The role of male scent in female attraction in the bank vole, Myodes glareolus.

Chemical signals are frequently utilised by male mammals for intersexual communication and females are often attracted to male scent. However, the mechanism underlying female attraction has only been identified in a small number of mammalian species. Mammalian scents contain airborne volatiles, that are detected by receivers at a distance from the scent source, as well as non-volatile molecules, such as proteins, that require physical contact for detection. Lipocalin proteins, produced within the scent secretions of many terrestrial mammals, are thought to be particularly important in chemical signalling. Here, we explore if the male-specific protein, glareosin, expressed by adult male bank voles, Myodes glareolus, stimulates female attraction to male scent. We show that female bank voles are more attracted to male compared to female scent, supporting the results of previous studies. Increased investigation and attraction to male scent occurred to both airborne volatiles and non-volatile proteins when they were presented separately. However, we found no evidence that attraction to male scent was driven by glareosin. Our results differ from those previously described in house mice, where a single protein induces female attraction to male scent, suggesting the mechanism underlying female attraction to male scent differs between species.

Cryptic kin discrimination during communal lactation in mice favours cooperation between relatives.

Breeding females can cooperate by rearing their offspring communally, sharing synergistic benefits of offspring care but risking exploitation by partners. In lactating mammals, communal rearing occurs mostly among close relatives. Inclusive fitness theory predicts enhanced cooperation between related partners and greater willingness to compensate for any partner under-investment, while females are less likely to bias investment towards own offspring. We use a dual isotopic tracer approach to track individual milk allocation when familiar pairs of sisters or unrelated house mice reared offspring communally. Closely related pairs show lower energy demand and pups experience better access to non-maternal milk. Lactational investment is more skewed between sister partners but females pay greater energetic costs per own offspring reared with an unrelated partner. The choice of close kin as cooperative partners is strongly favoured by these direct as well as indirect benefits, providing a driver to maintain female kin groups for communal breeding.

Unraveling female communication through scent marks in the Norway rat.

Chemical communication by females remains poorly understood, with most attention focused on female advertisement of sexual receptivity to males or mother-offspring communication. However, in social species, scents are likely to be important for mediating competition and cooperation between females determining individual reproductive success. Here, we explore chemical signaling by female laboratory rats (Rattus norvegicus) to test i) whether females target their deployment of scent information differentially according to their sexual receptivity and the genetic identity of both female and male conspecifics signaling in the local environment and ii) whether females are attracted to gain the same or different information from female scents compared to males. Consistent with targeting of scent information to colony members of similar genetic background, female rats increased scent marking in response to scents from females of the same strain. Females also suppressed scent marking in response to male scent from a genetically foreign strain while sexually receptive. Proteomic analysis of female scent deposits revealed a complex protein profile, contributed from several sources but dominated by clitoral gland secretion. In particular, female scent marks contained a series of clitoral-derived hydrolases and proteolytically truncated major urinary proteins (MUPs). Manipulated blends of clitoral secretion and urine from estrus females were strongly attractive to both sexes, while voided urine alone stimulated no interest. Our study reveals that information about female receptive status is shared between females as well as with males, while clitoral secretions containing a complex set of truncated MUPs and other proteins play a key role in female communication.

Dissection of schistosome tissues under LC-MS compatible preservative conditions for quantitative proteomics.

Schistosomes are blood flukes with specialised tissues and organs, each one playing a pivotal role in perpetuating the parasite life cycle. Herein, we describe a detailed methodology for preserving the proteome of adult Schistosoma mansoni worms during manual dissection for enrichment of tissues associated with the parasite's alimentary tract. We provide step-by-step directions for specimen storage and dissection while in preservative solution, tissue homogenisation, protein extraction and digestion using a methodology fully compatible with downstream quantitative liquid chromatography-mass spectrometry analysis. Our methodology uses label-free and QconCAT-based absolute quantification for detection of S. mansoni oesophageal gland products proposed as vaccine candidates. Through stabilisation of the proteome and minimising sample degradation during dissection our approach has allowed us to access the hidden proteome of target tissues not readily available from total lysates because of their small volume. This protocol can be replicated or adapted to other Schistosoma species lacking quantitative proteomics characterisation of specialised tissues for discovery of proteins with potential diagnostic and therapeutic utility.

Glycoproteins Involved in Sea Urchin Temporary Adhesion.

