Evolutionary patterns of major urinary protein scent signals in house mice and relatives

Scent marks are important mediators of territorial behaviour and sexual selection, especially among mammals. The evolution of compounds used in scent marks has the potential to inform our understanding of signal evolution in relation to social and sexual selection. A major challenge in studies of chemical communication is that the link between semiochemical compounds and genetic changes is often unclear. The major urinary proteins (MUPs) of house mice provide information on sex, status and individual identity. Importantly, MUPs are a direct protein product of genes, providing a clear link between genotype and phenotype. Here, we examine the evolution of urinary protein signals among house mice and relatives by examining the sequences and patterns of mRNA expression of Mup genes related to urinary scent marks. MUP patterns have evolved among mouse species both by gene duplication and variation in expression. Notably, protein scent signals that are male specific in well‐studied inbred laboratory strains vary in sex‐specificity among species. Our data reveal that individual identity signals in MUPs evolved prior to 0.35 million years ago and have rapidly diversified through recombining a modest number of amino acid variants. Amino acid variants are much more common on the exterior of the protein where they could interact with vomeronasal receptors, suggesting that chemosensory perception may have played a major role in shaping MUP diversity. These data highlight diverse processes and pressures shaping scent signals, and suggest new avenues for using wild mice to probe the evolution of signals and signal processing.     

The ownership signature in mouse scent marks is involatile

Male house mice advertise their territory ownership through urinary scent marks and use individual-specific patterns of major urinary proteins (MUPs) to discriminate between their own scent and that of other males. It is not clear whether recognition occurs through discrimination of the non-volatile proteins or protein-ligand complexes (direct model), or by the detection of volatile ligands that are released from MUPs (indirect model). To examine the mechanism underlying individual scent mark signatures, we compared investigatory and countermarking responses of male laboratory mice presented with male scent marks from a strain with a different MUP pattern, when they could contact the scent or when contact was prevented by a porous nitrocellulose sheet to which proteins bind. Mice investigated scent marks from other males whether these were covered or not, and biochemical analysis confirmed that the porous cover did not prevent the release of volatiles from scent marks. Having gained information through investigation, mice increased their own scent marking only if they had direct contact with another male's urine, failing to do this when contact was prevented. Individual signatures in scent marks thus appear to be carried by non-volatile proteins or by non-volatile protein-ligand complexes, rather than by volatiles emanating from the scent.     

The genetic basis of individual-recognition signals in the mouse

The major histocompatibility complex (MHC) is widely assumed to be a primary determinant of individual-recognition scents in many vertebrates [1-6], but there has been no functional test of this in animals with normal levels of genetic variation. Mice have evolved another polygenic and highly polymorphic set of proteins for scent communication, the major urinary proteins (MUPs) [7-12], which may provide a more reliable identity signature ([13, 14] and A.L. Sherborne, M.D.T., S. Paterson, F.J., W.E.R.O., P. Stockley, R.J.B., and J.L.H., unpublished data). We used female preference for males that countermark competitor male scents [15-17] to test the ability of wild-derived mice to recognize individual males differing in MHC or MUP type on a variable genetic background. Differences in MHC type were not used for individual recognition. Instead, recognition depended on a difference in MUP type, regardless of other genetic differences between individuals. Recognition also required scent contact, consistent with detection of involatile components through the vomeronasal system [6, 18]. Other differences in individual scent stimulated investigation but did not result in individual recognition. Contrary to untested assumptions of a vertebrate-wide mechanism based largely on MHC variation, mice use a species-specific [12] individual identity signature that can be recognized reliably despite the complex internal and external factors that influence scents [2]. Specific signals for genetic identity recognition in other species now need to be investigated.     

MHC odours are not required or sufficient for recognition of individual scent owners

To provide information about specific depositors, scent marks need to encode a stable signal of individual ownership. The highly polymorphic major histocompatibility complex (MHC) influences scents and contributes to the recognition of close kin and avoidance of inbreeding when MHC haplotypes are shared. MHC diversity between individuals has also been proposed as a primary source of scents used in individual recognition. We tested this in the context of scent owner recognition among male mice, which scent mark their territories and countermark scents from other males. We examined responses towards urine scent according to the scent owner's genetic difference to the territory owner (MHC, genetic background, both and neither) or genetic match to a familiar neighbour. While urine of a different genetic background from the subject always stimulated greater scent marking than own, regardless of familiarity, MHC-associated odours were neither necessary nor sufficient for scent owner recognition and failed to stimulate countermarking. Urine of a different MHC type to the subject stimulated increased investigation only when this matched both the MHC and genetic background of a familiar neighbour. We propose an associative model of scent owner recognition in which volatile scent profiles, contributed by both fixed genetic and varying non-genetic factors, are learnt in association with a stable involatile ownership signal provided by other highly polymorphic urine components.     

