Author response:
The following is the authors’ response to the original reviews.
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 Canton-S 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 Canton-S 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, 1) 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.
Recommendations for the authors:
Reviewing Editor Comments:
The major weaknesses raised by the reviewers, namely the flies used in the study (CsxBully compared to CsxCs) where the effect on lifespan could be explained by hybrid vigor and the use of only one Bully and one Cs line that does not allow to link unambiguously the observed effect to the selection for aggression, should be addressed by a different experimental design and additional lines to exclude the effect of non-aggression related factors.
We thank the Reviewing Editor for these comments.
(i) Experimental design and hybrid vigor:
Hybrid vigor typically arises from crosses between genetically divergent populations. In our study, Bully lines were derived from Canton-S background and are not therefore not genetically distant from controls. To directly address this concern, we included new data from Bully × Bully pairs (Figure 1), using independently selected Bully lines. These experiments reproduce the key aggression and courtship phenotypes observed in Cs × Bully assays, indicating that the effects are not attributable to hybrid vigor.
(ii) Use of additional selected lines:
We now include data from two independently selected lines (Bully A and Bully B), both derived from Cs, which show consistent behavioral phenotypes. This supports the conclusion that the observed effects are associated with selection for aggression rather than line-specific artifacts. We note that generating such lines is time- and labor-intensive, and only a few laboratories have established aggression-selected lines in Drosophila melanogaster (e.g., Penn et al., 2010; Dierick et al., 2006; Edwards et al., 2006). Accordingly, we have revised the manuscript to explicitly acknowledge this limitation and to frame our conclusions in terms of association rather than causation.
Reviewer #1 (Recommendations for the authors):
I can't see any way to interpret the data using CsxCs vs CsxBully comparisons.
We thank the reviewer for this important point. This concern appears to arise from the assumption that Bully and Cs represent genetically distinct or non-coevolved populations. However, Bully lines were directly derived from Cs and therefore share a common genetic background. We have clarified this point in the Introduction (lines 104-106) and Results (lines 132-137).
Importantly, we now include additional data showing that key phenotypes, including reduced mating duration, are also observed in Bully × Bully pairings and across independently selected Bully lines (new Figure 1). These results demonstrate that the observed effects are driven by the male genotype and do not depend on the female background.
Because selection for aggression was applied specifically to males, we used Cs females as a standardized background to isolate male-specific effects while minimizing variability arising from female genotype or behavior. This rationale is now explicitly stated in the Results (lines 163-166) and at the beginning of the Discussion (lines 326-330). This experimental design allows interpretation of male-specific effects, and the observed differences cannot be attributed to cross design artifacts or hybrid vigor.
Reviewer #2 (Recommendations for the authors):
Major comments:
(1) Several passages currently imply causality, whereas the data support correlations between selection and trait differences rather than direct causation. This overstatement also appears in section subheadings within the Results, such as "Hyper-aggressive males display reduced mate-guarding efficiency, without compromising female fertility." Please consider toning down the wording by replacing active causal verbs with more neutral phrasing. Additionally, it would be important to include a clear, explicit sentence in the Discussion acknowledging this caveat, as the existing phrase "is associated with changes in reproductive traits" does not fully convey this nuance.
We thank the reviewer for this important comment. We have revised the manuscript throughout, including Results subheadings, to replace causal language with association-based phrasing. We also rephrase the first sentence of the Discussion to clarify that our conclusions are correlational (see line 320).
(2) Figure 1 would benefit from a simple schematic of the behavioral paradigm and the arena, since the authors' arena design minimizes manual handling; a cartoon would help readers quickly grasp the assay flow and the conditions under which interactions occur.
We thank the reviewer for this helpful suggestion. We have added a schematic to Figure 1 illustrating the behavioral paradigm and arena design. Additional details are provided in the Materials and Methods (Trannoy et al., 2015). This improves clarity and accessibility of the experimental design.
(3) In Figures 2A-B and A'-B', the higher post-mating UWE in Bully males is intriguing, but these panels do not actually measure the refractory period. It would be helpful to include 'latency' in the first UWE after mating in these swapped-female conditions. This could also be repeated with pheromone-standardized (cVA/CHC-equalized) decapitated females to disentangle effects of female pheromone load from male sensory perception. In addition, a baseline courtship control (naive males with decapitated virgins) is necessary to test whether Bully males simply have a lower threshold for initiating courtship.
We thank the reviewer for this suggestion. The referenced panels are now shown in Figure 3. We quantified post-mating courtship latency; however, latencies were very short across conditions, and no differences were observed between genotypes. We therefore revised the text to interpret these results in terms of post-mating courtship motivation rather than refractory period. The baseline courtship control with decapitated virgins is provided in Fig 2G. These changes clarify the interpretation of post-mating behavior and address the reviewer’s concerns.
