Social context rapidly reshapes chemical signalling and endocrine profiles in two mouse subspecies

Social context rapidly reshapes chemical signalling and endocrine profiles in two mouse subspecies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Social context rapidly reshapes chemical signalling and endocrine profiles in two mouse subspecies Zuzana Hiadlovská, Barbora Vošlajerová Bímová, Kristina Daniszová, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9552933/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Chemical signalling and endocrine state are known to covary with social environment, but the speed of their response to social transitions under natural conditions remains unclear. We examined how major urinary proteins (MUPs) and faecal steroid hormone metabolites respond to shifts between solitary housing and a complex social setting in two house mouse subspecies, Mus musculus musculus and M. m. domesticus . Using repeated sampling in semi-natural enclosures, we measured MUP, testosterone, and corticosterone metabolite concentrations across three contexts: before social exposure, during the social phase, and after return to isolation. Reproductive output was quantified within a 44-day window around each sampling event. Entry into the enclosures triggered a strong, coordinated response: urinary MUP concentrations increased in both sexes and subspecies, paralleled by rises in testosterone and corticosterone metabolites during early social exposure. In males, this pattern reversed rapidly after removal, with MUPs and both hormone metabolites declining within 24 hours of renewed isolation. Under stable social conditions, urinary MUP concentrations increased with current reproductive output, whereas testosterone and corticosterone metabolites showed no such association. MUP concentrations were also unrelated to either hormone, suggesting that socially responsive MUP investment is not directly coupled to steroid levels. Despite baseline differences between subspecies and the expected male bias in MUP production, the main context-dependent patterns were consistent across subspecies. Our results demonstrate pronounced social plasticity in chemical signalling and endocrine state, and indicate that MUP concentrations more closely reflect reproductive outcomes than steroid metabolite concentrations. Major urinary proteins testosterone corticosterone Mus musculus musculus Mus musculus domesticus semi-natural enclosures Figures Figure 1 Figure 2 Figure 3 Introduction Group living often generates asymmetries in access to resources, promoting the emergence of dominance hierarchies. Higher-ranking individuals typically secure preferential access to food, space, or mates and therefore often achieve greater reproductive success (Clutton-Brock 1988 ; Holekamp and Strauss 2020 ). Once established, hierarchies can reduce the frequency and costs of repeated conflicts, thereby stabilising social systems (Lahn 2020 ). In mammals, where olfaction is the predominant sensory modality, chemical signalling plays a central role in social interactions (Feldhamer et al. 2007 ). Olfactory cues released via urine, faeces, and glandular secretions typically comprise complex mixtures of volatile and non-volatile compounds that convey information involving species identity, sex, health, and reproductive status (Hurst and Beynon 2013 ; Wyatt 2014 ). The formation of social hierarchies is often accompanied by dynamic changes in both endocrine secretion and the composition of chemical signals (Wyatt 2014 ; Koski et al. 2015 ; Thoß et al. 2019 ). Understanding how these signals change during the emergence of social structure requires comparisons across solitary and social contexts. However, this approach is methodologically challenging, as it requires standardised measurements under ecologically relevant conditions. Semi-natural enclosures combined with radio-frequency identification (RFID) tracking provide a powerful approach for such studies (Pereira et al. 2023 ), particularly in small, easily bred species. Among these, house mice ( Mus musculus ) represent a prominent model for investigating the links between semiochemicals, endocrinology, and social organisation (e.g., König et al. 2015 ; Thoß et al. 2015 ; Luzynski et al. 2021 ). They are relatively easy to keep and manipulate, have short generation times, and can breed year-round. In commensal populations, individuals form small social units known as demes (Berry 1981 ; Sage 1981 ; Mikula et al. 2022 ) typically comprising a dominant male that monopolises reproduction, several subordinate males, and multiple females with their juvenile offspring (Crowcroft 1955 ; van Zegeren and van Oortmerssen 1981 ). Females generally move freely within the deme’s territory, whereas subordinate males are typically restricted to its periphery (Wolff 1985 ), although they may occasionally reproduce (DeFries and McClearn 1972 ). Upon reaching sexual maturity, young males are often expelled, whereas females usually remain and reproduce within their natal deme (Lidicker 1976 ; van Zegeren 1980 ; Gerlach 1990 ; Vošlajerová Bímová et al. 2016 ). As social complexity increases, individuals must process more social information, which can favour greater signalling complexity (Rogers and Kaplan 2002 ; Bradbury and Vehrencamp 2011 ; Freeberg et al. 2012 ). In house mouse, chemical communication is the primary signalling modality (Arakawa et al. 2008 ; Hurst and Beynon 2013 ), although tactile and acoustic cues also contribute to social communication (Musolf and Penn 2012 ; Ehret 2013 ; Capas-Peneda et al. 2022 ; Diamond and Toso 2023 ). Chemical signals are detected via both the main olfactory and vomeronasal systems, and the mouse genome contains an exceptionally large repertoire of more than 1,100 functional olfactory receptors and approximately 250 pheromone receptors (Tan and Xie 2018 ; Barnes et al. 2020 ), reflecting the high informational complexity of its chemosensory system. Major urinary proteins (MUPs) are essential components of this signalling system. These lipocalins contribute to communication by binding and modulating the release of volatile ligands and by acting as proteinaceous pheromonal cues in their own right (Robertson et al. 1993 ; Beynon and Hurst 2003 ; Hurst and Beynon 2004 ). Once thought to be regulated primarily by testosterone, MUP expression is now known to involve multiple endocrine pathways, including growth hormone and thyroxine (Knopf et al. 1983 ; Noaín et al. 2013 ; Penn et al. 2022 ). Both total MUP output and isoform composition vary across the lifespan (Thoß et al. 2015 ) and are socially context-dependent in males (Stopka et al. 2007 , 2012 ; Thoß et al. 2015 , 2019 ; Luzynski et al. 2021 ). Certain MUP isoforms have been proposed as indicators of dominance status (Nelson et al. 2015 ; Lee et al. 2017 ; but see Hurst et al. 2001 ), modulators of aggressive behaviour, or predictors of aggression-related strategies (Rusu et al. 2008 ). Accordingly, urinary MUP levels have been proposed to function as honest signals of competitive ability and dominance (Rich and Hurst 1998 ; Garratt et al. 2011 , 2012 ; Nelson et al. 2015 ; but see Számadó 2011; Penn et al. 2022 ). MUP production is also sexually dimorphic: males excrete approximately three- to tenfold higher levels than females (Stopková et al. 2007 ; Cheetham et al. 2009 ; Janotová and Stopka 2009 ). Furthermore, two European house mouse subspecies, Mus musculus musculus and Mus musculus domesticus , differ markedly in both MUP abundance and isoform profiles (Stopková et al. 2007 ; Hurst et al. 2017 ). These differences may contribute to behavioural isolation and covary with endocrine dynamics (Smadja and Ganem 2002 ; Vošlajerová Bímová et al. 2011 ; Hiadlovská et al. 2015 ; Daniszová et al. 2017 ), as well as with social structure and hierarchy formation (Hiadlovská et al. 2021 ; Mikula et al. 2022 ). Comparative studies incorporating both subspecies may therefore provide deeper insight into the interplay between chemical signalling, endocrine regulation, and social organisation. Here, we used repeated measures from the same individuals to test how social environment and current reproductive output shape urinary MUP secretion and steroid hormone production. Specifically, we analysed samples collected before social exposure, during group living in semi-natural enclosures, and after return to isolation. To test generality across major axes of biological variation, we included both sexes from the two mouse subspecies. We predicted that MUP levels would increase in a complex social environment and decline after isolation, consistent with context-dependent MUP regulation and signalling functions (Lee et al. 2017 ; Penn et al. 2022 ). Because dominance hierarchies in mice are established through agonistic interactions (Oakeshott 1974 ; van Zegeren 1980 ), and endocrine state can both reflect and modulate competitive behaviour (Venetucci Gouveia et al. 2023 ), we also examined whether the production of two key hormones, testosterone and corticosterone, covaries with social context. Similarly to MUPs, we expected their concentrations to be elevated under social conditions and to correlate with social rank (Williamson et al. 2017 ), approximated here by current reproductive output. All three measures were therefore predicted to vary with reproductive success. Specifically, we expected MUP levels (in both sexes) and testosterone (in males) to be positively associated with reproduction. In contrast, corticosterone—used here as an indicator of physiological stress—was predicted to be elevated in females and subordinate males with lower reproductive output. Materials and methods The data analysed in this study were collected as part of a semi-natural enclosure project investigating population dynamics and the establishment and characterisation of social structure (Mikula et al. 2022 ). The present study specifically examines secretion patterns of MUPs and the two steroid hormones across changing social contexts – before, during, and after the semi-natural experiment. Animal origin, experimental design, enclosure setup, and parentage assignment procedures are described in Mikula et al. ( 2022 ). Details on sampling procedures, laboratory analyses, and statistical methods are provided below. Animals . Experimental animals were derived from wild-captured house mice, with founders representing both M. m. musculus and M. m. domesticus . These founders were collected in 2013 from multiple localities situated well outside the subspecies contact zone (see Mikula et al. 2022 for details). Within each subspecies, males and females originating from different populations were paired to produce first-generation (G1) offspring. After weaning at 20 days of age, juveniles were housed with same-sex littermates. At 55 days of age, 6 males and 6 females of each subspecies were transferred to single housing, where they remained until approximately 125 days of age, when they were introduced into the semi-natural enclosures as founders of the arena populations (Mikula et al. 2022 ). Numbers of mice examined during respective phases of the study (see below) are listed in Supplementary Table S1 . Semi-natural Enclosures . Two semi-natural arenas (2 × 4 m each) were established in neighbouring cabins. Each arena was initially populated with six males and six females of either M. m. musculus or M. m. domesticus (see Mikula et al. 2022 for details). Mice were provided with nest boxes, food, and water ad libitum and were allowed to establish social units and reproduce naturally. Each individual was implanted with an RFID microtransponder and marked by toe clipping at least 10 days prior to arena entry. All animals were introduced into the enclosures simultaneously after confirming good health and proper functioning of the RFID tags. Populations were monitored every 3–5 days, new animals were tagged when reaching proper body mass, shortly before leaving nest box. On day 195 of the experiment, the two enclosures were connected by two tubes, enabling inter-subspecific contact. The experiment concluded after 265 days for M. m. musculus and 272 days for M. m. domesticus . Sampling and Sample Processing . Before entry into the enclosures, and at least two weeks after transfer to single housing, founder animals were sampled for urine and faeces. Males were sampled twice and females three times, with intervals 3–4 days between sampling events. During the semi-natural experiment, both enclosures were monitored as described in Mikula et al. ( 2022 ). Urine and faecal samples were collected at six time points: days 8, 70, 140, 197, 212, and the final day of the experiment. In addition, all males were sampled 24 hours after the experiment concluded, once individuals had been returned to single housing. MUP Quantification . MUP concentrations were quantified from frozen urine samples collected by temporarily placing mice in clean cages. Proteins were separated using denaturing polyacrylamide gel electrophoresis (SDS–PAGE) on 4–15% Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad, Hercules, CA, USA). A concentration gradient of carbonic anhydrase (external standard) was loaded alongside the samples to enable absolute quantification of the separated proteins. Electrophoretograms were imaged using a GS-900 calibrated densitometer (Bio-Rad), and bands of interest were quantified with Image Lab™ software (Bio-Rad). Band volume was defined as the summed pixel intensity within the designated band boundary. Protein concentrations were calculated from a carbonic anhydrase calibration curve (Janotová and Stopka 2011). To account for variation in urine dilution among individuals and sampling occasions, MUP concentrations were normalised using a creatinine assay (LKreatinin Kit, BioVendor, Brno, Czechia). A dilution coefficient was calculated based on creatinine concentration following Stopka et al. ( 2007 ). Corrected MUP values were obtained by dividing each measured concentration by this coefficient, yielding creatinine-adjusted concentrations (ng ml⁻¹) for each sample. Hormone