Encoding choosiness: female attraction requires prior physical contact with individual male scents in mice

Scents, detected through both the main and vomeronasal olfactory systems, play a crucial role in regulating reproductive behaviour in many mammals. In laboratory mice, female preference for airborne urinary scents from males (detected through the main olfactory system) is learnt through association with scents detected through the vomeronasal system during contact with the scent source. This may reflect a more complex assessment of individual males than that implied by laboratory mouse studies in which individual variation has largely been eliminated. To test this, we assessed female preference between male and female urine using wild house mice with natural individual genetic variation in urinary identity signals. We confirm that females exhibit a general preference for male over female urine when able to contact urine scents. However, they are only attracted to airborne urinary volatiles from individual males whose urine they have previously contacted. Even females with a natural exposure to many individuals of both sexes fail to develop generalized attraction to airborne male scents. This implies that information gained through contact with a specific male's scent is essential to stimulate attraction, providing a new perspective on the cues and olfactory pathways involved in sex recognition and mate assessment in rodents.     

Murine scent mark microbial communities are genetically determined

Scent marking in mice allows males to communicate information such as territory ownership, male competitive ability and current reproductive, nutritional, social and health status. It has been suggested that female mice eavesdrop on these olfactory cues, using them as a means of selecting mates with dissimilar major histocompatibility complex (MHC) genes, known as H2 in mice. The mechanisms underpinning MHC-dependent olfactory communication remain unresolved. Using congenic mouse strains and molecular methods we explore the involvement of the microbial communities, a known source of odourants, in scent marks to test the hypothesis that the microbial communities and hence the olfactory signals are genetically determined. Here we show that the indigenous microbial community of murine scent marks is genetically determined. Both background genotype and H2 haplotype influence the community structure of the scent mark flora, removing the possibility that community composition is solely orchestrated by the MHC. Qualitative and quantitative components of the bacterial community associated with MHC haplotype and background genotype were identified. The analyses confirm that the four groups of congenic mice tested are distinguishable on basis of the microbiology of their scent marks alone, strengthening the role of microorganisms in the development of MHC-dependent odours.     

Individual recognition through olfactory–auditory matching in lemurs

Individual recognition can be facilitated by creating representations of familiar individuals, whereby information from signals in multiple sensory modalities become linked. Many vertebrate species use auditory–visual matching to recognize familiar conspecifics and heterospecifics, but we currently do not know whether representations of familiar individuals incorporate information from other modalities. Ring-tailed lemurs (Lemur catta) are highly visual, but also communicate via scents and vocalizations. To investigate the role of olfactory signals in multisensory recognition, we tested whether lemurs can recognize familiar individuals through matching scents and vocalizations. We presented lemurs with female scents that were paired with the contact call either of the female whose scent was presented or of another familiar female from the same social group. When the scent and the vocalization came from the same individual versus from different individuals, females showed greater interest in the scents, and males showed greater interest in both the scents and the vocalizations, suggesting that lemurs can recognize familiar females via olfactory–auditory matching. Because identity signals in lemur scents and vocalizations are produced by different effectors and often encountered at different times (uncoupled in space and time), this matching suggests lemurs form multisensory representations through a newly recognized sensory integration underlying individual recognition.     

Urinary proteins and the modulation of chemical scents in mice and rats

The urine of mice, rats and some other rodents contains substantial quantities of proteins that are members of the lipocalin family. The proteins are thought to be responsible for the binding and release of low molecular weight pheromones, and there is now good evidence that they discharge this role, providing a slow release mechanism for volatile components of scent marks. However, the proteins may function as chemosignalling molecules in their own right, contributing one or more roles in the communication of individual identity and scent mark ownership. In this review, we summarize current understanding of the structure and function of these urinary proteins, and speculate about their role as supporters or as key participants in the elaboration of the complex chemosensory properties of a rodent scent mark.     

