Aggressive trait selection influences life-history trade-offs, favoring survival over reproductive success in Drosophila melanogaster males

Selection for male aggression is associated with changes in reproductive traits, chemical signaling and lifespan in Drosophila melanogaster | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Selection for male aggression is associated with changes in reproductive traits, chemical signaling and lifespan in Drosophila melanogaster Anthony Defert , Romane Gout , Gaelle Pennot , Fanny Jamme , Amandine Castex , Anissa Handjar , Thomas Guilleman , Jean-Christophe Billeter , View ORCID Profile Séverine Trannoy doi: https://doi.org/10.1101/2025.06.02.657383 Anthony Defert 1 Research Center on Animal Cognition (CRCA), Center for Integrative Biology, Toulouse University, CNRS, UPS , Toulouse, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Romane Gout 1 Research Center on Animal Cognition (CRCA), Center for Integrative Biology, Toulouse University, CNRS, UPS , Toulouse, France 2 Institute for Zoology, Halle-Wittenberg University , Halle (Saale), Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gaelle Pennot 1 Research Center on Animal Cognition (CRCA), Center for Integrative Biology, Toulouse University, CNRS, UPS , Toulouse, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Fanny Jamme 1 Research Center on Animal Cognition (CRCA), Center for Integrative Biology, Toulouse University, CNRS, UPS , Toulouse, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Amandine Castex 1 Research Center on Animal Cognition (CRCA), Center for Integrative Biology, Toulouse University, CNRS, UPS , Toulouse, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anissa Handjar 1 Research Center on Animal Cognition (CRCA), Center for Integrative Biology, Toulouse University, CNRS, UPS , Toulouse, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas Guilleman 1 Research Center on Animal Cognition (CRCA), Center for Integrative Biology, Toulouse University, CNRS, UPS , Toulouse, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jean-Christophe Billeter 3 Groningen Institute for Evolutionary Life Sciences, University of Groningen , 9747AG Groningen, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Séverine Trannoy 1 Research Center on Animal Cognition (CRCA), Center for Integrative Biology, Toulouse University, CNRS, UPS , Toulouse, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Séverine Trannoy For correspondence: severine.trannoy{at}utoulouse.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Life history traits are all essential traits linked to development, survival and reproduction, directly influencing individual fitness. Limited environmental or physiological resources force organisms to balance competing demands, leading to fundamental trade-offs, particularly between survival and reproduction. Male aggression is often assumed to enhance reproductive success, yet its broader association with other fitness-related traits remains unclear. To address this, we used Drosophila melanogaster Bully lines selected for high levels of male aggression, to examine relationships between aggression, chemical signaling, and essential life-history traits including reproductive behaviors and lifespan. Our results reveal that increased male aggressiveness shifts the balance of a major life-history trade-off, favoring survival over reproductive success. Bully males exhibited lower mating success, shorter mating duration and less effective chemical mate-guarding. However, this was traded-off with increased lifespan, enabling more reproductive opportunities. We also uncover potential mechanistic underpinnings of the reproductive deficits. Bully males showed differences in their cuticular hydrocarbon (CHCs) profiles and transferred lower levels of cVA to females, a mate-guarding pheromone, thereby weakening their post-mating strategy. Overall, our findings indicate that selection for male aggression is associated with a shift in a key life-history trade-off, highlighting aggression as an evolutionary force shaping adaptation of life-history traits and providing a foundation for future genetic and mechanistic studies. INTRODUCTION Life history traits, such as development time, fertility, survival, and reproductive success, are key phenotypic components that directly influence individual fitness ( Braendle and Paaby, 2024 ; Roff, 1992 ; Stearns, 1992 ). These traits often face trade-offs: investing in one can compromise another ( Fisher, 1930 ; Stearns, 1989 ; Williams, 1966 ). For instance, high reproductive effort may reduce survival due to the energetic costs of gamete production or increased mortality associated with mating behaviors or competition ( Flatt, 2011 ; 2020 ). Trade-offs also emerge between pre- and post-copulatory strategies ( Simmons, 2001 ; Warner et al., 1995 ). Males prioritizing pre-copulatory investment, such as aggressive competition for mates, may have limited resources for post-copulatory investment linked to sperm competition such as mate guarding ( Andersson, 1994 ; Brennan and Orbach, 2021 ). Understanding how such trade-offs are shaped by natural selection offers insight into how organisms optimize fitness in competitive social environments ( Roff, 1992 ; Stearns, 1976 ). The fruit fly Drosophila melanogaster (D. mel . ) , provides a powerful model for studying life-history trade-offs, with well-characterized developmental, behavioral, and reproductive traits ( Flatt, 2020 ). These traits are shaped by both genetic and environmental factors and can evolve under ecological pressures. Faster development, earlier reproduction and high mating rate, for instance, are advantageous in changing environments but come at the cost of reduced longevity and fecundity ( Partridge and Farquhar, 1981 ; Stearns, 2000 ). Experimental manipulations of environmental conditions such as temperature and food availability reveal how external factors shape trade-offs between survival and reproduction ( Chippindale et al., 1996 ; Marshall and Sinclair, 2010 ). Beyond development and fecundity, D. mel . serves as a model to study a wide range of social behaviors, including aggregation, aggression, and courtship, that are largely mediated by chemical communication ( Kim et al., 2017 ; Kohl et al., 2015 ). Cuticular hydrocarbons (CHCs) such as some pheromones play diverse roles in social interaction: 7-Tricosene (7-T), a male-specific compound, increases female receptivity and modulates aggression ( Grillet et al., 2006 ; Wang et al., 2011 ); cis-vaccenyl acetate (cVA), a male-transferred pheromone, attracts females, repels other males, and promotes aggregation ( Kurtovic et al., 2007 ; Laturney and Billeter, 2016 ); and the female-specific hydrocarbon 7,11-heptacosadiene (7,11-HD) strongly elicits male courtship and supports species-specific mate recognition ( Toda et al., 2012 ). Among these interactions, male-male competition plays a central role in shaping reproductive strategies. Pre-copulatory competition involves resource defense, courtship displays, and aggressive interactions to secure mating opportunities. Courtship in D . mel . includes stereotyped behaviors such as orientation, wing extensions (producing courtship song), tapping, licking, and attempted copulation ( Yamamoto and Koganezawa, 2013 ). Successful courtship displays lead to female acceptance, outcompeting other males that fail to meet the female’s preferences ( Baxter et al., 2018 ; Hindmarsh Sten et al., 2025b ). Aggression intensifies this competition: more aggressive or dominant males often monopolize access to females ( Dow and von Schilcher, 1975 ; Filice and Dukas, 2019 ; Gao et al., 2024 ; Nandy et al., 2016 ; Prunier and Trannoy, 2024 ). Aggressive behaviors range from wing threats and lunging to high-intensity physical confrontations like tussling and boxing ( Chen et al., 2002 ). Beyond aggressive patterns, males also produce agonistic wing flicks when competing for access to females, which can interfere with a female’s auditory perception of rival males and thereby influence mate choice ( Hindmarsh Sten et al., 2025a ). These behaviors promote territorial dominance and enhance mating success ( Gao et al., 2024 ), although subordinate males may also achieve reproductive success through first access to females and prolonged copulation ( Filice and Dukas, 2019 ). Post-copulatory competition further shapes male reproductive success. After mating, males use multiple strategies to ensure paternity, including the transfer of seminal fluid proteins (SFPs) including Sex Peptide (SP) and Accessory gland proteins (Acps), which promote sperm storage, stimulate oviposition, and reduce female receptivity to further mating ( Chapman et al., 2003 ; Fricke et al., 2009 ). These factors also contribute to the formation of a mating plug that retains sperm, acting as a passive mate-guarding strategy ( Avila et al., 2011 ; Dunham and Rudolf, 2009 ). In parallel, chemical mate guarding via pheromone transfer (e.g. cVA and 7-T) reduce female attractiveness and their likelihood of re-mating ( Billeter et al., 2009 ; Gillott, 2003 ; Laturney and Billeter, 2016 ; Miyamoto and Amrein, 2008 ; Scott, 1986 ; Zawistowski and Richmond, 1986 ). Male reproductive behavior is plastic: in the presence of rivals, males extend their mating duration ( Bretman et al., 2009 ; Bretman et al., 2013 ; Kim et al., 2013 ), increase the number of sperm in their ejaculate ( Garbaczewska et al., 2013 ), adapt seminal fuid content in their ejaculate ( Hopkins et al., 2019 ), and present higher levels of aggression toward opponents to guard their mates ( Baxter et al., 2015 ; Bretman et al., 2011 ; Dore et al., 2021 ; Mazzi et al., 2009 ). Together, this demonstrates that male-male aggression influences access to mates and reproductive outcomes through both pre- and post-copulatory competition. While aggression can enhance male mating success in natural settings, the relationship between aggression and reproductive fitness is not always straightforward. In a previous study, Penn et al. ( Penn et al., 2010 ) generated “ Bully ” flies through 37 generations of selection for aggression starting from a Canton-S ( Cs ) population. In the first step, Cs winner males from aggressive encounters were allowed to mate with Cs females. In the resulting F1 progeny, males displaying higher levels of aggression were again selected and mated with their female siblings. The resulted hyper-aggressive males frequently escalated to boxing, the most intense aggression phenotype ( Chowdhury et al., 2017 ), but showed reduced reproductive success with virgin females compared to unselected control males ( Penn et al., 2010 ). This suggests that although aggression may enhance competitive ability, it