Biomedical adhesives, despite having been used increasingly in recent years, still face a major technological challenge: strong adhesion in wet environments. In this context, biological adhesives secreted by marine invertebrates have appealing characteristics to incorporate into new underwater biomimetic adhesives: water resistance, nontoxicity and biodegradability. Little is still known about temporary adhesion. Recently, a transcriptomic differential analysis of sea urchin Paracentrotus lividus tube feet pinpointed 16 adhesive/cohesive protein candidates. In addition, it has been demonstrated that the adhesive secreted by this species is composed of high molecular weight proteins associated with N-Acetylglucosamine in a specific chitobiose arrangement. As a follow-up, we aimed to investigate which of these adhesive/cohesive protein candidates were glycosylated through lectin pulldowns, protein identification by mass spectroscopy and in silico characterization. We demonstrate that at least five of the previously identified protein adhesive/cohesive candidates are glycoproteins. We also report the involvement of a third Nectin variant, the first adhesion-related protein to be identified in P. lividus. By providing a deeper characterization of these adhesive/cohesive glycoproteins, this work advances our understanding of the key features that should be replicated in future sea urchin-inspired bioadhesives.

AlacatDesigner─Computational Design of Peptide Concatamers for Protein Quantitation.

Protein quantitation via mass spectrometry relies on peptide proxies for the parent protein from which abundances are estimated. Owing to the variability in signal from individual peptides, accurate absolute quantitation usually relies on the addition of an external standard. Typically, this involves stable isotope-labeled peptides, delivered singly or as a concatenated recombinant protein. Consequently, the selection of the most appropriate surrogate peptides and the attendant design in recombinant proteins termed QconCATs are challenges for proteome science. QconCATs can now be built in a "a-la-carte" assembly method using synthetic biology: ALACATs. To assist their design, we present "AlacatDesigner", a tool that supports the peptide selection for recombinant protein standards based on the user's target protein. The user-customizable tool considers existing databases, occurrence in the literature, potential post-translational modifications, predicted miscleavage, predicted divergence of the peptide and protein quantifications, and ionization potential within the mass spectrometer. We show that peptide selections are enriched for good proteotypic and quantotypic candidates compared to empirical data. The software is freely available to use either via a web interface AlacatDesigner, downloaded as a Desktop application or imported as a Python package for the command line interface or in scripts.

Rapid identification of mosquito species and age by mass spectrometric analysis.

BACKGROUND: A rapid, accurate method to identify and to age-grade mosquito populations would be a major advance in predicting the risk of pathogen transmission and evaluating the public health impact of vector control interventions. Whilst other spectrometric or transcriptomic methods show promise, current approaches rely on challenging morphological techniques or simple binary classifications that cannot identify the subset of the population old enough to be infectious. In this study, the ability of rapid evaporative ionisation mass spectrometry (REIMS) to identify the species and age of mosquitoes reared in the laboratory and derived from the wild was investigated. RESULTS: The accuracy of REIMS in identifying morphologically identical species of the Anopheles gambiae complex exceeded 97% using principal component/linear discriminant analysis (PC-LDA) and 84% based on random forest analysis. Age separation into 3 different age categories (1 day, 5-6 days, 14-15 days) was achieved with 99% (PC-LDA) and 91% (random forest) accuracy. When tested on wild mosquitoes from the UK, REIMS data could determine the species and age of the specimens with accuracies of 91 and 90% respectively. CONCLUSIONS: The accuracy of REIMS to resolve the species and age of Anopheles mosquitoes is comparable to that achieved by infrared spectroscopy approaches. The processing time and ease of use represent significant advantages over current, dissection-based methods. Importantly, the accuracy was maintained when using wild mosquitoes reared under differing environmental conditions, and when mosquitoes were stored frozen or desiccated. This high throughput approach thus has potential to conduct rapid, real-time monitoring of vector populations, providing entomological evidence of the impact of alternative interventions.

The relationship between lung disease severity and the sputum proteome in cystic fibrosis.