Extraction and Enzyme Immunoassays . Testosterone and corticosterone metabolites were extracted from frozen faecal samples collected from mice temporarily placed in clean cages. Approximately 0.05 g of dried, finely ground faecal powder was vortexed in 1 ml of 80% methanol for 30 min, following Palme et al. ( 2013 ). After centrifugation (2500 ⋅ g, 15 min), supernatants were collected and stored at − 20°C until analysis. Corticosterone metabolites were measured using a polyclonal antibody (CJM006, 1:16,000 dilution), and testosterone metabolites using a polyclonal antibody (R156/7, 1:8,000 dilution), together with the corresponding horseradish peroxidase (HRP) conjugates (1:40,000 and 1:60,000 dilution, respectively), obtained from EndoLab (Department of Population Health and Reproduction, UC Davis, USA). Cross-reactivities and detailed assay procedures are described in Hiadlovská et al. ( 2015 ). Hormone metabolite concentrations are reported as ng g − 1 faeces. Inter- and intra-assay coefficients of variation for both assays were < 15%, consistent with recommended performance criteria for immunoassays (Findlay et al. 2000 ; Shah et al. 2000 ). Parentage Analysis and Reproductive Status . Genomic DNA was isolated from all individuals sampled prior to the experiment (enclosure founders) and during the semi-natural enclosure phase (pups were sampled shortly prior to RFID tagging). Panels of 25 and 26 microsatellite markers were genotyped in M. m. musculus and M. m. domesticus , respectively (see Mikula et al. 2022 for details). Parentage was estimated using CERVUS v.3.0.3 (Kalinowski et al. 2007 ) with a 95% confidence threshold. Only individuals meeting predefined criteria (i.e., alive and sexually mature at the estimated time of conception) were considered candidate parents. Parentage assignments were then used to determine reproductive status of individuals at each of the six enclosure sampling events. Current reproductive output for each individual was defined as the number of assigned offspring born within a 44-day window centred on a given sampling event (i.e., 22 days before to 22 days after the event). Under this definition, an individual was classified as reproductively active if it engaged in mating or conception, a female was pregnant (gestation ≈ 20 days) or nursing (weaning ≈ 20 days), or a male had genetically assigned offspring whose mother was pregnant or nursing during that period. Individuals with at least one assigned offspring within the 44-day window were classified as reproducing. Although this approach may not be entirely precise (particularly for males), it provides a better time-specific measure of reproductive success that can be directly aligned with contemporaneous measures of steroid hormone metabolites and urinary MUP concentrations, offering an improvement over a simple binary reproducing/non-reproducing classification. Statistical Analyses . The analyses were designed to address two complementary questions: (i) how the presence versus absence of social interactions affects urinary MUP concentrations and steroid hormone metabolite concentrations (Initial and Isolation phases), and (ii) how reproductive activity influences these traits under social conditions (Social phase). The ‘Initial phase’ compared measurements obtained under social isolation prior to arena entry (two sampling events in males, three in females) with those collected during the first enclosure sampling (day 8). The ‘Isolation phase’ compared measurements from the final enclosure sampling with those taken after 24 hours of solitary housing following the end of the experiment. For both phases, linear mixed-effects models (LMMs) were fitted with individual identity included as a random intercept, and social context (isolated vs. social) and subspecies specified as fixed effects. For the Initial phase, sex was included as an additional fixed effect. To evaluate effects of reproduction under complex social conditions, we analysed data from the enclosure phase (‘Social phase’), during which social organisation had been established. Current reproductive output of each individual was assessed using the 44-day window described above. LMMs included individual identity as a random intercept and current reproductive output, sex, and subspecies as fixed effects. For visualisation purposes, current reproductive output was represented as a binary factor: non-reproducing (no assigned offspring) versus reproducing (≥ 1 offspring within the 44-day window). Mice sampled only once in the arena were excluded from the Social dataset, as we presumed their presence in the population was only ephemeral. We further tested whether steroid hormone metabolite concentrations covaried with MUP concentrations in relation to current reproductive output, sex, and subspecies. Separate LMMs were fitted for each hormone, as testosterone was not measured in females. All models were estimated using restricted maximum likelihood (REML) in the R package nlme (Pinheiro and Bates 2025 ; R Core Team 2025 ). Response variables were natural log-transformed, normalised urinary MUP concentrations (ng ml⁻¹) and hormone metabolite concentrations (ng g⁻¹). Accordingly, fixed-effect estimates ( β ) represent effect sizes on the natural-log scale. Model assumptions were assessed by visual inspection of residual distributions and residual-versus-fitted plots. Predicted values were plotted to illustrate group effects while accounting for covariates; corresponding plots of the observed values are provided in the Supplementary Material (Fig. S1 –3). The difference between metabolite levels before and after isolation (Δ) was calculated for each individual in the Isolation phase. To investigate the potential effect of current reproductive output on both the magnitude and direction (decline/increase) of these changes (see Thoß et al. 2019 ), ΔMUP, ΔTestosterone and ΔCorticosterone were analysed using generalised least squares (GLS) models with subspecies and current reproductive output (CRO) as fixed effects. Current reproductive output was analysed either as a binary variable (reproducing: CRO > 0; non-reproducing: CRO = 0) or as a continuous variable (number of assigned offspring). Heteroscedasticity was accounted for by modelling group-specific standard deviations (varIdent structure) when CRO was treated as a binary variable, or by modelling residual variance as an exponential function of current reproductive output (varExp structure in the nlme package) when CRO was treated as a continuous variable. Results Initial Phase: Entry into the Social Environment . In this phase, we contrasted measurements obtained during individual housing with those collected on day 8 after entry into the semi-natural arena. At this time, no offspring had yet been born, although mating had already occurred. Urinary MUP concentrations increased significantly following the transition from individual to social housing, and this effect was observed in both sexes and in subspecies ( β = 1.385, P < 0.001, N obs = 82, N groups = 24; Fig. 1 A, Table 1 ). Across social contexts, males exhibited significantly higher MUP concentrations than females ( β = 1.196, P < 0.001), and M. m. musculus showed higher MUP concentrations than M. m. domesticus ( β = 1.452, P < 0.001). A similar response to the social environment was observed for both hormones. Testosterone concentrations increased significantly under social conditions in both subspecies ( β = 1.256, P < 0.001, N obs = 35, N groups = 12; Fig. 1 B, Table 1 ). Although M. m. musculus consistently showed higher testosterone concentrations than M. m. domesticus both before and after enclosure entry, this difference was not statistically significant ( β = 0.357, P = 0.143). Corticosterone concentrations were likewise significantly elevated under social conditions across all groups ( β = 1.254, P < 0.001, N obs = 82, N groups = 24; Fig. 1 C, Table 1 ). Females exhibited higher corticosterone concentrations than males ( β = -0.455, P = 0.016), whereas corticosterone did not differ significantly between subspecies ( β = -0.118, P = 0.503). Table 1 Fixed-effect estimates from linear mixed models evaluating MUP, corticosterone, and testosterone concentrations across the Initial, Social, and Isolation phases. Each model includes subspecies, sex (when applicable), and social context as fixed effects, with animal ID fitted as a random intercept (subspecies, sex, or context displaying higher values are given in parentheses). Estimates are presented on the natural-log scale of normalised concentrations. Columns report the estimate, standard error (SE), degrees of freedom ( df ), t -value, and p -value for each predictor. CRO = current reproductive output Phase Agent Effect Estimate ( β ) SE df t P Initial MUP Subspecies ( musculus ) 1.452 0.205 21 7.092 < 0.001 Sex (male) 1.196 0.207 21 5.777 < 0.001 Context (social) 1.385 0.197 57 7.018 < 0.001 Initial Corticosterone Subspecies ( musculus ) -0.118 0.174 21 -0.681 0.503 Sex (male) -0.455 0.174 21 -2.619 0.016 Context (social) 1.254 0.104 57 12.041 < 0.001 Initial Testosterone Subspecies ( musculus ) 0.358 0.225 10 1.589 0.143 Context (social) 1.256 0.211 22 5.944 < 0.001 Social MUP Subspecies ( musculus ) 0.643 0.101 98 6.352 < 0.001 Sex (male) 1.422 0.102 98 14.012 < 0.001 CRO 0.042 0.011 218 3.911 < 0.001 Social Corticosterone Subspecies ( musculus ) -0.126 0.093 99 -1.351 0.180 Sex (male) -0.563 0.093 99 -6.041 < 0.001 CRO -0.012 0.010 228 -1.25 0.215 Social Testosterone Subspecies ( musculus ) 0.642 0.121 56 5.317 < 0.001 CRO -0.005 0.012 114 -0.400 0.690 Isolation MUP Subspecies ( musculus ) 1.808 0.157 72 11.549 < 0.001 Context (post-social) -0.775 0.109 65 -7.083 < 0.001 Isolation Corticosterone Subspecies ( musculus ) 0.058 0.110 77 0.530 0.597 Context (post-social) -0.497 0.090 74 -5.506 < 0.001 Isolation Testosterone Subspecies ( musculus ) 0.463 0.113 74 4.104 < 0.001 Context (post-social) -0.607 0.110 73 -5.527 < 0.001 Isolation Phase: Exit from Social Environment . One day after removal from the complex social environment, urinary MUP concentrations decreased significantly in males of both subspecies ( β = -0.775, P < 0.001, N obs = 140, N groups = 74). Across social contexts, M. m. musculus males exhibited higher MUP concentrations than M. m. domesticus ( β = 1.808, P < 0.001; Fig. 2 A, Table 1 ). Both hormones likewise declined following removal from the social environment (testosterone: β = -0.607, P < 0.001, N obs = 150, N groups = 76; corticosterone: β = -0.497, P < 0.001 N obs = 154, N groups = 79; Fig. 2 B–C). Testosterone concentrations remained higher in M. m. musculus than in M. m. domesticus ( β = 0.463; P < 0.001), whereas corticosterone concentrations did not differ significantly between subspecies ( β = 0.058; P = 0.597). For testosterone, and to a lesser extent for MUPs, the decline was significantly stronger in M. m. musculus than in M. m. domesticus (Supplementary Information Tab. S1, Fig S4). Social Phase: Effect of Current Reproductive Output . Under established social conditions, urinary MUP concentrations increased with current reproductive output when modelled as a continuous variable ( β = 0.042, P < 0.001, N obs = 320, N groups = 101; Table 1 ). For visualisation, this pattern is also illustrated by contrasting two categories: reproducing vs. non-reproducing individuals (Fig. 3 A). Significant effects in MUP concentrations were also detected for sex (males > females; β = 1.416, P M. m. domesticus ; β = 0.650, P < 0.001). Current reproductive output was not significantly associated with testosterone or corticosterone metabolite concentrations (testosterone: β = -0.005, P = 0.690, N obs = 173, N groups = 58; corticosterone: β = -0.012; P = 0.215, N obs = 331, N groups = 102; Fig. 3 B–C, Table 1 ). Testosterone concentrations were higher in M. m. musculus than in M. m. domesticus ( β = 0.642, P < 0.001), whereas corticosterone concentrations did not differ significantly between subspecies ( β = -0.126, P = 0.180). In both subspecies, males displayed lower corticosterone concentrations than females ( β = -0.563, P < 0.001). After accounting for covariates, MUP concentrations were not significantly associated with testosterone ( β = 0.030; P = 0.678, N obs = 169, N groups = 58) or corticosterone concentrations ( β = 0.092; P = 0.135, N obs = 314, N groups = 100). Discussion This study examined how chemical signalling and endocrine outputs respond to transitions between solitary and socially complex environments. Both urinary MUP concentrations and steroid hormone metabolites were highly socially plastic under semi-natural conditions. We identified a coordinated “social-entry” response: MUP, testosterone, and corticosterone concentrations increased sharply after males entered enclosures, coinciding with the onset of hierarchy formation. A similarly rapid “social-exit” response followed removal from enclosures, with males downregulating urinary MUPs and both hormones within one day of isolation, consistent with rapid endocrine and signalling adjustment following the loss of social interactions (Creel 2001 , 2013; Eisenegger et al. 2011 ; Thoß et al. 2019 ). Under established social conditions, MUP concentrations – but not testosterone or corticosterone metabolites – tracked current reproductive output. MUP levels were not significantly associated with either hormone, supporting the view that their regulation is not directly coupled to steroid hormone dynamics (Knopf et al. 1983 ; Noaín et al. 2013 ; Penn et al. 2022 ). Entry into the Social Environment: Rapid Upregulation of MUPs and Endocrine Activation . The predicted increase in urinary MUP concentrations upon entry into the semi-natural enclosures was strongly supported and occurred in both sexes and both subspecies. This finding aligns with repeated evidence that MUP production is socially modulated and context-dependent, particularly under competitive conditions (Stopka et al. 2007 , 2012 ; Thoß et al. 2015 , 2019 ; Luzynski et al. 2021 ). The pronounced rise during the entry phase is consistent with increased signalling effort when social