaded quadrants). After 8 min, the number of larvae on each quadrant was counted to analyze the larval behavioral response to the compound. The short lines in A, B indicate larvae. C, Comparison of ingestion and choice preference behavioral responses for the twenty-two bitter compounds tested. Compounds are arranged in order of increasing I.I. values from left to right. Each data point was derived from 6 < n. Error bars are SEM. The following concentrations were used: 10 mM ATR, 5 mM BER, 100 mM CAF, 10 mM CAT, 10 mM COU, 1% DEET, 10 mM DEN, 1 mM ESC, 50 mM GAA, 10 mM GIA, 5 mM HAR, 10 mM LOB, 10 mM NIC, 1 mM PTU, 10 mM QUI, 0.1% SAP, 1 mM SOA, 100 mM STR, 1 mM TAA, 30 mM THB, 100 mM TPH, and 10 mM UMB (Extended Data Figure 1-1).
- Figure 2.
Measurement of larval behavioral responses to given tastants: mouth-hook contractions (left), bending (middle), and body wall contractions (right). Behavioral responses during 1 min for larvae placed on agarose plates containing the indicated tastants, compared with larvae placed on agarose-only control plates. A, Results for wCS larvae. For each data point, 16 < n < 40; *p < 0.05, **p < 0.01, ***p < 0.001 versus control, one-way ANOVA followed by Dunnett’s multiple comparison test. B, Results for Gr33a1 larvae. For each data point, 16 < n < 40; ***p < 0.001 versus control, one-way ANOVA followed by Dunnett’s multiple comparison test. C, Behavioral responses of wCS larvae to the indicated sugars and control. For each data point, 16 < n < 40.
- Figure 3.
A subset of pharyngeal neurons including DP1 mediates CAF-induced avoidance and changes in mouth-hook contraction and bending behaviors. A, Comparison of preference behavior in response to 100 mM CAF when Gr-GAL4 drivers specifically expressed in the GRNs listed under the underlines were used to block neuronal activity. For each data point, n = 6. *p < 0.05, **p < 0.01, Kruskal–Wallis test followed by Dunn’s multiple comparison test. B, Comparison of reduction of mouth-hook contractions in response to 100 mM CAF when Gr-GAL4 drivers specifically expressed in the TO or pharyngeal organs were used to block GRN activity. For each data point, 17 < n < 30; *p < 0.05, **p < 0.01, two-way ANOVA followed by the Bonferroni post hoc test. Gr33a>Kir2.1 larvae in response to 100 mM CAF was marked in a gray asterisk to distinguish it from other data, since mouth-hook contractions were increased compared with other data. ns, not significant. C, Comparison of increase of bending in response to 100 mM CAF when Gr-GAL4 drivers specifically expressed in the TO or pharyngeal organs were used to block GRN activity. For each data point, 17 < n < 30; *p < 0.05, **p < 0.01 versus control, two-way ANOVA followed by the Bonferroni post hoc test. ns, not significant. + and – indicate whether the transgenes are present or absent.
- Figure 4.
DP1 is sufficient for CAF-induced aversive responses in ingestion and choice preference. A, Choice preference in response to 100 mM CAF on expression of Gr33a in the Gr33a mutant using the indicated Gr-GAL4 drivers for specific expression in the GRNs listed under the underlines. For each data point, 6 < n < 12; ***p < 0.001 versus GAL4 and UAS control, one-way ANOVA followed by the Newman–Keuls method. B, Comparison of reduction of mouth-hook contractions in response to 100 mM CAF on expression of Gr33a in the Gr33a mutant using the indicated Gr-GAL4 drivers for specific expression in the GRNs. For each data point, 18 < n < 28; *p < 0.05, two-way ANOVA followed by the Bonferroni post hoc test. ns, not significant. C, Comparison of increase in bending in response to 100 mM CAF on expression of Gr33a in the Gr33a mutant using the indicated Gr-GAL4 drivers for specific expression in the GRNs. For each data point, 18 < n < 28; *p < 0.05, two-way ANOVA followed by the Bonferroni post hoc test. ns, not significant. + and – indicate whether transgenes are present or absent.
- Figure 5.
