Backbone chemical shift and secondary structure assignments for mouse siderocalin.

The lipocalin protein family is a structurally conserved group of proteins with a variety of biological functions defined by their ability to bind small molecule ligands and interact with partner proteins. One member of this family is siderocalin, a protein found in mammals. Its role is discussed in inflammatory processes, iron trafficking, protection against bacterial infections and oxidative stress, cell migration, induction of apoptosis, and cancer. Though it seems to be involved in numerous essential pathways, the exact mechanisms are often not fully understood. The NMR backbone assignments for the human siderocalin and its rat ortholog have been published before. In this work we describe the backbone NMR assignments of siderocalin for another important model organism, the mouse - data that might become important for structure-based drug discovery. Secondary structure elements were predicted based on the assigned backbone chemical shifts using TALOS-N and CSI 3.0, revealing a high content of beta strands and one prominent alpha helical region. Our findings correlate well with the known crystal structure and the overall conserved fold of the lipocalin family.

Epigenetic age estimation of wild mice using faecal samples.

Age is a key parameter in population ecology, with a myriad of biological processes changing with age as organisms develop in early life then later senesce. As age is often hard to accurately measure with non-lethal methods, epigenetic methods of age estimation (epigenetic clocks) have become a popular tool in animal ecology and are often developed or calibrated using captive animals of known age. However, studies typically rely on invasive blood or tissue samples, which limit their application in more sensitive or elusive species. Moreover, few studies have directly assessed how methylation patterns and epigenetic age estimates compare across environmental contexts (e.g. captive or laboratory-based vs. wild animals). Here, we built a targeted epigenetic clock from laboratory house mice (strain C57BL/6, Mus musculus) using DNA from non-invasive faecal samples, and then used it to estimate age in a population of wild mice (Mus musculus domesticus) of unknown age. This laboratory mouse-derived epigenetic clock accurately predicted adult wild mice to be older than juveniles and showed that wild mice typically increased in epigenetic age over time, but with wide variation in epigenetic ageing rate among individuals. Our results also suggested that, for a given body mass, wild mice had higher methylation across targeted CpG sites than laboratory mice (and consistently higher epigenetic age estimates as a result), even among the smallest, juvenile mice. This suggests wild and laboratory mice may display different CpG methylation levels from very early in life and indicates caution is needed when developing epigenetic clocks on laboratory animals and applying them in the wild.

Genetically identical mice express alternative reproductive tactics depending on social conditions in the field.

In many species, establishing and maintaining a territory is critical to survival and reproduction, and an animal's ability to do so is strongly influenced by the presence and density of competitors. Here we manipulate social conditions to study the alternative reproductive tactics displayed by genetically identical, age-matched laboratory mice competing for territories under ecologically realistic social environmental conditions. We introduced adult males and females of the laboratory mouse strain C57BL/6J into a large, outdoor field enclosure containing defendable resource zones under one of two social conditions. We first created a low-density social environment, such that the number of available territories exceeded the number of males. After males established stable territories, we introduced a pulse of intruder males and observed the resulting defensive and invasive tactics employed. In response to this change in social environment, males with large territories invested more in patrolling but were less effective at excluding intruder males as compared with males with small territories. Intruding males failed to establish territories and displayed an alternative tactic featuring greater exploration as compared with genetically identical territorial males. Alternative tactics did not lead to equal reproductive success-males that acquired territories experienced greater survival and had greater access to females.

Multiple Genomic Landscapes of Recombination and Genomic Divergence in Wild Populations of House Mice-The Role of Chromosomal Fusions and Prdm9.

