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.

Fitness Costs of Female Competition Linked to Resource Defense and Relatedness of Competitors.

AbstractFemale reproductive success is often limited by access to resources, and this can lead to social competition both within and between kin groups. Theory predicts that both resource availability and relatedness should influence the fitness consequences of social competition. However, testing key predictions requires differentiating the effects of these two factors. Here, we achieve this experimentally by manipulating the social environment of house mice, a facultative communal breeding species with known kin discrimination ability. This allows us to investigate (1) the reproductive costs of defending a limited resource in response to cues of social competition and (2) whether such costs, or their potential mitigation via cooperative behavior, are influenced by the relatedness of competitors. Our results support the hypothesis that resource defense can be costly for females, potentially trading off against maternal investment. When the availability of protected nest sites was limited, subjects (1) were more active, (2) responded more strongly to simulated territory intrusions via competitive signaling, and (3) produced smaller weaned offspring. However, we found no evidence that the propensity for kin to cooperate was influenced by the relatedness of rivals. Communal breeding between sisters occurred independently of the relatedness of competitors and communally breeding sisters weaned fewer offspring when competing with unrelated females, despite our study being designed to prevent infanticide between kin groups. Our findings thus demonstrate that female competition has fitness costs and that associating with kin is beneficial to avoid negative fitness consequences of competing with nonkin, in addition to more widely recognized kin-selected benefits.

Individual odour signatures that mice learn are shaped by involatile major urinary proteins (MUPs).

BACKGROUND: Reliable recognition of individuals requires phenotypic identity signatures that are both individually distinctive and appropriately stable over time. Individual-specific vocalisations or visual patterning are well documented among birds and some mammals, whilst odours play a key role in social recognition across many vertebrates and invertebrates. Less well understood, though, is whether individuals are recognised through variation in cues that arise incidentally from a wide variety of genetic and non-genetic differences between individuals, or whether animals evolve distinctive polymorphic signals to advertise identity reliably. As a bioassay to understand the derivation of individual-specific odour signatures, we use female attraction to the individual odours of male house mice (Mus musculus domesticus), learned on contact with a male's scent marks. RESULTS: Learned volatile odour signatures are determined predominantly by individual differences in involatile major urinary protein (MUP) signatures, a specialised set of communication proteins that mice secrete in their urine. Recognition of odour signatures in genetically distinct mice depended on differences in individual MUP genotype. Direct manipulation using recombinant MUPs confirmed predictable changes in volatile signature recognition according to the degree of matching between MUP profiles and the learned urine template. Both the relative amount of the male-specific MUP pheromone darcin, which induces odour learning, and other MUP isoforms influenced learned odour signatures. By contrast, odour recognition was not significantly influenced by individual major histocompatibility complex genotype. MUP profiles shape volatile odour signatures through isoform-specific differences in binding and release of urinary volatiles from scent deposits, such that volatile signatures were recognised from the urinary protein fraction alone. Manipulation using recombinant MUPs led to quantitative changes in the release of known MUP ligands from scent deposits, with MUP-specific and volatile-specific effects. CONCLUSIONS: Despite assumptions that many genes contribute to odours that can be used to recognise individuals, mice have evolved a polymorphic combinatorial MUP signature that shapes distinctive volatile signatures in their scent. Such specific signals may be more prevalent within complex body odours than previously realised, contributing to the evolution of phenotypic diversity within species. However, differences in selection may also result in species-specific constraints on the ability to recognise individuals through complex body scents.

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.

Darcin: a male pheromone that stimulates female memory and sexual attraction to an individual male's odour.

BACKGROUND: Among invertebrates, specific pheromones elicit inherent (fixed) behavioural responses to coordinate social behaviours such as sexual recognition and attraction. By contrast, the much more complex social odours of mammals provide a broad range of information about the individual owner and stimulate individual-specific responses that are modulated by learning. How do mammals use such odours to coordinate important social interactions such as sexual attraction while allowing for individual-specific choice? We hypothesized that male mouse urine contains a specific pheromonal component that invokes inherent sexual attraction to the scent and which also stimulates female memory and conditions sexual attraction to the airborne odours of an individual scent owner associated with this pheromone. RESULTS: Using wild-stock house mice to ensure natural responses that generalize across individual genomes, we identify a single atypical male-specific major urinary protein (MUP) of mass 18893Da that invokes a female's inherent sexual attraction to male compared to female urinary scent. Attraction to this protein pheromone, which we named darcin, was as strong as the attraction to intact male urine. Importantly, contact with darcin also stimulated a strong learned attraction to the associated airborne urinary odour of an individual male, such that, subsequently, females were attracted to the airborne scent of that specific individual but not to that of other males. CONCLUSIONS: This involatile protein is a mammalian male sex pheromone that stimulates a flexible response to individual-specific odours through associative learning and memory, allowing female sexual attraction to be inherent but selective towards particular males. This 'darcin effect' offers a new system to investigate the neural basis of individual-specific memories in the brain and give new insights into the regulation of behaviour in complex social mammals.See associated Commentary http://www.biomedcentral.com/1741-7007/8/71.

