DANCE provides an open-source and low-cost approach to quantify aggression and courtship in Drosophila
- Centre for Molecular Neurosciences, Kasturba Medical College, Manipal Academy of Higher Education, India
- Department of Biochemistry, Kasturba Medical College, Manipal Academy of Higher Education, India
Figures
Figure 1 with 2 supplements
The Drosophila Aggression and Courtship Evaluator (DANCE) assay provides an accessible approach for quantifying aggression and courtship behaviors.
(A) Comparison of existing machine-vision camera hardware (Dankert et al., 2009; Koemans et al., 2017) with the simplified, low-cost DANCE hardware for behavior acquisition. (B) Workflow for developing DANCE classifiers, including training, benchmarking against existing methods and manual ground-truth annotations to generate behavioral scores. (C) Behavioral classifiers developed to quantify male aggression (lunge) and courtship (wing extension, circling, following, attempted copulation, and copulation). (D) Representative raster plots comparing ground-truth, DANCE, CADABRA, and Divider assay performance for aggression. (E) Representative raster plots comparing ground-truth, DANCE, and MateBook performance for courtship. Created in BioRender.
Figure 1—figure supplement 1
Aggression chamber described by Dankert et al., 2009.
The setup consists of a bottom food plate, 12 wells (aggression arenas), and a top plate with fly-loading holes and a screw slot that allows sliding of the loading plate.
Figure 1—figure supplement 2
Courtship setup described by Koemans et al., 2017.
The setup consists of 18 wells (courtship arenas), a top cover plate, a sliding loading plate, and a sliding divider assembly used to separate male and female flies.
Figure 2 with 2 supplements
Validation of the Drosophila Aggression and Courtship Evaluator (DANCE) lunge classifier for quantifying male aggression.
(A) Lunge scores from 20-min-long videos scored using ground-truth annotations (gray), the DANCE lunge classifier (orange), CADABRA (purple), and Divider assay classifier (green). (B–E) Comparison of lunge scores across different aggression levels, based on manual scoring and predictions from DANCE, CADABRA, and Divider: (B) 0–70 lunges ‘low aggressive’ (n=10; ground-truth vs. DANCE ns, p>0.9951, ground-truth vs. CADABRA ns, p>0.3405, ground-truth vs. Divider assay classifier **p<0.0017, DANCE vs. CADABRA **p<0.0060, DANCE vs. Divider assay classifier ****p<0.0001, CADABRA vs. Divider assay classifier ns, p>0.4996). (C) 71–160 lunges, 'moderately aggressive’ (n=11; ground-truth vs. DANCE ns, p>0.9999, ground-truth vs. CADABRA ns, p>0.1247, ground-truth vs. Divider assay classifier ***p<0.0002, DANCE vs. CADABRA *p<0.0102, DANCE vs. Divider assay classifier ****p<0.0001, CADABRA vs. Divider assay classifier ns, p>0.4157). (D) 161–300 lunges, ‘highly aggressive’ (n=11; ground-truth vs. DANCE ns, p>0.9999, ground-truth vs. CADABRA, **p>0.0057, ground-truth vs. Divider assay classifier *p<0.0102, DANCE vs. CADABRA ***p<0.0002, DANCE vs. Divider assay classifier ***p<0.0004, CADABRA vs. Divider assay classifier ns, p>0.9999), and (E) >300 lunges, ‘hyper-aggressive’ (n=8; ground-truth vs. DANCE ns, p>0.9999, ground-truth vs. CADABRA, ***p>0.0006, ground-truth vs. Divider assay classifier **p<0.0029, DANCE vs. CADABRA, *p<0.0402, DANCE vs. Divider assay classifier *p<0.0102, CADABRA vs. Divider assay classifier ns, p>0.9999; Friedman’s ANOVA with Dunn’s test). (F) Regression analysis of the DANCE ‘lunge classifier’ vs. manual scores (R2=0.9760, n=40). (G) Regression of the CADABRA vs. the DANCE lunge classifier (R2=0.9, n=40). (H) Regression of the Divider assay lunge classifier score vs. manual score (R2=0.7739, n=40). (I) Precision, recall, and F1 scores of the DANCE lunge classifier compared with those of CADABRA and Divider.