can also incur fitness costs. This raises important questions about the role of aggression in shaping life-history trade-offs, such as: to what extent does aggression influence life history traits related to reproduction, and does it affect the balance reproduction vs survival? To address these questions, we investigated whether D. mel . males from Bully lines exhibit changes in life-history trade-offs compared to the unselected starting wild-type flies ( Cs lines). We found that hyper-aggressive Bully males show reduced reproductive success and shorter mating duration across their lifespan, indicating lower pre-copulatory success. Their mates present higher remating rate and increased attractiveness, suggesting that mate-guarding strategy of Bully males is less efficient. However, this poor pre- and post-copulatory success was traded-off with an increased lifespan, which is accompanied by late-life reproductive success. We then provided a mechanistic basis for the reduced copulatory success of Bully males. They showed distinct CHCs profiles compared to Cs males, which may contribute to their lower mating success. Moreover, they transferred to females lower levels of cVA, a mate-guarding pheromone, to females, potentially decreasing efficiency of their post-mating strategy. Our findings underscore the complex interplay between aggression, reproductive behavior, survival, and chemical signaling, providing insights into the evolutionary dynamics of life-history trade-offs in competitive social contexts. RESULTS Males exhibiting higher aggression intensity also exhibit shorter mating durations To determine whether selection for male aggressive traits may also be associated with changes in reproductive behaviors, we employed three male lines selected for increased aggression. In a previous study, Penn et al. ( Penn et al., 2010 ) generated the so-called “ Bully ” flies through 37 generations of artificial selection for male aggression starting from a Canton-S (Cs) population. Briefly, males that won aggressive encounters were allowed to mate with Cs females. In the resulting F1 progeny, males exhibiting higher levels of aggression were again selected and mated with their female siblings. Following the same experimental procedure, several independent selections were performed in parallel, giving rise to the Bully A, Bully B and Bully C lines. These Bully lines were originally derived from Cs flies and do not represent distinct populations per se , but rather result from male-specific selection on aggressive behavior. Males from these three lines ( Bully A, B, and C) were tested in standardized aggression assays ( Trannoy et al., 2015b ) ( Figure 1A ). Across same-line contests, males from all three Bully lines did not differ from Cs males in latency to lunge ( Figure 1B ), but displayed significant increase in the number of lunges ( Figure 1C ), the number of boxing events ( Figure 1D ), and boxing frequency (defined as the proportion of male pairs displaying at least one boxing event during the 10-min observation period) ( Figure 1E ) compared to Cs males. However, the magnitude of this phenotype differed across lines: Bully A and Bully B males exhibited higher aggression levels than Bully C males, which showed an intermediate phenotype ( Figure 1E ). Download figure Open in new tab Figure 1. Courtship and aggressive behaviors in Cs, Bully A, Bully B and Bully C lines. (A) Schema of the experimental set up to record aggressive and courtship behaviors. Couples of flies and presence of food cup are mentioned according to the figure of this article. (B) Latency to lunge of Cs, Bully A, Bully B and Bully C males in aggression assay with a territory against a male of the same line (n Cs =29, n BullyA =24, n BullyB =12, n Bully C =11, LM and permutation test (Monte-Carlo test): P=0.136). (C) Number of lunges of Cs, Bully A, Bully B and Bully C male pairs in aggression assays (n Cs =29, n Bully A =24, n Bully B =12, n Bully C =11, GLM quasi-Poisson and type II analysis of deviance table (Likelihood Ratio Test): P=1.622×10 −10 , LRχ 2 =48.556, post-hoc: P Cs_ Bully A <0.001, z ratio= −4.495, P Cs_ Bully B <0.001, z.ratio= −5.911, P Cs_ Bully C =0.018, z.ratio= −2.836, P Bully A_ Bully B =0.042, z ratio= −2.308, P Bully A_ Bully C =0.232, z ratio= 1.195, P Bully B_ Bully C =0.018, z ratio=2.797). (D) Number of boxing events of Cs, Bully A, Bully B and Bully C male pairs in aggression assays (n Cs =29, n Bully A =24, n Bully B =12, n Bully C =11, GLM Poisson and type II analysis of deviance table (Likelihood Ratio Test): P=2.2×10 −16 , LRχ 2 =2511.9, post-hoc: P Cs_ Bully A <0.001, z ratio= −6.819, P Cs_ Bully B <0.001, z.ratio= −6.839, P Cs_ Bully C <0.001, z.ratio= −3.649, P Bully A_ Bully B =0.728, z ratio= −0.347, P Bully A_ Bully C <0.001, z ratio=5.392, P Bully B_ Bully C <0.001, z ratio=5.366). (E) Non-competitive mating success of Cs, Bully A, Bully B and Bully C males paired with a virgin Cs female (n Cs =37, n Bully A =31, n Bully B =33, n Bully C =21, GLM binomial and type II analysis of deviance table (Likelihood Ratio Test): P=0.012, LRχ 2 =11.040, post-hoc: P Cs_ Bully A =0.934, z ratio=0.728, P Cs_ Bully B =0.099, z.ratio=2.395, P Cs_ Bully C =0.868, z.ratio=1.060, P Bully A_ Bully B =0.206, z ratio=2.043, P Bully A_ Bully C =0.934, z ratio=0.406, P Bully B_ Bully C =0.522, z ratio=-1.513). (F) Proportion of time spent by Cs and Cs, Bully A, Bully B and Bully C males doing Unilateral Wing Extension (UWE) toward a Cs virgin female in non-competitive courtship assays (n Cs =37, n Bully A =31, n Bully B =33, n Bully C =21, LM and type II Anova table: P=0.088, F=2.235). (G) Latency to court (time difference between first interaction and first UWE) of Cs, Bully A, Bully B and Bully C males paired with a virgin Cs female (n Cs =37, n Bully A =31, n Bully B =33, n Bully C =21, LM and type II Anova table: P=0.014, F=3.707, post-hoc: P Cs_ Bully A =0.338, t.ratio= −1.740, P Cs_ Bully B =0.433, t.ratio= 1.469, P Cs_ Bully C =0.524, t.ratio= −1.127, P Bully A_ Bully B =0.015, t.ratio=3.101, P Bully A_ Bully C =0.683, t.ratio=0.661, P Bully B_ Bully C =0.099, t.ratio= −2.364). (H) Latency to mate (time difference between first interaction and start of the mating) of Cs, Bully A, Bully B and Bully C males paired with a virgin Cs female (n Cs =36, n Bully A =29, n Bully B =24, n Bully C =19, LM and type II Anova table: P=8.527×10 −4 , F=5.970, post-hoc: P Cs_ Bully A =0.482, t.ratio= −1.413, P Cs_ Bully B <0.001, t.ratio= −4.191, P Cs_ Bully C =0.581, t.ratio= −1.062, P Bully A_ Bully B =0.038, t.ratio= −2.725, P Bully A_ Bully C =0.863, t.ratio=0.173, P Bully B_ Bully C =0.041, t.ratio= 2.615). (I) Mating duration of Cs, Bully A, Bully B and Bully C males paired with a virgin Cs female (n Cs =36, n Bully A =29, n Bully B =24, n Bully C =19, LM and type II Anova table: P=8.527×10 −4 , F=5.970, post-hoc: P Cs_ Bully A =0.482, t.ratio= −1.413, P Cs_ Bully B <0.001, t.ratio= −4.191, P Cs_ Bully C =0.581, t.ratio= −1.062, P Bully A_ Bully B =0.038, t.ratio= −2.725, P Bully A_ Bully C =0.863, t.ratio=0.173, P Bully B_ Bully C =0.041, t.ratio= 2.615). For all graphs, different letters mean a statistical difference. Statistical details are given in Figure 1-table supplement 1. We next assessed courtship and mating behaviors by pairing males and females from the same line and scored courtship metrics. The proportion of time spent by males displaying unilateral wing extension (UWE) and latency to court did not differ significantly between lines ( Figure 1F-G ), while Bully B males exhibited a longer latency to mate compared to other pairings ( Figure 1H ). In contrast, mating duration (MD) was significantly reduced in Bully A and Bully B pairings compared to Cs ( Figure 1I ), while Bully C males again show an intermediate phenotype ( Figure 1I ). These results indicate that males from the Bully A and Bully B lines, classified as hyper-aggressive in same-line competition ( Figure 1B-E ), also exhibit reduced mating duration when paired with same-line females, whereas Bully C males do not ( Figure 1I ). Because the Bully A line (hereafter named Bully ) has been already employed in previous studies to measure differential gene expression profile ( Chowdhury et al., 2017 ; Penn et al., 2010 ), we chose to conduct subsequent experiments on this line. Overall, these results support our initial hypothesis that selection for aggressive traits may have influenced other behavioral traits linked to reproductive behaviors. Highly aggressive males exhibit reduced mating success and have shorter mating duration As the selection for aggression was male-specific ( Penn et al., 2010 ), we next aimed to focus on examining male reproductive behaviors while avoiding confounding effects of female behavioral variation. Thus, all subsequent courtship and mating assays were performed by pairing Cs or Bully A males with Cs females, which served as a standardized female background. We first assessed mating success in competitive assays, where a Cs male and a Bully male competed for access to a single female. Under these conditions, no significant mating advantage was observed for Bully males ( Figure 2A ). We then assessed mating success in non-competitive assays by pairing each male with a virgin Cs female for 30 minutes. Under these conditions, Bully males showed a significantly reduced probability of mating compared to Cs males ( Figure 2B ), despite similar levels of courtship behavior, as measured by UWE ( Figure 2C ), courtship latency ( Figure 2D ), and mating latency among successful pairs ( Figures 2E ). We next examined copulation per se and found that MD was significantly shorter in Bully males, with a reduction of approximately 25% compared to Cs males ( Figure 2F ), consistent with the phenotype observed when males were paired with females from the same line ( Figure 1I ). Importantly, this reduction in MD persisted when Bully males were paired with decapitated Cs females ( Figure 2G ), ruling out female-driven termination of mating and demonstrating that the effect is male-specific. Download figure Open in new tab Figure 2. 