BACKGROUND: Proteomics can reveal molecular pathways of disease and provide translational perspectives to inform clinical decision making. Although several studies have previously reported the cystic fibrosis airway proteome, the relationship with severity of lung disease has not been characterised. The objectives of this observational study were to investigate differences in the CF sputum proteome associated with disease severity and identify potential markers of disease with translational potential. METHODS: Sputum samples from healthy volunteers and cystic fibrosis subjects (some prescribed modulator therapies) were analysed using liquid-chromatography tandem mass spectrometry. Severity of lung disease was based on baseline spirometry (percentage predicted forced expiratory volume in 1 s, FEV1%). RESULTS: Multiple sputum proteins (108 increased; 202 decreased) were differentially expressed in CF (n = 38) and healthy volunteers (n = 32). Using principal component analysis and hierarchical clustering, differences in sputum proteome were observed associated with progressive lung function impairment. In CF subjects, baseline FEV1% correlated with 87 proteins (positive correlation n = 20, negative n = 67); most were either neutrophil derived, or opposed neutrophil-driven oxidant and protease activity. CONCLUSION: Predictable and quantifiable changes in the CF sputum proteome occurred associated with progressive lung function impairment, some of which might have value as markers of disease severity in CF sputum. Further work validating these markers in other patient cohorts and exploring their clinical utility is needed.

Rapid Evaporative Ionization Mass Spectrometry (REIMS): a Potential and Rapid Tool for the Identification of Insecticide Resistance in Mosquito Larvae.

Insecticide resistance is a significant challenge facing the successful control of mosquito vectors globally. Bioassays are currently the only method for phenotyping resistance. They require large numbers of mosquitoes for testing, the availability of a susceptible comparator strain, and often insectary facilities. This study aimed to trial the novel use of rapid evaporative ionization mass spectrometry (REIMS) for the identification of insecticide resistance in mosquitoes. No sample preparation is required for REIMS and analysis can be rapidly conducted within hours. Temephos resistant Aedes aegypti (Linnaeus) larvae from Cúcuta, Colombia and temephos susceptible larvae from two origins (Bello, Colombia, and the lab reference strain New Orleans) were analyzed using REIMS. We tested the ability of REIMS to differentiate three relevant variants: population source, lab versus field origin, and response to insecticide. The classification of these data was undertaken using linear discriminant analysis (LDA) and random forest. Classification models built using REIMS data were able to differentiate between Ae. aegypti larvae from different populations with 82% (±0.01) accuracy, between mosquitoes of field and lab origin with 89% (±0.01) accuracy and between susceptible and resistant larvae with 85% (±0.01) accuracy. LDA classifiers had higher efficiency than random forest with this data set. The high accuracy observed here identifies REIMS as a potential new tool for rapid identification of resistance in mosquitoes. We argue that REIMS and similar modern phenotyping alternatives should complement existing insecticide resistance management tools.

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals.

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope-labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.

Quantitative proteomic analysis of bronchoalveolar lavage fluid in West Highland white terriers with canine idiopathic pulmonary fibrosis.

BACKGROUND: Canine idiopathic pulmonary fibrosis (CIPF) is a chronic, progressive, interstitial fibrosing lung disease, manifesting as cough, exercise intolerance and ultimately, dyspnea and respiratory failure. It mainly affects West Highland white terriers (WHWTs), lacks curable treatment and has a poor prognosis. Aspiration of gastroesophageal refluxate may play a role in the development of CIPF. In the first part of this study, we completed label-free quantitative proteomic analysis of bronchoalveolar lavage fluid (BALF) from CIPF and healthy WHWTs. In the second part, we evaluated potential protein markers of reflux aspiration from canine gastric juice and vomitus and whether these were present in BALF from the two groups. RESULTS: Across all BALF samples, 417 proteins were identified, and of these, 265 proteins were identified by two or more unique tryptic peptides. Using the 265 high confidence assignments, the quantitative proteome profiles were very similar in the two cohorts, but they could be readily resolved by principal component analysis on the basis of differential protein expression. Of the proteins that were differentially abundant in the two groups, several (including inflammatory and fibrotic markers) were elevated in CIPF, and a smaller, more diverse group of proteins were diminished in CIPF. No protein markers indicative of reflux aspiration were identified. CONCLUSIONS: Label-free proteomics allowed discrimination between CIPF and healthy WHWTs, consistent with fibrotic process but did not provide clear evidence for gastrointestinal aspiration. The measurement of proteins may provide a proteomics signature of CIPF that could be used to evaluate treatment options.

Decoding the Absolute Stoichiometric Composition and Structural Plasticity of α-Carboxysomes.

