Amanda J. Davidson

Pheromonal induction of spatial learning in mice.

Spots to Remember Scent marking is an essential component of communication for most mammals. Individuals remember the location of scent marks and regularly revisit marked sites, presumably to assess the condition and status of the animal doing the marking. It is known that individuals can follow odor or pheromone gradients to locate another individual, but relocating scent marks is a much more difficult task given the small amount of volatile compounds deposited, and their static nature. Roberts et al. (p. 1462) show that a nonvolatile component of male urine, the protein pheromone darcin, stimulates spatial preference and learning in mice. Female mice preferred locations where male urine (or synthesized darcin) had been found, and remembered these spatial locations for 2 weeks post-exposure. A male sex pheromone rapidly stimulates learning of place preference that is remembered for about 2 weeks. 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.

Immunisation of cattle with recombinant acetylcholinesterase from Dictyocaulus viviparus and with adult worm ES products

Dictyocaulus viviparus causes a serious lung disease of cattle. For over 30 years, a radiation-attenuated larval vaccine has been used with success; however, this vaccine has several disadvantages. A more stable vaccine against D. viviparus, capable of stimulating prolonged protective immunity, would be beneficial. Recent research has been directed at adult worm ES components that may be involved in parasite survival in the host. One component is the secreted enzyme, acetylcholinesterase (AChE), a target for circulating antibody in infected calves. Here, we describe a study where protection was investigated in calves immunised with either native adult ES products or a recombinant parasite AChE. These antigens were administered twice with Freunds incomplete adjuvant. Subsequently, all calves were challenged with 700 L3 and their worm burdens and immune responses compared with those in calves that received an anthelmintic-abbreviated infection and challenge control calves. Significant levels of protection were not obtained in the immunised groups but significant immunity was achieved in the calves that received the anthelmintic abbreviated infection. Antibody responses amongst the groups were different, with significantly higher IgG1 responses in the immune, infected group and in adult ES recipients. Significantly higher IgG2 responses were found in the latter group. Following challenge, the groups that received the abbreviated infection and the fusion protein produced specific antibody that bound the native enzyme. No differences were observed between groups in peripheral blood mononuclear cell responsiveness to either antigen. However, adult ES products appeared to have a mitogenic effect on these cells, whilst the fusion protein exhibited an inhibitory effect. These results suggest that in this form, AChE is not a potential vaccine candidate and that adult ES products, in contrast to previous experiments in guinea pigs, do not contain protective components.

Female attraction to male scent and associative learning: the house mouse as a mammalian model

Many territorial mammals invest heavily in competitive scent marks that advertise their location, identity and current social and physiological status. Here we review the behavioural and molecular components of scent marking in house mice, Mus musculus domesticus , that influence female attraction to males and discuss how pheromone-induced learning among females and differential scent investment among males both influence female attraction to specific scent owners. Although mouse urine scents contain numerous sex-specific and individual-specific components, female attraction to spend more time near urine from males depends on contact with an involatile protein pheromone, darcin. This is an atypical major urinary protein (MUP) expressed only by males. On contact, this pheromone acts as a highly potent stimulus for associative learning, such that females learn similar attraction to the individual males airborne odour associated with darcin; they also learn attraction to spatial cues where the pheromone was encountered. This targets female attraction to both the odour and location of individual male scent mark owners. However, the concentration and quality of airborne volatiles emitted from scent marks influence approach and contact with male scents. Under competitive pressure, males invest heavily in refreshment of scent marks at a high rate and deposit a high concentration of MUPs that bind urinary volatiles and extend the duration of volatile release. Females also gain information from airborne volatiles, including the social and infection status of the owner, which can alter their attraction to contact his scent. The ability of females to learn about individual males from their scent marks means that most decisions about preferred males are likely to be made before females are ready to mate. We are just starting to understand how different information in male scents is integrated in making these decisions.

The application of mass spectrometry to identify immunogenic components of excretory/secretory products from adult Dictyocaulus viviparus.

Proteomics has come to the forefront in the post-genomic era. The ability to compare and identify proteins expressed in a particular cell type under specific physiological or pathological states requires a range of technologies, including separation of complex protein or peptide mixtures, densitometry-based or isotope-coded methods for comparison of multiple proteomes, and mass spectrometric methods for identification of individual low abundance proteins. Although an emergent technology, thus far, proteomics has provided new perspectives on many problems in biomedical science. In parasitology, proteomics has been used to answer specific biological questions relating to survival and development, and also to identify candidates for vaccines. Here, we describe an ongoing research programme in which proteomics is being used to identify potential vaccine candidates for the bovine lungworm, Dictyocaulus viviparus. This work is focusing on antibody responses to the adult parasite excretory/secretory (ES) products, with selection of candidate antigens based on differential screening with serum from immune versus non-immune animals to simplify the proteome and the ensuing analytical challenges. Thus far, we have identified seven candidate proteins using this strategy. Of these, one protein showed significant identity to a previously cloned gene from D. viviparus, whilst the other six proteins have shown no significant identities. Isolation of further peptide sequences is now warranted to facilitate cloning of the genes encoding these antigens.

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.

Characterisation of urinary WFDC12 in small nocturnal basal primates, mouse lemurs (Microcebus spp.)

Mouse lemurs are basal primates that rely on chemo- and acoustic signalling for social interactions in their dispersed social systems. We examined the urinary protein content of two mouse lemurs species, within and outside the breeding season, to assess candidates used in species discrimination, reproductive or competitive communication. Urine from Microcebus murinus and Microcebus lehilahytsara contain a predominant 10 kDa protein, expressed in both species by some, but not all, males during the breeding season, but at very low levels by females. Mass spectrometry of the intact proteins confirmed the protein mass and revealed a 30 Da mass difference between proteins from the two species. Tandem mass spectrometry after digestion with three proteases and sequencing de novo defined the complete protein sequence and located an Ala/Thr difference between the two species that explained the 30 Da mass difference. The protein (mature form: 87 amino acids) is an atypical member of the whey acidic protein family (WFDC12). Seasonal excretion of this protein, species difference and male-specific expression during the breeding season suggest that it may have a function in intra- and/or intersexual chemical signalling in the context of reproduction, and could be a cue for sexual selection and species recognition.

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

BackgroundReliable 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.ResultsLearned 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.ConclusionsDespite 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.

The Application of Proteomics to the Discovery and Quantification of Proteins in Scent Signals

The increasing awareness of the occurrence and role of proteins in scent secretions has created a requirement for analytical approaches that can define the protein complexity of a scent secretion and identify and quantify those proteins. In this brief review, we summarise current approaches to the characterisation of proteins in scent secretion. By applying analytical methodologies of progressively increasing complexity, it is possible for behavioural labs to complete analytical surveys of scent complexity and protein component prior to the move to more specialised methods based on protein and peptide mass spectrometry. Whilst protein mass spectrometry is most easily delivered when the cognate gene or mRNA sequence is known, it is possible to recover most or all of the protein sequence by mass spectrometry, improving the chances of a cross-species identification. Finally, once the identity of the protein(s) is known, new analytical approaches permit high-quality quantification of the proteins, an essential pre-requisite to assessment of the regulation of the protein in scent secretions.