The predation risks of interspecific eavesdropping: weasel–vole interactions

Competing species benefit from eavesdropping on each other's signals by learning about shared resources or predators. But conspicuous signals are also open to exploitation by eavesdropping predators and should also pose a threat to other sympatric prey species. In western Finland, sibling voles Microtus rossiameridionalis and field voles M. agrestis compete for food and space, and both species rely upon scent marks for intraspecific communication. Both vole species are prey to a range of terrestrial scent hunting predators such as least weasels, however, the competitively superior sibling voles are taken preferentially. We tested in large out‐door enclosures whether field voles eavesdrop on the signals of its competitor, and whether they behave as though this eavesdropping carries a risk of predation. We presented field voles with scent marks from unknown conspecifics and sibling voles and measured their visitation, activity and scent marking behaviours at these scents under high (weasel present) and low (weasel absent) predation risk. Field voles readily visited both field and sibling vole scents under both high and low predation risk; however their activity at sibling vole scent marks declined significantly under increased predation risk. In contrast, predation risk did not affect field voles’ activity at conspecific scents. Thus, field voles were compelled to maintain eavesdropping on heterospecific scents under an increased risk of predation, however they compensated for this additional risk by reducing their activity at these risky scents. Scent marking rates declined significantly under high predation risk. Our results therefore reveal a hidden complexity in the use of social signals within multi‐species assemblages that is clearly sensitive to the potential for increased predation risk. The predation risks of interspecific eavesdropping demonstrated here represents a significant generalisation of the concept of associational susceptibility.     

The consequences of inbreeding for recognizing competitors

Extreme inbreeding will compromise an animal's ability to discriminate between individuals and, thus, assess familiarity and kinship with conspecifics. In rodents, a large component of individual recognition is mediated through chemical communication. The counter-marking of competitor males' scent marks provides a measure of discrimination between their own scent and that from other individuals. We investigated whether males in common outbred (ICR(CD-1) and TO) and inbred (BALB/c) strains of laboratory mice could recognize the urinary scents of other individuals by measuring their investigation and counter-marking responses. Dominant males of outbred strains investigated and counter-marked scents from other males, whether of the same or another strain. Dominant inbred BALB/c males investigated but did not counter-mark their own strain scents, counter-marking only those from another strain. They did not use environmentally induced status differences in odours to recognize scents from other males. The inability of the inbred mice to discriminate between their own scent marks and those of other males is likely to alter their competitive behaviour, which could influence responses in experiments and the welfare of caged laboratory mice.     

The competing countermarks hypothesis: reliable assessment of competitive ability by potential mates

Scent marking on top of (overmarking), or in the vicinity of, a scent mark already present is commonly termed countermarking. Scent marks and countermarks provide a continuous record of competitive challenges between conspecifics, thus providing a reliable advertisement of an individual's ability to dominate or defend an area to other competitors and potential mates. To test the hypothesis that females should prefer males that countermark competing scent marks in their territory over those whose own marks are partially countermarked by a competing male, we manipulated scent marks in the territories of neighbouring male house mice (captive-bred Mus domesticus). As predicted, oestrous females were more strongly attracted to approach territory owners that countermarked the scent mark challenges of competitors than those that had been countermarked, and females themselves deposited more scent marks near the scents of these males. To investigate whether female mice use scent age, overlap or intrinsic qualitative or quantitative differences between scent marks and countermarks to make this discrimination, we redeposited male scent marks artificially as marks and partially overlapping countermarks, with or without a 24-h age difference between them. While the intrinsic quality or quantity of countermarks did not affect discrimination, an age difference between the original mark and subsequent countermark was important for consistent discrimination. The ultimate function of such competitive scent signalling thus may be to provide potential mates with a reliable indicator of the competitive ability of individuals advertising their high status. Since scent marks remain in the environment and are continuously available to challenge and investigation, they may provide a particularly effective and reliable means of dominance advertisement. Copyright 1999 The Association for the Study of Animal Behaviour.     

The direct assessment of genetic heterozygosity through scent in the mouse

The role of individual genetic heterozygosity in mate choice is the subject of much current debate. Several recent studies have reported female preference for more heterozygous males, but the mechanisms underlying heterozygote preference remain largely unknown. Females could favor males that are more successful in intrasexual competition, but they could also assess male heterozygosity directly at specific polymorphic genetic markers. Here, we use a breeding program to remove the intrinsic correlation between genome-wide heterozygosity and two highly polymorphic gene clusters that could allow direct assessment of heterozygosity through scent in mice: the major histocompatibility complex (MHC) and the major urinary proteins (MUPs). When other sources of variation are controlled and intrasexual competition is minimized, female mice prefer to associate with MUP heterozygous over MUP homozygous males. MHC heterozygosity does not influence preference, and neither does heterozygosity across the rest of the genome when intrasexual competition between males is restricted. Female mice thus assess male heterozygosity directly through multiple MUP isoforms expressed in scent signals, independently of the effects of genome-wide heterozygosity on male competitiveness. This is the first evidence that animals may use signals of genetic heterozygosity that have no direct association with individual vigour.     

