Michael D. Thom

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identity signature ([13, 14] and A.L. Sherborne, M.D.T., S. Paterson, F.J., W.E.R.O., P. Stockley, R.J.B., and J.L.H., unpublished data).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.

The Genetic Basis of Inbreeding Avoidance in House Mice

Summary Animals might be able to use highly polymorphic genetic markers to recognize very close relatives and avoid inbreeding [1, 2]. The major histocompatibility complex (MHC) is thought to provide such a marker [1, 3–6] because it influences individual scent in a broad range of vertebrates [6–10]. However, direct evidence is very limited [1, 6, 10, 11]. In house mice (Mus musculus domesticus), the major urinary protein (MUP) gene cluster provides another highly polymorphic scent signal of genetic identity [8, 12–15] that could underlie kin recognition. We demonstrate that wild mice breeding freely in seminatural enclosures show no avoidance of mates with the same MHC genotype when genome-wide similarity is controlled. Instead, inbreeding avoidance is fully explained by a strong deficit in successful matings between mice sharing both MUP haplotypes. Single haplotype sharing is not a good guide to the identification of full sibs, and there was no evidence of behavioral imprinting on maternal MHC or MUP haplotypes. This study, the first to examine wild animals with normal variation in MHC, MUP, and genetic background, demonstrates that mice use self-referent matching of a species-specific [16, 17] polymorphic signal to avoid inbreeding. Recognition of close kin as unsuitable mates might be more variable across species than a generic vertebrate-wide ability to avoid inbreeding based on MHC.

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.

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 owners 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.

Baculum morphology predicts reproductive success of male house mice under sexual selection

BackgroundDiversity in penile morphology is characterised by extraordinary variation in the size and shape of the baculum (penis bone) found in many mammals. Although functionally enigmatic, diversity in baculum form is hypothesised to result from sexual selection. According to this hypothesis, the baculum should influence the outcome of reproductive competition among males within promiscuous mating systems. However, a test of this key prediction is currently lacking.ResultsHere we show that baculum size explains significant variation in the reproductive success of male house mice under competitive conditions. After controlling for body size and other reproductive traits, the width (but not length) of the house mouse baculum predicts both the mean number of offspring sired per litter and total number of offspring sired.ConclusionsBy providing the first evidence linking baculum morphology to male reproductive success, our results support the hypothesis that evolutionary diversity in baculum form is driven by sexual selection.

Protein turnover: Measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non‐invasively. Time‐series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.

The Effect of Familiarity on Mate Choice

The ability to recognize familiar conspecifics appears to be widespread among vertebrates and influences a variety of behavioural interactions including mate selection. Female choice of males has been shown to vary according to male familiarity, but interestingly in some species this favours familiar males, while in others unfamiliar males are preferred. Preference for unfamiliar partners might result from the attempt to minimise inbreeding costs by avoiding mating with individuals encountered during development, or with those sharing relatedness cues. Conspecifics that are familiar through prior mating experience might be avoided in species that benefit from a promiscuous mating system, again resulting in preference for unfamiliar mates. Conversely, familiar mates may be favoured in monogamous species where formation of a pair bond is important for parental investment, and when familiarity provides an opportunity for females to assess the quality and compatibility of potential mates. Thus different types of familiarity may have differing effects on mate choice, with the direction of preference being determined by other aspects of life history, such as the likelihood of inbreeding, the importance of polyandry, and the role of social dominance and territoriality in reproductive success.

Scent, Mate Choice and Genetic Heterozygosity

Females of many species choose to mate with relatively unrelated males in order to ensure outbred, heterozygous offspring. There is some evidence to suggest that the MHC is involved in mate choice decisions, either because MHC heterozygous offspring are more resistant to disease, or because the highly detectable odours associated with this region allow it to act as a marker of general inbreeding. To determine which role the MHC plays it is necessary to disentangle this region from the genetic background, a requirement which has generally proven difficult to achieve. We argue that the emphasis on MHC’s role in mate choice has resulted in other potential markers of inbreeding being neglected, and discuss the evidence for MHC disassortative mating, the interaction with genetic background, and a possible role for alternative markers of inbreeding.

The “scents” of ownership

Scent marks need to encode a reliable signal of ownership to inform about the specific owner. Although tests of discrimination have told us much about the abilities of animals to detect differences in conspecific scents deriving from a wide range of sources, we know little about the components involved in scent ownership recognition or individual recognition when animals meet because this requires functional tests of individual recognition. Ownership signals in scent marks need to be stable and persistent, ideally genetically determined and sufficiently polymorphic. Recent work from our laboratory, using functional tests of scent mark recognition, suggest that the pattern of polymorphic MUPs in the urinary scent marks of male house mice provides an ownership signal. The ownership signal is involatile, requiring investigatory contact with the scent source, and involves either involatile complexes between MUPs and their bound odorants or the MUPs themselves, probably detected through the vomeronasal system. However, mice also detect non-MUP related differences in urinary volatiles. We propose a model of learnt association between involatile and volatile components that would allow mice to recognize previously encountered volatile profiles from familiar individuals or animals of the same sex without requiring close contact investigation. Investigation of fresh scent marks deposited around the environment would allow animals to update the proposed association between an individual’s stable involatile profile with any changes in its volatile profile. Further research is required to test this model and to establish its generality in other mammalian species.

The role of the major histocompatibility complex in scent communication

There is incontrovertible evidence that the MHC region is associated with type-specific odours in a number of species. Existing experimental paradigms have demonstrated that animals can discriminate between classes of MHC-associated odour with great acuity. Furthermore, experiments on mate choice and kin selection provide convincing evidence of a functional role for these odours. At the moment, evidence for genuine individual recognition or discrimination, meaning the “learned discrimination among conspecific individuals” (Halpin, 1986; p. 44), is lacking. This is partly because the four main experimental paradigms have not been used explicitly to address the question of individual recognition, but rather to test whether broader subgroups of MHC-associated odours exist and are distinguishable. These types of experiment can certainly be adapted to test individual recognition. For example Gheusi et al., (1997) found evidence for individual discrimination by rats using operant training, although this study did not examine MHC-associated odours specifically. The experimental paradigms described here have been vital in narrowing down the search for potential sources of individual odours, and testing odour discrimination abilities. Ultimately however, they can only allow an assessment of olfactory acuity, rather than whether this acuity is put to any use. In order to demonstrate that MHC-associated odours have an adaptive role in individual recognition, functional tests in a realistic behavioural context are required. Such functional tests exist (e.g. see Rich and Hurst, 1999; Hurst et al., 2001; Lai and Johnston, 2002) and now need to be applied to the unresolved issue of MHC-associated odours and individual recognition.