relationships are being established and competition for mates and space is most intense (Bradbury and Vehrencamp 2011 ; Számadó 2011). This temporal pattern accords with general signalling theory: the costs and benefits of signalling – and thus selection on signal investment – vary as individuals move between social conditions, as hierarchies form or destabilise, and as roles change over time (Bradbury and Vehrencamp 2011 ; Számadó 2011). The increase in MUP concentrations in both males and females further supports the view that MUPs both influence and respond to social interactions beyond male–male competition alone (Beynon and Hurst 2003 ; Chamero et al. 2007 ; Rusu et al. 2008 ; Roberts et al. 2010 , 2012 ; Nelson et al. 2015 ). Endocrine responses during entry mirrored the MUP pattern. Testosterone and corticosterone metabolites both increased significantly. For testosterone, this is consistent with context-dependent frameworks in which testosterone biases motivation and information processing toward status-relevant goals, with behavioural outcomes shaped by the social environment (Eisenegger et al. 2011 ; Knight et al. 2020 ). Under the challenge hypothesis, testosterone rises transiently during social challenges and may decline when effort shifts toward parenting or bonding (Wingfield 1990; Bell 2020 ). The observed increase therefore matches expectations for the early, unstable phase of hierarchy formation (Wingfield 1990; Eisenegger et al. 2011 ; Bell 2020 ). The concurrent rise in corticosterone metabolites is likewise expected under novel social conditions, instability, and elevated interaction rates. In mammals, glucocorticoid–status relationships may reflect either “subordinate stress” or the “cost of dominance”, depending on hierarchy stability and the distribution of physiological costs (Creel 2001 ; Goymann and Wingfield 2004 ; Creel et al. 2013 ). Although our design does not allow direct rank-specific inference, the increase observed during entry supports the broader conclusion that glucocorticoids track changes in the social environment (Creel 2001 ; Creel et al. 2013 ; Wyatt 2014 ). Exit from the Social Environment: Rapid Downregulation of Signalling and Hormones . Plasticity was equally evident upon exit from the social environment. Within one day of isolation, MUP, testosterone, and corticosterone concentrations decreased significantly in males of both subspecies. This rapid reversal is consistent with the expectation that signalling investment should decline when opportunities for territorial advertisement associated with social and reproductive competition diminish (Bradbury and Vehrencamp 2011 ; Számadó 2011). Our results parallel those of Thoß et al. ( 2019 ), who showed that dominant males upregulated MUP expression under competitive conditions and that differences between dominant and subordinate males disappeared after removal from the enclosure, largely through downregulation by dominant individuals. Although we did not assign dominance behaviourally, our data support the broader conclusion that increased MUP investment is favoured under competitive social conditions and rapidly reduced when competition ceases (Stopka et al. 2007 , 2012 ; Thoß et al. 2019 ; Luzynski et al. 2021 ). The parallel endocrine declines are also consistent with reciprocal causality perspectives, in which hormones both shape and respond to social context (Eisenegger et al. 2011 ). In contrast to Thoß et al. ( 2019 ), who reported rank-dependent differences in the magnitude of MUP regulation, we detected no significant effect of reproductive output on the direction or extent of change in any measured variable (Supplementary Material). A striking feature of these responses is their speed: MUP concentrations and steroid metabolites shifted markedly within 24 hours. This has important methodological implications, as animals captured in the field are often held in isolation prior to sampling, potentially leading to systematic underestimation of MUP and hormone expression. This highlights the need for caution when comparing studies with different housing histories or sampling protocols. Social Phase: Reproductive Output but not Steroid Metabolites Predicts MUP Concentrations . Rather than dyadic tests or direct behavioural observations, we used current reproductive output as a proxy for competitive success, given that dominant males typically secure greater access to females and reproduction. Under established social conditions, urinary MUP concentrations increased with reproductive output, whereas testosterone and corticosterone metabolites did not. This divergence is consistent with the hypothesis that urinary MUPs function as honest signals of competitive ability within complex social networks (Rich and Hurst 1998 ; Garratt et al. 2011 , 2012 ; Nelson et al. 2015 ; Thoß et al. 2019 ; Luzynski et al. 2021 ). However, as emphasised by Számadó (2011) and Penn et al. ( 2022 ), signal honesty depends on context-dependent trade-offs and the potential costs of cheating. The absence of associations between steroid metabolites and reproductive output does not preclude their role in competition or mating. Plasma hormones fluctuate rapidly in response to acute social interactions, whereas faecal metabolites integrate endocrine activity over longer periods. Consequently, metabolite measures may better reflect broad environmental transitions – including entry into or exit from social environments – than short-lived endocrine responses mediating agonistic or reproductive behaviour (Wingfield 1990; Creel 2001 ; Eisenegger et al. 2011 ; Creel et al. 2013 ). Under these conditions, urinary MUPs appear to be more sensitive indicators of realised reproductive outcomes than time-integrated hormone metabolites. No Direct Association between MUPs and Testosterone: Multi-layered Endocrine Regulation . Interestingly, MUP concentrations were not significantly associated with testosterone or corticosterone after accounting for covariates. This supports the view that MUP regulation is not governed by testosterone alone. Although traditionally considered androgen-dependent, MUP expression is now known to be under multi-hormonal control, including a key role for growth hormone (Knopf et al. 1983 ; Noaín et al. 2013 ; Penn et al. 2022 ). Our results are therefore consistent with the idea that social context shapes MUP investment through regulatory pathways that are not captured by simple linear relationships with androgen metabolites (Penn et al. 2022 ). Sex and Subspecies Patterns . Across contexts, males exhibited substantially higher urinary MUP concentrations than females, consistent with established sexual dimorphism (Stopková et al. 2007 ; Cheetham et al. 2009 ; Janotová and Stopka 2009 ; Penn et al. 2022 ). This male bias is likely maintained by sexual selection, either through male-male competition or via female mate choice (Meagher et al. 2000 ; Hurst and Beynon 2004 ; Kaur et al. 2014 ; Penn et al. 2022 ). Importantly, females showed the same direction of context dependence during the entry phase, indicating that MUP signalling is socially responsive in both sexes, even when absolute levels differ (Beynon and Hurst 2003 ; Chamero et al. 2007 ; Roberts et al. 2010 , 2012 ; Nelson et al. 2015 ). Together with studies by Garratt et al. ( 2011 ) and Stockley et al. ( 2013 ), which report increased urinary protein excretion during female–female aggression, it appears that the MUP-based signalling system is as important for females as it is for males. We observed higher MUP concentrations in M. m. musculus than in M. m. domesticus across all three phases, consistent with known differences in MUP abundance and isoform profiles between these subspecies (Stopková et al. 2007 ; Hurst et al. 2017 ; Macholán et al. 2023 ). According to Stopková et al. ( 2007 ) and Janotová and Stopka ( 2009 ), this pattern may be related to stronger assortative mate preferences in M. m. musculus females compared with M. m. domesticus females (Munclinger and Frynta 1997 ; Christophe and Baudoin 1998 ; Smadja and Ganem 2002 ; Smadja et al. 2004 ; Bímová et al. 2005 ; Vošlajerová Bímová et al. 2011 ). Alternatively, as suggested by Janotová and Stopka ( 2009 ), MUP production may be important in both subspecies, but mate choice in M. m. domesticus females could be constrained by a conflict between selecting a high-quality male with high MUP output and selecting a mate of the same subspecies. The lower MUP output found in M. m. domesticus may also reflect the greater aggressiveness of M. m. domesticus males (Thuessen 1977; van Zegeren and van Oortmerssen 1981 ; Piálek et al. 2008 ; Ďureje et al. 2011 ), such that reproductive success is determined primarily through male–male competition rather than through female choice. Adding further complexity, other communication-related body fluids, such as tears, show the opposite pattern – higher protein output in M. m. domesticus – which may represent a complementary signalling mechanism (Vošlajerová Bímová et al. 2026 ). Despite baseline differences, the main effects relevant to our focal hypotheses – social entry and exit responses and the association between MUPs and reproductive output – were consistent across subspecies, suggesting a general pattern in how complex social environments shape MUP signalling investment. Regarding steroid metabolites, we again observed higher testosterone concentrations in M. m. musculus than in M. m. domesticus (Hiadlovská et al. 2015 ), whereas corticosterone did not differ significantly between subspecies. This pattern coincides with variation in dominance dynamics rather than a fixed endocrine difference: prolonged testosterone production in M. m. musculus males may reflect longer periods of unsettled dominance relationships (Hiadlovská et al. 2015 ), which was later confirmed under semi-natural conditions (Mikula et al. 2022 ). Corticosterone levels were consistently higher in females than in males, suggesting greater physiological demands or social challenges. Baseline levels did not differ between subspecies; however, M. m. domesticus females showed a stronger increase after entering the enclosure than M. m. musculus females. Two non-mutually exclusive mechanisms may explain this pattern. First, the higher aggressiveness of M. m. domesticus males may result in accidental or misdirected attacks on females, as occasionally reported by animal keepers. Second, aggression is not restricted to male mice (Crowcroft and Rowe 1963 ; van Zegeren 1980 ; Frynta et al. 2005 ), and the social structure of M. m. domesticus populations appears more closed than that of M. m. musculus (Mikula et al. 2022 ). This may increase female social pressure. However, whether these mechanisms underlie the observed endocrine differences following enclosure entry remains to be tested. Conclusions Our repeated-measures design demonstrates strong social plasticity in both chemical signalling and endocrine outputs. Entry into a complex social environment elicited coordinated increases in MUP secretion and steroid metabolites, whereas removal rapidly reduced both signalling and endocrine activity. Under established social conditions, MUP concentrations – but not steroid metabolites – tracked current reproductive output, and were not significantly associated with either hormone. Two findings deserve emphasis. First, although most studies treat MUPs primarily as male honest signals, our results show that females modulate MUP production across social transitions in parallel with males, highlighting a broader role of this signalling system beyond male competition. Second, despite divergence between M. m. musculus and M. m. domesticus in social behaviour, the key patterns relevant to our focal questions were consistent across subspecies. This suggests general principles governing how complex social environments regulate signalling investment and endocrine state in house mice. Declarations Competing Interests – The authors have no relevant financial or non-financial interests to disclose. Ethics statement – The breeding facility of the Institute of Vertebrate Biology, Czech Academy of Sciences, in Studenec, has been licensed for keeping small mammals according to the Czech law (Licenses No. 227203/2011-MZE- 17214 2011–2016). Animals were handled by authorised persons only (ZH Licenses No. CZ 00548, CZ 0127; PH as a university student under supervision). This study was performed in accordance with Czech law, implementing all corresponding EU regulations and approved by the IVB Ethical Committee (N 145/2010). Author Contributions – The project was conceptualized and supervised by Barbora Vošlajerová and (temporarily) by Miloš Macholán. Ľudovít Ďureje, Zuzana Hiadlovská, and Iva Pospíšilová were responsible for semi-natural enclosure management. Data were curated by Barbora Vošlajerová Bímová, Ľudovít Ďureje, and Zuzana Hiadlovská. Kristina Daniszová was responsible for MUP and hormone quantification, basic data processing, and paternity analysis; Iva Pospíšilová participated in hormone quantification and basic data processing; Kateřina Janotová participated in MUP data acquisition. Statistical analyses were performed by Natália Martínková with the participation of Zuzana Hiadlovská. The first draft was written by Zuzana Hiadlovská, Miloš Macholán, Natália Martínková, Barbora Vošlajerová Bímová, and Kristina Daniszová. All authors participated in the final editing of the manuscript. Data Availability – The data are available in zenodo with the identifier doi : 10.5281/zenodo.19454221 at zenodo.org/records/19454221. Funding – The study was funded by the Czech Science Foundation grant (GAP506/11/1792 to BVB) and the institutional funding of the Institute of Vertebrate Biology (RVO:68081766). The authors also thank the RECETOX Research Infrastructure (No LM2023069), financed by the Ministry of Education, Youth and Sports, for its supportive background. This work was supported by the European Union’s Horizon 2020 research and innovation programme under grant agreements No 857560 (CETOCOEN Excellence). This publication reflects only the author’s view, and the European Commission is not responsible for any use that may be made of the information it contains. Author Contribution The project was conceptualized and supervised by Barbora Vošlajerová and (temporarily) by Miloš Macholán. Ľudovít Ďureje, Zuzana Hiadlovská, and Iva Pospíšilová were responsible for semi-natural enclosure management. Data were curated by Barbora Vošlajerová Bímová, Ľudovít Ďureje, and Zuzana Hiadlovská. Kristina Daniszová was responsible for MUP and hormone quantification, basic data processing, and paternity analysis; Iva Pospíšilová participated in hormone quantification and basic data processing; Kateřina Janotová participated in MUP data acquisition. Statistical analyses were performed by Natália Martínková with the participation of Zuzana Hiadlovská. The first draft was written by Zuzana Hiadlovská, Miloš Macholán, Natália Martínková, Barbora Vošlajerová Bímová, and Kristina Daniszová. All authors participated in the final editing of the manuscript. 