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Figure 2—source data 1
Source data for Figure 2 showing quantitative aggressive lunge counts and performance metrics for DANCE and existing methods used to quantify aggressive behavior in male flies.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Comparison of annotations by two independent evaluators to assess observer bias during ground-truthing.
(A) Aggressive lunges, ns, p=0.8789, n=15. (B) Courtship wing extension, ns, p=0.9999, n=13. (C) Attempted copulation, ns, p=0.0571, n=16. (D) Circling, ns, p=0.4343, n=12. (E) Following, ns, p=0.4405, n=13. (F) Copulation, ns, p=0.9221, n=25. (A–F) All comparisons were performed using the Mann‒Whitney U test.
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Figure 2—figure supplement 1—source data 1
Source data for Figure 2—figure supplement 1 comparing behavioral annotations across multiple behaviors (lunges, wing extension, attempted copulation, following, circling, and copulation indices) independently scored by two observers to assess observer bias during ground-truthing.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Evaluation of the Drosophila Aggression and Courtship Evaluator (DANCE) lunge classifier predictions across training videos.
(A–D) Scatter plots showing the correlation between manual ground-truth annotations and DANCE-predicted frame counts across four independent training videos (Videos 8–11). (A) R2=0.9794, (B) R2=0.9760, (C) R2=0.9847, and (D) R2=0.9893, demonstrating close agreement between automated classification and manual scoring. (E–H) Bar plots showing the precision, recall, and F1 score of the DANCE classifier for the corresponding videos. Video 9, which yielded the highest overall performance, was also used in Figure 2 for inter-method comparison with CADABRA and the Divider assay to maintain consistency across analyses.
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Figure 2—figure supplement 2—source data 1
Source data for Figure 2—figure supplement 2 comparing aggressive lunge metrics and performance of different classifiers across training videos to assess robustness and reproducibility.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 1 supplement
Evaluation of the Drosophila Aggression and Courtship Evaluator (DANCE) wing extension classifier for quantifying courtship behavior.
(A) Wing extension index of males from 15-min-long videos scored using manual ground-truth annotations (gray), the DANCE wing extension classifier (orange), and MateBook (purple), with decapitated virgin females. MateBook underscored wing extension across multiple videos (Friedman’s ANOVA with Dunn’s test: ground-truth vs. MateBook **p=0.0020, ground-truth vs. DANCE ns, p>0.9999; n=15). (B) Comparison of ground-truth, DANCE, and MateBook wing extension scores (Kruskal‒Wallis ANOVA with Dunn’s test, ground-truth vs. DANCE ns, p>0.9999, ground-truth vs. MateBook *p=0.0436; n=15). (C) Regression analysis of the DANCE wing extension classifier vs. ground-truth (R2=0.9831, n=15). (D) Regression of MateBook vs. ground-truth (R2=0.1054, n=15). (E) Precision, recall, and F1 score of the DANCE wing extension classifier and MateBook relative ground-truth scores.
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Figure 3—source data 1
Source data for Figure 3 showing quantitative behavioral indices and performance metrics (bout-level analysis) for DANCE and existing methods used to quantify wing-extension behavior in decapitated virgin female flies.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Evaluation of the Drosophila Aggression and Courtship Evaluator (DANCE) wing extension classifier in the mated-female dataset.