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 was scored when paired with a single virgin Cs female (n Cs =19, n Bully =19, GLM binomial and type II analysis of deviance table (Likelihood Ratio): Test, P=0.329, LRχ 2 =0.951). (B) In non-competitive courtship assays, the mating success of Cs and Bully males paired with a virgin Cs female were analyzed (n Cs =64, n Bully =41, GLM binomial and type II analysis of deviance table (Likelihood Ratio Test): P=0.037, LRχ 2 =4.347). (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 (n Cs =64, n Bully =41, Fisher Pitman permutation test: P=0.877, t= −0.131). (D) Latency to court (time difference between first interaction and first UWE) of Cs and Bully males paired with a virgin Cs female (n Cs =64, n Bully =41, Fisher Pitman permutation test: P=0.319, Z=1.043). (E) Latency to mate (time difference between first interaction and start of the mating) of Cs and Bully males paired with a virgin Cs female (n Cs =61, n Bully =34, Fisher Pitman permutation test: P=0.266, Z=1.170). (F) Mating duration of Cs and Bully males paired with a virgin Cs female (n Cs =61, n Bully =34, T-test: P=6.009×10 −10 , t= −6.917) or (G) a virgin Cs decapitated female (n Cs =26, n Bully =20, T-test: P=0.022, t= −2.367). (H) Mating duration of Cs and Bully males that were previously raised in group of 10 males or in social isolation (n Cs-isolated =22, n Cs-group =32, n Bully -isolated =23, n Bully -group =25, LM and type II ANOVA: F-test, P=7.582×10 −11 , F=21.669, post-hoc: P Cs-isolated_Cs-group =0.013, F=6.583, P Bully -isolated_ Bully -group =2.532×10 −4 , F=15.731, P Cs-isolated_ Bully -isolated =4.263×10 −6 , F=27.697, P Cs-group_ Bully -group =1.884×10 −4 , F=16.032). 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 1. As group-housed males have lengthened MD ( Bretman et al., 2013 ; Kim et al., 2013 ), we compared MD of singly and group-housed males. We found that both Cs and Bully group-housed males exhibited significantly extended mating durations compared to single-housed males ( Figure 1H ). However, the MD in Bully males remained shorter compared to Cs males in group-housed condition ( Figure 2H ), indicating that Cs and Bully males respond equally to early social experience Together, these results show that Bully males display reduced mating success and shorter copulation duration, indicating differences in pre-copulatory outcomes and copulatory investment. This supports our main hypothesis that selecting males for aggressive traits may also had influenced one or both pre- and post-copulatory strategies. Post-mating behaviors are affected in hyper-aggressive males Given these differences in mating performance, we asked whether they compensate through distinct post-mating strategies. To investigate this, we first quantified courtship behavior immediately after copulation by measuring unilateral wing extension (UWE) directed toward the just-mated female. While Cs males performed minimal UWE towards just-mated females ( Figure 3A ’), Bully males displayed significantly higher levels of post-mating courtship in this context ( Figure 3A ’). This effect persisted when females were swapped after mating, with Bully males showing increased UWE towards Cs -mated females ( Figure 3B–B’ ). This indicates that this phenotype reflects an intrinsic increase in courtship drive rather than a female-dependent effect. This elevated post-mating motivation led us to hypothesize that Bully males might achieve higher remating rates. To test this, we conducted sequential mating assays in which a single Cs or Bully male was consecutively paired with six virgin females over a 3-hour period and we scored the number of mating. Contrary to our expectation, Bully males did not mate with more females than Cs controls ( Figure 3C ), despite maintaining consistently shorter MD across successive mating ( Figures 3D ). As mating accompanies ejaculate transfer and incurs a reduction in sperm and seminal fluid reserve in D. mel . males ( Hihara, 1981 ; Sirot et al., 2009 ), we further examined whether shorter MD in Bully males could reflect more efficient allocation, allowing fertilization of a greater number of females. However, analysis of progeny production revealed no significant differences between Bully - and Cs -mated females across successive matings ( Figure 3E ). Thus, increased post-mating courtship in Bully males does not translate into improved reproductive output. Download figure Open in new tab Figure 3. Hyper-aggressive males exhibit changes in mate-guarding features. (A, B, F, G). Mating duration of Cs and Bully males paired with a virgin Cs female (A: n Cs =21, n Bully =22, T-test: P=2.912×10 −11 , t= −8.759, B - n Cs =24, n Bully =24, T-test: P=5.425×10 −7 , t= −5.818, F - n Cs =25, n Bully =25, T-test: P=7.139×10 −4 , t= −3.617, G - n Cs =30, n Bully =24, T-test: P=8.743×10 −5 , t= −4.255). (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 (n Cs =21, n Bully =22, Fisher Pitman permutation test: P<0.001, Z=3.239). (B’) The proportion of time spent by the males performing UWE an unfamiliar mated female was scored for 10 minutes (n Cs =24, n Bully =24, Fisher Pitman permutation test: P=0.007, Z= −2.679). (C) Total number of mating reached by Cs and Bully males over six consecutive mating with virgin Cs females (type II analysis of deviance table (n Cs =18⍰, n Bully =18⍰, Wald Chi-square test): P=0.659, χ 2 =0.195). (D) Mating duration of Cs and Bully males over six consecutive mating with virgin Cs females (n Cs =18⍰, n Bully =18⍰, LMM and Satterthwaite T-test: P interaction =0.001, t= −3.261). 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 (n Cs =18⍰, n Bully =18⍰, GLMM and Wald Chi-square test: P male_genotype =0.279, χ 2 =1.173, P mating =0.847, χ 2 =2.019). (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 ( n Cs-Cs-mated =12, n Cs- Bully - mated =15, n Bully-Cs-mated =9, n Bully - Bully - mated =10 , LMM and permutation test (Monte-Carlo test): P male_genotype <0.001, P mated_female =0.457). (G’) Proportion of Cs-mated and Bully -mated females that re-mated 12 or 14 days after a first mating (n Cs-12days =19, n Bully - 12days =15, n Cs-14days =11, n Bully - 14days =9, GLM binomial and type II analysis of deviance table (Likelihood Ratio Test): P mated_female =3.639×10 −4 , LRχ 2 =12.708, P day =0.385, LRχ 2 =0.756). 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 1. We next investigated whether Bully males differ in their ability to modify female post-mating state. During mating, males transfer pheromonal cues, including cuticular hydrocarbons (CHCs), that reduce female attractiveness to rival males. We reasoned that the shorter MD of Bully males might limit the transfer of such mate-guarding signals, thereby reducing the extent of female unattractiveness. To test this independently of female behavior, we performed binary choice assays using decapitated females previously mated with either Cs or Bully males. Both Cs and Bully males spent significantly more time courting females that had mated with Bully males ( Figure 3F ’, green vs yellow bars), indicating that these females remain more attractive and suggesting reduced transfer of inhibitory cues. To validate the experimental procedure and rule out potential deficits in sensory perception, we confirmed that both Cs and Bully males spent significantly more time performing UWE toward a decapitated female than toward a decapitated male (Figure 3-figure supplement 1). Finally, we assessed the functional consequences of this change in female state on remating behavior. Virgin Cs females were first mated with either Cs or Bully males and subsequently paired with naïve Cs males several days later. Females previously mated with Bully males, remated significantly more than females that had mated with Cs males ( Figure 3G ’, green), consistent with weaker mate-guarding. Together, these results indicate that Bully males fail to offset their poor pre-copulatory success with more efficient post-mating strategies. Despite increased post-mating courtship motivation, they do not achieve higher remating rates or reproductive output, and their mates remain more attractive and more likely to remate. This dissociation indicates that increased courtship motivation is not predictive of reproductive success in this context. These findings suggest that selection for aggressive traits in males appears to compromise both pre- and post-copulatory aspects of male reproductive performance. Hyper-aggressive males present differences in their CHCs profiles and transfer lower quantities of mate-guarding pheromones to females To investigate mechanisms underlying reduced mating success ( Figure 2B ) and changes in mate-guarding behaviors ( Figure 3F-G ) in Bully males, we quantified the CHCs profiles of naïve Cs and Bully males. PCA analysis revealed a clear separation between the two groups, indicating distinct CHCs profiles between naïve Cs and Bully males ( Figure 4A ). Quantification of individual compounds showed that Bully males exhibited significantly higher levels of alkanes, monoenes, and methyl-alkanes ( Figure 4B , Table 1 ). View this table: View inline View popup Download powerpoint Table 1: 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 986 are given in Figure 1-table supplement 1. Download figure Open in new tab Figure 4. 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 (n Cs =10⍰, n Bully =10⍰, PerMANOVA: P=0.002, F=6.789, R 2 =0.274). (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 (n Cs =10⍰, n Bully =10⍰, LMM and permutation test (Monte-Carlo test): P=0.009, post-hoc: P Alkanes =0.036, P Monoenes =0.002, P Methyl alkanes =0.048, P nC21 <0.001, P C22:1(9) =0.892, P C22:1(7) =0.568, P cVA =0.731, P nC22 =0.854, P C23:1(9) =0.175, P C23:1(7) =0.062, P C23:1(5) <0.001, P nC23 =0.014, P C24:1(9) =0.345, P C24:1(7 <0.001, P 24:1(5) =0.015, P nC24 =0.002, P 2MeC24 =0.548, P C25:1(9) =0.524, P C25:1(7) <0.001, P C25:1(5) <0.001, P nC25 <0.001, P 2MeC26 =0.159, P C27:1(7) <0.001, P nC27 =0.078, P 2MeC28 =0.003, P nC29 =0.005). (C) PCA analysis on mean concentration for each CHCs compound of virgin, Cs-mated and Bully -mated females (n virgin =10⍰, n Cs-mated =16⍰, n Bully - mated =14⍰, PerMANOVA: P=0.017, F=2.594, R 2 =0.123, post-hoc: P virgin_Cs-mated =0.001, F=19.434, R 2 =0.447, P virgin_ Bully - mated =0.034, F=2.266, R 2 =0.093, P Cs-mated_ Bully - mated =0.746, F=0.428, R 2 =0.015). (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 (LMM and permutation test (n virgin =10⍰, n Cs-mated =16⍰, n Bully - mated =14⍰, Monte-Carlo test: P=0.618) Even though the p-value was not significant, post-hoc: P Alkanes =0.705, P Monoenes =0.294, P Dienes =0.715, P Methyl alkanes =0.750, P nC21 =0.158, Pn C22 =0.927, P 7,11C23:2 <0.001, P 2MeC22 <0.001, P C23:1(9) =0.023, P C23:1(7) =0.001, P C23:1(5) =0.016, P nC23 =0.908, P nC24 =0.899, P 7,11C25:2 <0.001, P 2MeC24 <0.001, P C25:1(9) =0.832, P C25:1(7) =0.047, P C25:1(5) <0.001, P nC25 =0.769, P 9,13C27:2 <0.001, P 7,11C27:2 =0.747, P 2MeC26 =0.884, P C27:1(9) =0.016, P C27:1(7) =0.625, P C27:1(5) <0.001, P nC27 =0.367, P 9,13C29:2 <0.001, P 7,11C23:2 =0.899, P 2MeC28 =0.413, P C29:1(9) <0.001, P C29:1(7) <0.001, P nC29 =0.022). 