Bully males exhibit reduced mating success and allocate less time to mating.

(A) in competitive courtship assays, the mating success of Cs and Bully males were scored when paired time with a single virgin Cs female. (B) In non-competitive courtship assays, the mating success of Cs and Bully males paired with a virgin Cs female were analyzed. (C) Proportion of time spent by Cs and Bully males doing Unilateral Wing Extension (UWE) toward a Cs virgin female in non-competitive courtship assays. (D-E) Latency to court (time difference between first interaction and first UWE) and to mate (time difference between first interaction and start of the mating) of Cs and Bully males paired with a virgin Cs female. (F) Mating duration of Cs and Bully males paired with a virgin Cs female or (G) a virgin Cs decapitated female. (H) Mating duration of Cs and Bully males that were previously raised in group of 10 males or in social isolation. For all graphs, stars indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001) and statistical details are given in Figure 1-table supplement 2.

Hyper-aggressive males exhibit lower mate-guarding efficiency, yet this does not affect female fertility.

(A-G). Mating duration of Cs and Bully males paired with a virgin Cs female. (A’) After mating ended, the proportion of time spent by each male to court performing UWE towards the just mated female was scored for 10 minutes. (B’) The proportion of time spent by the males performing UWE an unfamiliar mated female was scored for 10 minutes. (C) Total number of mating reached by Cs and Bully males over six consecutive mating with virgin Cs females. (D) Mating duration of Cs and Bully males over six consecutive mating with virgin Cs females. A significant interaction between males’ genotype and number of mating was found, indicating that Cs decreased their mating duration with the number of mating, while Bully males did not. (E) Proportion of mating that gave rise to progeny in consecutive mating assay. (F’) Proportion of time spent by naïve Cs and Bully males to display UWE either toward Cs-mated or Bully-mated decapitated female over an observation period of 2 minutes. (G’) Proportion of Cs-mated and Bully-mated females that re-mated 12 or 14 days after a first mating. For graphs A, A’, B, B’, F, F’, G and G’, stars indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001) and statistical details are given in Figure 1-table supplement 2.

Hyper-aggressive males exhibit altered CHCs profiles and transfer reduced amounts of mate-guarding pheromones to females.

(A) PCA analysis on mean concentration for each Cuticular Hydrocarbon (CHCs) compound of naïve Cs and Bully. Their CHCs profiles were found similar. (B) Relative concentrations of Bully’s CHCs were calculated with respect to those of Cs males. The CHCs levels in Cs males were normalized to 1, and Bully’s CHC concentrations were expressed relative to this reference. (C) PCA analysis on mean concentration for each CHCs compound of virgin, Cs- and Bully-mated females. (D) Relative concentrations of CHCs compounds in mated females compared to virgin females. CHCs levels in virgin females were normalized to 1, and the concentrations in mated females were expressed relative to this baseline. Significant differences are indicated by letters: a indicates a significant difference between virgin and Cs-mated females; b indicates a significant difference between virgin and Bully-mated females; and c indicates a significant difference between Cs- and Bully-mated females (a P < 0.05; aa P < 0.01; aaa P < 0.001). (E) Mean ± SE concentration in ng of CHCs compounds that were not present in virgin females. (F) Mating duration of Cs and Bully males with a Cs female. (F’) cVA concentration measured within Cs- and Bully-mated genitalia tract right after mating. For graphs E, F and F’, stars indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001) and statistical details are given in Figure 1-table supplement 2.

Comparison of CHCs concentrations between naïve Cs and Bully males.

Mean ± SE concentrations are provided in ng for each compound and each group of compounds. In statistics columns, stars represent significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001). Statistical details are given in Figure 1-table supplement 2.

Comparison of CHCs concentrations among virgin, Cs-mated, and Bully-mated females.

Mean ± SE concentrations are provided in ng for each compound and each group of compounds. In the statistics columns, stars indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001). Statistical details are given in Figure 1-table supplement 2.

Aggressive males offset their reduced reproductive success with a longer lifespan and preserved reproductive success in later life.

(A) Mating duration of Cs and Bully males with virgin Cs females across their lifespan. A significant interaction between male genotype and age was observed: Bully males maintained a stable mating duration over time, whereas Cs males showed an increase in mating duration with successive mating events. (B) Number of mating throughout the lifespan of Cs and Bully males. Numbers next to data points indicate the proportion of mating. Arrows denote the last mating event and the time of death for each genotype. The number of offspring produced from the final Bully mating is indicated next to the corresponding arrow. (C) Survival curves of progeny from ♀Cs × ♂Cs and ♀Cs × ♂Bully crosses. Progeny from Bully males had a median survival (EC₅₀) of 79 days, with the last individual dying at day 106 (green line), while progeny from Cs males showed an EC₅₀ of 57 days, with the final death at day 90 (yellow line). (D) Survival curves of males raised in group of 12♀Cs & 10♂Cs or 12♀Cs & 10♂Bully. Bully males had a median survival (EC₅₀) of 56 days, with the last individual dying at day 71 (green line), while Cs males showed an EC₅₀ of 43 days, with the final death at day 67 (yellow line). Statistical details are given in Figure 1-table supplement 2.

Courtship and aggressive behaviors in Cs, Bully A, and Bully B lines.

(A) Non-competitive mating success of Cs, Bully A and Bully B males paired with a virgin Cs female. (B) Mating duration of Cs, Bully A and Bully B males paired with a virgin Cs female. (C) Latency to lunge of Cs, Bully A and Bully B males in aggression assay with a territory against a male of the same line. (D) Number of lunges and (E) boxing events of Cs, Bully A and Bully B male pairs in aggression assays. For all graphs, stars indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001). Statistical details are given in Figure 1-table supplement 2.

Statistical details of all analyses.

This table gathers all statistical analyses performed in all the Figures. The first column refers to the panels of each Figure, the second one gives the statistical model used to analyze data and the last one provides the names of statistical tests and corrections performed as well as all p-values and statistical values. Statistical details are given in Figure 1-table supplement 2.

More details on PCA analysis for virgin and mated females.

(A) Eigenvectors and associated cos² for each CHCs compounds. Contribution of each CHCs compound in the difference between (B) virgin and Cs-mated, (C) virgin and Bully-mated and (D) Cs-mated and Bully-mated CHCs profiles. Compounds contributing significantly to the difference between CHCs profiles are represented in red. Numbers above bars refer to the mean contribution of each compound in the difference between CHCs profiles. Statistical details are given in Figure 1-table supplement 2.

Interindividual variability across multiple mating events throughout the flies’ lifespan.

Interindividual variability in mating duration for each (A) Cs and (B) Bully male when mated with a virgin Cs female across their lifespan. Each panel represents the mating durations (in minutes) for a single individual, with each dot corresponding to a single mating event. Statistical details are given in Figure 1-table supplement 2.