(A) Wing extension of males from 15 min videos scored using manual ground-truth annotations (gray), DANCE classifier (orange), and MateBook (purple) with mated females. MateBook underscored wing extension across multiple videos (Friedman’s ANOVA with Dunn’s test: ground-truth vs. DANCE ns, p=0.3582, ground-truth vs. MateBook p<0.0001; n=25). (B) Comparison of wing extension scores from ground-truth, DANCE, and MateBook datasets (Kruskal–Wallis ANOVA with Dunn’s test: ground-truth vs. DANCE ns, p>0.9999, ground-truth vs. MateBook ns, p=0.1039; n=25). (C) Regression of DANCE classifier scores vs. ground-truth (R2=0.9951, n=25). (D) Regression of MateBook vs. ground-truth (R2=0.8282, n=25). (E) Precision, recall, and F1 scores of the DANCE classifier and MateBook relative to ground-truth annotations.
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1 showing quantitative behavioral indices and performance metrics (bout-level analysis) for DANCE and existing methods used to quantify wing-extension behavior in mated female flies.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 1 supplement
Validation of the Drosophila Aggression and Courtship Evaluator (DANCE) attempted-copulation classifier.
(A) Attempted copulation index of males from 15-min-long videos scored using manual ground-truth annotations (gray), the ‘DANCE attempted copulation classifier’ (orange), and the MateBook (purple) with both mated and decapitated females (Friedman’s ANOVA with Dunn’s test: ground-truth vs. MateBook ****p<0.0001, ground-truth vs. DANCE ns, p>0.9999; n=32). (B) Comparison of ground-truth, DANCE attempted-copulation classifier, and MateBook scores (Kruskal‒Wallis ANOVA with Dunn’s test, ground-truth vs. DANCE ns, p>0.9999; ground-truth vs. MateBook ****p<0.0001, n=32). (C) Regression analysis of the attempted-copulation classifier vs. ground-truth (R2=0.9565, n=32). (D) Regression analysis of MateBook vs. ground-truth (R2=2115, n=32). (E) Precision, recall, and F1 score of the DANCE and MateBook attempted-copulation classifiers relative to the ground-truth scores.
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Figure 4—source data 1
Source data for Figure 4 showing quantitative behavioral indices and performance metrics (bout-level analysis) for DANCE and existing methods used to quantify attempted-copulation behavior in both decapitated virgin and mated female flies.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Evaluation of the Drosophila Aggression and Courtship Evaluator (DANCE) copulation classifier in the mixed-female dataset.
(A) Copulation scores from 15 min videos obtained using manual ground-truth annotations (gray), the DANCE copulation classifier (orange), and MateBook (purple) (Friedman’s ANOVA with Dunn’s test: ground-truth vs. DANCE ns, p>0.9999; ground-truth vs. MateBook ns, p>0.9999; n=21). (B) Box plot comparison of the manual ground-truth, DANCE copulation classifier, and MateBook (Kruskal‒Wallis ANOVA with Dunn’s test: ground-truth vs. DANCE ns, p>0.9999; ground-truth vs. MateBook ns, p>0.9999; n=21). (C) Regression of DANCE classifier scores vs. manual ground-truth (R2=0.98, n=21). (D) Regression of MateBook vs. manual ground-truth (R2=0.81, n=21). (E) Precision, recall, and F1 score of the DANCE copulation classifier and MateBook relative to the ground-truth score.
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1 showing quantitative behavioral indices and performance metrics (bout-level analysis) for DANCE and existing methods used to quantify copulation behavior in mixed female dataset.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig4-figsupp1-data1-v1.xlsx
Figure 5 with 3 supplements
Evaluation of the Drosophila Aggression and Courtship Evaluator (DANCE) circling classifier.
(A) Circling index of males from 15-min-long videos scored using manual ground-truth annotations (gray), ‘DANCE circling classifier’ (orange), and MateBook (purple) with decapitated virgin females (Friedman’s ANOVA with Dunn’s test: ground-truth vs. DANCE ns, p=0.2049, ground-truth vs. MateBook ****p<0.0001, n=12). (B) Comparison of the ground-truth, DANCE, and MateBook circling classifiers (ordinary one-way ANOVA with Dunnett’s test, ground-truth vs. DANCE ns, p=0.8014; ground-truth vs. MateBook *p=0.0157, n=12). (C) Regression analysis of the DANCE circling classifier vs. ground-truth (R2=0.92, n=12). (D) Regression of MateBook vs. ground-truth (R2=0.88, n=12). (E) Precision, recall, and F1 score of the DANCE and MateBook circling classifiers relative to the ground-truth score.
