Author response:
eLife Assessment
This valuable study addresses the effects of selection on aggression on fitness and life-history trade-offs in Drosophila melanogaster. However, the evidence presented is incomplete and does not support the claims proposed in the study of increased survival of highly aggressive males at the expense of reproductive success and shorter mating duration. The main limitation of the study is the choice to use males from only one aggressive Drosophila line in combination with CantonS females, that do not allow disambiguation between nonaggression-related factors, such as hybrid vigor and aggression-related factors influencing mating and lifespan.
We would like to clarify the points raised in the eLife assessment.
The report states that we relied on a single line of hyper-aggressive males tested with CantonS females, and implies that Bully and Cs have not co-evolved. This is a misunderstanding: Bully flies were derived from Cs population. Thus, Bully and Cs have co-evolved. In addition to the Bully A line presented in the main figures of the manuscript, we replicated several of our findings with a second independent selected line, Bully B. Results from courtship assays involving both Bully A and Bully B couples males and females were presented in Figure Supp1. We apologies for not having made this more explicit in the original manuscript, which we will correct. These experiments should alleviate the concerns from the reviewers; they demonstrate that our conclusions are supported by two independent hyper-aggressive lines, and these include assays with selected male and female flies.
Public Reviews:
Reviewer #1 (Public review):
Summary:
This study asks how selection for male aggressiveness affects life-history and reproductive fitness traits in Drosophila melanogaster males.
Strengths:
Multiple comprehensive assays are used to address the question.
We thank the reviewer for recognizing these strengths.
Weaknesses:
(1) The flies used for comparisons are inadequate. Behavioral assays compare Bully males mated to non-coevolved Cs females with Cs males mated to coevolved Cs females.
We thank the reviewer for this comment, which made us realize that we had not sufficiently highlighted some of our experiments. The Bully lines used in our work were derived from Canton-S flies and thus did co-evolve with Cs. As originally described by Penn et al. (2010), highly aggressive “Bully” lines were generated through selective breeding from Canton-S males that consistently won aggressive encounters. After 34–37 generations, stable Bully lines were established. Thus, Bully and Cs flies have co-evolved and 2) the selection applied was male-specific. Independent selection replicates produced distinct lines, including Bully A and Bully B. Previous studies only characterized Bully A (Penn et al., 2010; Chowdhury et al., 2017), but our work includes both Bully A and Bully B (Fig. S1).
The rationale for pairing Bully or Cs males with Cs females (with which both male types co-evolved) follows the approach used by Dierick et al. (2006), who investigated how the male-specific selection for aggression affected courtship and mating behaviors by testing them with standard Canton-S females. This design allows to isolate the effects of male genotype and behavior on courtship and mating outcomes, avoiding confounding effects from female behavioral changes.
We initially compared selected Bully pairs (Bully males × Bully females) (Fig. S1) with Cs pairs and observed similarly shortened mating durations in both Bully × Bully and Bully × Cs matings (Fig. S1, Fig. 1F and G). Thus, the reduction in mating duration arises specifically from Bully males. We therefore chose to use Cs females as a standard background to assess the consequences of male-specific selection for aggression on reproductive behaviors.
(2) Lifespan analysis is done on male progeny of Cs females mated to either genetically more distant Bully or co-evolved Cs males; the longer lifespan and performance on the former is interpreted as a trade-off with aggressiveness, rather than a simple explanation of hybrid vigor.
We appreciate this comment, which again stems from a poor explanation from our part about the origin of the Bully line in the original manuscript. The Bully flies were derived from the same original population as the Cs line. Hybrid vigor typically arises when crossing individuals from distinct populations, which is not the case here as both Bully and CS come from the same population.
To further support our conclusions, we conducted additional experiments using progeny from within-line crosses (Bully males × Bully females) and results revealed the same phenotype: the progeny of these flies also exhibited significantly longer lifespans than Cs males x Cs females progeny. This finding argues against hybrid vigor as the main explanation for the observed phenotype, since both the Bully and Cs crosses result in inbreeding, yet give longer lifespan in Bully. We will include these additional longevity data (currently not included in the manuscript) to strengthen our results and reinforce our interpretation.
(3) Differences in CHCs between Bully and Cs males and Cs females mated to those males are not shown to cause differences in measured behavioral outcomes.