Wake up and smell the conflict: odour signals in female competition

Odour signals used in competitive and aggressive interactions between males are well studied in the context of sexual selection. By contrast, relatively little is known about comparable signals used by females, despite current interest in the evolution of female ornaments and weaponry. Available evidence suggests that odour signals are important in competitive interactions between female mammals, with reductions or reversals of male-biased sexual dimorphism in signalling where female competition is intense. Scent marking is often associated with conflict between females over access to resources or reproductive opportunities. Female scent marks may therefore provide reliable signals of competitive ability that could be used both by competitors and potential mates. Consistent with this hypothesis, we report that aggressive behaviour of female house mice is correlated with the amount of major urinary protein (MUP) excreted in their urine, a polymorphic set of proteins that are used in scent mark signalling. Under semi-natural conditions, females with high MUP output are more likely to produce offspring sired by males that have high reproductive success, and less likely to produce offspring by multiple different sires, suggesting that females with strong MUP signals are monopolized by males of particularly high quality. We conclude that odour signals are worthy of more detailed investigation as mediators of female competition.     

The scent of senescence: sexual signalling and female preference in house mice

Sexual signals are expected to be costly to produce and maintain, thus ensuring that only males in good condition can sustain their expression at high levels. When males reach senescence they lose physiological function and condition, which could constrain their ability to invest in costly sexual signals, decreasing their attractiveness to mates. Furthermore, females may have evolved mating preferences that cause avoidance of senesced males to enhance fertilization success and viability of offspring. Among mammals, the size of antlers and other weapons can decrease with senescence, but changes in olfactory sexual signals have been largely unexplored. We examined changes in olfactory signals with senescence in house mice (Mus musculus domesticus), where males excrete volatile and involatile molecules in scent marks that elicit behavioural and priming responses in females. Compared to middle-aged males, the urine of senesced males contained a lower concentration of involatile signalling proteins (major urinary proteins or MUPs), and associated volatiles that bind to these proteins. The reduced intensity of male scent will affect the longevity of scent signals deposited in the environment and, accordingly, females were less attracted to urine from senesced males deposited 12 h previously. Females also discriminated against senesced males encountered behind a mesh barrier. These results reveal that investment in olfactory signalling is reduced during senescence and suggest that senesced males and their scent may be less attractive to females.     

1H, 15N and 13C resonance assignment of darcin, a mouse major urinary protein

Darcin is an important lipocalin of the urinary MUP family. These beta-barrel structures differ subtly in sequence and function and facilitate communication between members of the mouse population via scent marks. Polymorphism within the family has led to the hypothesis that individual MUPs can also contribute to social and physiological information of the scent owner and thus demonstrates the necessity for structural investigation of these variations. Using conventional triple resonance experiments, ¹H ¹⁵N and ¹³C assignment of recombinant N terminal hexa-histidine tagged Darcin has been achieved. The corresponding chemical shifts have been deposited in the BioMagResBank; Accession No. 16840.     

Individual Odors in Djungarian Hamsters (Phodopus campbelli)

The sources of scent in Djungarian hamsters (Phodopus campbelli) that may be individually discriminated were investigated using an habituation paradigm. Male Djungarian hamsters were exposed to five presentations of a particular scent from one individual, and then to the same scent from a novel individual. Increased investigation of the scent from the novel individual indicated discrimination of scents from different individuals. Male hamsters distinguished individual differences in scents of other males from the midventral gland, urine, feces, mouth, and the corner of the mouth, which includes the sacculi; they did not discriminate among odors of different individuals when the scents were from the genital region, hindfeet, fur from behind ears or fur from the back. The results indicate that Djungarian hamsters have a repertoire of individually distinctive scents that are located in specific places on the body; these scents are not actively distributed to, nor passively picked up on, other parts of the body. The fact that scents from some areas do not contain individually distinctive information suggests that some sources may be specialized for producing individually distinctive scents.     