Chromosomal fusions represent one of the most common types of chromosomal rearrangements found in nature. Yet, their role in shaping the genomic landscape of recombination and hence genome evolution remains largely unexplored. Here, we take advantage of wild mice populations with chromosomal fusions to evaluate the effect of this type of structural variant on genomic landscapes of recombination and divergence. To this aim, we combined cytological analysis of meiotic crossovers in primary spermatocytes with inferred analysis of recombination rates based on linkage disequilibrium using single nucleotide polymorphisms. Our results suggest the presence of a combined effect of Robertsonian fusions and Prdm9 allelic background, a gene involved in the formation of meiotic double strand breaks and postzygotic reproductive isolation, in reshaping genomic landscapes of recombination. We detected a chromosomal redistribution of meiotic recombination toward telomeric regions in metacentric chromosomes in mice with Robertsonian fusions when compared to nonfused mice. This repatterning was accompanied by increased levels of crossover interference and reduced levels of estimated recombination rates between populations, together with high levels of genomic divergence. Interestingly, we detected that Prdm9 allelic background was a major determinant of recombination rates at the population level, whereas Robertsonian fusions showed limited effects, restricted to centromeric regions of fused chromosomes. Altogether, our results provide new insights into the effect of Robertsonian fusions and Prdm9 background on meiotic recombination.

Exploring patterns of female house mouse spatial organisation among outbreaking and stable populations.

The size and distribution of home ranges reflect how individuals within a population use, defend, and share space and resources, and may thus be an important predictor of population-level dynamics. Eruptive species, such as the house mouse in Australian grain-growing regions, are an ideal species in which to investigate variations in space use and home range overlap between stable and outbreaking populations. In this study, we use spatially explicit capture-recapture models to explore if space use and home range overlap among female mice could serve as indicators of changes in population density leading into summer. Additionally, we assess the sensitivity of space use and home range estimates to reduced recapture rates. Our analysis did not reveal variations in the spring spatial organisation of female mice based on existing capture-mark-recapture data. However, our study highlights the need to balance monitoring efforts within regions, emphasising the importance of exploring studies that can improve spatial recaptures by optimising trapping efforts. This is particularly important in Australian agricultural systems, where varying farm management practices may drive differences in population dynamics.

Germ-free status but not subacute polychlorinated biphenyl (PCB) exposure altered hepatic phosphatidylcholine and ether-phosphatidylcholine levels in mice.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that pose a current ecosystem and human health concern. PCB exposure impacts the gut microbiome in animal models, suggesting a mechanistic link between PCB exposure and adverse health outcomes. The presence and absence of the microbiome and exposure to PCBs independently affect the lipid composition in the liver, which in turn affects the PCB disposition in target tissues, such as the liver. Here, we investigated microbiome × subacute PCB effects on the hepatic lipid composition of conventional and germ-free female mice exposed to 0, 6, or 30 mg/kg body weight of an environmental PCB mixture in sterile corn oil once daily for 3 consecutive days. Hepatic triacylglyceride and polar lipid levels were quantified using mass spectrometric methods following the subacute PCB exposure. The lipidomic analysis revealed no PCB effect on the hepatic levels. No microbiome effect was observed on levels of triacylglyceride and most polar lipid classes. The total hepatic levels of phosphatidylcholine (PC) and ether-phosphatidylcholine (ePC) lipids were lower in germ-free mice than the conventional mice from the same exposure group. Moreover, levels of several unsaturated PCs, such as PC(36:5) and PC(42:10), and ePCs, such as ePC(36:2) and ePC(4:2), were lower in germ-free than conventional female mice. Based on a KEGG pathway meta-analysis of RNA sequencing data, the ether lipid metabolism pathway is altered in the germ-free mouse liver. In contrast to the liver, extractable lipid levels, determined gravimetrically, differed in several tissues from naïve conventional vs. germ-free mice. Overall, microbiome × subacute PCB exposure effects on hepatic lipid composition are unlikely to affect PCB distribution into the mouse liver. Further studies are needed to assess how the different extractable lipid levels in other tissues alter PCB distribution in conventional vs. germ-free mice.

Noncanonical usage of stop codons in ciliates expands proteins with structurally flexible Q-rich motifs.

Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

Histological evidence of hypothyroidism in mice chronically exposed to conventional farming.