We thank the reviewer for raising this important point regarding causality. One way to establish a causal link between differences in CHCs observed in Bully and Cs flies and the corresponding behavioral outcomes would be to experimentally manipulate CHC profiles. For instance, one could perfume oenocyte-less males with the compounds found in higher abundance in Bully flies, then perform behavioral assays to assess causality. We agree that such experiments would be highly informative in determining the functional roles of specific CHCs elevated in Bully males. However, this approach is technically challenging, as the perfuming technique must be optimized to transfer precise amounts of each compound. For example, this method can be used to gradually perfume flies to assess dose–response behavioral effects, whereas matching exactly the natural concentrations found in individuals, especially given inter-individual variability, remains difficult.
We considered conducting such experiments during our study but did not pursue them for these technical reasons. Nevertheless, we can include a statement in the Discussion acknowledging this as an important future direction to test the causal relationship between CHC variation and behavior.
Reviewer #2 (Public review):
Summary:
The authors compare "Bully" lines, selected for male aggression, to Canton-S controls and find that Bully males have lower mating success, shorter mating durations, and remate sooner. Chemical analyses show Bully males have distinct cuticular hydrocarbons (CHC) signatures and transfer markedly less cVA to females, offering a plausible mechanistic link to weaker mate-guarding.
Paradoxically, Bully males live longer and remain fertile at older ages when CS males no longer mate, indicating a shift in the reproduction-survival trade-off in aggression-selected populations.
Importantly, the work sheds light on proximate mechanisms, demonstrating that shifts in CHCs and pheromone transfer co-occur with changes in fitness traits, thus offering new entry points for understanding life-history evolution.
We thank the reviewer for this positive summary of our work.
Strengths:
The manuscript's strengths lie in its comprehensive and integrative approach framed within an evolutionary context. By combining behavioral assays, chemical profiling, and lifespan measurements, the authors reveal a coherent pattern linking aggression selection to life-history trade-offs. The direct quantification of cVA in female reproductive tracts after mating provides a particularly compelling mechanistic correlate, strengthening the link between behavior and chemical signaling. Findings on altered 5-T and 5-P levels further highlight how chemical communication shapes mating and mate-guarding strategies. Analytical approaches are largely rigorous, and the results provide valuable insights into the pleiotropic effects of selection on socially relevant traits. The study will be of interest to Drosophila biologists working on sexual selection, behavioral evolution, and aging.
We thank the reviewer for recognizing the integrative design and mechanistic contributions of our study.
Weaknesses:
The weaknesses are primarily conceptual rather than procedural. The generality of the findings is uncertain, as selection appears to be represented by only one (and a second closely related) Bully line, limiting conclusions about selection responses versus line-specific drift or founder effects. The causal link between aggression selection and increased longevity is not established: the data show a correlated shift but do not identify mechanisms underlying lifespan extension. In several places, the manuscript uses causal language (e.g., that selection 'influences' longevity or mating strategy) where association would be more accurate; this should be toned down to avoid overstatement. Ecological relevance is also not addressed, since laboratory conditions may bias the balance between costs and benefits of aggression compared with variable natural environments. Addressing these points would strengthen both the impact and clarity of the study.
(1) Generality of findings and potential line effects
We agree that our results presented in the main figures of the manuscript relied mainly on one Bully line (Bully A). To address potential line-specific effects, we replicated key courtship experiments with another independent line, Bully B, selected in parallel from the same Canton-S stock but through distinct selection replicates. The results obtained from Bully B closely matched those from Bully A, suggesting that the observed phenotypes are consistent consequences of aggression selection rather than random drift or founder effects.
(2) Causality versus correlation
We concur that some sentences in the manuscript could overstate causal interpretations. We will revise the text to clearly distinguish correlation from causation and to avoid implying direct causal relationships where data only support association.
(3) Ecological relevance
We appreciate this point. Our experiments were performed under controlled laboratory conditions, which may not fully capture the ecological contexts shaping the costs and benefits of aggression. We will acknowledge this limitation and expand the Discussion to consider how environmental variability could modulate the fitness trade-offs associated with aggression in natural populations.
We thank both reviewers for their constructive feedback, which will help us strengthen the rigor and clarity of the manuscript. We believe that the additional results and revisions will satisfactorily address their concerns.
