Animal Behaviour 97 (2014) 313e321

Contents lists available at ScienceDirect

Animal Behaviour
journal homepage: www.elsevier.com/locate/anbehav

Special Issue: Biochemistry & Animal Communication

Female attraction to male scent and associative learning: the house

mouse as a mammalian model
Sarah A. Roberts a, *, Amanda J. Davidson a, Robert J. Beynon b, Jane L. Hurst a
a
Mammalian Behaviour & Evolution Group, Institute of Integrative Biology, University of Liverpool, Neston, U.K.
b
Proteomics & Functional Genomics Group, Institute of Integrative Biology, University of Liverpool, Liverpool, U.K.

a r t i c l e i n f o
Many territorial mammals invest heavily in competitive scent marks that advertise their location,
Article history: identity and current social and physiological status. Here we review the behavioural and molecular
Received 31 March 2014 components of scent marking in house mice, Mus musculus domesticus, that influence female attraction
Initial acceptance 2 May 2014 to males and discuss how pheromone-induced learning among females and differential scent investment
Final acceptance 22 July 2014 among males both influence female attraction to specific scent owners. Although mouse urine scents
Available online 11 October 2014 contain numerous sex-specific and individual-specific components, female attraction to spend more time
MS. number: 14-00274R 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
Keywords: highly potent stimulus for associative learning, such that females learn similar attraction to the indi-
associative learning
vidual male's airborne odour associated with darcin; they also learn attraction to spatial cues where the
darcin
pheromone was encountered. This targets female attraction to both the odour and location of individual
female choice
house mouse
male scent mark owners. However, the concentration and quality of airborne volatiles emitted from
male investment scent marks influence approach and contact with male scents. Under competitive pressure, males invest
pheromone heavily in refreshment of scent marks at a high rate and deposit a high concentration of MUPs that bind
scent marking urinary volatiles and extend the duration of volatile release. Females also gain information from airborne
sexual attraction 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.
© 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Scent communication is widely used for sexual communication
and attraction across the animal kingdom. Some of the most well-
known examples include the simple species- and sex-specific
mating attractant pheromones emitted by moths to attract the
opposite sex, often over large distances (e.g. Butenandt, Beckmann,
Stamm, & Hecker, 1959; Howard & Blomquist, 2005; Karlson &
Butenandt, 1959; Witzgall, Kirsch, & Cork, 2010). However, chem-
ical signals play much wider roles in mate assessment and selection
that underpin sexual attraction (e.g. see Johansson & Jones, 2007;
Wyatt, 2014). Among terrestrial mammals scent marks deposited
around an animal's home area or territory are widely used to
advertise an individual's identity, location and current status to
other conspecifics in the locality (Gosling & Roberts, 2001; Johnson,
1973). These scents are highly complex, typically consisting of many
* Correspondence: S. A. Roberts, Mammalian Behaviour & Evolution Group,
Institute of Integrative Biology, University of Liverpool, Leahurst Campus, Neston
CH64 7TE, U.K.
E-mail address: sacheet@liv.ac.uk (S. A. Roberts).
hundreds of components that encode information about the ani-
mal's species, sex and individual identity as well as a broad range of
information about the owner's current reproductive status, social
status and health, all things that might influence their attractive-
ness to potential mates. This provides other animals using the area
or visiting a scent-marked site with ample opportunity to learn
information about individual scent owners, their spatial locations
and defended territories that may be used in current and future
mate choice decisions (Drea, Vignieri, Kim, Weldele, & Glickman,
2002; Hurst & Beynon, 2004; 2013; Vogt, Zimmerman, Ko €lliker &
Breitenmoser, 2014).
The best-studied model of mammalian communication is the
house mouse, Mus musculus domesticus, for which studies have
been able to integrate behavioural, molecular and neurophysio-
logical approaches under strictly controlled laboratory and semi-
natural conditions to understand the functions and mechanisms of
scent signalling. Although house mouse social structure can vary
across different habitats, given a choice most house mice live in
agricultural or human-built environments where food resources