Two pairs of GRNs in the TO, C1 and C7, detect denatonium to induce avoidance. A, Comparison of preference behavior in response to 10 mm denatonium when GAL4 drivers specifically expressed in C1 or C7, GRNs in the TO, were used to block neuronal activity. For each data point, 6 < n < 10; *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA followed by uncorrected Fisher’s LSD test. Symbols § and ¶ each represent the significance versus C7>Kir2.1 and GrX>Kir2.1. B, Comparison of reduction of mouth-hook contractions in response to 10 mM denatonium when GAL4 drivers specifically expressed in the TO were used to block GRN activity in C1 and/or C7. For each data point, 18 < n < 20. Two-way ANOVA followed by the Bonferroni post hoc test. ns, not significant. C, Comparison of increase of bending in response to 10 mm denatonium when GAL4 drivers specifically expressed in the TO were used to block GRN activity in C1 and/or C7. For each data point, 18 < n < 20; *p < 0.05, two-way ANOVA followed by the Bonferroni post hoc test. ns, not significant. “+” and “-” indicate whether transgenes are present or absent. D, E, Calcium currents can be measured in the C1 (D) and C7 (E) neurons before and during the application of 10 mM denatonium using the genetically encoded calcium sensor GCaMP6m. F, TO C1 and C7 neurons, labeled by Gr59c-GAL4 and C7-GAL4, respectively, showed neuronal activity to 10 mM denatonium. For each data point, 6 < n < 30; *p < 0.05, **p < 0.01, Mann–Whitney U test pair-wise comparisons of water control and 10 mM denatonium.
- Figure 6.
Detection of denatonium by DP1, a pair of dorsal pharyngeal neurons, causes suppression of ingestion. A, Comparison of ingestion in response to 10 mm denatonium when Gr22d-GAL4 and Gr39b-GAL4 drivers were used to ectopically express Gr59c in DP1. For each data point, 6 < n < 14; **p < 0.01, ***p < 0.001 versus GAL4 and UAS control, one-way ANOVA followed by the Newman–Keuls method. B, Comparison of reduction of mouth-hook contractions in response to 10 mM denatonium when Gr22d-GAL4 and Gr39b-GAL4 drivers were used to ectopically express Gr59c in DP1. For each data point, 18 < n < 20; ***p < 0.001, two-way ANOVA followed by the Bonferroni post hoc test. ns, not significant. + and – indicate whether transgenes are present or absent. C, D, Calcium currents were measured in DP1 neurons with Gr39b-GAL4 used to express GCaMP6m only (C) or GCaMP6m and Gr59c together (D) before and during the application of 10 mm denatonium. E, Changes in neuronal activity in response to 10 mM denatonium was measured in the DP1 neuron with ectopically expressed Gr59c. For each data point, 6 < n < 11; **p < 0.01, Mann–Whitney U test pair-wise comparisons of water control and 10 mM denatonium.
Tables
Figure Test comparison Statistical test Result Post hoc test post-hoc comparison Adjusted P value 2A Mouth-hook contraction Comparisons between tastants One-way ANOVA F(3,102) = 7.901, P < 0.0001 Dunnett's multiple comparison test Control vs CAF 0.001 Control vs DEN >0.999 Control vs NIC <0.001 Bending F(3,102) = 8.846, P < 0.0001 Control vs CAF 0.092 Control vs DEN 0.026 Control vs NIC 0.026 Body wall contraction F(3,102) = 16.94, P < 0.0001 Control vs CAF 0.598 Control vs DEN 0.628 Control vs NIC <0.001 2B Mouth-hook contraction Comparisons between tastants One-way ANOVA F(3,76) = 4.355, P = 0.0069 Dunnett's multiple comparison test Control vs CAF 0.473 Control vs DEN 0.060 Control vs NIC 0.995 Bending F(3,76) = 4.773, P = 0.0042 Control vs CAF 0.312 Control vs DEN 0.923 Control vs NIC 0.078 Body wall contraction F(3,76) = 11.90, P < 0.0001 Control vs CAF >0.999 Control vs DEN 0.765 Control vs NIC <0.001 2C Mouth-hook