Worldwide, disorders of the thyroid gland are a growing concern; such can be caused by exposure to contaminants, including agrochemicals used in conventional agriculture, which act as endocrine disruptors. The purpose of this study is to evaluate whether or not exposure to an environment with conventional agriculture leads to thyroid disruption. Mus musculus were used as bioindicator species, captured in two sites: a farm where conventional agriculture is practiced, and a place without agriculture. Thyroid histomorphometric and morphologic data were analyzed. The impacts of the agricultural environment over the thyroid were revealed, as indications of hypothyroidism were observed in exposed mice: the area and volume of epithelial cells were much lower. Alterations in thyroid histomorphology were also observed: lower follicular sphericity, irregularly delimited epithelium and increased exfoliation into the colloid. These results highlight the need for transition from current conventional agricultural systems towards organic systems.

Microbial, proteomic, and metabolomic profiling of the estrous cycle in wild house mice.

Symbiotic microbial communities affect the host immune system and produce molecules contributing to the odor of an individual. In many mammalian species, saliva and vaginal fluids are important sources of chemical signals that originate from bacterial metabolism and may act as honest signals of health and reproductive status. In this study, we aimed to define oral and vaginal microbiomes and their dynamics throughout the estrous cycle in wild house mice. In addition, we analyzed a subset of vaginal proteomes and metabolomes to detect potential interactions with microbiomes. 16S rRNA sequencing revealed that both saliva and vagina are dominated by Firmicutes and Proteobacteria but differ at the genus level. The oral microbiome is more stable during the estrous cycle and most abundant bacteria belong to the genera Gemella and Streptococcus, while the vaginal microbiome shows higher bacterial diversity and dynamics during the reproductive cycle and is characterized by the dominance of Muribacter and Rodentibacter. These two genera cover around 50% of the bacterial community during estrus. Proteomic profiling of vaginal fluids revealed specific protein patterns associated with different estrous phases. Highly expressed proteins in estrus involve the keratinization process thus providing estrus markers (e.g., Hrnr) while some proteins are downregulated such as immune-related proteins that limit bacterial growth (Camp, Clu, Elane, Lyz2, and Ngp). The vaginal metabolome contains volatile compounds potentially involved in chemical communication, for example, ketones, aldehydes, and esters of carboxylic acids. Data integration of all three OMICs data sets revealed high correlations, thus providing evidence that microbiomes, host proteomes, and metabolomes may interact.IMPORTANCEOur data revealed dynamic changes in vaginal, but not salivary, microbiome composition during the reproductive cycle of wild mice. With multiple OMICs platforms, we provide evidence that changes in microbiota in the vaginal environment are accompanied by changes in the proteomic and metabolomics profiles of the host. This study describes the natural microbiota of wild mice and may contribute to a better understanding of microbiome-host immune system interactions during the hormonal and cellular changes in the female reproductive tract. Moreover, analysis of volatiles in the vaginal fluid shows particular substances that can be involved in chemical communication and reproductive behavior.

Natural variation in the zinc-finger-encoding exon of Prdm9 affects hybrid sterility phenotypes in mice.

PRDM9-mediated reproductive isolation was first described in the progeny of Mus musculus musculus (MUS) PWD/Ph and Mus musculus domesticus (DOM) C57BL/6J inbred strains. These male F1 hybrids fail to complete chromosome synapsis and arrest meiosis at prophase I, due to incompatibilities between the Prdm9 gene and hybrid sterility locus Hstx2. We identified 14 alleles of Prdm9 in exon 12, encoding the DNA-binding domain of the PRDM9 protein in outcrossed wild mouse populations from Europe, Asia, and the Middle East, 8 of which are novel. The same allele was found in all mice bearing introgressed t-haplotypes encompassing Prdm9. We asked whether 7 novel Prdm9 alleles in MUS populations and the t-haplotype allele in 1 MUS and 3 DOM populations induce Prdm9-mediated reproductive isolation. The results show that only combinations of the dom2 allele of DOM origin and the MUS msc1 allele ensure complete infertility of intersubspecific hybrids in outcrossed wild populations and inbred mouse strains examined so far. The results further indicate that MUS mice may share the erasure of PRDM9msc1 binding motifs in populations with different Prdm9 alleles, which implies that erased PRDM9 binding motifs may be uncoupled from their corresponding Prdm9 alleles at the population level. Our data corroborate the model of Prdm9-mediated hybrid sterility beyond inbred strains of mice and suggest that sterility alleles of Prdm9 may be rare.