http://dx.doi.org/10.1016/j.anbehav.2014.08.010
0003-3472/© 2014 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
314 S. A. Roberts et al. / Animal Behaviour 97 (2014) 313e321
are concentrated (Barnard, Hurst, & Aldhous, 1991; Berry, 1981;
Bronson, 1979). Adult males typically defend small territories,
which may cover only a few square metres in high-density pop-
ulations. Successful territory owners ensure that their own scent
marks predominate throughout their defended area by continually
and deliberately depositing their urine in many small streaks and
spots as they move around the territory. Discovery of competing
scents from other males provokes not only aggression towards the
owners but also a rapid and elevated countermarking response,
resulting in a particularly high rate of scent marking at borders
between neighbouring male territories (Hurst & Beynon, 2004).
Adult females typically range over several male territories, even
though they may choose to nest in the territory of one particular
resident male according to the quality and protection offered by
available nest sites (Rich & Hurst, 1998; Wolff, 1985). Females mate
with the owners of these scent-marked territories, although they
frequently mate with more than one male, visiting selected males
when ready to mate (there is little opportunity for male coercion
under free-ranging conditions, with males mating only within their
own territories). Here, we review the mechanisms that underlie
female sexual attraction to male scents in this well-studied system
and discuss the implications of an important new mechanism of
pheromone-induced associative learning that we recently discov-
ered for targeting female sexual attraction to specific individual
males of high quality.
MECHANISM OF SCENT ATTRACTION
Investigation and Processing of Scents
Mammalian scent marks are multicomponent signals that
contain both volatile components, which can become airborne and
are gradually lost from the scent source, and nonvolatile (or much
less volatile) components that can often persist in the environment
for an extended period of time. On detecting an airborne scent that
is unfamiliar or has not been encountered for some time, animals
typically approach the scent source to investigate further at close
contact, spending longer sniffing to process information when the
scent is more unfamiliar and distinct from scents recently
encountered (Todrank & Heth, 2003). This general investigatory
response is not sex specific though, reflecting a general motivation
to gain further information about whether scent cues come from
the same or opposite sex. Airborne volatiles are detected through
the main olfactory system as air is taken into the nasal cavity during
normal breathing and active sniffing. However, most terrestrial
vertebrates (except catarrhine primates and birds) also have an
accessory olfactory system that is activated only on close contact
with scents. Activation of a vascular pump (or active flehmen
response in species such as ungulates or felids) is required to
deliver molecules, in solution, from the scent source to the vom-
eronasal organ which is sited in a blind-ended mucus-filled capsule
at the base of the nasal cavity (Breer, Fleischer, & Strotmann, 2006;
Halpern & Martinez-Marcos, 2003). While odorant receptors (ORs)
in the main olfactory epithelium are typically broadly tuned to
detect a wide range of social and nonsocial odours alongside more
specialized receptors sensitive to volatile amines (TAARs, Liberles &
Buck, 2006), vomeronasal receptors respond more specifically to
particular lipophilic low molecular weight odorants (V1Rs),
nonvolatile peptides and proteins (V2Rs) and N-formyl peptides
that are produced largely by bacteria (FPRs) (Dulac & Axel, 1995;
Herrada & Dulac, 1997; Matsunami & Buck, 1997; Riviere, Challet,
Fleggue, Spehr & Rodriguez, 2009; Ryba & Tirindelli, 1997). Dur-
ing close contact investigation, scents may be processed in parallel
through the main and accessory olfactory systems, both of which