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Supplementary Files SocialmodulationpaperSupplement.pdf SupplementaryTableS1.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 22 May, 2026 Reviewers invited by journal 07 May, 2026 Editor assigned by journal 07 May, 2026 Submission checks completed at journal 01 May, 2026 First submitted to journal 28 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9552933","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":641582821,"identity":"3269d498-30b4-4603-a25f-a78c194f403c","order_by":0,"name":"Zuzana Hiadlovská","email":"","orcid":"","institution":"Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zuzana","middleName":"","lastName":"Hiadlovská","suffix":""},{"id":641582823,"identity":"56b3e365-5ef4-49c8-a58a-32773cbbc664","order_by":1,"name":"Barbora Vošlajerová Bímová","email":"","orcid":"","institution":"Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Barbora","middleName":"Vošlajerová","lastName":"Bímová","suffix":""},{"id":641582824,"identity":"b47e57b6-02e8-4762-bb50-8da466619898","order_by":2,"name":"Kristina Daniszová","email":"","orcid":"","institution":"Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kristina","middleName":"","lastName":"Daniszová","suffix":""},{"id":641582829,"identity":"b9992937-09af-4faf-99aa-66be5b861b2e","order_by":3,"name":"Natália Martínková","email":"","orcid":"","institution":"Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Natália","middleName":"","lastName":"Martínková","suffix":""},{"id":641582833,"identity":"3932df3a-f597-4548-bcca-95320b2d6f11","order_by":4,"name":"Iva Pospíšilová","email":"","orcid":"","institution":"Masaryk University","correspondingAuthor":false,"prefix":"","firstName":"Iva","middleName":"","lastName":"Pospíšilová","suffix":""},{"id":641582838,"identity":"3fb83cbc-f473-4aa9-a9e6-6b34320e3114","order_by":5,"name":"Kateřina Janotová","email":"","orcid":"","institution":"Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kateřina","middleName":"","lastName":"Janotová","suffix":""},{"id":641582843,"identity":"7d86870c-4937-421d-b558-7d7e1ecc9334","order_by":6,"name":"Ľudovít Ďureje","email":"","orcid":"","institution":"Czech Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ľudovít","middleName":"","lastName":"Ďureje","suffix":""},{"id":641582844,"identity":"580fbac5-469c-4afc-9014-3fed882ec2e8","order_by":7,"name":"Miloš Macholán","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYHACNgaGAgYGfmYGBmYStBgwMEg2k6zF4ACxWnTbDz978MHALs/4OPPDzwUMdvYEtZidSTM3nGGQXGx2mM1YegZDMmGbzA7ksEnzGDAnbjvMYMbMw3CAjbCW829AWuoTNzezfwNp4SGs5QbYlsOJG5h5wLZIEKHlmZnkDIPjiTMO8xQD9SYbEOGw5GcSHyqqE/v7j2/8zFNBRIihAcJ2jIJRMApGwSggBgAAEBQxH7fBrjIAAAAASUVORK5CYII=","orcid":"","institution":"Czech Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Miloš","middleName":"","lastName":"Macholán","suffix":""}],"badges":[],"createdAt":"2026-04-28 10:54:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9552933/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9552933/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109462537,"identity":"8f79db2d-9673-40b9-9fcc-23c09140497d","added_by":"auto","created_at":"2026-05-18 11:12:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":282242,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted concentrations of MUPs (A), testosterone (B), and corticosterone (C) in the Initial phase. Desaturated colours depict singly housed mice before enclosure entrance (pre-social context), whereas saturated colours display mice socialised for 8 days in the enclosure (social context). Boxplots represent the interquartile range (Q1–Q3), with the bold horizontal line indicating the median. Whiskers extend to the most extreme data points within 1.5 × Interquartile range (IQR), outlier values outside this range are shown as empty circles\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9552933/v1/be4d21d823eb029a6613d136.png"},{"id":109462534,"identity":"8e70ee18-9d7e-4bb6-9dbe-4ce01160d18b","added_by":"auto","created_at":"2026-05-18 11:12:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227794,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted concentrations of MUPs (A), testosterone (B), and corticosterone (C) in the Isolation phase. Desaturated colours represent mice on their final day in the complex social environment (social context), whereas saturated colours represent mice 24 h after transfer to solitary housing (post-social context). Boxplots display the interquartile range (Q1–Q3), with the bold horizontal line indicating the median. Whiskers extend to the most extreme data points within 1.5 × IQR, and values outside the range are shown as empty circles\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9552933/v1/9908de333d65c420485017c1.png"},{"id":109759308,"identity":"8b690a69-6ebd-4fc1-be99-5ae8d3e53e5b","added_by":"auto","created_at":"2026-05-22 07:26:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":319257,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted concentrations of MUPs (A), testosterone (B), and corticosterone (C) during the Social phase. Desaturated colours display non-reproducing mice (i.e., those to which no offspring within the 44-day window surrounding sampling could be assigned), whereas saturated colours represent reproducing mice (≥ 1 assigned offspring). Boxplots display the median and IQR. Whiskers extend to the most extreme data points within 1.5 × IQR; outliers are shown as empty circles\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9552933/v1/56c4e0419761dcceee197ac4.png"},{"id":109763985,"identity":"981f308e-1b30-446d-ab41-06473cb56212","added_by":"auto","created_at":"2026-05-22 07:36:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1176317,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9552933/v1/2e8aa5ad-5662-4308-b866-36fd1a7d7aa5.pdf"},{"id":109462533,"identity":"f6d7cb1d-1781-436b-8f4c-1a4de70c7b52","added_by":"auto","created_at":"2026-05-18 11:12:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":778751,"visible":true,"origin":"","legend":"","description":"","filename":"SocialmodulationpaperSupplement.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9552933/v1/b349a7abda75d282d591a52a.pdf"},{"id":109759839,"identity":"23ada05c-a368-4da7-b258-351b134e3c08","added_by":"auto","created_at":"2026-05-22 07:27:49","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18760,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9552933/v1/9215f463ece6b6d775cd718e.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Social context rapidly reshapes chemical signalling and endocrine profiles in two mouse subspecies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGroup living often generates asymmetries in access to resources, promoting the emergence of dominance hierarchies. Higher-ranking individuals typically secure preferential access to food, space, or mates and therefore often achieve greater reproductive success (Clutton-Brock \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Holekamp and Strauss \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Once established, hierarchies can reduce the frequency and costs of repeated conflicts, thereby stabilising social systems (Lahn \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn mammals, where olfaction is the predominant sensory modality, chemical signalling plays a central role in social interactions (Feldhamer et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Olfactory cues released via urine, faeces, and glandular secretions typically comprise complex mixtures of volatile and non-volatile compounds that convey information involving species identity, sex, health, and reproductive status (Hurst and Beynon \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wyatt \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The formation of social hierarchies is often accompanied by dynamic changes in both endocrine secretion and the composition of chemical signals (Wyatt \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Koski et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tho\u0026szlig; et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUnderstanding how these signals change during the emergence of social structure requires comparisons across solitary and social contexts. However, this approach is methodologically challenging, as it requires standardised measurements under ecologically relevant conditions. Semi-natural enclosures combined with radio-frequency identification (RFID) tracking provide a powerful approach for such studies (Pereira et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), particularly in small, easily bred species.\u003c/p\u003e \u003cp\u003eAmong these, house mice (\u003cem\u003eMus musculus\u003c/em\u003e) represent a prominent model for investigating the links between semiochemicals, endocrinology, and social organisation (e.g., K\u0026ouml;nig et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tho\u0026szlig; et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Luzynski et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). They are relatively easy to keep and manipulate, have short generation times, and can breed year-round. In commensal populations, individuals form small social units known as demes (Berry \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Sage \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) typically comprising a dominant male that monopolises reproduction, several subordinate males, and multiple females with their juvenile offspring (Crowcroft \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1955\u003c/span\u003e; van Zegeren and van Oortmerssen \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Females generally move freely within the deme\u0026rsquo;s territory, whereas subordinate males are typically restricted to its periphery (Wolff \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), although they may occasionally reproduce (DeFries and McClearn \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). Upon reaching sexual maturity, young males are often expelled, whereas females usually remain and reproduce within their natal deme (Lidicker \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; van Zegeren \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Gerlach \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Vošlajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs social complexity increases, individuals must process more social information, which can favour greater signalling complexity (Rogers and Kaplan \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Bradbury and Vehrencamp \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Freeberg et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In house mouse, chemical communication is the primary signalling modality (Arakawa et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hurst and Beynon \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), although tactile and acoustic cues also contribute to social communication (Musolf and Penn \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ehret \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Capas-Peneda et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Diamond and Toso \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Chemical signals are detected via both the main olfactory and vomeronasal systems, and the mouse genome contains an exceptionally large repertoire of more than 1,100 functional olfactory receptors and approximately 250 pheromone receptors (Tan and Xie \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Barnes et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), reflecting the high informational complexity of its chemosensory system.