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria and some chemoautotrophs. This self-assembling organelle encapsulates the key CO2-fixing enzymes, Rubisco, and carbonic anhydrase using a polyhedral protein shell that is constructed by hundreds of shell protein paralogs. The α-carboxysome from the chemoautotroph Halothiobacillus neapolitanus serves as a model system in fundamental studies and synthetic engineering of carboxysomes. In this study, we adopted a QconCAT-based quantitative mass spectrometry approach to determine the stoichiometric composition of native α-carboxysomes from H. neapolitanus. We further performed an in-depth comparison of the protein stoichiometry of native α-carboxysomes and their recombinant counterparts heterologously generated in Escherichia coli to evaluate the structural variability and remodeling of α-carboxysomes. Our results provide insight into the molecular principles that mediate carboxysome assembly, which may aid in rational design and reprogramming of carboxysomes in new contexts for biotechnological applications. IMPORTANCE A wide range of bacteria use special protein-based organelles, termed bacterial microcompartments, to encase enzymes and reactions to increase the efficiency of biological processes. As a model bacterial microcompartment, the carboxysome contains a protein shell filled with the primary carbon fixation enzyme Rubisco. The self-assembling organelle is generated by hundreds of proteins and plays important roles in converting carbon dioxide to sugar, a process known as carbon fixation. In this study, we uncovered the exact stoichiometry of all building components and the structural plasticity of the functional α-carboxysome, using newly developed quantitative mass spectrometry together with biochemistry, electron microscopy, and enzymatic assay. The study advances our understanding of the architecture and modularity of natural carboxysomes. The knowledge learned from natural carboxysomes will suggest feasible ways to produce functional carboxysomes in other hosts, such as crop plants, with the overwhelming goal of boosting cell metabolism and crop yields.

Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.

BACKGROUND: QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. RESULTS: We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. CONCLUSIONS: We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

The Impacts of Surgery and Intracerebral Electrodes in C57BL/6J Mouse Kainate Model of Epileptogenesis: Seizure Threshold, Proteomics, and Cytokine Profiles.

Intracranial electroencephalography (EEG) is commonly used to study epileptogenesis and epilepsy in experimental models. Chronic gliosis and neurodegeneration at the injury site are known to be associated with surgically implanted electrodes in both humans and experimental models. Currently, however, there are no reports on the impact of intracerebral electrodes on proteins in the hippocampus and proinflammatory cytokines in the cerebral cortex and plasma in experimental models. We used an unbiased, label-free proteomics approach to identify the altered proteins in the hippocampus, and multiplex assay for cytokines in the cerebral cortex and plasma of C57BL/6J mice following bilateral surgical implantation of electrodes into the cerebral hemispheres. Seven days following surgery, a repeated low dose kainate (KA) regimen was followed to induce status epilepticus (SE). Surgical implantation of electrodes reduced the amount of KA necessary to induce SE by 50%, compared with mice without surgery. Tissues were harvested 7 days post-SE (i.e., 14 days post-surgery) and compared with vehicle-treated mice. Proteomic profiling showed more proteins (103, 6.8% of all proteins identified) with significantly changed expression (p < 0.01) driven by surgery than by KA treatment itself without surgery (27, 1.8% of all proteins identified). Further, electrode implantation approximately doubled the number of KA-induced changes in protein expression (55, 3.6% of all identified proteins). Further analysis revealed that intracerebral electrodes and KA altered the expression of proteins associated with epileptogenesis such as inflammation (C1q system), neurodegeneration (cystatin-C, galectin-1, cathepsin B, heat-shock protein 25), blood-brain barrier dysfunction (fibrinogen-α, serum albumin, α2 macroglobulin), and gliosis (vimentin, GFAP, filamin-A). The multiplex assay revealed a significant increase in key cytokines such as TNFα, IL-1ß, IL-4, IL-5, IL-6, IL-10, IL12p70, IFN-γ, and KC/GRO in the cerebral cortex and some in the plasma in the surgery group. Overall, these findings demonstrate that surgical implantation of depth electrodes alters some of the molecules that may have a role in epileptogenesis in experimental models.

Lymphocytic Choriomeningitis Virus Alters the Expression of Male Mouse Scent Proteins.