are necessary to fully process the entire scent cue (Restrepo,
Arellano, Oliva, Schaefer, & Lin, 2004; Spehr et al., 2006), with in-
puts from the two systems converging at the level of the amygdala
(Brennan & Kendrick, 2006). However, the main olfactory system is
likely to be particularly important for the detection of airborne
scents at a distance from the source and for recognition when
scents require further close contact investigation, while the
accessory olfactory system appears to be particularly important for
detection of nonvolatile components held within scent marks or on
an animal's body (Luo, Fee, & Katz, 2003); these components
include proteins and peptides but also low molecular weight
ligands that are bound to involatile molecules or have not yet
evaporated from the scent source.
Attraction to Male Scents among Female House Mice
When female house mice encounter unfamiliar urinary scents
from adult male or female conspecifics, they are stimulated to
approach and investigate both. However, following brief close
contact investigation, females reliably prefer to spend more time
near scents from intact adult males than near those from females
or castrated males. This attraction to male scents is shown by both
sexually experienced and naïve females, and by laboratory mice
and wild-stock house mice, even when naïve females have never
previously encountered scent from an adult male (Moncho-Bogani,
Lanuza, Hernandez, Novejarque, & Martinez-Garcia, 2002; Ramm,
Cheetham, & Hurst, 2008; Roberts et al., 2010). Indeed, wild-stock
female house mice are attracted to spend time near male odours
even when these are placed in open areas where wild mice nor-
mally choose to spend very little time (Roberts et al., 2010).
However, if direct contact with scent is prevented, females fail to
spend any more time near airborne volatiles from male urine
versus female urine (Moncho-Bogani et al., 2002), whether or not
females are naïve to male odours or sexually experienced (Ramm
et al., 2008). This is somewhat surprising given that there are a
large number of differences in volatiles that emanate from male
and female mouse urine. Simple discrimination tests show that
females readily discriminate these urinary volatile differences (e.g.
Martel & Baum, 2009). None the less, normal female mice fail to
show any inherent attraction to spend time near androgen-
dependent volatiles beyond their normal investigation of an
unfamiliar odour. This suggests that females detect an androgen-
dependent signal on contact that is essential to stimulate attrac-
tion to spend more time near a male's scent, most likely perceived
through the accessory olfactory system. Consistent with this,
lesion of the accessory olfactory bulbs eliminates female attraction
on contact with male soiled bedding (Martinez-Ricos, Agustin-
Pavon, Lanuza & Martinez-Garcia, 2008). It should be noted,
though, that studies using laboratory mice in which hormonal
levels of female subjects have been artificially manipulated
through ovariectomy and chronic treatment with oestradiol im-
plants have found differential attraction to urinary volatiles from
intact males compared with either females or castrated males,
even without direct contact with male urine (reviewed by Baum,
2012). Currently, the relevance of this to mouse behaviour under
natural conditions is difficult to interpret, but variation in female
sensitivity to sex-specific cues during normal hormonal cycling
deserves further research. None the less, adult females are
attracted to spend time near male urine not only when in oestrus
and ready to mate but also at random stages with respect to the
oestrous cycle (Moncho-Bogani et al., 2002) and continue to be
attracted even through pregnancy (Fig. 1); however, prepubertal
females avoid unfamiliar adult male urine scents (Drickamer, 1989;
Lanuza et al., 2014) while 2e3-week-old juveniles secrete a pep-
tide, ESP22, in tear fluids that inhibits adult male mating behaviour
(Ferrero et al., 2013).
 S. A. Roberts et al. / Animal Behaviour 97 (2014) 313e321 315