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 (n virgin =10⍰, n Cs-mated =16⍰, n Bully-mated =14⍰, P C23:1(6) <0.001, P C25:1(6) <0.001, P cVA =0.001). (F) Mating duration of Cs and Bully males with a Cs female (n Cs-mated =17, n Bully -mated =14, T-test: P=0.003, t= −3.359). (F’) cVA concentration measured within Cs- and Bully -mated genitalia tract right after mating (n Cs-mated =17, n Bully - mated =14, Fisher Pitman permutation test: P=0.001, Z= −2.390). 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 1. We next examined whether mating with Bully males induces changes in CHC profiles of females compared to those that have mated with Cs males. To test this hypothesis, we extracted CHCs from the cuticles of Cs virgin females and Cs females mated to either a Cs or Bully males and compared their CHCs profile. PCA analysis showed that Cs virgin females form a distinct cluster from mated females ( Figure 4C ), indicating that mating induces substantial changes in female CHC profiles. The first two principal components accounted for most of the variance, and several compounds, including C23:1(6), C25:1(6), and cVA, contributed strongly to this separation (Figure 4-figure supplement 1). Consistent with this, individual compound analysis revealed that male-specific CHCs, such as 7-tricosene (7-T), 9-tricosene (9-T), and cVA, were significantly increased in mated females compared to virgins ( Figure 4D–E , Table 2 ). These results are consistent with previous studies showing that 7-T and 9-T are acquired by females during mating ( Everaerts et al., 2010 ; Laturney and Billeter, 2016 ). View this table: View inline View popup Download powerpoint Table 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 1. We then compared CHC profiles between females mated with Cs or Bully males. While both groups exhibited the expected increase in male-derived compounds, females mated with Bully males showed significantly higher levels of 5-tricosene (5-T) and 5-pentacosene (5-P) ( Figure 4D , Table 2 ). As Bully males also showed higher levels of both 5-P and 5-T compared to naïve Cs males ( Figure 4B ), it is possible that the elevated levels of these compounds observed in females could result from their transfer during mating. cVA, a male-specific compound that acts as an anti-aphrodisiac and a mate-guarding molecule, is primarily transferred to the female’s genital tract during mating, with only trace amounts deposited on the cuticle ( Laturney and Billeter, 2016 ). Females with higher cVA levels ( Kurtovic et al., 2007 ), combined with 7-T, are less attractive to males ( Laturney and Billeter, 2016 ). Given that remating rate of Bully -mated females was higher than that of Cs -mated females ( Figure 3G ’), we hypothesized that Bully -mated females would receive less cVA in their genital tract during mating, thus decreasing their attractiveness than when mating with Cs males. To test this, we conducted mating assays with Cs females paired with either Cs or Bully males, followed by dissection of the females’ genital tracts to quantify the amount of cVA transferred. We found that females mated with Bully males had about three times lower cVA quantities in their genital tracts compared to those mated with Cs males ( Figure 4F ’). Combined with higher quantities of 5-T and 5-P, reduced cVA level in genital tract likely contribute to the increased attractiveness and higher remating rates observed in females that previously mated with Bully males. Overall, our findings demonstrate that naïve Bully males differ from Cs males in their CHC profiles and transfer a distinct quantity of chemical compounds to females during mating. These observations are consistent with a model in which changes in chemical signaling in Bully males is associated with both reduced mating success and weaker mate-guarding-related behaviors. In particular, reduced cVA transfer, combined with elevated levels of specific hydrocarbons such as 5-T and 5-P in mated females, may contribute to the increased attractiveness and remating propensity observed in females that previously mated with Bully males. Reduced copulatory investment in aggressive males is associated with extended lifespan Based on the main life-history trade-off between reproduction and survival, we next examined whether reduced pre- and post-copulatory investment observed in Bully males would be accompanied by increased longevity. To this end, we conducted longevity assays and monitored lifespan in Cs and Bully males. When housed individually, Bully males survived significantly longer than Cs males ( Figure 5A , green vs yellow curves). Median survival (EC50) was increased from 57 days in Cs males to 82 days in Bully males ( Figure 5A ), and maximum lifespan was also extended from 90 days in Cs males to 100 days in Bully ( Figure 5A ). To assess whether this difference could reflect hybrid vigor, we measured lifespan in heterozygous Bully males generated by crossing Cs females with Bully males. The survival of these heterozygous males was comparable to that of homozygous Bully males ( Figure 5A , red curve), with males showing an EC50 of 79 days and a maximum lifespan of 106 days. This result indicates that Bully background positively influence males’ lifespan and argue against hybrid vigor as the main explanation for the observed phenotype. We next evaluated survival under more naturalistic social conditions by maintaining males in mixed-sex groups. Under these conditions, Bully males again showed increased survival compared to Cs males, with both higher median lifespan and extended maximum lifespan ( Figure 5B ). Bully males had a median survival of 56 days and a maximum lifespan of 71 days, compared to Cs males, which had a median survival of 43 days and all died by day 67 ( Figure 5B ). Download figure Open in new tab Figure 5. Aggressive males offset their reduced reproductive success with a longer lifespan and preserved reproductive success in later life. (A) Survival curves of Cs (yellow) and homozygous (green) and heterozygous (red) Bully males (Weibull regression model (n =50, n =50, n =51, Likelihood Ratio Test): χ 2 =23.91, p=2.6×10 −5 , post-hoc: P Cs – Bully _homo <0.001, t.ratio= 4.061, P Cs – Bully _hetero < 0.001, t.ratio=5.088, P Bully _homo – Bully _hetero =0.318, t.ratio = −1.001). Cs males had a median survival (EC 50 ) of 57 days, with the last individual dying at day 90 (yellow), while progeny from homozygous Bully males showed an EC 50 of 82 days, with the final death at day 101 (green) and heterozygous Bully males showed an EC 50 of 79 days, with the final death at day 106 (red). (B) Survival curves of group of 10 Cs and 10 homozygous Bully males raised in group with 12 Cs females. Bully males had a median survival (EC 50 ) of 56 days, with the last individual dying at day 71 (red line), while Cs males showed an EC 50 of 43 days, with the final death at day 67 (yellow line) (n Cs =50, n Bully -hetero =50, Cox proportional hazards regression model with stratification by group (Likelihood Ratio Test): P=7×10 −5 , χ 2 =15.94). Statistical details are given in Figure 1-table supplement. (C) Mating duration of Cs and Bully males with virgin Cs females across their lifespan (n Cs-J0 =24, n Bully -J14 =23, LMM and Satterthwaite T-test: P interaction =3.360×10 −5 , t=4.227). 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. (D) Number of mating throughout the lifespan of Cs and Bully males (n Cs-J0 =24, n Bully -J14 =23, GLM binomial and Wald Z-test: P interaction =0.039, Z= −2.068). Numbers in bars indicate the proportion of mating. Arrows denote the last mating event and the number of offspring produced. Statistical details are given in Figure 1-table supplement 1. Table 1 : 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 1. We then assessed how reproductive performance varies across the lifespan. Individual males were paired weekly with virgin Cs females until death, and mating success and mating duration were recorded. Throughout their lifespan, Bully males consistently displayed shorter MD than Cs males ( Figure 5C ). While mating duration increased with age in Cs males, a pattern reported in another Drosophila species ( Dhole and Pfennig, 2014 ), this effect was not observed in Bully males, whose MD remained stable across mating and throughout their lives ( Figure 5C , green). Analysis of individual male slopes confirmed this divergence: most Cs males showed increasing MD across mating events, while Bully males did not (Figure 5-figure supplement 1). Mating success followed a similar age-dependent pattern. Bully males showed reduced mating success compared to Cs males during early and mid-adulthood (until 70 days), but continued to mate at ages when Cs males had ceased reproductive activity ( Figure 5D ). While all Cs males stopped mating by day 78 and died by day 97 ( Figure 5D , yellow), Bully males maintained mating activity until day 98 and survived up to 119 days ( Figure 5D , green). In addition, late-life mating involving 98-days-old Bully males produced viable offspring ( Figure 5D , green), demonstrating that Bully males remain fertile at older ages. Together, our findings demonstrate that Bully males exhibit both reduced copulatory investment and extended lifespan, as well as a shift in the temporal pattern of reproductive activity. These observations are consistent with a scenario in which reduced investment in mating success and mating duration is associated with extended lifespan and sustained reproductive output later in life. Rather than enhancing early-life reproductive success, Bully males maintain reproductive capacity over a longer time window, suggesting a redistribution of reproductive effort across the lifespan. DISCUSSION Our study reveals that selection for aggressive traits in D. mel . males correlates with changes in key life-history trade-off, in which survival is extended while reproductive success is reduced. This pattern challenges the hypothesis that aggression necessarily enhances mating success ( Andersson, 1994 ; West-Eberhard, 1983 ), and indicates that aggressive phenotypes may incur substantial reproductive costs. Consistent phenotypes across independent Bully lines support a link between aggression selection and the reproductive traits described here. However, the absence of additional selected lines limits our ability to fully exclude contributions of genetic drift or line-specific effects. Because selection for aggression was applied specifically to males, we used a standardized female background ( Cs ) to isolate male-driven effects. While this design minimizes female variability, it does not fully disentangle male and female genotype interactions. However, the persistence of key phenotypes in Bully × Bully pairings and across independent selected lines supports a primary contribution of male genotype. Aggression therefore does not act as an isolated fitness-enhancing trait, but rather as one