Gonadal control of male scent in the tarmarin Saguinus fuscicollis (Callitrichidae, Primates)

Spontaneous responses to the scent marks of male Saguinus fuscicolliswere tested in experiments where the subjects received a choicebetween scent of intact males and castrated males. Male tamarinsdiscriminated between the odors of intact and castrated males,both on the basis of fresh individual scent marks and on thebasis of marks from several donors pooled in methanol. Theirdiscriminatory response to pooled odors was more pronouncedthan that to individual odors. Females discriminated betweenintact and castrated male scents only on the basis of pooledsamples. This result shows that castration causes changes inthe odor of males. The difference in the response of the tamarinsto individual scent marks as compared to pooled samples is discussedin terms of quantitative changes caused by extraction and ofindividual recognition.     

Unravelling the chemical basis of competitive scent marking in house mice

Major urinary proteins (MUPs) in the urine of male house mice, Mus domesticus, bind the male signalling volatiles 2- sec -butyl-4,5-dihydrothiazole (thiazole) and 3,4-dehydro- exo -brevicomin (brevicomin) and slowly release these volatiles from urinary scent marks. To examine the role of urinary proteins and volatiles, either attached or unattached to the proteins, in competitive scent marking, we fractionated urine from isolated male BALB/c laboratory mice, Mus musculus, by size-exclusion chromatography into three pools. Pool I contained all of the urinary proteins and their bound ligands while pools II and III contained lower molecular weight components including unbound signalling volatiles. In experiment 1, pools I-III were streaked out on to absorbent paper (Benchkote) and introduced into enclosures housing single wild-caught male mice, together with a clean control surface. Each male was tested with fresh stimuli and with aged stimuli deposited 24 h previously. Only pool I stimulated significantly more countermarking and investigation than the control, attracting mice to investigate from a distance even when the rate of ligand release was considerably reduced after 24 h. Experiment 2 examined responses to pool I when this was fresh, aged by 7 days, or had been mixed with menadione to displace ligands from the proteins. Although all three protein stimuli were investigated and countermarked more than a clean control, the aged and menadione-treated pool I stimulated the strongest responses, despite containing the lowest levels of thiazole and brevicomin. Thus competitive countermarking is stimulated by proteins or by nonvolatile protein-ligand complexes in male urine, while release of volatile ligands attracts attention to a competitor's scent marks. Copyright 1999 The Association for the Study of Animal Behaviour.     

Cross-species Discrimination of Individual Odors by Hamsters (Muridae: Mesocricetus auratus,Phodopus campbelli)

Golden hamsters, Mesocricetus auratus, and Djungarian hamsters, Phodopus campbelli, Muridae, were tested for the ability to discriminate between individual odors from both their own species and the other species. Hamsters were tested using an habituation technique, in which differences in time spent investigating familiar and novel scents indicate discrimination of individual differences. Both species showed similar patterns of results when tested with flank gland scents from golden hamsters and ventral gland scents from Djungarian hamsters: The duration of investigation of samples from the same indivídual scent donor declined over trials, indicating recognition of this scent, but then increased when a scent from a novel individual was presented, indicating discrimination of individual differences. Thus, the capacity and tendency to respond differentially to signals from different individuals is not confined to a single species, suggesting the lack of species-specificity in the signals and perceptual mechanisms involved in individual recognition by scent. This capacity may be common in mammals, and if so suggests the possibility for considerable knowledge of heterospecific individuals.     

Dogs discriminate identical twins

Earlier studies have shown variation among experimental attempts to establish whether human monozygotic twins that are genetically identical also have identical individual scents. In none of the cases were the dogs able to distinguish all the individual scents of monozygotic twins living in the same environment if the scents were presented to them separately. Ten specially trained police German Shepherd dogs of three Czech Republic Police Regional Headquarters were used for scent identification in our study. The dogs were supposed to match scents of two monozygotic pairs (5 and 7 years old) and two dizygotic twin pairs (8 and 13 years old). Scents were collected on cotton squares stored in glass jars. Dog handlers were blind to the experiment details. In each trial (line-up), one scent was used as a starting scent and the dog was then sent to determine if any of the 7 presented glass jars contained a matching scent. Scents of children of similar ages were used as distractors. In the matching procedure, the dogs matched correctly the scent of one twin with the other, as well as two scents collected from every single identical and non-identical twin to prove their efficacy and likewise, the presence of the matching twin scent in any given glass jar. All dogs in all trials distinguished correctly the scents of identical as well as non-identical twins. All dogs similarly matched positively two scents collected from the same individuals. Our findings indicated that specially trained German Shepherd dogs are able to distinguish individual scents of identical twins despite the fact that they live in the same environment, eat the same food and even if the scents are not presented to them simultaneously.