P = 0.001 P = 0.003
60

M F

Time near (s)

40

20

M F M F

0
Cycling Pregnant
females females

Figure 1. Time spent by females near unfamiliar male (M) or female (F) urine (mean ± SEM). Female wild-stock house mice were given full scent contact with 10 ml urine samples
from wild-stock male house mice versus control female urine (here from BALB/c strain females although the same response is seen using wild-stock female urine, see Ramm et al.,
2008 or Roberts et al., 2010). Urine samples were streaked on glass microfibre filters stuck to the underside of the arena lid (for full details of the test procedure, see Ramm et al.,
2008; Roberts et al., 2010). Matched-pair t tests assessed greater attraction to spend more total time near to male versus female scent among cycling females (t13 ¼ 3.85, P ¼ 0.001)
or pregnant females 2e5 days prior to giving birth (t12 ¼ 3.40, P ¼ 0.003), with no difference in the strength of preference for male scent between cycling and pregnant females
(independent samples t test: t25 ¼ 0.16, P ¼ 0.87).

Urine from healthy mice contains many involatile proteins and
peptides at very low concentration that may be detected on contact
with the scent; however, mice make substantial investment in a set
of small communicatory proteins called major urinary proteins
(MUPs), expressed in the liver and deliberately excreted at high
concentration in urine (>99% of urinary protein excreted by male
mice, Humphries, Robertson, Beynon, & Hurst, 1999). MUPs are a
group of 18e20 kDa proteins that are members of the lipocalin
superfamily; these proteins have a characteristic b-barrel structure
surrounding a central cavity that is capable of binding low molec-
ular weight lipophilic molecules (see Beynon & Hurst, 2004). MUPs
are produced by both sexes but adult male urine typically contains
an approximately three to four times higher concentration than
female urine under laboratory housing conditions (Cheetham,
Smith, Armstrong, Beynon, & Hurst, 2009); under more natural
social conditions in seminatural populations, the investment
among wild-stock house mice increases greatly, with males
continuing to produce approximately twice as much as females but
with considerable overlap in investment between the sexes (Hurst
& Beynon, 2013). MUPs bind a number of ligands, including several
androgen-specific volatiles that have known reproductive priming
effects on females (Bacchini, Gaetani, & Cavaggioni, 1992;
Mucignat-Caretta, Caretta, & Cavaggioni, 1995; Novotny, Ma,
Wiesler, & Zidek, 1999) as well as a much wider range of volatile
organic urinary metabolites (Kwak et al., 2012), which appear to
include the volatile odours associated with MHC type (Singer,
Tsuchiya, Wellington, Beauchamp, & Yamazaki, 1993). The bind-
ing of the MUPs to the volatiles slows the release of bound volatiles
from deposited scent marks (Armstrong, Robertson, Cheetham,
Hurst, & Beynon, 2005; Hurst, Robertson, Tolladay, & Beynon,
1998; Robertson, Marie, Veggerby, Hurst, & Beynon, 2001), which
both increases the duration over which airborne volatiles are
emitted and can be detected at a distance and holds volatiles in
scents for longer where they may be detected on contact through
the accessory olfactory system.
To identify the components of male mouse urine that stimulate
attraction on contact, we fractionated urine into high molecular
weight components (containing proteins and low molecular weight
bound ligands) and low molecular weight components not bound
to MUPs (Roberts et al. 2010). This indicated that all activity was in
the high molecular weight fraction, which was as effective as intact
urine. Further separation of the high molecular weight fraction by
anion exchange chromatography revealed that attraction was eli-
cited by a single subfraction. We showed that the active component
in this subfraction was a single atypical urinary MUP (MGI:3651981,
Mup20) expressed only by males and encoded by a gene in the
peripheral region of the Mup gene cluster where there is consid-
erable divergence between Mup genes (Mudge et al., 2008;
who referred to this as Mup gene 17 using a different numbering
scheme from MGI). This MUP is responsible for binding most of the
male-specific urinary volatile pheromone 2-sec-butyl-4,5-dihy-
drothiazole (Armstrong et al., 2005), which is known to stimulate
receptors in the vomeronasal organ with high sensitivity (Leinders-
Zufall et al., 2000). However, there was no reduction in female
attraction response when urine was aged to severely deplete any
volatile components, whereas a recombinant protein tested
without any natural mouse ligands stimulated a response very
similar to normal male mouse urine. Thus, the protein itself rather
than any bound ligands stimulates the reliable inherent attraction
of female mice to spend time near male urine (Roberts et al., 2010).
In recognition of its key role in female sexual attraction to spend
time near male scent, we named this MUP darcin after Jane Austen's
romantic hero Mr Darcy in Pride and Prejudice (Roberts et al., 2010).
All wild-stock adult male house mice appear to excrete darcin in
their urine, but it is notable that it is missing or present at only trace
levels in male urine from many common strains of inbred labora-
tory mice (Cheetham et al., 2009). Perhaps unsurprisingly given its
importance for female attraction, darcin has recently been shown
also to stimulate competitive aggression between males (Kaur et al.,
2014); thus there is likely to have been strong selection against
expression of this pheromone for survival among laboratory males
confined together in small cages where attractiveness to females is
not a requirement. Wild-stock females fail to show their normal
reliable attraction to urine from a laboratory strain male that does
not express darcin, but addition of recombinant darcin at a normal
male level stimulates a normal attraction response (Roberts et al.,
2010). Thus darcin is effective whether encountered in the
context of other male urinary components or on its own. However,
although this pheromone is effective on its own within the confines
of a small test arena, under natural conditions volatile urinary
components are likely to be essential to alert females to the pres-
ence of this involatile pheromone. It is also information in urinary
volatiles that will stimulate females to contact the scent source and
deliver the pheromone to receptors in the vomeronasal organ.
316 S. A. Roberts et al. / Animal Behaviour 97 (2014) 313e321