Do housing-induced changes in brain activity cause stereotypic behaviours in laboratory mice?

Abnormal repetitive stereotypic behaviours (SBs) (e.g. pacing, body-rocking) are common in animals with poor welfare (e.g. socially isolated/in barren housing). But how (or even whether) poor housing alters animals' brains to induce SBs remains uncertain. To date, there is little evidence for environmental effects on the brain that also correlate with individual SB performance. Using female mice from two strains (SB-prone DBA/2s; SB-resistant C57/BL/6s), displaying two forms of SB (route-tracing; bar-mouthing), we investigated how housing (conventional laboratory conditions vs. well-resourced 'enriched' cages) affects long-term neuronal activity as assessed via cytochrome oxidase histochemistry in 13 regions of interest (across cortex, striatum, basal ganglia and thalamus). Conventional housing reduced activity in the cortex and striatum. However, DBA mice had no cortical or striatal differences from C57 mice (just greater basal ganglia output activity, independent of housing). Neural correlates for individual levels of bar-mouthing (positive correlations in the substantia nigra and thalamus) were also independent of housing; while route-tracing levels had no clear neural correlates at all. Thus conventional laboratory housing can suppress cortico-striatal activity, but such changes are unrelated to SB (since not mirrored by congruent individual and strain differences). Furthermore, the neural correlates of SB at individual and strain levels seem to reflect underlying predispositions, not housing-mediated changes. To aid further work, hypothesis-generating model fit analyses highlighted this unexplained housing effect, and also suggested several regions of interest across cortex, striatum, thalamus and substantia nigra for future investigation (ideally with improved power to reduce risks of Type II error).

Inner ear morphology in wild versus laboratory house mice.

The semicircular canals of the inner ear are involved in balance and velocity control. Being crucial to ensure efficient mobility, their morphology exhibits an evolutionary conservatism attributed to stabilizing selection. Release of selection in slow-moving animals has been argued to lead to morphological divergence and increased inter-individual variation. In its natural habitat, the house mouse Mus musculus moves in a tridimensional space where efficient balance is required. In contrast, laboratory mice in standard cages are severely restricted in their ability to move, which possibly reduces selection on the inner ear morphology. This effect was tested by comparing four groups of mice: several populations of wild mice trapped in commensal habitats in France; their second-generation laboratory offspring, to assess plastic effects related to breeding conditions; a standard laboratory strain (Swiss) that evolved for many generations in a regime of mobility reduction; and hybrids between wild offspring and Swiss mice. The morphology of the semicircular canals was quantified using a set of 3D landmarks and semi-landmarks analyzed using geometric morphometric protocols. Levels of inter-population, inter-individual (disparity) and intra-individual (asymmetry) variation were compared. All wild mice shared a similar inner ear morphology, in contrast to the important divergence of the Swiss strain. The release of selection in the laboratory strain obviously allowed for an important and rapid drift in the otherwise conserved structure. Shared traits between the inner ear of the lab strain and domestic pigs suggested a common response to mobility reduction in captivity. The lab-bred offspring of wild mice also differed from their wild relatives, suggesting plastic response related to maternal locomotory behavior, since inner ear morphology matures before birth in mammals. The signature observed in lab-bred wild mice and the lab strain was however not congruent, suggesting that plasticity did not participate to the divergence of the laboratory strain. However, contrary to the expectation, wild mice displayed slightly higher levels of inter-individual variation than laboratory mice, possibly due to the higher levels of genetic variance within and among wild populations compared to the lab strain. Differences in fluctuating asymmetry levels were detected, with the laboratory strain occasionally displaying higher asymmetry scores than its wild relatives. This suggests that there may indeed be a release of selection and/or a decrease in developmental stability in the laboratory strain.

Urinary VOCs as biomarkers of early stage lung tumour development in mice.