\u003c/p\u003e \u003cp\u003eMajor urinary proteins (MUPs) are essential components of this signalling system. These lipocalins contribute to communication by binding and modulating the release of volatile ligands and by acting as proteinaceous pheromonal cues in their own right (Robertson et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Beynon and Hurst \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hurst and Beynon \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Once thought to be regulated primarily by testosterone, MUP expression is now known to involve multiple endocrine pathways, including growth hormone and thyroxine (Knopf et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Noa\u0026iacute;n et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Penn et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Both total MUP output and isoform composition vary across the lifespan (Tho\u0026szlig; et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and are socially context-dependent in males (Stopka et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tho\u0026szlig; et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Luzynski et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Certain MUP isoforms have been proposed as indicators of dominance status (Nelson et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; but see Hurst et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), modulators of aggressive behaviour, or predictors of aggression-related strategies (Rusu et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Accordingly, urinary MUP levels have been proposed to function as honest signals of competitive ability and dominance (Rich and Hurst \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Garratt et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nelson et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; but see Sz\u0026aacute;mad\u0026oacute; 2011; Penn et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMUP production is also sexually dimorphic: males excrete approximately three- to tenfold higher levels than females (Stopkov\u0026aacute; et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Cheetham et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Janotov\u0026aacute; and Stopka \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Furthermore, two European house mouse subspecies, \u003cem\u003eMus musculus musculus\u003c/em\u003e and \u003cem\u003eMus musculus domesticus\u003c/em\u003e, differ markedly in both MUP abundance and isoform profiles (Stopkov\u0026aacute; et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hurst et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These differences may contribute to behavioural isolation and covary with endocrine dynamics (Smadja and Ganem \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Vošlajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Hiadlovsk\u0026aacute; et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Daniszov\u0026aacute; et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), as well as with social structure and hierarchy formation (Hiadlovsk\u0026aacute; et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Comparative studies incorporating both subspecies may therefore provide deeper insight into the interplay between chemical signalling, endocrine regulation, and social organisation.\u003c/p\u003e \u003cp\u003eHere, we used repeated measures from the same individuals to test how social environment and current reproductive output shape urinary MUP secretion and steroid hormone production. Specifically, we analysed samples collected before social exposure, during group living in semi-natural enclosures, and after return to isolation. To test generality across major axes of biological variation, we included both sexes from the two mouse subspecies. We predicted that MUP levels would increase in a complex social environment and decline after isolation, consistent with context-dependent MUP regulation and signalling functions (Lee et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Penn et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBecause dominance hierarchies in mice are established through agonistic interactions (Oakeshott \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; van Zegeren \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1980\u003c/span\u003e), and endocrine state can both reflect and modulate competitive behaviour (Venetucci Gouveia et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we also examined whether the production of two key hormones, testosterone and corticosterone, covaries with social context. Similarly to MUPs, we expected their concentrations to be elevated under social conditions and to correlate with social rank (Williamson et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), approximated here by current reproductive output. All three measures were therefore predicted to vary with reproductive success. Specifically, we expected MUP levels (in both sexes) and testosterone (in males) to be positively associated with reproduction. In contrast, corticosterone\u0026mdash;used here as an indicator of physiological stress\u0026mdash;was predicted to be elevated in females and subordinate males with lower reproductive output.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe data analysed in this study were collected as part of a semi-natural enclosure project investigating population dynamics and the establishment and characterisation of social structure (Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The present study specifically examines secretion patterns of MUPs and the two steroid hormones across changing social contexts \u0026ndash; before, during, and after the semi-natural experiment. Animal origin, experimental design, enclosure setup, and parentage assignment procedures are described in Mikula et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Details on sampling procedures, laboratory analyses, and statistical methods are provided below.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnimals\u003c/em\u003e. Experimental animals were derived from wild-captured house mice, with founders representing both \u003cem\u003eM. m. musculus\u003c/em\u003e and \u003cem\u003eM. m. domesticus\u003c/em\u003e. These founders were collected in 2013 from multiple localities situated well outside the subspecies contact zone (see Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e for details). Within each subspecies, males and females originating from different populations were paired to produce first-generation (G1) offspring. After weaning at 20 days of age, juveniles were housed with same-sex littermates. At 55 days of age, 6 males and 6 females of each subspecies were transferred to single housing, where they remained until approximately 125 days of age, when they were introduced into the semi-natural enclosures as founders of the arena populations (Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Numbers of mice examined during respective phases of the study (see below) are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSemi-natural Enclosures\u003c/em\u003e. Two semi-natural arenas (2 \u0026times; 4 m each) were established in neighbouring cabins. Each arena was initially populated with six males and six females of either \u003cem\u003eM. m. musculus\u003c/em\u003e or \u003cem\u003eM. m. domesticus\u003c/em\u003e (see Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e for details). Mice were provided with nest boxes, food, and water \u003cem\u003ead libitum\u003c/em\u003e and were allowed to establish social units and reproduce naturally. Each individual was implanted with an RFID microtransponder and marked by toe clipping at least 10 days prior to arena entry. All animals were introduced into the enclosures simultaneously after confirming good health and proper functioning of the RFID tags. Populations were monitored every 3\u0026ndash;5 days, new animals were tagged when reaching proper body mass, shortly before leaving nest box. On day 195 of the experiment, the two enclosures were connected by two tubes, enabling inter-subspecific contact. The experiment concluded after 265 days for \u003cem\u003eM. m. musculus\u003c/em\u003e and 272 days for \u003cem\u003eM. m. domesticus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSampling and Sample Processing\u003c/em\u003e. Before entry into the enclosures, and at least two weeks after transfer to single housing, founder animals were sampled for urine and faeces. Males were sampled twice and females three times, with intervals 3\u0026ndash;4 days between sampling events. During the semi-natural experiment, both enclosures were monitored as described in Mikula et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Urine and faecal samples were collected at six time points: days 8, 70, 140, 197, 212, and the final day of the experiment. In addition, all males were sampled 24 hours after the experiment concluded, once individuals had been returned to single housing.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMUP Quantification\u003c/em\u003e. MUP concentrations were quantified from frozen urine samples collected by temporarily placing mice in clean cages. Proteins were separated using denaturing polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE) on 4\u0026ndash;15% Mini-PROTEAN\u0026reg; TGX\u0026trade; Precast Gels (Bio-Rad, Hercules, CA, USA). A concentration gradient of carbonic anhydrase (external standard) was loaded alongside the samples to enable absolute quantification of the separated proteins. Electrophoretograms were imaged using a GS-900 calibrated densitometer (Bio-Rad), and bands of interest were quantified with Image Lab\u0026trade; software (Bio-Rad). Band volume was defined as the summed pixel intensity within the designated band boundary. Protein concentrations were calculated from a carbonic anhydrase calibration curve (Janotov\u0026aacute; and Stopka 2011).\u003c/p\u003e \u003cp\u003eTo account for variation in urine dilution among individuals and sampling occasions, MUP concentrations were normalised using a creatinine assay (LKreatinin Kit, BioVendor, Brno, Czechia). A dilution coefficient was calculated based on creatinine concentration following Stopka et al. (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Corrected MUP values were obtained by dividing each measured concentration by this coefficient, yielding creatinine-adjusted concentrations (ng ml⁻\u0026sup1;) for each sample.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHormone Extraction and Enzyme Immunoassays\u003c/em\u003e. Testosterone and corticosterone metabolites were extracted from frozen faecal samples collected from mice temporarily placed in clean cages. Approximately 0.05 g of dried, finely ground faecal powder was vortexed in 1 ml of 80% methanol for 30 min, following Palme et al. (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). After centrifugation (2500 \u0026sdot; g, 15 min), supernatants were collected and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eCorticosterone metabolites were measured using a polyclonal antibody (CJM006, 1:16,000 dilution), and testosterone metabolites using a polyclonal antibody (R156/7, 1:8,000 dilution), together with the corresponding horseradish peroxidase (HRP) conjugates (1:40,000 and 1:60,000 dilution, respectively), obtained from EndoLab (Department of Population Health and Reproduction, UC Davis, USA). Cross-reactivities and detailed assay procedures are described in Hiadlovsk\u0026aacute; et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Hormone metabolite concentrations are reported as ng g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e faeces. Inter- and intra-assay coefficients of variation for both assays were \u0026lt;\u0026thinsp;15%, consistent with recommended performance criteria for immunoassays (Findlay et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Shah et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eParentage Analysis and Reproductive Status\u003c/em\u003e. Genomic DNA was isolated from all individuals sampled prior to the experiment (enclosure founders) and during the semi-natural enclosure phase (pups were sampled shortly prior to RFID tagging). Panels of 25 and 26 microsatellite markers were genotyped in \u003cem\u003eM. m. musculus\u003c/em\u003e and \u003cem\u003eM. m. domesticus\u003c/em\u003e, respectively (see Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e for details). Parentage was estimated using CERVUS v.3.0.3 (Kalinowski et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) with a 95% confidence threshold. Only individuals meeting predefined criteria (i.e., alive and sexually mature at the estimated time of conception) were considered candidate parents. Parentage assignments were then used to determine reproductive status of individuals at each of the six enclosure sampling events.\u003c/p\u003e \u003cp\u003eCurrent reproductive output for each individual was defined as the number of assigned offspring born within a 44-day window centred on a given sampling event (i.e., 22 days before to 22 days after the event). Under this definition, an individual was classified as reproductively active if it engaged in mating or conception, a female was pregnant (gestation\u0026thinsp;\u0026asymp;\u0026thinsp;20 days) or nursing (weaning\u0026thinsp;\u0026asymp;\u0026thinsp;20 days), or a male had genetically assigned offspring whose mother was pregnant or nursing during that period. Individuals with at least one assigned offspring within the 44-day window were classified as reproducing.\u003c/p\u003e \u003cp\u003eAlthough this approach may not be entirely precise (particularly for males), it provides a better time-specific measure of reproductive success that can be directly aligned with contemporaneous measures of steroid hormone metabolites and urinary MUP concentrations, offering an improvement over a simple binary reproducing/non-reproducing classification.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical Analyses\u003c/em\u003e. The analyses were designed to address two complementary questions: (i) how the presence versus absence of social interactions affects urinary MUP concentrations and steroid hormone metabolite concentrations (Initial and Isolation phases), and (ii) how reproductive activity influences these traits under social conditions (Social phase).\u003c/p\u003e \u003cp\u003eThe \u0026lsquo;Initial phase\u0026rsquo; compared measurements obtained under social isolation prior to arena entry (two sampling events in males, three in females) with those collected during the first enclosure sampling (day 8). The \u0026lsquo;Isolation phase\u0026rsquo; compared measurements from the final enclosure sampling with those taken after 24 hours of solitary housing following the end of the experiment. For both phases, linear mixed-effects models (LMMs) were fitted with individual identity included as a random intercept, and social context (isolated vs. social) and subspecies specified as fixed effects. For the Initial phase, sex was included as an additional fixed effect.