(4) Related to my above point, the results in Figure 2B-B′ raise the possibility that Bully males have reduced perception or neural sensitivity to anti-aphrodisiac pheromones deposited by CS males, which could account for their elevated post-mating courtship; the authors might consider experiments that directly test male sensory responsiveness to these cues or mention this possibility in the Discussion.
We thank the reviewer for this point. We performed additional assays to test males’ sensory responsiveness using binary choice assays and measured the time spent performing UWE towards decapitated females versus males. These results were added in Figure 3-Figure Supp 1, and indicate that both Cs and Bully males displayed courtship preferentially towards females, providing a control for sensory perception.
(5) In multiple figure panels, virgin and mated females are depicted with the same symbols, which makes interpretation confusing.
Thank you for pointing this out. We have updated the figure panels to use distinct symbols for virgin and mated females to improve clarity.
(6) For Figure 4, it would be helpful to provide standalone KM curves for Bully versus CS males, including a separate panel for isolated (never-mated) males, and present mating counts in a separate panel while reporting survival models that incorporate mating frequency (or use it as a time-dependent covariate). Although Figures 4C-D report median survivals, full KM plots and an isolated-male curve are important since mating itself elevates mortality and can otherwise confound intrinsic lifespan differences.
Thank you for this important point to improve clarity of this figure. We have reorganized this figure (now Figure 5) to now, present survival curves first (isolated and group-housed males), followed by lifetime mating counts. This reorganization separates survival from mating activity and addresses the potential confounding effect of mating on lifespan. For the lifetime mating data, we used bar plots rather than curves to better visualize individual mating events.
(7) The following sentences overstate the results and imply causality; consider toning down: "These findings suggest that 5-P and 5-T might contribute to promoting remating in females that have previously mated with Bully males (Figure 2F' and G'). Given that Bully males also showed higher levels of both 5-P and 5-T compared to naïve Cs males (Figure 3B), it is likely that the elevated levels of these compounds observed in females result from their transfer during mating."
We thank the reviewer for this important point. We have rephrased these sentences to remove causal language and instead describe associations between CHC profiles and behavioral outcomes. In particular, statements implying that 5-P and 5-T promote remating or are directly transferred during mating have been revised to reflect correlational evidence only (see lines 253-256).
(8) It is not entirely clear how aggression was quantified in each generation, what proportion of males were selected to breed, and whether the findings generalize beyond a single Bully line (Figure Supplement 1 shows data from a closely related Bully line). Without independent replicate lines or sham-selected controls, it remains difficult to rule out drift or line-specific artifacts, and this limitation should be explicitly acknowledged.
We thank the reviewer for this important point. We have clarified the aggression selection procedure by adding methodological details from Penn et al., including how aggression was quantified and how breeders were selected (lines 104-106 and 131-137). Briefly, independent selection replicates were initiated from the same Canton-S population, generating three lines (Bully A, B, and C), which were maintained separately.
To address generality, we now include data from multiple lines. In particular, a new Figure 1 presents aggression and courtship phenotypes across Bully A, B, and C, and key behavioral results are consistent across independent lines.
We acknowledge that additional independent lines would further strengthen generality; this limitation is now explicitly stated in the Discussion (lines 325-326).
These additions clarify the selection procedure and support that the observed phenotypes are associated with aggression selection rather than line-specific artifacts.
Minor Comments:
(1) Exact sample sizes for every experiment should be included in the main figure legends.
Thank you. We have added the number of replicates in each figure legends.
(2) In Figure 1-Supplement 1, the orientation for depicting mating success is reversed compared to Figure 1, which is a bit jarring; it would be clearer to keep the orientation consistent with the main figure.
Thank you. We have incorporated the results initially presented in Figure 1-Sup 1 into a new Figure 1 with additional results, and have taken into account reviewers’ comment.
(3) For multivariate analyses, I suggest including important details such as group sample sizes, p-value, and the percent variance, etc., in the figure legend rather than keeping this only in Supplementary Table S1.
We have inserted these details directly into the figure legends for clarity.
(4) Why was the food cup used for arenas where decapitated virgins were used in mating assays?
Thank you for pointing this. We now have clarified the experimental procedure in the M&M of the revised manuscript (lines 465-469).
(5) For cartoons in Figure 2, the current yellow background makes it very difficult to distinguish flies drawn in yellow or green. Please adjust to a higher-contrast background or add darker outlines so that the cartoons are clearly legible.
Thank you. We have increased the contrast of the female bodies to ensure the cartoons are clearly distinguishable (now figure 3).
(6) Addition of line numbers in the manuscript would be helpful during the review process.
Line numbers have been added throughout the manuscript.
(7) I noticed a few typos in the manuscript. For example, in the Introduction, "seminal fuids" should be corrected to "seminal fluid." In the Discussion, the phrase "CHCs profiles compared those" requires a "to" before "those." Please carefully review the manuscript for similar errors.
Thank you for pointing this out. We carefully reviewed the manuscript for typos and corrected all identified errors.