BACKGROUND: Lung cancer is the primary cause of cancer-induced death. In addition to prevention and improved treatment, it has increasingly been established that early detection is critical to successful remission. OBJECTIVE: The aim of this study was to identify volatile organic compounds (VOCs) in urine that could help diagnose mouse lung cancer at an early stage of its development. METHODS: We analysed the VOC composition of urine in a genetically engineered lung adenocarcinoma mouse model with oncogenic EGFR doxycycline-inducible lung-specific expression. We compared the urinary VOCs of 10 cancerous mice and 10 healthy mice (controls) before and after doxycycline induction, every two weeks for 12 weeks, until full-blown carcinomas appeared. We used SPME fibres and gas chromatography - mass spectrometry to detect variations in cancer-related urinary VOCs over time. RESULTS: This study allowed us to identify eight diagnostic biomarkers that help discriminate early stages of cancer tumour development (i.e., before MRI imaging techniques could identify it). CONCLUSION: The analysis of mice urinary VOCs have shown that cancer can induce changes in odour profiles at an early stage of cancer development, opening a promising avenue for early diagnosis of lung cancer in other models.

Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer.

IMPORTANCE: H. pylori infects half of the world population and is the leading cause of gastric cancer. We previously demonstrated that gastric cancer risk is associated with gastric microbiota. Specifically, gastric urease-positive Staphylococcus epidermidis and Streptococcus salivarius had contrasting effects on H. pylori-associated gastric pathology and immune responses in germ-free INS-GAS mice. As gastritis progresses to gastric cancer, the oncogenic transcription factor Foxm1 becomes increasingly expressed. In this study, we evaluated the gastric commensal C. acnes, certain strains of which produce thiopeptides that directly inhibit FOXM1. Thiopeptide-positive C. acnes was isolated from Nicaraguan patient gastric biopsies and inoculated into germ-free INS-GAS mice with H. pylori. We, therefore, asked whether coinfection with C. acnes expressing thiopeptide and H. pylori would decrease gastric Foxm1 expression and pro-inflammatory cytokine mRNA and protein levels. Our study supports the growing literature that specific non-H. pylori gastric bacteria affect inflammatory and cancer biomarkers in H. pylori pathogenesis.

The effect of host admixture on wild house mouse gut microbiota is weak when accounting for spatial autocorrelation.

The question of how interactions between the gut microbiome and vertebrate hosts contribute to host adaptation and speciation is one of the major problems in current evolutionary research. Using bacteriome and mycobiome metabarcoding, we examined how these two components of the gut microbiota vary with the degree of host admixture in secondary contact between two house mouse subspecies (Mus musculus musculus and M. m. domesticus). We used a large data set collected at two replicates of the hybrid zone and model-based statistical analyses to ensure the robustness of our results. Assuming that the microbiota of wild hosts suffers from spatial autocorrelation, we directly compared the results of statistical models that were spatially naive with those that accounted for spatial autocorrelation. We showed that neglecting spatial autocorrelation can strongly affect the results and lead to misleading conclusions. The spatial analyses showed little difference between subspecies, both in microbiome composition and in individual bacterial lineages. Similarly, the degree of admixture had minimal effects on the gut bacteriome and mycobiome and was caused by changes in a few microbial lineages that correspond to the common symbionts of free-living house mice. In contrast to previous studies, these data do not support the hypothesis that the microbiota plays an important role in host reproductive isolation in this particular model system.

Identification of SARS-CoV-2 in urban rodents from Southern Mexico City at the beginning of the COVID-19 pandemic

ABSTRACT Currently, there are some concerns about the situation and, in particular, about the future of the COVID-19 pandemic and the new emerging variants of SARS-CoV-2. Rodents are an example of synanthropic animals in urban environments that harbor important zoonoses. Although the molecular identification of SARS-CoV-2 in Rattus norvegicus from New York City had been reported, in other studies, urban wild rodents infected with this virus have not been found. This study aimed to molecularly identify the presence of SARS-CoV-2 in urban wild rodents from Mexico City, trapped along a water channel of a public park as part of a pest control program, at the beginning of the COVID-19 pandemic, during the fall and winter of 2020. Up to 33 Mus musculus and 52 R. norvegicus were captured and euthanized, large intestine samples with feces from the animals were obtained. RNAs were obtained and subjected to qRT-PCR for SARS-CoV-2 identification and threshold cycle (Ct) values were obtained. Four mice (12.1%) and three rats (5.8%) were positive, three rodents exhibited Ct<30. Our results on the frequency of SARS-CoV-2 in urban rats are in line with other previous reports. Thus, similar to other authors, we suggest that surveillance for the detection of SARS-CoV-2 in urban wild rodents, as sentinel animals, should be maintained.