contraction Comparisons between tastants One-way ANOVA F(2,56) = 0.2104, P = 0.8109 Dunnett's multiple comparison test Control vs SUC >0.999 Control vs FRU 0.793 Bending Kruskal–Wallis test 0.9784, P = 0.6131 Dunn's multiple comparison test Control vs SUC >0.05 Control vs FRU >0.05 Body wall contraction One-way ANOVA F(2,56) = 0.1099, P = 0.8961 Dunnett's multiple comparison test Control vs SUC 0.990 Control vs FRU 0.863 3A Comparisons between genotypes Kruskal–Wallis test 39.44, P < 0.0001 Dunn's multiple comparison test UAS-Kir2.1/+ vs Gr33a>Kir2.1 <0.05 Gr33a-GAL4/+ vs Gr33a>Kir2.1 <0.05 UAS-Kir2.1/+ vs Gr39b>Kir2.1 <0.05 Gr39b-GAL4/+ vs Gr39b>Kir2.1 <0.01 UAS-Kir2.1/+ vs Gr23a>Kir2.1 >0.05 Gr23a-GAL4/+ vs Gr23a>Kir2.1 >0.05 UAS-Kir2.1/+ vs Gr22a>Kir2.1 >0.05 Gr22a-GAL4/+ vs Gr22a>Kir2.1 >0.05 UAS-Kir2.1/+ vs Gr36b>Kir2.1 >0.05 Gr36b-GAL4/+ vs Gr36b>Kir2.1 >0.05 3B Interaction between genotype and chemical Two-way ANOVA F(9,356) = 4.149, P < 0.0001 Bonferroni's multiple comparison test Control vs 100 mM CAF (Gr33a-GAL4/+) <0.05 Control vs 100 mM CAF (Gr33a>Kir2.1) <0.05 Control vs 100 mM CAF (Gr39b-GAL4/+) <0.05 Control vs 100 mM CAF (Gr39b>kir2.1) >0.05 Control vs 100 mM CAF (Gr23a-GAL4/+) <0.01 Control vs 100 mM CAF (Gr23a>kir2.1) <0.01 Control vs 100 mM CAF (Gr22a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22a>kir2.1) >0.05 Control vs 100 mM CAF (Gr36b-GAL4/+) <0.01 Control vs 100 mM CAF (Gr36b>kir2.1) >0.05 3C Interaction between genotype and chemical Two-way ANOVA F(9,356) = 1.837, P = 0.0606 Bonferroni's multiple comparison test Control vs 100 mM CAF (Gr33a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr33a>Kir2.1) >0.05 Control vs 100 mM CAF (Gr39b-GAL4/+) >0.05 Control vs 100 mM CAF (Gr39b>kir2.1) >0.05 Control vs 100 mM CAF (Gr23a-GAL4/+) <0.01 Control vs 100 mM CAF (Gr23a>kir2.1) <0.05 Control vs 100 mM CAF (Gr22a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22a>kir2.1) >0.05 Control vs 100 mM CAF (Gr36b-GAL4/+) >0.05 Control vs 100 mM CAF (Gr36b>kir2.1) >0.05 4A Comparisons between genotypes One-way ANOVA F(10,64) = 15.38, P < 0.0001 Newman–Keuls multiple comparison test UAS-Gr33a/+ vs Gr33a>Gr33a <0.001 Gr33a-GAL4/+ vs Gr33a>Gr33a <0.001 UAS-Gr33a/+ vs Gr39b>Gr33a <0.001 Gr39b-GAL4/+ vs Gr39b>Gr33a <0.001 UAS-Gr33a/+ vs Gr22d>Gr33a <0.001 Gr22d-GAL4/+ vs Gr22d>Gr33a <0.001 UAS-Gr33a/+ vs Gr22a>Gr33a >0.05 Gr22a-GAL4/+ vs Gr22a>Gr33a >0.05 UAS-Gr33a/+ vs Gr36b>Gr33a >0.05 Gr36b-GAL4/+ vs Gr36b>Gr33a >0.05 4B Interaction between genotype and chemical Two-way ANOVA F(9,402) = 2.597, P = 0.0064 Bonferroni's multiple comparison test Control vs 100 mM CAF (Gr33a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr33a>Gr33a) <0.05 Control vs 100 mM CAF (Gr39b-GAL4/+) >0.05 Control vs 100 mM CAF (Gr39b>Gr33a) >0.05 Control vs 100 mM CAF (Gr22d-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22d>Gr33a) >0.05 Control vs 100 mM CAF (Gr22a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22a>Gr33a) >0.05 Control vs 100 mM CAF (Gr36b-GAL4/+) >0.05 Control vs 100 mM CAF (Gr36b>Gr33a) >0.05 4C Interaction between genotype and chemical Two-way ANOVA F(9,402) = 2.084, P = 0.0299 Bonferroni's multiple comparison test Control vs 100 mM CAF (Gr33a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr33a>Gr33a) >0.05 Control vs 100 mM CAF (Gr39b-GAL4/+) >0.05 Control vs 100 mM CAF (Gr39b>Gr33a) <0.05 Control vs 100 mM CAF (Gr22d-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22d>Gr33a) >0.05 Control vs 100 mM CAF (Gr22a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22a>Gr33a) >0.05 Control vs 100 mM CAF (Gr36b-GAL4/+) >0.05 Control vs 100 mM CAF (Gr36b>Gr33a) >0.05 5A Comparisons between genotypes