Establishing a paradigm for tussling behavior in male Drosophila.

(A) Diagrams of two fighting paradigms for Drosophila tussling behavior. The left diagram shows a traditional fighting paradigm with no female (NF) in the food area and a new fighting paradigm with a fixed female (FF) in the center of the food area. Before the experiment began, two male flies were placed on the upper layer, separated from the food by a film. After a 30-minute adaptation, the film was removed to start recording. The right diagram shows a pair of tussling male Drosophila.

(B) The influence of age and female factors on male tussling behavior. From top to bottom, the tussle latency, the number of tussles, and the proportion of tussles occurring are shown respectively. n=30, 30, 30, 46, 46, 46 from left to right. Bars sharing the same letter are not significantly different according to Kruskal-Wallis test (tussle latency, number of tussles) and Chi-square test (proportion of tussles occurring).

(C) Sucrose concentration of food have no effect on male tussling behavior. n=30 for each group, n.s., not significant, Kruskal-Wallis test (tussle latency, number of tussles), and Chi-square test (proportion of tussles occurring).

(D) The influence of yeast concentration on male tussling behavior. n=30 for each group, n.s., not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001, Kruskal-Wallis test (tussle latency, number of tussles), and Chi-square test (proportion of tussles occurring). Error bars correspond to SEM.

Aging suppresses lunging behavior in male Drosophila.

(A) The lunge latency in S7, S14, G7 and G14 males, n=12 for each group, n.s., not significant, *P < 0.05, Mann-Whitney test.

(B) The number of lunges in S7, S14, G7 and G14 males within 10 minutes, n=12 for each group, *P < 0.05, ****P < 0.0001, Mann-Whitney test. Error bars correspond to SEM.

Social enrichment inhibits lunging behavior but promotes tussling behavior in males.

(A) Schematic diagram of Drosophila rearing conditions and experimental procedure.

(B) Representative raster plots illustrating lunge events in the first 10 minutes and tussle events over 2 hours in single-housed (SH) and group-housed (GH) males.

(C, D) Latency (C) and number (D) of tussling behavior in SH and GH males, n=27, 30 for SH and GH males, respectively, ****P < 0.0001, Mann-Whitney test for both.

(E, F) Latency (E) and number (F) of lunging behavior in SH and GH males, n=27, 30 for SH and GH males, respectively, ****P < 0.0001, Mann-Whitney test for both. Error bars correspond to SEM.

Or47b neurons are required for male tussling but not lunging.

(A, B) The effect of inactivating different sensory neurons on latency (A) and number

(B) of male tussling behavior, n=30 for each group, n.s., not significant, **P < 0.01, Kruskal-Wallis test.

(C, D) The effect of inactivating different sensory neurons on the latency (C) and number (D) of male lunging behavior, n=30 for each group, n.s., not significant, **P < 0.01, ***P < 0.001, Kruskal-Wallis test.

(E, F) The effect of knocking down Or47b or fruM expression in Or47b ORNs on the latency (E) and number (F) of male tussling behavior, respectively. n=30 for each group, n.s., not significant, ****P < 0.0001, Kruskal-Wallis test.

(G, H) The effect of activating Or47b ORNs on latency (G) and number (H) of tussling behavior under conditions of GH or SH for 14 days, n=60, 60, 60, 49 respectively, n.s., not significant, ****P < 0.0001, Kruskal-Wallis test. Error bars correspond to SEM.

Validation of the Or47b and fruM RNAi efficiency.

Validation of UAS-Or47b-RNAi (A) and UAS-fruMi (B) efficiency by quantitative qPCR. n=9 for each of three replicates, ****P < 0.0001, ***P < 0.001, Mann-Whitney test. Error bars correspond to SEM.

Group housing increases downstream signals of Or47b ORNs.

(A) Projection of Or47b ORNs (green) and their downstream neurons (red) in GH and SH conditions. Scale bar, 100μm.

(B) The fluorescence intensity of neurons downstream of Or47b ORNs is stronger under GH conditions compared to SH conditions. Both groups of male testers were 14 days old, n=10 and 8, respectively, ****P < 0.0001, Mann-Whitney test. Error bars correspond to SEM.

A subset of pC1 neurons specifically promote male tussling behavior.

(A, B) The effect of inactivating candidate interneurons on latency (A) and number (B) of male tussling behavior, n=30 for each group, bars sharing the same letter are not significantly different according to Kruskal-Wallis test.

(C-E) The expression pattern of P1a-spGAL4 (C) and pC1SS2-spGAL4 (D) in male brain, and (E) the registration of P1a (magenta) and pC1SS2 (green) neurons in a standard brain. The GFP expression indicates P1a (magenta) and pC1SS2 (green) neurons in the male brain counterstained with nc82 (blue). Scale bars, 100μm.

(F-H) Statistics on tussling (F), lunging (G) and unilateral wing extension (UWE, H) by UAS-CsChrimson/+ males. Red light activation with a light intensity of 0.02 mW/mm2 for 1 minute was applied at the 2nd and 5th minutes of this and subsequent experiments. n=32 for each group, n.s., not significant, Friedman test.

(I-K) Statistics on tussling (I), lunging (J) and UWE (K) in P1a>UAS-CsChrimson males. n=32 for each group, n.s., not significant, ****P < 0.0001, Friedman test.

(L-N) Statistics on tussling (L), lunging (M) and UWE (N) in pC1SS2>UAS-CsChrimson males. n=32 for each group, n.s., not significant, ***P < 0.001, ****P < 0.0001, Friedman test.

Light-dependent initiation of male tussling behavior.

(A) Schematic diagram of the thermal activation experiment.

(B) The percentage of tussling occurrence (left) and total tussling time (right) within 10 mins. n=18 for each group, bars sharing the same letter are not significantly different according to Kruskal-Wallis test based on the data of total tussling time.

Social enrichment enhances male territorial control and mating competition.

(A) Schematic diagram of the paradigm for testing territorial control in males.

(B) The territorial control test of 7-day-old GH (G7) and SH (S7) males. G7a and G7b stand for marked G7 male and unmarked G7 male, respectively. n=12 for each group, n.s., not significant, one sample t test.

(C) The territorial control test of 14-day-old GH (G14) and SH (S14) males. n=12 for each group, n.s., not significant, *P < 0.05, one sample t test.

(D) Schematic diagram of the paradigm for testing male mating advantage.

(E) The mating competition test between G7 and S7 males. n=12 for each group, n.s., not significant, one sample t test.

(F) The mating competition test between G14 and S14 males. n=12 for each group, n.s., not significant, *P < 0.05, one sample t test.

(G and H) The mating competition test between young males and old males, n=12 for each group, n.s., not significant, *P < 0.05, ****P < 0.0001, one sample t test for comparison within group (red labeling), Mann-Whitney test for comparison between groups (black labeling). Error bars correspond to SEM.

Winning events of representative samples for territorial competition.

The blue and red bars represent the wins for S14 males and G14 males, respectively. An example formula is provided for calculating the winning index in the dashed box.

Summary model of fighting strategies and reproductive success in male Drosophila.

Age and social enrichment inhibit the low-intensity, high-frequency lunging while promoting the low-frequency, high-intensity tussling. This shift in fighting strategies among experienced, aged males enhances their territorial control and mating competition, even offsetting aging-related mating disadvantages. Lunging and tussling behaviors are regulated by distinct sensory and central neurons.