Maintenance of neuronal identity in C. elegans and beyond: Lessons from transcription and chromatin factors.

Neurons are remarkably long-lived, non-dividing cells that must maintain their functional features (e.g., electrical properties, chemical signaling) for extended periods of time - decades in humans. How neurons accomplish this incredible feat is poorly understood. Here, we review recent advances, primarily in the nematode C. elegans, that have enhanced our understanding of the molecular mechanisms that enable post-mitotic neurons to maintain their functionality across different life stages. We begin with "terminal selectors" - transcription factors necessary for the establishment and maintenance of neuronal identity. We highlight new findings on five terminal selectors (CHE-1 [Glass], UNC-3 [Collier/Ebf1-4], LIN-39 [Scr/Dfd/Hox4-5], UNC-86 [Acj6/Brn3a-c], AST-1 [Etv1/ER81]) from different transcription factor families (ZNF, COE, HOX, POU, ETS). We compare the functions of these factors in specific neuron types of C. elegans with the actions of their orthologs in other invertebrate (D. melanogaster) and vertebrate (M. musculus) systems, highlighting remarkable functional conservation. Finally, we reflect on recent findings implicating chromatin-modifying proteins, such as histone methyltransferases and Polycomb proteins, in the control of neuronal terminal identity. Altogether, these new studies on transcription factors and chromatin modifiers not only shed light on the fundamental problem of neuronal identity maintenance, but also outline mechanistic principles of gene regulation that may operate in other long-lived, post-mitotic cell types.

A conserved role for frizzled in sleep architecture.

Previous studies of natural variants in Drosophila melanogaster implicated the Wnt signaling receptor frizzled in sleep. Given that the Wnt signaling pathway is highly conserved across species, we hypothesized that frizzled class receptor 1 (Fzd1), the murine homolog of frizzled, would also have a role in sleep. Using a CRISPR transgenic approach, we removed most of the Fzd1 coding region from C57BL/6N mice. We used a video assay to measure sleep characteristics in Fzd1-deficient mice. As Wnt signaling is known to affect visuospatial memory, we also examined the impact of the deletion on learning and memory using the novel object recognition (NOR) paradigm. Fzd1-deficient mice had altered sleep compared to littermate controls. The mice did not respond differently to the NOR paradigm compared to controls but did display anxiety-like behavior. Our strategy demonstrates that the study of natural variation in Drosophila sleep translates into candidate genes for sleep in vertebrate species such as the mouse.

The Expansion of House Mouse Major Urinary Protein Genes Likely Did Not Facilitate Commensalism with Humans.

Mouse wild-derived strains (WDSs) combine the advantages of classical laboratory stocks and wild animals, and thus appear to be promising tools for diverse biomedical and evolutionary studies. We employed 18 WDSs representing three non-synanthropic species (Mus spretus, Mus spicilegus, and M. macedonicus) and three house mouse subspecies (Mus musculus musculus, M. m. domesticus, M. m. castaneus), which are all important human commensals to explore whether the number of major urinary protein (MUP) genes and their final protein levels in urine are correlated with the level of commensalism. Contrary to expectations, the MUP copy number (CN) and protein excretion in the strains derived from M. m. castaneus, which is supposed to be the strongest commensal, were not significantly different from the non-commensal species. Regardless of an overall tendency for higher MUP amounts in taxa with a higher CN, there was no significant correlation at the strain level. Our study thus suggests that expansion of the Mup cluster, which appeared before the house mouse diversification, is unlikely to facilitate commensalism with humans in three house mouse subspecies. Finally, we found considerable variation among con(sub)specific WDSs, warning against generalisations of results based on a few strains.