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Figure 5—source data 1
Source data for Figure 5 showing quantitative behavioral indices and performance metrics (bout-level analysis) for DANCE and existing methods used to quantify circling behavior in decapitated virgin female flies.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Evaluation of the Drosophila Aggression and Courtship Evaluator (DANCE) circling classifier in the mated-female dataset.
(A) Circling index of males from 15 min videos scored using manual ground-truth annotations (gray), the DANCE circling classifier (orange), and the MateBook (purple) with mated females (Friedman’s ANOVA with Dunn’s test: ground-truth vs. DANCE ns, p>0.9999; ground-truth vs. MateBook, p<0.0001; n=19). (B) Comparison of circling scores from ground-truth, DANCE, and MateBook datasets (Kruskal‒Wallis ANOVA, ground-truth vs. DANCE ns, p>0.9999; ground-truth vs. MateBook ns, p=0.0822; n=19). (C) Regression of DANCE classifier scores vs. ground-truth (R2=0.9494, n=19). (D) Regression of MateBook vs. ground-truth (R2=0.6938, n=19). (E) Precision, recall, and F1 scores of the DANCE and MateBook circling classifiers relative to the ground-truth scores.
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1 showing quantitative behavioral indices and performance metrics (bout-level analysis) for DANCE and existing methods used to quantify circling behavior in mated female flies.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
Evaluation of the Drosophila Aggression and Courtship Evaluator (DANCE) following classifier in the mated-female dataset.
(A) Following the index of males from 15 min videos scored using manual ground-truth annotations (gray), DANCE following classifier (orange), and MateBook (purple) with mated females (Friedman’s ANOVA with Dunn’s test: ground-truth vs. DANCE ns, p=0.1794; ground-truth vs. MateBook p=0.0029; n=25). (B) Box plot comparison of the following scores from the ground-truth, DANCE, and MateBook datasets (Kruskal‒Wallis ANOVA with Dunn’s test: ground-truth vs. DANCE ns, p>0.9999; ground-truth vs. MateBook ns, p=0.5287; n=25). (C) Regression analysis of the following classifier vs. ground-truth (R2=0.9894, n=25). (D) Regression of MateBook vs. ground-truth (R2=0.9204, n=25). (E) Precision, recall, and F1 scores of DANCE and MateBook following classifiers relative to ground-truth annotations.
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Figure 5—figure supplement 2—source data 1
Source data for Figure 5—figure supplement 2 showing quantitative behavioral indices and performance metrics (bout-level analysis) for DANCE and existing methods used to quantify following behavior in mated female flies.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig5-figsupp2-data1-v1.xlsx
Figure 5—figure supplement 3
Frame-level analysis of duration-based courtship classifiers.
(A–G) Bar plots showing precision, recall, and F1 scores for Drosophila Aggression and Courtship Evaluator (DANCE) (orange) and MateBook (purple) across different behaviors: (A) wing extension toward decapitated females, (B) wing extension toward mated females, (C) copulation, (D) attempted copulation (mixed dataset), (E) circling toward decapitated females, (F) circling toward mated females, and (G) following. Percent error rates are shown above each bar.
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Figure 5—figure supplement 3—source data 1
Source data for Figure 5—figure supplement 3 showing performance metrics (frame-level analysis) for DANCE and existing methods used to quantify multiple courtship behaviors.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig5-figsupp3-data1-v1.xlsx
Figure 6 with 4 supplements
Drosophila Aggression and Courtship Evaluator (DANCE) hardware and recording setup.
(A) DANCE aggression setup. (B) 3D-rendered components of the aggression setup. (C) DANCE courtship setup. (D) 3D-rendered components of the courtship setup, showing males and females separated by an X-ray film separator or ‘divider comb’. (E–G) Top and side views of the DANCE setup with a smartphone camera for recording and an electronic tablet as the backlight. Created in BioRender.