component of a coordinated life-history strategy in which gains in competitive ability are coupled to constraints on reproductive performance. The increased survival of highly aggressive males further raises important questions regarding the evolutionary consequences of aggression. While Bully males have reduced mating success compared to Cs males, their extended lifespan may allow them to sustain mating activity over longer periods and produce viable offspring at later ages, challenging the classical view that early reproductive success is the primary driver of male fitness ( Flatt, 2011 ). This shift suggests that alternative reproductive strategies, even those associated with lower mating success and mate-guarding, can persist if they confer long-term survival advantages under laboratories conditions. At the behavioral level, highly aggressive Bully A males exhibit changes in both pre- and post-copulatory phases. They achieve lower mating success and engage in shorter copulations than less aggressive Cs males, suggesting reduced efficiency in mate acquisition and potentially in sperm transfer. Despite higher courtship motivation after mating, Bully males do not achieve higher remating success. Instead, females previously mated with these males remate more readily, indicating reduced mate-guarding efficiency and possibly increased attractiveness. These observations point to a decoupling between courtship intensity and mating outcome, indicating that increased behavioral drive does not translate into reproductive advantage. While our study did not show a causal link, our results suggest that artificial selection applied to male aggressive traits to produce the Bully A line, is associated with changes in reproductive traits, enhanced survival, and increased CHCs levels. These behavioral differences are closely associated with significant modifications in chemical signaling. Cuticular hydrocarbons (CHCs), which are crucial to mate recognition and sexual communication ( Billeter and Wolfner, 2018 ; Yew and Chung, 2017 ) differ significantly between Bully A and Cs males. We show that naïve highly aggressive individuals present distinct chemical profiles compared to unselected wild-type flies, with higher levels of most CHCs. Importantly, CHCs also contribute to physiological functions such as protection against desiccation ( Nayal et al., 2024 ), suggesting that changes in CHC composition may simultaneously influence both reproductive performances and survival. Thus, selection on aggressive traits may have been accompanied by modifications in overall chemical signaling that would impact both physiological resilience and social behaviors. Although higher CHC level might be expected to enhance male attractiveness, Bully males display reduced mating success, suggesting a non-linear or dose-dependent relationship between CHC levels and female acceptance. Female mate choice relies on finely tuned blends of chemical cues, and changes in these optimal ratios may impair signal interpretation ( Billeter and Wolfner, 2018 ). In this context, excessive levels of specific CHCs could reduce courtship efficiency rather than enhance it. In addition to major compounds such as 7-tricosene, male attractiveness and courtship performance are influenced by other CHC, including palmitoleic acid, which signals through the Or47b receptor and modulates courtship behavior in an age-dependent manner ( Dweck et al., 2015 ; Kohlmeier et al., 2021 ; Lin et al., 2016 ). This pathway enables older males to initiate courtship more rapidly and with greater intensity, potentially compensating for age-related declines in competitiveness. Although palmitoleic acid levels were not measured here, change in level of such compounds could further contribute to decreasing mating success despite overall higher CHC level. Modifications in chemical communication extend into the post-mating context, where they potentially intersect with male reproductive performance. Females mated with Bully males display distinct CHC profiles, with higher levels of 5-tricosene (5-T) and 5-pentacosene (5-P), together with reduced transfer of cis-vaccenyl acetate (cVA) into the female genital tract, which may fail to efficiently decrease their attractiveness, thus triggering higher remating rate. This interpretation is consistent with previous studies showing that lower cVA levels decrease the effectiveness of chemical mate guarding and accelerate the recovery of female attractiveness ( Ejima, 2015 ). At the same time, the behavioral effects of cVA depend on its interaction with other CHCs ( Laturney and Billeter, 2016 ; Verschut et al., 2023 ), suggesting that changes in the overall chemical profile may further modulate how mated females are perceived by subsequent males. These results indicate that selection on aggressive traits can indirectly shape post-copulatory sexual selection by altering female chemical signatures. While causal manipulation of CHC profiles would be required to directly test their functional role, the consistent association between CHC composition, cVA transfer, and behavioral outcomes across assays of our study identifies chemical signaling as a strong candidate mechanism. Taken together, these observations support the idea that cuticular hydrocarbons constitute a central mechanistic substrate linking aggression, reproduction, and survival. Rather than hypothezising selection on multiple behavioral traits, the phenotypic differences observed in our study may alternatively arise from selection acting on genes involved in chemical signaling. Because CHCs influence both social communication and physiological resilience ( Nayal et al., 2024 ), selection that have acted on CHC production or emission would be expected to generate correlated effects across social behavior, reproductive success, and survival, and physiological resilience, thereby producing the unique life-history pattern observed in Bully males. At the genetic level, this integration may be supported by pleiotropic factors or by the co-selection of partially independent loci affecting multiple traits. In this study, we mainly focused on Bully A males, as transcriptomic analyses have identified a limited set of genes differentially expressed between Bully A and Cs males, including CG13646 , a putative transmembrane transporter whose down-regulation increases aggressive behavior ( Chowdhury et al., 2017 ). These candidate genes provide an entry point for dissecting the molecular and genetic logic underlying the observed phenotypes, particularly in determining whether changes in aggression, CHC composition, survival and life-history traits arise from common genetic regulation. In summary, our findings indicate that selection for male aggression is associated with a coordinated shift in reproductive investment, chemical signaling and lifespan, consistent with a redistribution of reproductive effort across the lifespan. This highlights aggression as a potential entry point into the coordinated evolution of reproductive and physiological traits. MATERIAL AND METHODS EXPERIMENTAL MODEL AND SUBJECT DETAILS Fly stocks Drosophila melanogaster strains were raised at 25°C under a 12h:12h light/dark cycle (LD= 9:00a.m. – 9:00p.m.) on standard fly food (for 1l of water, 74g corn flour, 28g yeast, 40g sugar, 7.5g agar, 10ml Moldex). Canton-S ( Cs ) flies (from Edward Kravitz’s laboratory, RRID: BDSC_64349) were used as wild-type. The Bully A, B and C lines were originally selected in Edward Kravitz’s laboratory from Cs during 37 generations ( Penn et al., 2010 ), resulting in hyper-aggressive male flies ( Garbaczewska et al., 2013 ; Hopkins et al., 2019 ). Experimental arena Experimental arenas were described in details in Trannoy et al ., 2015 ( Trannoy et al., 2015a ). Briefly, resin blocks containing three circular behavioral arenas (dimension: 2.3 cm diameter, 1.7 cm height) were used to conduct behavioral assays. Each arena was divided in two equal sizes by a plastic divider allowing flies to acclimate without physical interactions with the second fly. Then, dividers are removed from the arenas to start the behavioral assays. All behavioral assays were performed in empty arenas, expect for aggression assays and courtship assays with decapitated females, in which a food cup (1.5 cm diameter, 1 cm height) containing fresh standard fly food with a drop of fresh yeast paste on the surface was placed in the center of each arena. METHOD DETAILS Commonalities between all behavioral assays Late-stage pupae were collected from stocks raised at 25°C. Male pupae were individually placed in 5ml vials (Dutscher, ref 390597) containing about 1ml of standard fly food while female pupae were placed in group of 15 in ≈47ml vials (Dutscher, ref 789001B) containing about 15ml of standard fly food. All vials were placed at 25°C for 7 days. Seven-day-old individuals were randomly inserted in arenas by negative geotaxis, and allow to acclimate for 5 min before experiments. All behavioral assays were performed at 25°C. All tests were recorded with Basler camera Ace1970 through the StreamPix8 multi video recording software ( norpix.com ) and analyzed with BORIS software ( Friard and Gamba, 2016 ). After 15 min or 30 min, if pairs of flies had not yet lunged or mated respectively, we considered that no aggression or mating occurred and arenas were not considered for analysis. No power analyses were performed prior to the experiments, as we chose to use sample sizes of approximately 20 replicates, which is considered standard practice in the field. Aggression assays Two days prior to the aggression assays, males were anesthetized with CO 2 , and half were marked with a small white dot on the thorax before being returned to their original isolation vials. Aggression tests were conducted between ZT0 and ZT3 using size-matched male pairs. Males competed for access to a central food cup containing yeast. The following behaviors were recorded for 10 minutes following the first lunge: latency to lunge (i.e., the time between the initial encounter on the food cup and the first lunge), total number of lunges, and number of boxing events. Mating assays Seven-day-old individuals were tested in arenas without any food source. Latency to court and to mate, defined as the time between the first encounter and either the first Unilateral Wing Extension (UWE) or the initiation of mating, respectively, were measured. Total UWE duration was recorded over a 10-minute period starting from the first encounter. Mating proportion (proportion of fly pairs that succeeded to mate) and mating duration were also scored. In all assays, males were reared in social isolation, except for Figure 1H , where they were housed in groups of 10 males for 7 days. Mating assays