\u003c/p\u003e \u003cp\u003eTo evaluate effects of reproduction under complex social conditions, we analysed data from the enclosure phase (\u0026lsquo;Social phase\u0026rsquo;), during which social organisation had been established. Current reproductive output of each individual was assessed using the 44-day window described above. LMMs included individual identity as a random intercept and current reproductive output, sex, and subspecies as fixed effects. For visualisation purposes, current reproductive output was represented as a binary factor: non-reproducing (no assigned offspring) versus reproducing (\u0026ge;\u0026thinsp;1 offspring within the 44-day window). Mice sampled only once in the arena were excluded from the Social dataset, as we presumed their presence in the population was only ephemeral.\u003c/p\u003e \u003cp\u003eWe further tested whether steroid hormone metabolite concentrations covaried with MUP concentrations in relation to current reproductive output, sex, and subspecies. Separate LMMs were fitted for each hormone, as testosterone was not measured in females.\u003c/p\u003e \u003cp\u003eAll models were estimated using restricted maximum likelihood (REML) in the R package \u003cem\u003enlme\u003c/em\u003e (Pinheiro and Bates \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; R Core Team \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Response variables were natural log-transformed, normalised urinary MUP concentrations (ng ml⁻\u0026sup1;) and hormone metabolite concentrations (ng g⁻\u0026sup1;). Accordingly, fixed-effect estimates (\u003cem\u003eβ\u003c/em\u003e) represent effect sizes on the natural-log scale. Model assumptions were assessed by visual inspection of residual distributions and residual-versus-fitted plots. Predicted values were plotted to illustrate group effects while accounting for covariates; corresponding plots of the observed values are provided in the Supplementary Material (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u0026ndash;3).\u003c/p\u003e \u003cp\u003eThe difference between metabolite levels before and after isolation (Δ) was calculated for each individual in the Isolation phase. To investigate the potential effect of current reproductive output on both the magnitude and direction (decline/increase) of these changes (see Tho\u0026szlig; et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), ΔMUP, ΔTestosterone and ΔCorticosterone were analysed using generalised least squares (GLS) models with subspecies and current reproductive output (CRO) as fixed effects. Current reproductive output was analysed either as a binary variable (reproducing: CRO\u0026thinsp;\u0026gt;\u0026thinsp;0; non-reproducing: CRO\u0026thinsp;=\u0026thinsp;0) or as a continuous variable (number of assigned offspring). Heteroscedasticity was accounted for by modelling group-specific standard deviations (varIdent structure) when CRO was treated as a binary variable, or by modelling residual variance as an exponential function of current reproductive output (varExp structure in the \u003cem\u003enlme\u003c/em\u003e package) when CRO was treated as a continuous variable.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eInitial Phase: Entry into the Social Environment\u003c/em\u003e. In this phase, we contrasted measurements obtained during individual housing with those collected on day 8 after entry into the semi-natural arena. At this time, no offspring had yet been born, although mating had already occurred.\u003c/p\u003e \u003cp\u003eUrinary MUP concentrations increased significantly following the transition from individual to social housing, and this effect was observed in both sexes and in subspecies (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.385, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 82, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 24; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Across social contexts, males exhibited significantly higher MUP concentrations than females (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.196, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and \u003cem\u003eM. m. musculus\u003c/em\u003e showed higher MUP concentrations than \u003cem\u003eM. m. domesticus\u003c/em\u003e (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.452, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA similar response to the social environment was observed for both hormones. Testosterone concentrations increased significantly under social conditions in both subspecies (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.256, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 35, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 12; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although \u003cem\u003eM. m. musculus\u003c/em\u003e consistently showed higher testosterone concentrations than \u003cem\u003eM. m. domesticus\u003c/em\u003e both before and after enclosure entry, this difference was not statistically significant (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.357, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.143).\u003c/p\u003e \u003cp\u003eCorticosterone concentrations were likewise significantly elevated under social conditions across all groups (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.254, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 82, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 24; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Females exhibited higher corticosterone concentrations than males (\u003cem\u003eβ\u003c/em\u003e = -0.455, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016), whereas corticosterone did not differ significantly between subspecies (\u003cem\u003eβ\u003c/em\u003e = -0.118, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.503).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFixed-effect estimates from linear mixed models evaluating MUP, corticosterone, and testosterone concentrations across the Initial, Social, and Isolation phases. Each model includes subspecies, sex (when applicable), and social context as fixed effects, with animal ID fitted as a random intercept (subspecies, sex, or context displaying higher values are given in parentheses). Estimates are presented on the natural-log scale of normalised concentrations. Columns report the estimate, standard error (SE), degrees of freedom (\u003cem\u003edf\u003c/em\u003e), \u003cem\u003et\u003c/em\u003e-value, and \u003cem\u003ep\u003c/em\u003e-value for each predictor. CRO\u0026thinsp;=\u0026thinsp;current reproductive output\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhase\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAgent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEffect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEstimate (\u003cem\u003eβ\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003edf\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003et\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMUP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7.092\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.196\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eContext (social)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.197\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorticosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.503\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-2.619\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eContext (social)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTestosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eContext (social)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.944\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMUP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.643\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.352\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e14.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCRO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorticosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex (male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-6.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCRO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.215\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSocial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTestosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.317\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCRO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.690\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMUP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11.549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eContext (post-social)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.775\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-7.083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorticosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.597\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eContext (post-social)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-5.506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTestosterone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSubspecies (\u003cem\u003emusculus\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.463\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4.104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eContext (post-social)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-5.527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eIsolation Phase: Exit from Social Environment\u003c/em\u003e. One day after removal from the complex social environment, urinary MUP concentrations decreased significantly in males of both subspecies (\u003cem\u003eβ\u003c/em\u003e = -0.775, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 140, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 74). Across social contexts, \u003cem\u003eM. m. musculus\u003c/em\u003e males exhibited higher MUP concentrations than \u003cem\u003eM. m. domesticus\u003c/em\u003e (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.808, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Both hormones likewise declined following removal from the social environment (testosterone: \u003cem\u003eβ\u003c/em\u003e = -0.607, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 150, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 76; corticosterone: \u003cem\u003eβ\u003c/em\u003e = -0.497, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 154, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 79; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;C). Testosterone concentrations remained higher in \u003cem\u003eM. m. musculus\u003c/em\u003e than in \u003cem\u003eM. m. domesticus\u003c/em\u003e (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.463; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas corticosterone concentrations did not differ significantly between subspecies (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.058; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.597). For testosterone, and to a lesser extent for MUPs, the decline was significantly stronger in \u003cem\u003eM. m. musculus\u003c/em\u003e than in \u003cem\u003eM. m. domesticus\u003c/em\u003e (Supplementary Information Tab. S1, Fig S4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eSocial Phase: Effect of Current Reproductive Output\u003c/em\u003e. Under established social conditions, urinary MUP concentrations increased with current reproductive output when modelled as a continuous variable (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.042, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 320, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 101; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For visualisation, this pattern is also illustrated by contrasting two categories: reproducing vs. non-reproducing individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Significant effects in MUP concentrations were also detected for sex (males\u0026thinsp;\u0026gt;\u0026thinsp;females; \u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.416, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and subspecies (\u003cem\u003eM. m. musculus\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eM. m. domesticus\u003c/em\u003e; \u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.650, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eCurrent reproductive output was not significantly associated with testosterone or corticosterone metabolite concentrations (testosterone: \u003cem\u003eβ\u003c/em\u003e = -0.005, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.690, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 173, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 58; corticosterone: \u003cem\u003eβ\u003c/em\u003e = -0.012; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.215, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 331, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 102; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;C, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Testosterone concentrations were higher in \u003cem\u003eM. m. musculus\u003c/em\u003e than in \u003cem\u003eM. m. domesticus\u003c/em\u003e (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.642, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas corticosterone concentrations did not differ significantly between subspecies (\u003cem\u003eβ\u003c/em\u003e = -0.126, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.180). In both subspecies, males displayed lower corticosterone concentrations than females (\u003cem\u003eβ\u003c/em\u003e = -0.563, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter accounting for covariates, MUP concentrations were not significantly associated with testosterone (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.678, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 169, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 58) or corticosterone concentrations (\u003cem\u003eβ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.092; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.135, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003eobs\u003c/em\u003e\u003c/sub\u003e = 314, \u003cem\u003eN\u003c/em\u003e\u003csub\u003e\u003cem\u003egroups\u003c/em\u003e\u003c/sub\u003e = 100).