Figure 6—video 1
3D-rendered Drosophila Aggression and Courtship Evaluator (DANCE) aggression hardware.
Figure 6—video 2
Using the Drosophila Aggression and Courtship Evaluator (DANCE) hardware setup for recording aggression.
Figure 6—video 3
3D-rendered Drosophila Aggression and Courtship Evaluator (DANCE) courtship hardware.
Figure 6—video 4
Using the Drosophila Aggression and Courtship Evaluator (DANCE) hardware setup for recording courtship.
Figure 7 with 6 supplements
Benchmarking Drosophila Aggression and Courtship Evaluator (DANCE) hardware and application to neurogenetic tools.
(A–B) Courtship behaviors recorded using a pre-existing circular setup (Koemans et al., 2017) and DANCE setup in group-housed (GH) and single-housed (SH) flies for (C–D) wing extension, (C) GH vs. SH ***p<0.0010, n=23; (D) GH vs. SH ****p<0.0001, GH, n=22 and SH, n=33. (E–F) Attempted copulation, (E) GH vs. SH ***p<0.0002, n=23; (F) GH vs. SH **p<0.0022, GH, n=21 and SH, n=33. (G–H) Following, (G) GH vs. SH ns, p>0.0959, n=23; (H) GH vs. SH ns, p<0.2537, GH, n=22 and SH, n=32. (I–J) Circling, (I) GH vs. SH *p<0.012, n=23; (J) GH vs. SH *p<0.0104, GH, n=24 and SH, n=31. (K–L) Aggressive lunges were recorded using a pre-existing circular setup (Dankert et al., 2009) and a DANCE setup. (M‒N) Lunges of SH flies compared with those of GH flies reared on food with yeast granules. (M) GH vs. SH **p<0.0138, n=36; (N) GH vs. SH **p<0.0372, n=40. (O) Effect of yeast extract food on aggressive behavior; GH vs. SH ****p<0.0001, n=38–39. (P–Q) Genetic knockdown of the neuropeptide Drosulfakinin (Dsk) in insulin-producing neurons using dilp2-GAL4. (P) Dilp2-GAL4-GAL4>attp2 GH vs. SH ns, p<0.0502; Dilp2-GAL4-GAL4>attp2 GH vs. Dilp2-GAL4-GAL4>Dsk RNAi GH ns, p>0.9999; Dilp2-GAL4-GAL4>attp2 SH vs. Dilp2-GAL4-GAL4>Dsk RNAi SH ****p<0.0001; Dilp2-GAL4-GAL4>Dsk RNAi SH vs. Dilp2-GAL4-GAL4>Dsk RNAi SH ****p<0.0001; n=24. (Q) Dilp2-GAL4-GAL4>attp2 GH vs. SH ****p<0.0001, Dilp2-GAL4-GAL4>attp2 GH vs. Dilp2-GAL4-GAL4>Dsk RNAi GH ns, p>0.9999, Dilp2-GAL4-GAL4>Dsk RNAi SH vs. Dilp2-GAL4-GAL4>Dsk RNAi SH ****p<0.0001, Dilp2-GAL4-GAL4>attp2 SH vs. Dilp2-GAL4-GAL4>Dsk RNAi SH *p>0.0210, n=24. (R) Optogenetic silencing of dopaminergic neurons with UAS-GtACR1 driven by the TH-GAL4 driver; UAS-GtACR1 SH vs. GH ns, p=0.0986; TH-GAL4 SH vs. GH ns, p=0.9999; TH-GAL4>UAS-GtACR1 SH vs. GH ****p<0.0001; UAS-GtACR1 SH vs. TH-GAL4>UAS-GtACR1 SH **p<0.0012; TH-GAL4 SH vs. TH-GAL4>UAS-GtACR1 SH **p<0.0013; n=21–24. (C‒J and M‒O) Mann‒Whitney U test; (P‒R) Kruskal‒Wallis test with Dunn’s multiple comparisons.