with decapitated females Females were anesthetized with CO 2 and placed under the binocular for head removal. Experiments with decapitated females were performed in arenas with food cup filled with fresh fly food but without yeast. The females’ bodies were gently pressed into the food cup to ensure they remained completely immobile, as males can move their bodies during courtship. This allows to maintain a constant distance between the two decapitated females across trials, thereby improving the reproducibility of the assay. Female bodies were positioned in the food cup with the abdomen raised, allowing males to initiate and complete copulation. Multiple consecutive mating events assays Single naïve Cs and Bully males were sequentially paired with six virgin Cs females. For each male, number of mating and each mating duration were recorded. After each mating, the mated female was transferred to a 5 ml vial containing fly food to assess progeny production. Mating assays throughout the lifespan 24 Cs and Bully males were reared in isolation and paired with seven-day-old virgin Cs females for mating assays, conducted every seven days until all males had died. At each time point, mating duration and mating success (proportion of mating) were recorded. After all Cs males had died, females that had mated with Bully males were transferred to 5 ml vials containing fly food to assess progeny production. Attractiveness assays Measure of post-mating UWE Mating assays were performed by pairing naïve Cs and Bully males with a Cs female. Right after mating end, total time that males displayed UWE for 10 minutes towards the just-mated females was scored. Measure of post-mating UWE with switched females Mating assays were performed with naive Cs or Bully males and virgin Cs females. Immediately after mating ends, females were switched: Cs males interacted with Bully -mated females and Bully males interacted with Cs -mated females. Then total time of UWE towards the mated females was scored during 10 min. Measure of post-mating UWE with decapitated females Mating assays were conducted using naïve Cs or Bully males paired with virgin Cs females. Following copulation, mated females were decapitated. One decapitated Cs -mated and one Bully -mated female were then placed on the surface of the food cup. A new naïve Cs or Bully male was introduced and allowed to interact with both decapitated females. The duration of Unilateral Wing Extensions (UWE) directed toward each female was recorded over a 2-minute period following the first encounter. Arenas in which males interacted with only one of the two females were excluded from the analysis, as no choice could be determined. Males’ perception assays A decapitated Cs male and female were placed on a food cup in presence of either a naïve Cs or a Bully male. The time spent by the focal male courting each decapitated fly was quantified over a 2-minute period following the first unilateral wing extension (UWE). Arenas in which the male interacted with only one of the two decapitated flies were excluded from the analysis, as no choice could be determined. Re-mating assays Mating assays were performed with naïve Cs or Bully males and virgin Cs females. Mated-females were then individually placed in 5ml vials containing about 1ml of standard fly food and kept at 25°C. 12 or 14 days after the initial mating, mated-females were paired with a new naïve Cs male in empty arena and the proportion of re-mating during 30 min was scored. Survival assays Survival with isolated males Cs , homozygous and heterozygous Bully late-stage pupae were individually isolated in 5 ml vials containing 1ml of standard fly food and kept at 25⍰°C until adult emergence. The number of dead individuals was recorded daily until all flies had died. To maintain food quality, flies were transferred weekly to fresh vials containing approximately 1⍰ml of new fly food. Survival with group-raised males Late-stage Cs and homozygous Bully male pupae were placed in group with Cs female pupae in ≈47ml vials containing 15mL standard fly food and kept at 25⍰°C until adult emergence. Groups were composed of 10 males (either Cs or Bully ) and 12 Cs females. The number of dead males was recorded daily until they all had died. To maintain food quality, flies were transferred twice a week to fresh vials containing approximately 15⍰mL of new fly food. CHCs analysis Extraction and analysis of cuticular hydrocarbons (CHCs) of naive Cs and Bully males, as well as virgin Cs, Cs - and Bully -mated females were performed in Jean-Christophe Billeter’s lab, Groningen, Netherlands, with a Gas Chromatography coupled with Flame Ionization Detection (GC-FID) as already described ( Laturney and Billeter, 2016 ). Briefly, single whole flies were put in a 2ml glass vial (Supelco certified vial kit, 2ml clear glass vial 12×32mm and Supelco inserts) containing 50µl of nC18+nC26 10 ng/µl standard solution (made in Billeter’s lab) diluted in n-Hexane 99%+ (Acrōs Organic®). Vials were then vortexed at the lowest speed for 2min, afterward flies’ body were gently removed from vials using a “J-shape” paper clip sliced at the end, taking care not to damage the body to prevent from hemolymph contamination. Finally, samples were placed in GC-FID for apolar compounds analysis only. cVA quantification into genital tract Mating assays were performed with naïve Cs or Bully males and virgin Cs female. Right after mating, females were transferred into a 5ml vial previously put in ice in −80°C fridge to make them fall asleep quickly and prevent from sperm ejection. Mated-females’ genital tract was dissected in PBS with precision forceps under a binocular loupe. Protocol was already employed in ( Verschut et al., 2022 ). Briefly, abdomen was torn to make ovaries and genital tract out and visible, then ovaries, oviduct, spermathecae, parovaria and cuticula were removed to keep only the uterus, taking care not to injure it to prevent from sperm leak. Each uterus was inserted in a 2ml glass vial (Supelco certified vial kit, 2ml clear glass vial 12×32mm and Supelco inserts) containing 50µl of n-Hexane 99%+ (Acrōs Organic) for 1h at ambient temperature. Hexane solution was then evaporated under helium flow to prevent from cVA oxidation with dioxygen. Samples were sent to Jean-Christophe Billeter’s lab for GC-FID analysis of cVA, as describe above in CHCs analysis method part. QUANTIFICATION AND STATISTICAL ANALYSIS No data have been removed from the statistical analysis (except arenas where no choice was observed within the males’ perception assays, as described below). Some experiments were independently replicated by different authors to reduce potential bias. All statistical analyses were performed on R software version 4.0. ( Team, 2020 ) with packages ( Fox and Weisberg, 2019 ), coin ( Hothorn et al., 2006 ), dplyr ( Wickham et al., 2023 ), factoextra ( Kassambara and Mundt, 2020 ), FactoMineR ( Lê et al., 2008 ), lme4 ( Bates et al., 2015 ), lmerTest ( Kuznetsova et al., 2017 ), MASS ( Venables and Ripley, 2002 ), MuMIn ( Bartoń, 2023 ), nlme ( Pinheiro et al., 2022 ), performance ( Lüdecke et al., 2021 ), pgirmess ( Giraudoux. P, 2018 ), predictmeans ( Luo et al., 2021 ), survival ( Therneau, 2022 ), vegan ( Oksanen J, 2022 ). Comparisons of means (k =2) were performed with a T-test when possible ( i . e . mating duration) otherwise a Fisher Pitman permutation test was used ( i . e . UWE, latency to court and to mate). For comparisons with more than 2 conditions, a linear model (LM) was used and tested either with a type II ANOVA when possible ( i . e . mating duration) or a permutation test instead ( i . e . latency to lunge and CHC concentrations). For comparison of proportions and counting events, a type II analysis of deviance table with a Likelihood Ratio Test ( i . e . mating success, proportion of female re-mating, number of lunge and boxing events) or a Wald Chi-square test ( i . e . number of consecutive mating events) was performed. A Principal Component Analysis (PCA) was performed to see different CHC profiles and tested with a PerMANOVA and a SIMPER analysis. Survival data were analysed with a Weibull regression model and a Likelihood Ratio Test. When paired data, dependency was considered thanks to linear mixed model (LMM) with the source of dependency as random factor. Data following a distribution other than a normal distribution ( e . g . binomial or Poisson distribution) were computed in a generalized linear model (GLM) ( i . e . mating success, number of consecutive mating events, number of mating giving progeny, proportion of females re-mating, number of lunges and boxing events). When necessary, interactions were considered within models and tested. All post-hoc comparisons were performed with a sequential Bonferroni correction on the alpha level using either Holm method ( Holm, 1979 ) when few numbers of comparisons or Benjamini and Hochberg method ( Benjamini and Hochberg, 1995 ) when more comparisons. All statistical details are given in Figure 1-table supplement 1. RESOURCE AVAILABILITY Further information and requests for resources and reagents are accessible from the lead contact, Séverine Trannoy ( severine.trannoy{at}univ-tlse3.fr or severine.trannoy{at}utoulouse.fr ). AUTHOR CONTRIBUTIONS Conceptualization: S.T., J.C.B., A.D. Data curation: A.D., R.G., G.P., F.J., A.C., A.H., T.G., S.T. Formal analysis: A.D., R.G., G.P., F.J., A.C., A.H., T.G., S.T. Funding acquisition: S.T., J.C.B., A.D. Project administration: S.T. Supervision: S.T., J.C.B. Writing: S.T., J.C.B., A.D. DECLARATION OF INTERESTS Authors declare that they have no competing interests. SUPPORTING INFORMATIONS Figure 1-table supplement 1. 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. Figure 3-figure supplement 1. Cs and Bully males discrimination between males and females CHC profiles . Time spent by Cs and Bully males to court either a naïve decapitated Cs male or a virgin Cs decapitated female (LMM and Analysis of Deviance Table (Type II Wald chisquare tests): P male_genotype =0.327, χ 2 =0.960, P mated_female =5.518×10 −8 , χ 2 =29.626, post-hoc: P Cs_male =2.064×10 −4 , χ 2 =13.772, P Bully _male =6.308×10 −5 , χ 2 =16.008). Stars represent significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001). Statistical details are given in Figure 1-table supplement 1. n Cs =10, n Bully =13 . Figure 4-figure supplement 1. Details on PCA analysis for virgin and mated females . ( A) Eigenvectors and associated cos 2 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 1. n virgin =10⍰, n Cs-mated =16⍰, n Bully -mated =14⍰. Figure 5-figure supplement 1. 