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study examined how chemical signalling and endocrine outputs respond to transitions between solitary and socially complex environments. Both urinary MUP concentrations and steroid hormone metabolites were highly socially plastic under semi-natural conditions. We identified a coordinated \u0026ldquo;social-entry\u0026rdquo; response: MUP, testosterone, and corticosterone concentrations increased sharply after males entered enclosures, coinciding with the onset of hierarchy formation. A similarly rapid \u0026ldquo;social-exit\u0026rdquo; response followed removal from enclosures, with males downregulating urinary MUPs and both hormones within one day of isolation, consistent with rapid endocrine and signalling adjustment following the loss of social interactions (Creel \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, 2013; Eisenegger et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Tho\u0026szlig; et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Under established social conditions, MUP concentrations \u0026ndash; but not testosterone or corticosterone metabolites \u0026ndash; tracked current reproductive output. MUP levels were not significantly associated with either hormone, supporting the view that their regulation is not directly coupled to steroid hormone dynamics (Knopf et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Noa\u0026iacute;n et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Penn et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eEntry into the Social Environment: Rapid Upregulation of MUPs and Endocrine Activation\u003c/em\u003e. The predicted increase in urinary MUP concentrations upon entry into the semi-natural enclosures was strongly supported and occurred in both sexes and both subspecies. This finding aligns with repeated evidence that MUP production is socially modulated and context-dependent, particularly under competitive conditions (Stopka et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tho\u0026szlig; et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Luzynski et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The pronounced rise during the entry phase is consistent with increased signalling effort when social relationships are being established and competition for mates and space is most intense (Bradbury and Vehrencamp \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sz\u0026aacute;mad\u0026oacute; 2011).\u003c/p\u003e \u003cp\u003eThis temporal pattern accords with general signalling theory: the costs and benefits of signalling \u0026ndash; and thus selection on signal investment \u0026ndash; vary as individuals move between social conditions, as hierarchies form or destabilise, and as roles change over time (Bradbury and Vehrencamp \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sz\u0026aacute;mad\u0026oacute; 2011). The increase in MUP concentrations in both males and females further supports the view that MUPs both influence and respond to social interactions beyond male\u0026ndash;male competition alone (Beynon and Hurst \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Chamero et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rusu et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Roberts et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nelson et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEndocrine responses during entry mirrored the MUP pattern. Testosterone and corticosterone metabolites both increased significantly. For testosterone, this is consistent with context-dependent frameworks in which testosterone biases motivation and information processing toward status-relevant goals, with behavioural outcomes shaped by the social environment (Eisenegger et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Knight et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Under the challenge hypothesis, testosterone rises transiently during social challenges and may decline when effort shifts toward parenting or bonding (Wingfield 1990; Bell \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The observed increase therefore matches expectations for the early, unstable phase of hierarchy formation (Wingfield 1990; Eisenegger et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bell \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe concurrent rise in corticosterone metabolites is likewise expected under novel social conditions, instability, and elevated interaction rates. In mammals, glucocorticoid\u0026ndash;status relationships may reflect either \u0026ldquo;subordinate stress\u0026rdquo; or the \u0026ldquo;cost of dominance\u0026rdquo;, depending on hierarchy stability and the distribution of physiological costs (Creel \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Goymann and Wingfield \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Creel et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although our design does not allow direct rank-specific inference, the increase observed during entry supports the broader conclusion that glucocorticoids track changes in the social environment (Creel \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Creel et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wyatt \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eExit from the Social Environment: Rapid Downregulation of Signalling and Hormones\u003c/em\u003e. Plasticity was equally evident upon exit from the social environment. Within one day of isolation, MUP, testosterone, and corticosterone concentrations decreased significantly in males of both subspecies. This rapid reversal is consistent with the expectation that signalling investment should decline when opportunities for territorial advertisement associated with social and reproductive competition diminish (Bradbury and Vehrencamp \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sz\u0026aacute;mad\u0026oacute; 2011).\u003c/p\u003e \u003cp\u003eOur results parallel those of Tho\u0026szlig; et al. (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), who showed that dominant males upregulated MUP expression under competitive conditions and that differences between dominant and subordinate males disappeared after removal from the enclosure, largely through downregulation by dominant individuals. Although we did not assign dominance behaviourally, our data support the broader conclusion that increased MUP investment is favoured under competitive social conditions and rapidly reduced when competition ceases (Stopka et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tho\u0026szlig; et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Luzynski et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The parallel endocrine declines are also consistent with reciprocal causality perspectives, in which hormones both shape and respond to social context (Eisenegger et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In contrast to Tho\u0026szlig; et al. (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), who reported rank-dependent differences in the magnitude of MUP regulation, we detected no significant effect of reproductive output on the direction or extent of change in any measured variable (Supplementary Material).\u003c/p\u003e \u003cp\u003eA striking feature of these responses is their speed: MUP concentrations and steroid metabolites shifted markedly within 24 hours. This has important methodological implications, as animals captured in the field are often held in isolation prior to sampling, potentially leading to systematic underestimation of MUP and hormone expression. This highlights the need for caution when comparing studies with different housing histories or sampling protocols.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSocial Phase: Reproductive Output but not Steroid Metabolites Predicts MUP Concentrations\u003c/em\u003e. Rather than dyadic tests or direct behavioural observations, we used current reproductive output as a proxy for competitive success, given that dominant males typically secure greater access to females and reproduction. Under established social conditions, urinary MUP concentrations increased with reproductive output, whereas testosterone and corticosterone metabolites did not. This divergence is consistent with the hypothesis that urinary MUPs function as honest signals of competitive ability within complex social networks (Rich and Hurst \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Garratt et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nelson et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tho\u0026szlig; et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Luzynski et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, as emphasised by Sz\u0026aacute;mad\u0026oacute; (2011) and Penn et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), signal honesty depends on context-dependent trade-offs and the potential costs of cheating.\u003c/p\u003e \u003cp\u003eThe absence of associations between steroid metabolites and reproductive output does not preclude their role in competition or mating. Plasma hormones fluctuate rapidly in response to acute social interactions, whereas faecal metabolites integrate endocrine activity over longer periods. Consequently, metabolite measures may better reflect broad environmental transitions \u0026ndash; including entry into or exit from social environments \u0026ndash; than short-lived endocrine responses mediating agonistic or reproductive behaviour (Wingfield 1990; Creel \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Eisenegger et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Creel et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Under these conditions, urinary MUPs appear to be more sensitive indicators of realised reproductive outcomes than time-integrated hormone metabolites.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNo Direct Association between MUPs and Testosterone: Multi-layered Endocrine Regulation\u003c/em\u003e. Interestingly, MUP concentrations were not significantly associated with testosterone or corticosterone after accounting for covariates. This supports the view that MUP regulation is not governed by testosterone alone. Although traditionally considered androgen-dependent, MUP expression is now known to be under multi-hormonal control, including a key role for growth hormone (Knopf et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Noa\u0026iacute;n et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Penn et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Our results are therefore consistent with the idea that social context shapes MUP investment through regulatory pathways that are not captured by simple linear relationships with androgen metabolites (Penn et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSex and Subspecies Patterns\u003c/em\u003e. Across contexts, males exhibited substantially higher urinary MUP concentrations than females, consistent with established sexual dimorphism (Stopkov\u0026aacute; et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Cheetham et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Janotov\u0026aacute; and Stopka \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Penn et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This male bias is likely maintained by sexual selection, either through male-male competition or via female mate choice (Meagher et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Hurst and Beynon \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kaur et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Penn et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Importantly, females showed the same direction of context dependence during the entry phase, indicating that MUP signalling is socially responsive in both sexes, even when absolute levels differ (Beynon and Hurst \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Chamero et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Roberts et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Nelson et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Together with studies by Garratt et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Stockley et al. (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which report increased urinary protein excretion during female\u0026ndash;female aggression, it appears that the MUP-based signalling system is as important for females as it is for males.\u003c/p\u003e \u003cp\u003eWe observed higher MUP concentrations in \u003cem\u003eM. m. musculus\u003c/em\u003e than in \u003cem\u003eM. m. domesticus\u003c/em\u003e across all three phases, consistent with known differences in MUP abundance and isoform profiles between these subspecies (Stopkov\u0026aacute; et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hurst et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Machol\u0026aacute;n et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to Stopkov\u0026aacute; et al. (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and Janotov\u0026aacute; and Stopka (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), this pattern may be related to stronger assortative mate preferences in \u003cem\u003eM. m. musculus\u003c/em\u003e females compared with \u003cem\u003eM. m. domesticus\u003c/em\u003e females (Munclinger and Frynta \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Christophe and Baudoin \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Smadja and Ganem \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Smadja et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; B\u0026iacute;mov\u0026aacute; et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Vošlajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Alternatively, as suggested by Janotov\u0026aacute; and Stopka (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), MUP production may be important in both subspecies, but mate choice in \u003cem\u003eM. m. domesticus\u003c/em\u003e females could be constrained by a conflict between selecting a high-quality male with high MUP output and selecting a mate of the same subspecies.