Molecular heterogeneity in major urinary proteins of Mus musculus subspecies: potential candidates involved in speciation.

When hybridisation carries a cost, natural selection is predicted to favour evolution of traits that allow assortative mating (reinforcement). Incipient speciation between the two European house mouse subspecies, Mus musculus domesticus and M.m.musculus, sharing a hybrid zone, provides an opportunity to understand evolution of assortative mating at a molecular level. Mouse urine odours allow subspecific mate discrimination, with assortative preferences evident in the hybrid zone but not in allopatry. Here we assess the potential of MUPs (major urinary proteins) as candidates for signal divergence by comparing MUP expression in urine samples from the Danish hybrid zone border (contact) and from allopatric populations. Mass spectrometric characterisation identified novel MUPs in both subspecies involving mostly new combinations of amino acid changes previously observed in M.m.domesticus. The subspecies expressed distinct MUP signatures, with most MUPs expressed by only one subspecies. Expression of at least eight MUPs showed significant subspecies divergence both in allopatry and contact zone. Another seven MUPs showed divergence in expression between the subspecies only in the contact zone, consistent with divergence by reinforcement. These proteins are candidates for the semiochemical barrier to hybridisation, providing an opportunity to characterise the nature and evolution of a putative species recognition signal.

The Genetic Basis of Kin Recognition in a Cooperatively Breeding Mammal.

Cooperation between relatives yields important fitness benefits, but genetic loci that allow recognition of unfamiliar kin have proven elusive. Sharing of kinship markers must correlate strongly with genome-wide similarity, creating a special challenge to identify specific loci used independently of other shared loci. Two highly polymorphic gene complexes, detected through scent, have been implicated in vertebrates: the major histocompatibility complex (MHC), which could be vertebrate wide, and the major urinary protein (MUP) cluster, which is species specific. Here we use a new approach to independently manipulate sharing of putative genetic kin recognition markers, with the animal itself or known family members, while genome-wide relatedness is controlled. This was applied to wild-stock outbred female house mice, which nest socially and often rear offspring cooperatively with preferred nest partners. Females preferred to nest with sisters, regardless of prior familiarity, confirming the use of phenotype matching. Among unfamiliar relatives, females strongly preferred nest partners that shared their own MUP genotype, though not those with only a partial (single-haplotype) MUP match to themselves or known family. In the absence of MUP sharing, females preferred related partners that shared multiple loci across the genome to unrelated females. However, MHC sharing was not used, even when MHC type completely matched their own or that of known relatives. Our study provides empirical evidence that highly polymorphic species-specific kinship markers can evolve where reliable recognition of close relatives is an advantage. This highlights the potential for identifying other genetic kinship markers in cooperative species and calls for better evidence that MHC can play this role.

Cloning and expression of two secretory acetylcholinesterases from the bovine lungworm, Dictyocaulus viviparus.

We describe the molecular cloning, expression and biochemical characterisation of recombinant forms of two secreted acetylcholinesterases from adult Dictyocaulus viviparus. The two variants (designated Dv-ACE-1 and Dv-ACE-2) were 613 and 615 amino acids long and showed 94.7% identity to one another. The highest level of identity to other cholinesterases was with ACE-2 of Caenorhabditis elegans. Dv-ACE-1 and Dv-ACE-2 showed 48.0 and 47.7% identity to C. elegans ACE-2 over 577 amino acids, respectively. The primary structure of both enzymes showed conservation of the catalytic triad and of a tryptophan residue known to be critical for the choline-binding site, but differed in the number of potential glycosylation sites and at one amino acid in the peripheral anionic site. Southern blotting and PCR experiments indicated that the genes encoding these enzymes are distinct. When expressed in Pichia pastoris, the enzymes were active, but differed subtly in their biochemical characteristics. Both enzymes exhibited a preference for acetylcholine as substrate, but differed in the extent of excess substrate inhibition and in their optimal pH for activity. The lack of an obvious carboxy-terminal membrane anchor and the presence of an insertion at the molecular surface were other features which, thus far, appear to be characteristic of parasite secreted acetylcholinesterases.

Pheromonal induction of spatial learning in mice.

Many mammals use scent marking for sexual and competitive advertisement, but little is known about the mechanism by which scents are used to locate mates and competitors. We show that darcin, an involatile protein sex pheromone in male mouse urine, can rapidly condition preference for its remembered location among females and competitor males so that animals prefer to spend time in the site even when scent is absent. Learned spatial preference is conditioned through contact with darcin in a single trial and remembered for approximately 14 days. This pheromone-induced learning allows animals to relocate sites of particular social relevance and provides proof that pheromones such as darcin can be highly potent stimuli for social learning.