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 1. n Cs =24, n Bully =23. ACKNOWLEDGEMENTS We thank the members of EXPLAIN and IVEP team at the CRCA for their helpful discussion and support on this study. We thank Kravitz’s lab for sharing the Bully lines. We also thank all the member of Billeter’s lab to host A. Defert and train him to GC-FID methodology. We acknowledge support from the French Research National Agency (ANR) (ANR-19-CE37-0018-01 to S.T.), the Fondation Fyssen (190573 to S.T.), and the Doctoral Mobility Campaign of Toulouse III University in 2023 (to A.D.). Funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Footnotes New data on Figure 1 Update on Figure 5 for clarification Update throughout the text REFERENCES ↵ Andersson , M. ( 1994 ). Sexual Selection ( Princeton University Press ). doi: 10.1515/9780691207278 . OpenUrl CrossRef ↵ Avila , F.W. , Sirot , L.K. , LaFlamme , B.A. , Rubinstein , C.D. , and Wolfner , M.F. ( 2011 ). Insect seminal fluid proteins: identification and function . Annu Rev Entomol 56 : 21 - 40 . doi: 10.1146/annurev-ento-120709-144823 . OpenUrl CrossRef PubMed Web of Science ↵ Bartoń , K. ( 2023 ). MuMIn: Multi-Model Inference . R package version 1.47.5 ↵ Bates , D. , Mächler , M. , Bolker , B. , and Walker , S. ( 2015 ). Fitting Linear Mixed-Effects Models Using lme4 . Journal of Statistical Software 67 : 1 - 48 . doi: 10.18637/jss.v067.i01 . OpenUrl CrossRef PubMed ↵ Baxter , C. , Mentlik , J. , Shams , I. , and Dukas , R. ( 2018 ). Mating success in fruit flies: courtship interference versus female choice . Animal Behaviour 138 : 101 – 108 . doi: 10.1016/j.anbehav.2018.02.010 . OpenUrl CrossRef ↵ Baxter , C.M. , Barnett , R. , and Dukas , R. ( 2015 ). Aggression, mate guarding and fitness in male fruit flies . Animal Behaviour 109 : 235 – 241 . doi: 10.1016/j.anbehav.2015.08.023 . OpenUrl CrossRef ↵ Benjamini , Y. , and Hochberg , Y. ( 1995 ). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing . Journal of the Royal Statistical Society: Series B (Methodological) 57 : 289 – 300 . doi: 10.1111/j.2517-6161.1995.tb02031.x . OpenUrl CrossRef PubMed ↵ Billeter , J.C. , Atallah , J. , Krupp , J.J. , Millar , J.G. , and Levine , J.D. ( 2009 ). Specialized cells tag sexual and species identity in Drosophila melanogaster . Nature 461 : 987 – 991 . doi: 10.1038/nature08495 . OpenUrl CrossRef PubMed Web of Science ↵ Billeter , J.C. , and Wolfner , M.F. ( 2018 ). Chemical Cues that Guide Female Reproduction in Drosophila melanogaster . J Chem Ecol 44 : 750 – 769 . doi: 10.1007/s10886-018-0947-z . OpenUrl CrossRef PubMed ↵ Braendle , C. , and Paaby , A. ( 2024 ). Life history in Caenorhabditis elegans: from molecular genetics to evolutionary ecology . Genetics 228 : doi: 10.1093/genetics/iyae151 . OpenUrl CrossRef ↵ Brennan , P. , and Orbach , D. ( 2021 ). Post-Copulatory Sexual Selection. In . doi: 10.1093/obo/9780199941728-0131 . OpenUrl CrossRef ↵ Bretman , A. , Fricke , C. , and Chapman , T. ( 2009 ). Plastic responses of male Drosophila melanogaster to the level of sperm competition increase male reproductive fitness . Proc Biol Sci 276 : 1705 – 1711 . doi: 10.1098/rspb.2008.1878 . OpenUrl CrossRef PubMed Web of Science ↵ Bretman , A. , Gage , M.J. , and Chapman , T. ( 2011 ). Quick-change artists: male plastic behavioural responses to rivals . Trends Ecol Evol 26 : 467 – 473 . doi: 10.1016/j.tree.2011.05.002 . OpenUrl CrossRef PubMed Web of Science ↵ Bretman , A. , Westmancoat , J.D. , and Chapman , T. ( 2013 ). Male control of mating duration following exposure to rivals in fruitflies . J Insect Physiol 59 : 824 – 827 . doi: 10.1016/j.jinsphys.2013.05.011 . OpenUrl CrossRef PubMed ↵ Chapman , T. , Bangham , J. , Vinti , G. , Seifried , B. , Lung , O. , Wolfner , M.F. , Smith , H.K. , and Partridge , L. ( 2003 ). The sex peptide of Drosophila melanogaster: female post-mating responses analyzed by using RNA interference . Proc Natl Acad Sci U S A 100 : 9923 – 9928 . doi: 10.1073/pnas.1631635100 . OpenUrl Abstract / FREE Full Text ↵ Chen , S. , Lee , A.Y. , Bowens , N.M. , Huber , R. , and Kravitz , E.A. ( 2002 ). Fighting fruit flies: a model system for the study of aggression . Proc Natl Acad Sci U S A 99 : 5664 – 5668 . doi: 10.1073/pnas.082102599 . OpenUrl Abstract / FREE Full Text ↵ Chippindale , A.K. , Chu , T.J.F. , and Rose , M.R. ( 1996 ). Complex trade-offs and the evolution of starvation resistance in drosophila melanogaster . Evolution 50 : 753 – 766 . doi: 10.1111/j.1558-5646.1996.tb03885.x . OpenUrl CrossRef ↵ Chowdhury , B. , Chan , Y.B. , and Kravitz , E.A. ( 2017 ). Putative transmembrane transporter modulates higher-level aggression in Drosophila . Proc Natl Acad Sci U S A 114 : 2373 – 2378 . doi: 10.1073/pnas.1618354114 . OpenUrl Abstract / FREE Full Text ↵ Dhole , S. , and Pfennig , K.S. ( 2014 ). Age-dependent male mating investment in Drosophila pseudoobscura . PLoS One 9 : e88700 . doi: 10.1371/journal.pone.0088700 . OpenUrl CrossRef PubMed ↵ Dore , A.A. , Rostant , W.G. , Bretman , A. , and Chapman , T. ( 2021 ). Plastic male mating behavior evolves in response to the competitive environment . Evolution 75 : 101 – 115 . doi: 10.1111/evo.14089 . OpenUrl CrossRef ↵ Dow , M.A. , and von Schilcher , F. ( 1975 ). Aggression and mating success in Drosophila melanogaster . Nature 254 : 511 – 512 . OpenUrl CrossRef PubMed ↵ Dunham , A.E. , and Rudolf , V.H. ( 2009 ). Evolution of sexual size monomorphism: the influence of passive mate guarding . J Evol Biol 22 : 1376 – 1386 . doi: 10.1111/j.1420-9101.2009.01768.x . OpenUrl CrossRef PubMed ↵ Dweck , H.K. , Ebrahim , S.A. , Thoma , M. , Mohamed , A.A. , Keesey , I.W. , Trona , F. , Lavista-Llanos , S. , Svatos , A. , Sachse , S. , Knaden , M. , and Hansson , B.S. ( 2015 ). Pheromones mediating copulation and attraction in Drosophila . Proc Natl Acad Sci U S A 112 : E2829 – 2835 . doi: 10.1073/pnas.1504527112 . OpenUrl Abstract / FREE Full Text ↵ Ejima , A. ( 2015 ). Pleiotropic actions of the male pheromone cis-vaccenyl acetate in Drosophila melanogaster . J Comp Physiol A Neuroethol Sens Neural Behav Physiol 201 : 927 – 932 . doi: 10.1007/s00359-015-1020-9 . OpenUrl CrossRef PubMed ↵ Everaerts , C. , Farine , J.P. , Cobb , M. , and Ferveur , J.F. ( 2010 ). Drosophila cuticular hydrocarbons revisited: mating status alters cuticular profiles . PLoS One 5 : e9607 . doi: 10.1371/journal.pone.0009607 . OpenUrl CrossRef PubMed ↵ Filice , D.C.S. , and Dukas , R. ( 2019 ). Winners have higher pre-copulatory mating success but losers have better post-copulatory outcomes . Proc Biol Sci 286 : 20182838 . doi: 10.1098/rspb.2018.2838 . OpenUrl CrossRef PubMed ↵ Fisher , R.A. ( 1930 ). The genetical theory of natural selection ( The Clarendon Press ). ↵ Flatt , T. ( 2011 ). Survival costs of reproduction in Drosophila . Exp Gerontol 46 : 369 – 375 . doi: 10.1016/j.exger.2010.10.008 . OpenUrl CrossRef PubMed ↵ Flatt , T. ( 2020 ). Life-History Evolution and the Genetics of Fitness Components in Drosophila melanogaster . Genetics 214 : 3 – 48 . doi: 10.1534/genetics.119.300160 . OpenUrl Abstract / FREE Full Text ↵ Fox , J. , and Weisberg , S. ( 2019 ). An R Companion to Applied Regression . Sage, Thousand Oaks CA . Third edition: ↵ Friard , O. , and Gamba , M. ( 2016 ). BORIS: a free, versatile open-source event-logging software for video/audio coding and live observations . Methods in Ecology and Evolution 7 : 1325 – 1330 . OpenUrl ↵ Fricke , C. , Wigby , S. , Hobbs , R. , and Chapman , T. ( 2009 ). The benefits of male ejaculate sex peptide transfer in Drosophila melanogaster . J Evol Biol 22 : 275 – 286 . doi: 10.1111/j.1420-9101.2008.01638.x . OpenUrl CrossRef PubMed Web of Science ↵ Gao , C. , Ma , M. , Chen , J. , Ji , X. , Peng , Q. , and Pan , Y. ( 2024 ). Social Experience Shapes Fighting Strategies for Reproductive Success . eLife Sciences Publications, Ltd . ↵ Garbaczewska , M. , Billeter , J.C. , and Levine , J.D. ( 2013 ). Drosophila melanogaster males increase the number of sperm in their ejaculate when perceiving rival males . J Insect Physiol 59 : 306 – 310 . doi: 10.1016/j.jinsphys.2012.08.016 . OpenUrl CrossRef PubMed Web of Science ↵ Gillott , C. ( 2003 ). Male accessory gland secretions: modulators of female reproductive physiology and behavior . Annu Rev Entomol 48 : 163 – 184 . doi: 10.1146/annurev.ento.48.091801.112657 . OpenUrl CrossRef PubMed Web of Science ↵ Giraudoux . P ( 2018 ). pgirmess: Spatial Analysis and Data Mining for Field Ecologists . R package version 2.0.0 ↵ Grillet , M. , Dartevelle , L. , and Ferveur , J.F. ( 2006 ). A Drosophila male pheromone affects female sexual receptivity . Proc Biol Sci 273 : 315 – 323 . doi: 10.1098/rspb.2005.3332 . OpenUrl CrossRef PubMed Web of Science ↵ Hihara , F. ( 1981 ). Effects of the male accessory gland secretion on oviposition and remating in females of Drosophila melanogaster . Zool. Mag . 90 : 307 – 316 . OpenUrl ↵ Hindmarsh Sten , T. , Li , R. , Hollunder , F. , Eleazer , S. , and Ruta , V. ( 2025a ). Male-male interactions shape mate selection in Drosophila . Cell 188 : 1486 – 1503.e1425 . doi: 10.1016/j.cell.2025.01.008 . OpenUrl CrossRef PubMed ↵ Hindmarsh Sten , T. , Li , R. , Hollunder , F. , Eleazer , S. , and Ruta , V. ( 2025b ). Male-male interactions shape mate selection in Drosophila . Cell 188 : 1486 – 1503.e1425 . doi: 10.1016/j.cell.2025.01.008 . OpenUrl CrossRef PubMed ↵ Holm , S. ( 1979 ). A Simple Sequentially Rejective Multiple Test Procedure . Scandinavian Journal of Statistics 6 : 65 – 70 . OpenUrl CrossRef PubMed Web of Science ↵ Hopkins , B.R. , Sepil , I. , Thézénas , M.L. , Craig , J.F. , Miller , T. , Charles , P.D. , Fischer , R. , Kessler , B.M. , Bretman , A. , Pizzari , T. , and Wigby , S. ( 2019 ). Divergent allocation of sperm and the seminal proteome along a competition gradient in Drosophila melanogaster . Proc Natl Acad Sci U S A 116 : 17925 – 17933 . doi: 10.1073/pnas.1906149116 . OpenUrl Abstract / FREE Full Text ↵ Hothorn , T. , Kurt , H. , A., v.d.W.M., and and Zeileis , A. ( 2006 ). A Lego System for Conditional Inference . The American Statistician 60 : 257 – 263 . doi: 10.1198/000313006X118430 . OpenUrl CrossRef Web of Science ↵ Kassambara , A. , and Mundt , F. ( 2020 ). Factoextra: Extract and Visualize the Results of Multivariate Data Analyses . ↵ Kim , S.M. , Su , C.Y. , and Wang , J.W. ( 2017 ). Neuromodulation of Innate Behaviors in Drosophila . Annu Rev Neurosci 40 : 327 – 348 . doi: 10.1146/annurev-neuro-072116-031558 . OpenUrl CrossRef PubMed ↵ Kim , W.J. , Jan , L.Y. , and Jan , Y.N. ( 2013 ). A PDF/NPF neuropeptide signaling circuitry of male Drosophila melanogaster controls rival-induced prolonged mating . Neuron 80 : 1190 – 1205 . doi: 10.1016/j.neuron.2013.09.034 . OpenUrl CrossRef PubMed Web of Science ↵ Kohl , J. , Huoviala , P. , and Jefferis , G.S. ( 2015 ). Pheromone processing in Drosophila . Curr Opin Neurobiol 34 : 149 – 157 . doi: 10.1016/j.conb.2015.06.009 . OpenUrl CrossRef PubMed ↵ Kohlmeier , P. , Zhang , Y. , Gorter , J.A. , Su , C.Y. , and Billeter , J.C. ( 2021 ). Mating increases Drosophila melanogaster females’ choosiness by reducing olfactory sensitivity to a male pheromone . Nat Ecol Evol 5 : 1165 – 1173 . doi: 10.1038/s41559-021-01482-4 . OpenUrl CrossRef PubMed ↵ Kurtovic , A. , Widmer , A. , and Dickson , B.J. ( 2007 ). A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone . Nature 446 : 542 – 546 . doi: 10.1038/nature05672 . OpenUrl CrossRef PubMed Web of Science ↵ Kuznetsova , A. , Brockhoff , P.B. , and Christensen , R.H.B. ( 2017 ). lmerTest Package: Tests in Linear Mixed Effects Models . Journal of Statistical Software 82 : 1 – 26 . doi: 10.18637/jss.v082.i13 . OpenUrl CrossRef ↵ Laturney , M. , and Billeter , J.C. ( 2016 ). Drosophila melanogaster females restore their attractiveness after mating by removing male anti-aphrodisiac pheromones . Nat Commun 7 : 12322 . doi: 10.1038/ncomms12322 . OpenUrl CrossRef PubMed ↵ Lê , S. , Josse , J. , and Husson , F. ( 2008 ). FactoMineR: An R Package for Multivariate Analysis . Journal of Statistical Software 25 : 1 – 18 . doi: 10.18637/jss.v025.i01 . OpenUrl CrossRef ↵ Lin , H.H. , Cao , D.S. , Sethi , S. , Zeng , Z. , Chin , J.S.R. , Chakraborty , T.S. , Shepherd , A.K. , Nguyen , C.A. , Yew , J.Y. , Su , C.Y. , and Wang , J.W. ( 2016 ). Hormonal Modulation of Pheromone Detection Enhances Male Courtship Success . Neuron 90 : 1272 – 1285 . doi: 10.1016/j.neuron.2016.05.004 . OpenUrl CrossRef PubMed ↵ Lüdecke , D. , Ben-Shachar , M.S. , Patil , I. , Waggoner , P. , and Makowski , D. ( 2021 ). performance: An R Package for Assessment, Comparison and Testing of Statistical Models . Journal of Open Source Software 6 ( 60 ): 31 – 39 . doi: 10.21105/joss.03139 . OpenUrl CrossRef ↵ Luo , D. , Ganesh , S. , and Koolaard , J. ( 2021 ). predictmeans: Calculate Predicted Means for Linear Models . R package version 1.0.6 ↵ Marshall , K.E. , and Sinclair , B.J. ( 2010 ). Repeated stress exposure results in a survival-reproduction trade-off in Drosophila melanogaster . Proc Biol Sci 277 : 963 – 969 . doi: 10.1098/rspb.2009.1807 . OpenUrl CrossRef PubMed ↵ Mazzi , D. , Kesaniemi , J. , Hoikkala , A. , and Klappert , K. ( 2009 ). Sexual conflict over the duration of copulation in Drosophila montana: why is longer better? BMC Evol Biol 9 : 132 . doi: 10.1186/1471-2148-9-132 . OpenUrl CrossRef PubMed ↵ Miyamoto , T. , and Amrein , H. ( 2008 ). Suppression of male courtship by a Drosophila pheromone receptor . Nat Neurosci 11 : 874 – 876 . doi: 10.1038/nn.2161 . OpenUrl CrossRef PubMed ↵ Nandy , B. , Dasgupta , P. , Halder , S. , and Verma , T. ( 2016 ). Plasticity in aggression and the correlated changes in the cost of reproduction in male Drosophila melanogaster . Animal Behaviour 114 : 3 – 9 . doi: 10.1016/j.anbehav.2016.01.019 . OpenUrl CrossRef ↵ Nayal , K. , Krupp , J.J. , Abdalla , O. , and Levine , J.D. ( 2024 ). Cuticular hydrocarbons promote desiccation resistance by preventing transpiration in Drosophila melanogaster . J Exp Biol 227 : doi: 10.1242/jeb.247752 . OpenUrl CrossRef ↵ Oksanen J S.G. , Blanchet F , Kindt R , Legendre P , Minchin P , O’Hara R , Solymos P , Stevens M , Szoecs E , Wagner H , Barbour M , Bedward M , Bolker B , Borcard D , Carvalho G , Chirico M , De Caceres M , Durand S , Evangelista H , FitzJohn R , Friendly M , Furneaux B , Hannigan G , Hill M , Lahti L , McGlinn D , Ouellette M , Ribeiro Cunha E , Smith T , Stier A , Ter Braak C , Weedon J ( 2022 ). vegan: Community Ecology Package . R package version 2.6-4 ↵ Partridge , L. , and Farquhar , M. ( 1981 ). Sexual activity reduces lifespan of male fruitflies . Nature 294 : 580 – 582 . doi: 10.1038/294580a0 . OpenUrl CrossRef ↵ Penn , J.K. , Zito , M.F. , and Kravitz , E.A. ( 2010 ). A single social defeat reduces aggression in a highly aggressive strain of Drosophila . Proc Natl Acad Sci U S A 107 : 12682 – 12686 . doi: 10.1073/pnas.1007016107 . OpenUrl Abstract / FREE Full Text ↵ Pinheiro , J. , Bates , D. , and Team , R.C. ( 2022 ). nlme: Linear and Nonlinear Mixed Effects Models . R package version 3.1-160 ↵ Prunier , A. , and Trannoy , S. ( 2024 ). Learning from fights: Males’ social dominance status impact reproductive success in Drosophila melanogaster . PLoS One 19 : e0299839 . doi: 10.1371/journal.pone.0299839 . OpenUrl CrossRef PubMed ↵ Roff , D.A. ( 1992 ). The evolution of life histories: theory and analysis ( Chapman & Hall ). ↵ Scott , D. ( 1986 ). Sexual mimicry regulates the attractiveness of mated Drosophila melanogaster females . Proc Natl Acad Sci U S A 83 : 8429 – 8433 . doi: 10.1073/pnas.83.21.8429 . OpenUrl Abstract / FREE Full Text ↵ Simmons , L.W. ( 2001 ). Sperm Competition and Its Evolutionary Consequences in the Insects ( Princeton University Press ). doi: 10.2307/j.ctvs32sr2 . OpenUrl CrossRef ↵ Sirot , L.K. , Buehner , N.A. , Fiumera , A.C. , and Wolfner , M.F. ( 2009 ). Seminal fluid protein depletion and replenishment in the fruit fly, Drosophila melanogaster: an ELISA-based method for tracking individual ejaculates . Behav Ecol Sociobiol 63 : 1505 – 1513 . doi: 10.1007/s00265-009-0806-6 . OpenUrl CrossRef PubMed Web of Science ↵ Stearns , S. ( 1992 ). The Evolution Of Life Histories ( Oxford University Press , Oxford .). doi: 10.1093/oso/9780198577416.001.0001 . OpenUrl CrossRef ↵ Stearns , S.C. ( 1976 ). Life-history tactics: a review of the ideas . Q Rev Biol 51 : 3 – 47 . doi: 10.1086/409052 . OpenUrl CrossRef PubMed Web of Science ↵ Stearns , S.C. ( 1989 ). Trade-Offs in Life-History Evolution . Functional Ecology 3 : 259 – 268 . doi: 10.2307/2389364 . OpenUrl CrossRef Web of Science ↵ Stearns , S.C. ( 2000 ). Life history evolution: successes, limitations, and prospects . Naturwissenschaften 87 : 476 – 486 . doi: 10.1007/s001140050763 . OpenUrl CrossRef PubMed Web of Science ↵ Team , R.C. ( 2020 ). R: A Language and Environment for Statistical Computing . R Foundation for Statistical Computing ↵ Therneau , T. ( 2022 ). A Package for Survival Analysis in R . R package version 3.4-0 ↵ Toda , H. , Zhao , X. , and Dickson , B.J. ( 2012 ). The Drosophila female aphrodisiac pheromone activates ppk23(+) sensory neurons to elicit male courtship behavior . Cell Rep 1 : 599 – 607 . doi: 10.1016/j.celrep.2012.05.007 . OpenUrl CrossRef PubMed Web of Science ↵ Trannoy , S. , Chowdhury , B. , and Kravitz , E.A. ( 2015a ). A New Approach that Eliminates Handling for Studying Aggression and the “Loser” Effect in Drosophila melanogaster . J Vis Exp e53395 . doi: 10.3791/53395 . OpenUrl CrossRef ↵ Trannoy , S. , Chowdhury , B. , and Kravitz , E.A. ( 2015b ). A New Approach that Eliminates Handling for Studying Aggression and the “Loser” Effect in Drosophila melanogaster . J. Vis. Exp . 106 : e53395 . doi: 10.3791/53395 . OpenUrl CrossRef ↵ Venables , W.N. , and Ripley , B.D. ( 2002 ). Modern Applied Statistics with S . Springer , New York Fourth edition: ↵ D. Yamamoto Verschut , T.A. , Kohlmeier , P. , and Billeter , J.-C. ( 2022 ). Bioassaying the Function of Pheromones in Drosophila melanogaster’s Social Behavior . In Behavioral Neurogenetics , D. Yamamoto , ed. ( Springer US ), pp. 123 – 156 . doi: 10.1007/978-1-0716-2321-3_9 . OpenUrl CrossRef ↵ Verschut , T.A. , Ng , R. , Doubovetzky , N.P. , Le Calvez , G. , Sneep , J.L. , Minnaard , A.J. , Su , C.-Y. , Carlsson , M.A. , Wertheim , B. , and Billeter , J.-C. ( 2023 ). Aggregation pheromones have a non-linear effect on oviposition behavior in Drosophila melanogaster . Nature Communications 14 : 1544 . doi: 10.1038/s41467-023-37046-2 . OpenUrl CrossRef PubMed ↵ Wang , L. , Han , X. , Mehren , J. , Hiroi , M. , Billeter , J.C. , Miyamoto , T. , Amrein , H. , Levine , J.D. , and Anderson , D.J. ( 2011 ). Hierarchical chemosensory regulation of male-male social interactions in Drosophila . Nat Neurosci 14 : 757 – 762 . doi: 10.1038/nn.2800 . OpenUrl CrossRef PubMed ↵ Warner , R.R. , Shapiro , D.Y. , Marcanato , A. , and Petersen , C.W. ( 1995 ). Sexual conflict: males with highest mating success convey the lowest fertilization benefits to females . Proc Biol Sci 262 : 135 – 139 . doi: 10.1098/rspb.1995.0187 . OpenUrl CrossRef PubMed ↵ West-Eberhard , M.J. ( 1983 ). Sexual Selection, Social Competition, and Speciation . The Quarterly Review of Biology 58 : 155 – 183 . OpenUrl CrossRef Web of Science ↵ Wickham , H. , François , R. , Henry , L. , Müller , K. , and Vaughan , D. ( 2023 ). dplyr: A Grammar of Data Manipulation . ↵ Williams , G.C. ( 1966 ). Natural Selection, the Costs of Reproduction, and a Refinement of Lack’s Principle . The American Naturalist 100 : 687 – 690 . doi: 10.1086/282461 . OpenUrl CrossRef Web of Science ↵ Yamamoto , D. , and Koganezawa , M. ( 2013 ). Genes and circuits of courtship behaviour in Drosophila males . Nature Reviews Neuroscience 14 : 681 – 692 . doi: 10.1038/nrn3567 . OpenUrl CrossRef PubMed ↵ Yew , J.Y. , and Chung , H. ( 2017 ). Drosophila as a holistic model for insect pheromone signaling and processing . Curr Opin Insect Sci 24 : 15 – 20 . doi: 10.1016/j.cois.2017.09.003 . OpenUrl CrossRef PubMed ↵ Zawistowski , S. , and Richmond , R.C. ( 1986 ). Inhibition of courtship and mating of Drosophila melanogaster by the male-produced lipid, cis-vaccenyl acetate . Journal of Insect Physiology 32 : 189 – 192 . doi: 10.1016/0022-1910(86)90057-0 . OpenUrl CrossRef Web of Science View the discussion thread. Back to top Previous Next Posted May 05, 2026. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Selection for male aggression is associated with changes in reproductive traits, chemical signaling and lifespan in Drosophila melanogaster Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Selection for male aggression is associated with changes in reproductive traits, chemical signaling and lifespan in Drosophila melanogaster Anthony Defert , Romane Gout , Gaelle Pennot , Fanny Jamme , Amandine Castex , Anissa Handjar , Thomas Guilleman , Jean-Christophe Billeter , Séverine Trannoy bioRxiv 2025.06.02.657383; doi: https://doi.org/10.1101/2025.06.02.657383 Share This Article: Copy Citation Tools Selection for male aggression is associated with changes in reproductive traits, chemical signaling and lifespan in Drosophila melanogaster Anthony Defert , Romane Gout , Gaelle Pennot , Fanny Jamme , Amandine Castex , Anissa Handjar , Thomas Guilleman , Jean-Christophe Billeter , Séverine Trannoy bioRxiv 2025.06.02.657383; doi: https://doi.org/10.1101/2025.06.02.657383 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Animal Behavior and Cognition Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40364) Molecular Biology (17163) Neuroscience (88537) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)