Mature male mice produce a particularly high concentration of major urinary proteins (MUPs) in their scent marks that provide identity and status information to conspecifics. Darcin (MUP20) is inherently attractive to females and, by inducing rapid associative learning, leads to specific attraction to the individual male's odour and location. Other polymorphic central MUPs, produced at much higher abundance, bind volatile ligands that are slowly released from a male's scent marks, forming the male's individual odour that females learn. Here, we show that infection of C57BL/6 males with LCMV WE variants (v2.2 or v54) alters MUP expression according to a male's infection status and ability to clear the virus. MUP output is substantially reduced during acute adult infection with LCMV WE v2.2 and when males are persistently infected with LCMV WE v2.2 or v54. Infection differentially alters expression of darcin and, particularly, suppresses expression of a male's central MUP signature. However, following clearance of acute v2.2 infection through a robust virus-specific CD8 cytotoxic T cell response that leads to immunity to the virus, males regain their normal mature male MUP pattern and exhibit enhanced MUP output by 30 days post-infection relative to uninfected controls. We discuss the likely impact of these changes in male MUP signals on female attraction and mate selection. As LCMV infection during pregnancy can substantially reduce embryo survival and lead to lifelong infection in surviving offspring, we speculate that females use LCMV-induced changes in MUP expression both to avoid direct infection from a male and to select mates able to develop immunity to local variants that will be inherited by their offspring.

Probing the biogenesis pathway and dynamics of thylakoid membranes.

How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.

The characteristic response of domestic cats to plant iridoids allows them to gain chemical defense against mosquitoes.

Domestic cats and other felids rub their faces and heads against catnip (Nepeta cataria) and silver vine (Actinidia polygama) and roll on the ground as a characteristic response. While this response is well known, its biological function and underlying mechanism remain undetermined. Here, we uncover the neurophysiological mechanism and functional outcome of this feline response. We found that the iridoid nepetalactol is the major component of silver vine that elicits this potent response in cats and other felids. Nepetalactol increased plasma ß-endorphin levels in cats, while pharmacological inhibition of µ-opioid receptors suppressed the classic rubbing response. Rubbing behavior transfers nepetalactol onto the faces and heads of respondents where it repels the mosquito, Aedes albopictus Thus, self-anointing behavior helps to protect cats against mosquito bites. The characteristic response of cats to nepetalactol via the µ-opioid system provides an important example of chemical pest defense using plant metabolites in nonhuman mammals.

The application of rapid evaporative ionization mass spectrometry in the analysis of Drosophila species-a potential new tool in entomology.

There is increasing emphasis on the use of new analytical approaches in subject analysis and classification, particularly in respect to minimal sample preparation. Here, we demonstrate that rapid evaporative ionization mass spectrometry (REIMS), a method that captures metabolite mass spectra after rapid combustive degradation of an intact biological specimen, generates informative mass spectra from several arthropods, and more specifically, is capable of discerning differences between species and sex of several adult Drosophila species. A model including five Drosophila species, built using pattern recognition, achieves high correct classification rates (over 90%) using test datasets and is able to resolve closely related species. The ease of discrimination of male and female specimens also demonstrates that sex-specific differences reside in the REIMS metabolite patterns, whether analysed across all five species or specifically for D. melanogaster. Further, the same approach can correctly discriminate and assign Drosophila species at the larval stage, where these are morphologically highly similar or identical. REIMS offers a novel approach to insect typing and analysis, requiring a few seconds of data acquisition per sample and has considerable potential as a new tool for the field biologist.

Social status and ejaculate composition in the house mouse.

Sperm competition theory predicts that males should tailor ejaculates according to their social status. Here, we test this in a model vertebrate, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our analyses reveal that both sperm production and the composition of proteins found in seminal vesicle secretions differ according to social status. Dominant males invested more in ejaculate production overall. Their epididymides contained more sperm than those of subordinate or control males, despite similar testes size between the groups. Dominant males also had larger seminal vesicle glands than subordinate or control males, despite similar body size. However, the seminal vesicle secretions of subordinate males had a significantly higher protein concentration than those of dominant males. Moreover, detailed proteomic analysis revealed subtle but consistent differences in the composition of secreted seminal vesicle proteins according to social status, involving multiple proteins of potential functional significance in sperm competition. These findings have significant implications for understanding the dynamics and outcome of sperm competition, and highlight the importance of social status as a factor influencing both sperm and seminal fluid investment strategies. This article is part of the theme issue 'Fifty years of sperm competition'.

Revealing mechanisms of mating plug function under sexual selection.

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus We show that, although the first male's plug is usually dislodged, it can be retained throughout the second male's copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male's sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.