One-way ANOVA F(13,106) = 7.609, P < 0.0001 Uncorrected Fisher's LSD test C7-GAL4/+ vs C7>Kir2.1 0.032 Gr22a-GAL4/+ vs Gr22a>Kir2.1 0.002 C7,Gr22a-GAL4/+ vs C7,Gr22a>kir2.1 0.001 Gr28b.e-GAL4/+ vs Gr28b.e>Kir2.1 0.005 C7,Gr28b.e-GAL4/+ vs C7,Gr28b.e>kir2.1 <0.001 Gr59c-GAL4/+ vs Gr59c>Kir2.1 0.368 C7,Gr59c-GAL4/+ vs C7,Gr59c>kir2.1 <0.001 C7>Kir2.1 vs C7,Gr22a>Kir2.1 0.019 Gr22a>Kir2.1 vs C7,Gr22a>Kir2.1 0.002 C7>Kir2.1 vs C7,Gr28b.e>Kir2.1 0.044 Gr28b.e>Kir2.1 vs C7,Gr28b.e>Kir2.1 0.012 C7>Kir2.1 vs C7,Gr59c>Kir2.1 0.245 Gr59c>Kir2.1 vs C7,Gr59c>Kir2.1 0.018 5B Interaction between genotype and chemical Two-way ANOVA F(13,521) = 0.7760, P = 0.6860 Bonferroni's multiple comparison test Control vs 100 mM CAF (C7-GAL4/+) >0.05 Control vs 100 mM CAF (C7>Kir2.1) >0.05 Control vs 100 mM CAF (Gr22a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22a>Kir2.1) >0.05 Control vs 100 mM CAF (Gr22a,C7-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22a,C7>Kir2.1) >0.05 Control vs 100 mM CAF (Gr28b.e-GAL4/+) >0.05 Control vs 100 mM CAF (Gr28b.e>Kir2.1) >0.05 Control vs 100 mM CAF (Gr28b.e,C7-GAL4/+) >0.05 Control vs 100 mM CAF (Gr28b.e,C7>Kir2.1) >0.05 Control vs 100 mM CAF (Gr59c-GAL4/+) >0.05 Control vs 100 mM CAF (Gr59c>Kir2.1) >0.05 Control vs 100 mM CAF (Gr59c,C7-GAL4/+) >0.05 Control vs 100 mM CAF (Gr59c,C7>Kir2.1) >0.05 5C Interaction between genotype and chemical Two-way ANOVA F(13,521) = 1.619, P = 0.0761 Bonferroni's multiple comparison test Control vs 100 mM CAF (C7-GAL4/+) >0.05 Control vs 100 mM CAF (C7>Kir2.1) >0.05 Control vs 100 mM CAF (Gr22a-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22a>Kir2.1) >0.05 Control vs 100 mM CAF (Gr22a,C7-GAL4/+) >0.05 Control vs 100 mM CAF (Gr22a,C7>Kir2.1) >0.05 Control vs 100 mM CAF (Gr28b.e-GAL4/+) <0.05 Control vs 100 mM CAF (Gr28b.e>Kir2.1) >0.05 Control vs 100 mM CAF (Gr28b.e,C7-GAL4/+) >0.05 Control vs 100 mM CAF (Gr28b.e,C7>Kir2.1) >0.05 Control vs 100 mM CAF (Gr59c-GAL4/+) >0.05 Control vs 100 mM CAF (Gr59c>Kir2.1) >0.05 Control vs 100 mM CAF (Gr59c,C7-GAL4/+) <0.05 Control vs 100 mM CAF (Gr59c,C7>Kir2.1) >0.05 5F Comparisons between tastants Mann–Whitney U test Water vs 10 mM denatonium (Gr59c>GCaMP6m) 0.009 Water vs 10 mM denatonium (C7>GCaMP6m) 0.013 6A Comparisons between genotypes One-way ANOVA F(4,35) = 10.73, P < 0.0001 Newman–Keuls multiple comparison test UAS-Gr59c/+ vs Gr22d>Gr59c <0.01 Gr22d-GAL4 vs Gr22d>Gr59c <0.01 UAS-Gr59c/+ vs Gr39b>Gr59c <0.001 Gr39b-GAL4 vs Gr39b>Gr59c <0.01 6B Interaction between genotype and chemical Two-way ANOVA F(4,182) = 6.100, P = 0.0001 Bonferroni's multiple comparison test Control vs 10 mM denatonium (UAS-Gr59c/+) >0.05 Control vs 10 mM denatonium (Gr22d-GAL4/+) >0.05 Control vs 10 mM denatonium (Gr22d>Gr59c) <0.001 Control vs 10 mM denatonium (Gr39b-GAL4/+) >0.05 Control vs 10 mM denatonium (Gr39b>Gr59c) <0.001 6E Comparisons between tastants Mann–Whitney U test Water vs 10 mM denatonium (Gr39b>GCaMP6m) >0.05 Water vs 10 mM denatonium (Gr39b>Gr59c,GCaMP6m) <0.01
Extended Data Figure 1-1
The toxic effects of nicotine that block larval movement. As marked in the left uppermost panel, two quadrants of the plate are the agarose-only control, and the remaining two quadrants contain agarose with nicotine. Twenty larvae were placed in the center of the plate, and images of the plate were taken, starting from the upper left corner and moving to the lower right corner, at 40-s intervals. Therefore, the image of the upper left corner is at 0 s, and the image of the lower right corner at 7 min 20 s. The asterisks indicate larvae that have almost stopped moving. Download Figure 1-1, EPS file.
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