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Figure 7—source data 1
Source data for Figure 7 showing for quantitative behavioral counts across aggression and courtship assays used to benchmark DANCE hardware and neurogenetic manipulations.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Drosophila Aggression and Courtship Evaluator (DANCE) optogenetic recording setup for behavioral experiments.
(A) Schematic of the setup showing green LEDs (520–540 nm) controlled by an Arduino and powered via a PC, with illumination directed onto blister-pack arenas placed on an electronic tablet. The tablet provides an adjustable backlight via screen-light software and a smartphone camera records behavior. (B) Photograph of the complete setup, showing the smartphone, tablet, LED stands, Arduino controller, and arenas under green LED illumination. Created in BioRender.
Figure 7—figure supplement 2
Effect of optogenetic silencing of dopaminergic neurons on daytime activity.
(A–B) Transient silencing of dopaminergic neurons using UAS-GtACR1 did not affect daytime activity between single-housing (SH) and group-housed (GH) flies. (A) On day 1 (no green-light silencing), one-way ANOVA with Tukey’s multiple comparisons revealed no differences for TH-GAL4 (ns, p=0.7383; GH, n=43; SH, n=43), UAS-GtACR1 (ns, p=0.4812; GH, n=51; SH, n=42), and TH-GAL4>UAS-GtACR1 (ns, p=0.9942; GH, n=54; SH, n=51). (B) On day 2 (with green-light silencing), no differences were observed for TH-GAL4 (ns, p=0.9976; GH, n=43; SH, n=43), UAS-GtACR1 (ns, p=0.9779; GH, n=51; SH, n=42), and TH-GAL4>UAS-GtACR1 (ns, p=0.9974; GH, n=54; SH, n=51). Two-way ANOVA across days revealed no significant interaction or main effects for TH-GAL4 (interaction: ns, p=0.5504; silencing: ns, p=0.5172; housing: ns, p=0.1602), UAS-GtACR1 (interaction: ns, p=0.4533; silencing: ns, p=0.3602; housing: p=0.255; GH, n=51; SH, n=42), or TH-GAL4>UAS-GtACR1 (interaction: ns, p=0.9977; silencing: ns, p=0.2454; housing: ns, p=0.5868). Within-day comparisons were performed using one-way ANOVA with Tukey’s multiple comparisons; across-day comparisons were performed using two-way ANOVA.
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Figure 7—figure supplement 2—source data 1
Source data for Figure 7—figure supplement 2 showing daytime activity counts across days and housing conditions during optogenetic silencing of dopaminergic neurons.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig7-figsupp2-data1-v1.xlsx
Figure 7—figure supplement 3
Effect of optogenetic silencing of dopaminergic neurons on male aggression across arena sizes.
(A–C) Number of lunges performed in 20 min by TH-GAL4>UAS-GtACR1 males tested in arenas of different diameters: (A) 13 mm (n=29–38), UAS-GtACR1 SH vs. GH ns, p=0.9741; TH-GAL4 SH vs. GH ns, p=0.9866; TH-GAL4>UAS-GtACR1 SH vs. GH ****p<0.0001; UAS-GtACR1 SH vs. TH-GAL4>UAS-GtACR1 SH ****p<0.0001; TH-GAL4 SH vs. TH-GAL4>UAS-GtACR1 SH ****p<0.0001. (B) 17 mm (n=27–32), UAS-GtACR1 SH vs. GH ns, p=0.9807; TH-GAL4 SH vs. GH ns, p=0.8195; TH-GAL4>UAS-GtACR1 SH vs. GH ****p<0.0001; UAS-GtACR1 SH vs. TH-GAL4>UAS-GtACR1 SH ****p<0.0001; TH-GAL4 SH vs. TH-GAL4>UAS-GtACR1 SH ****p<0.0001. (C) 21 mm (n=12–16), UAS-GtACR1 SH vs. GH ns, p=0.9981; TH-GAL4 SH vs. GH ns, p=0.9999; TH-GAL4>UAS-GtACR1 SH vs. GH ****p<0.0001; UAS-GtACR1 SH vs. TH-GAL4>UAS-GtACR1 SH ****p<0.0001; TH-GAL4 SH vs. TH-GAL4>UAS-GtACR1 SH ****p<0.0001. Lunges of flies with GtACR1-mediated silencing were significantly greater than those of GH and control flies across all arena sizes. Statistical comparisons are indicated: ****p<0.0001; ns, not significant; ordinary one-way ANOVA followed by Tukey’s multiple comparisons test.