\u003c/p\u003e \u003cp\u003eThe lower MUP output found in \u003cem\u003eM. m. domesticus\u003c/em\u003e may also reflect the greater aggressiveness of \u003cem\u003eM. m. domesticus\u003c/em\u003e males (Thuessen 1977; van Zegeren and van Oortmerssen \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1981\u003c/span\u003e; Pi\u0026aacute;lek et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ďureje et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), such that reproductive success is determined primarily through male\u0026ndash;male competition rather than through female choice. Adding further complexity, other communication-related body fluids, such as tears, show the opposite pattern \u0026ndash; higher protein output in \u003cem\u003eM. m. domesticus\u003c/em\u003e \u0026ndash; which may represent a complementary signalling mechanism (Vošlajerov\u0026aacute; B\u0026iacute;mov\u0026aacute; et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). Despite baseline differences, the main effects relevant to our focal hypotheses \u0026ndash; social entry and exit responses and the association between MUPs and reproductive output \u0026ndash; were consistent across subspecies, suggesting a general pattern in how complex social environments shape MUP signalling investment.\u003c/p\u003e \u003cp\u003eRegarding steroid metabolites, we again observed higher testosterone concentrations in \u003cem\u003eM. m. musculus\u003c/em\u003e than in \u003cem\u003eM. m. domesticus\u003c/em\u003e (Hiadlovsk\u0026aacute; et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), whereas corticosterone did not differ significantly between subspecies. This pattern coincides with variation in dominance dynamics rather than a fixed endocrine difference: prolonged testosterone production in \u003cem\u003eM. m. musculus\u003c/em\u003e males may reflect longer periods of unsettled dominance relationships (Hiadlovsk\u0026aacute; et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which was later confirmed under semi-natural conditions (Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCorticosterone levels were consistently higher in females than in males, suggesting greater physiological demands or social challenges. Baseline levels did not differ between subspecies; however, \u003cem\u003eM. m. domesticus\u003c/em\u003e females showed a stronger increase after entering the enclosure than \u003cem\u003eM. m. musculus\u003c/em\u003e females. Two non-mutually exclusive mechanisms may explain this pattern. First, the higher aggressiveness of \u003cem\u003eM. m. domesticus\u003c/em\u003e males may result in accidental or misdirected attacks on females, as occasionally reported by animal keepers. Second, aggression is not restricted to male mice (Crowcroft and Rowe \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1963\u003c/span\u003e; van Zegeren \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e1980\u003c/span\u003e; Frynta et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and the social structure of \u003cem\u003eM. m. domesticus\u003c/em\u003e populations appears more closed than that of \u003cem\u003eM. m. musculus\u003c/em\u003e (Mikula et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This may increase female social pressure. However, whether these mechanisms underlie the observed endocrine differences following enclosure entry remains to be tested.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur repeated-measures design demonstrates strong social plasticity in both chemical signalling and endocrine outputs. Entry into a complex social environment elicited coordinated increases in MUP secretion and steroid metabolites, whereas removal rapidly reduced both signalling and endocrine activity. Under established social conditions, MUP concentrations \u0026ndash; but not steroid metabolites \u0026ndash; tracked current reproductive output, and were not significantly associated with either hormone.\u003c/p\u003e \u003cp\u003eTwo findings deserve emphasis. First, although most studies treat MUPs primarily as male honest signals, our results show that females modulate MUP production across social transitions in parallel with males, highlighting a broader role of this signalling system beyond male competition. Second, despite divergence between \u003cem\u003eM. m. musculus\u003c/em\u003e and \u003cem\u003eM. m. domesticus\u003c/em\u003e in social behaviour, the key patterns relevant to our focal questions were consistent across subspecies. This suggests general principles governing how complex social environments regulate signalling investment and endocrine state in house mice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests \u0026ndash;\u003c/h2\u003e \u003cp\u003e \u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e \u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eEthics statement \u0026ndash;\u003c/h2\u003e \u003cp\u003e\u003cem\u003e The breeding facility of the Institute of Vertebrate Biology, Czech Academy of Sciences, in Studenec, has been licensed for keeping small mammals according to the Czech law (Licenses No. 227203/2011-MZE- 17214 2011\u0026ndash;2016). Animals were handled by authorised persons only (ZH Licenses No. CZ 00548, CZ 0127; PH as a university student under supervision). This study was performed in accordance with Czech law, implementing all corresponding EU regulations and approved by the IVB Ethical Committee (N 145/2010).\u003c/em\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAuthor Contributions \u0026ndash;\u003c/strong\u003e \u003cp\u003e \u003cem\u003eThe project was conceptualized and supervised by Barbora Vošlajerov\u0026aacute; and (temporarily) by Miloš Machol\u0026aacute;n. Ľudov\u0026iacute;t Ďureje, Zuzana Hiadlovsk\u0026aacute;, and Iva Posp\u0026iacute;šilov\u0026aacute; were responsible for semi-natural enclosure management. Data were curated by Barbora Vošlajerov\u0026aacute; B\u0026iacute;mov\u0026aacute;, Ľudov\u0026iacute;t Ďureje, and Zuzana Hiadlovsk\u0026aacute;. Kristina Daniszov\u0026aacute; was responsible for MUP and hormone quantification, basic data processing, and paternity analysis; Iva Posp\u0026iacute;šilov\u0026aacute; participated in hormone quantification and basic data processing; Kateřina Janotov\u0026aacute; participated in MUP data acquisition. Statistical analyses were performed by Nat\u0026aacute;lia Mart\u0026iacute;nkov\u0026aacute; with the participation of Zuzana Hiadlovsk\u0026aacute;. The first draft was written by Zuzana Hiadlovsk\u0026aacute;, Miloš Machol\u0026aacute;n, Nat\u0026aacute;lia Mart\u0026iacute;nkov\u0026aacute;, Barbora Vošlajerov\u0026aacute; B\u0026iacute;mov\u0026aacute;, and Kristina Daniszov\u0026aacute;. All authors participated in the final editing of the manuscript.\u003c/em\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eData Availability \u0026ndash;\u003c/strong\u003e \u003cp\u003e \u003cem\u003eThe data are available in zenodo with the identifier doi\u003c/em\u003e: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.5281/zenodo.19454221\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.19454221\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cem\u003eat zenodo.org/records/19454221.\u003c/em\u003e\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding \u0026ndash;\u003c/h2\u003e \u003cp\u003eThe study was funded by the Czech Science Foundation grant (GAP506/11/1792 to BVB) and the institutional funding of the Institute of Vertebrate Biology (RVO:68081766). The authors also thank the RECETOX Research Infrastructure (No LM2023069), financed by the Ministry of Education, Youth and Sports, for its supportive background. This work was supported by the European Union\u0026rsquo;s Horizon 2020 research and innovation programme under grant agreements No 857560 (CETOCOEN Excellence). This publication reflects only the author\u0026rsquo;s view, and the European Commission is not responsible for any use that may be made of the information it contains.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe project was conceptualized and supervised by Barbora Vošlajerov\u0026aacute; and (temporarily) by Miloš Machol\u0026aacute;n. Ľudov\u0026iacute;t Ďureje, Zuzana Hiadlovsk\u0026aacute;, and Iva Posp\u0026iacute;šilov\u0026aacute; were responsible for semi-natural enclosure management. Data were curated by Barbora Vošlajerov\u0026aacute; B\u0026iacute;mov\u0026aacute;, Ľudov\u0026iacute;t Ďureje, and Zuzana Hiadlovsk\u0026aacute;. Kristina Daniszov\u0026aacute; was responsible for MUP and hormone quantification, basic data processing, and paternity analysis; Iva Posp\u0026iacute;šilov\u0026aacute; participated in hormone quantification and basic data processing; Kateřina Janotov\u0026aacute; participated in MUP data acquisition. Statistical analyses were performed by Nat\u0026aacute;lia Mart\u0026iacute;nkov\u0026aacute; with the participation of Zuzana Hiadlovsk\u0026aacute;. The first draft was written by Zuzana Hiadlovsk\u0026aacute;, Miloš Machol\u0026aacute;n, Nat\u0026aacute;lia Mart\u0026iacute;nkov\u0026aacute;, Barbora Vošlajerov\u0026aacute; B\u0026iacute;mov\u0026aacute;, and Kristina Daniszov\u0026aacute;. All authors participated in the final editing of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data are available in zenodo with the identifier doi: 10.5281/zenodo.19454221 at zenodo.org/records/19454221\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArakawa H, Blanchard DC, Arakawa K, Dunlap C, Blanchard R (2008) Scent marking behavior as an odorant communication in mice. 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Cambridge University Press, Cambridge. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1017/CBO9781139030748\u003c/span\u003e\u003cspan address=\"10.1017/CBO9781139030748\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-chemical-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joce","sideBox":"Learn more about [Journal of Chemical Ecology](https://www.springer.com/journal/10886)","snPcode":"10886","submissionUrl":"https://submission.nature.com/new-submission/10886/3","title":"Journal of Chemical Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Major urinary proteins, testosterone, corticosterone, Mus musculus musculus, Mus musculus domesticus, semi-natural enclosures","lastPublishedDoi":"10.21203/rs.3.rs-9552933/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9552933/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChemical signalling and endocrine state are known to covary with social environment, but the speed of their response to social transitions under natural conditions remains unclear. We examined how major urinary proteins (MUPs) and faecal steroid hormone metabolites respond to shifts between solitary housing and a complex social setting in two house mouse subspecies, \u003cem\u003eMus musculus musculus\u003c/em\u003e and \u003cem\u003eM. m. domesticus\u003c/em\u003e. Using repeated sampling in semi-natural enclosures, we measured MUP, testosterone, and corticosterone metabolite concentrations across three contexts: before social exposure, during the social phase, and after return to isolation. Reproductive output was quantified within a 44-day window around each sampling event. Entry into the enclosures triggered a strong, coordinated response: urinary MUP concentrations increased in both sexes and subspecies, paralleled by rises in testosterone and corticosterone metabolites during early social exposure. In males, this pattern reversed rapidly after removal, with MUPs and both hormone metabolites declining within 24 hours of renewed isolation. Under stable social conditions, urinary MUP concentrations increased with current reproductive output, whereas testosterone and corticosterone metabolites showed no such association. MUP concentrations were also unrelated to either hormone, suggesting that socially responsive MUP investment is not directly coupled to steroid levels. Despite baseline differences between subspecies and the expected male bias in MUP production, the main context-dependent patterns were consistent across subspecies. Our results demonstrate pronounced social plasticity in chemical signalling and endocrine state, and indicate that MUP concentrations more closely reflect reproductive outcomes than steroid metabolite concentrations.\u003c/p\u003e","manuscriptTitle":"Social context rapidly reshapes chemical signalling and endocrine profiles in two mouse subspecies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 11:12:43","doi":"10.21203/rs.3.rs-9552933/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"9491996150555106383784350649302883721","date":"2026-05-22T20:26:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-07T15:49:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-07T15:38:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-02T00:14:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Chemical Ecology","date":"2026-04-28T10:38:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-chemical-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"joce","sideBox":"Learn more about [Journal of Chemical Ecology](https://www.springer.com/journal/10886)","snPcode":"10886","submissionUrl":"https://submission.nature.com/new-submission/10886/3","title":"Journal of Chemical Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"259aeba3-8528-4cac-a96e-bbb7ed719ab3","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"9491996150555106383784350649302883721","date":"2026-05-22T20:26:49+00:00","index":16,"fulltext":""},{"type":"reviewersInvited","content":"8","date":"2026-05-07T15:49:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-07T15:38:35+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T11:12:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 11:12:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9552933","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9552933","identity":"rs-9552933","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}