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Figure 7—figure supplement 3—source data 1
Source data for Figure 7—figure supplement 3 showing aggressive lunge counts during optogenetic silencing of dopaminergic neurons across different arena sizes.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig7-figsupp3-data1-v1.xlsx
Figure 7—figure supplement 4
Quantification of wild-type Drosophila courtship behavior in Drosophila Aggression and Courtship Evaluator (DANCE) chambers of varying diameters.
(A–C) Wing extension bouts (A: n=22, 33, ****p<0.0001; B: n=25, 28, ****p<0.0001; C: n=20, 25, ****p<0.0001). (D–F) Attempted copulation bouts (D: n=21, 33, **p<0.0022; E: n=25, 28, ****p<0.0001; F: n=20, 25, ****p<0.0001). (G–I) Following bouts (G: n=22, 32, ns, nonsignificant p>0.2537; H: n=25, 28, ns, nonsignificant p>0.7917; I: n=21, 24, ns, nonsignificant p>0.0705). (J–L) Circling bouts (J: n=24, 31, *p<0.0104; K: n=26, 24, **p<0.0048; L: n=18, 23, **p<0.0032). Data were obtained from Canton-S (CS) males paired with either group-housed (GH) or single-housed (SH) females over 15 min in chambers of 11 mm, 13 mm, or 17 mm diameter. Each bar represents the median. Statistical differences are indicated as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant (Mann–Whitney U test).
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Figure 7—figure supplement 4—source data 1
Source data for Figure 7—figure supplement 4 showing behavioral counts across multiple courtship behaviors in wild-type Drosophila measured in arenas of different sizes.
- https://cdn.elifesciences.org/articles/105465/elife-105465-fig7-figsupp4-data1-v1.xlsx
Figure 7—video 1
Aggression and courtship behaviors recorded in Drosophila Aggression and Courtship Evaluator (DANCE) hardware.
Figure 7—video 2
Optogenetic silencing of dopaminergic neurons in Drosophila Aggression and Courtship Evaluator (DANCE) shows increased aggression.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | Sigmacote | Sigma-Aldrich | Cat#: SL2 | |
| Chemical compound, drug | Alcojet | Alconox | Cat#: 1401-1 | |
| Chemical compound, drug | Sucrose | HiMedia | Cat#: GRM601 | |
| Chemical compound, drug | Agar | HiMedia | Cat#: GRM026 | |
| Chemical compound, drug | Yeast extract powder | HiMedia | Cat#: RM0271 | |
| Chemical compound, drug | Yeast granules | AB Mauri, India | ||
| Chemical compound, drug | Apple juice | Commercial | ||
| Chemical compound, drug | Fluon (Insect-a-Slip) | BioQuip | Cat#: 2871B | |
| Strain, strain background (Drosophila melanogaster, male) | Canton-S | Ulrike Heberlein (HHMI Janelia) | Wild-type strain | |
| Genetic reagent (Drosophila melanogaster) | TH-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_51982 | |
| Genetic reagent (Drosophila melanogaster) | dilp2-GAL4 | Bloomington Drosophila Stock Center | RRID:BDSC_37516 | |
| Genetic reagent (Drosophila melanogaster) | Dsk-RNAi | Bloomington Drosophila Stock Center | RRID:BDSC_25869 | |
| Genetic reagent (Drosophila melanogaster) | attP2 empty vector control | Bloomington Drosophila Stock Center | RRID:BDSC_36303 | |
| Genetic reagent (Drosophila melanogaster) | UAS-GtACR1 | Mohammad et al., 2017 | RRID:BDSC_92983 | Gift from Gaurav Das, (NCCS, Pune) |
| Software, algorithm | JAABA | Kabra et al., 2013 | RRID:SCR_027430 | https://jaaba.sourceforge.net/ |
| Software, algorithm | Caltech FlyTracker | Eyjolfsdottir et al., 2014 | RRID:SCR_027431 | https://kristinbranson.github.io/FlyTracker/ |
| Software, algorithm | MateBook | Ribeiro et al., 2018 | https://github.com/Dicksonlab/MateBook | |
| Software, algorithm | CADABRA | Dankert et al., 2009 | https://www.vision.caltech.edu/cadabra/ | |
| Software, algorithm | GraphPad Prism 8 | GraphPad Software | RRID:SCR_002798 | http://www.graphpad.com/ |
| Software, algorithm | DANCE classifiers and code | This paper | RRID:SCR_027812 | https://github.com/agrawallab/DANCE |
| Software, algorithm | BioRender | BioRender Software | RRID:SCR_018361 | https://www.biorender.com/ |
| Software, algorithm | Inkscape | Inkscape Software | RRID:SCR_014479 | https://github.com/inkscape/inkscape |
| Other | LEDs (520–540 nm) | Lumileds, SM Electronic Technologies Pvt Ltd. Bangalore | Model: 2835 | Hardware and equipment |
| Other | DMK 33UX252 USB 3.0 monochrome camera | Menzel Vision and Robotics Pvt Ltd. Mumbai | Model: DMK 33UX252 | Hardware and equipment |
| Other | Metaphase backlight | Alpha Techsys, Pune | Model: TMS, BHS4-00100-X-W-24V | Hardware and equipment |
| Other | Huawei Y9 2019 smartphone | Huawei | Model: Y9 2019 | Hardware and equipment |
| Other | OnePlus Nord CE 2 Lite 5G smartphone | OnePlus | Model: CPH2381 | Hardware and equipment |
| Other | Redmi Note 11 Pro+ 5G smartphone | Xiaomi | Model: 221116SI | Hardware and equipment |
| Other | iPad Air (5th Generation) | Apple | Model: iPad Air 5 | Hardware and equipment |
| Other | iPhone 13 | Apple | Model: 13 | Hardware and equipment |
| Other | White-screen light app | App Store/Play Store | Hardware and equipment |
Additional files
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Supplementary file 1
Bill of materials for the Drosophila Aggression and Courtship Evaluator (DANCE) setup and comparison with existing setups.
- https://cdn.elifesciences.org/articles/105465/elife-105465-supp1-v1.xlsx
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Supplementary file 2
Comparison between the Drosophila Aggression and Courtship Evaluator (DANCE) lunge classifier, ground-truth, and existing methods.
- https://cdn.elifesciences.org/articles/105465/elife-105465-supp2-v1.xlsx
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Supplementary file 3
Definitions of the behavioral classifiers.
- https://cdn.elifesciences.org/articles/105465/elife-105465-supp3-v1.xlsx
-
Supplementary file 4
Comparison between the Drosophila Aggression and Courtship Evaluator (DANCE) courtship classifiers, ground-truth, and MateBook.
- https://cdn.elifesciences.org/articles/105465/elife-105465-supp4-v1.xlsx
-
Supplementary file 5
Quantifying identity swaps to validate tracking across setups.
- https://cdn.elifesciences.org/articles/105465/elife-105465-supp5-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/105465/elife-105465-mdarchecklist1-v1.pdf
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
DANCE provides an open-source and low-cost approach to quantify aggression and courtship in Drosophila
eLife 14:RP105465.
https://doi.org/10.7554/eLife.105465.3