Pheromone-induced Learned Attraction to Specific Males
Although female mice show reliable attraction to spend time
near scent marks from unfamiliar males if these contain darcin,
under natural conditions females settle within the scent-marked
territories of one or a small number of neighbouring males. They
will encounter scents from these males and those in nearby terri-
tories repeatedly, particularly as resident owners scent-mark their
territories liberally (Garratt et al., 2012; Hurst & Beynon, 2004; Zala,
Potts, & Penn, 2004). This provides females with ample opportunity
to learn about potential mates in their local area. Importantly, on
contact with the male sex pheromone darcin, females very rapidly
learn a similar attraction to airborne scents associated with this
pheromone (Moncho-Bogani et al., 2002; Roberts et al., 2010).
However, females do not learn a generalized attraction to
androgen-dependent urinary volatiles that represent ‘male’ even
after they have gained substantial experience of odour cues from
many different males. Instead, their learned attraction is to the
specific airborne scent signature that was detected during simul-
taneous contact with darcin (Ramm et al., 2008; Roberts et al.,
2010; Fig. 2a, b). If females are unable to contact the scent source
to detect darcin, they fail to learn any attraction to the male's
airborne signature regardless of its familiarity (Ramm et al., 2008).
Similarly, females fail to learn any attraction to airborne volatiles
when there is no darcin or only trace amounts in urine; however, if
recombinant darcin is added artificially, this now induces a strong
learned attraction to an airborne signature that previously was not
attractive whereas addition of other recombinant MUPs has no
effect (Roberts et al., 2010; Fig. 2b). Thus, contact with darcin in-
duces females to immediately learn an attraction to the male's
airborne volatile signature that can then be detected at a distance
from the scent source, drawing females to spend time near that
particular male's scent regardless of further contact. If females
contact urine scent marks from two equivalent males, they learn an
attraction to both individual airborne odour signatures (relative to
unfamiliar male airborne odour), indicating that females are able to
remember attraction to multiple individuals (Cheetham, 2006). The
outcome of this pheromone-induced learning is to target female
attraction to odours from specific males (including their scent-
marked territories) following close contact investigation of their
scents. This learned attraction to individual-specific airborne vol-
atile signatures is remembered for a surprisingly long time: even
after only brief contact with a male's scent, females are attracted to
the remembered airborne signature for at least 4 weeks (Fig. 3).
However, it is worth noting that similar contact with darcin in urine
from males of an inbred laboratory strain (where all individuals are
genetically identical) results in attraction to an airborne signature
that female wild mice generalize to other males of the same strain,
as these all share very similar ‘individual’ signatures. In normal
outbred populations, though, each male has an individually
distinctive signature (Cheetham et al., 2007; Hurst, Beynon, et al.,
2001).
In addition to inducing attraction to individual scent signatures,
pheromone-induced learning is also important in allowing animals
to remember locations where they have previously encountered
scents. Although we usually assume that animals detect scent cues
from a distance through molecules emanating from the source and
orient towards these, learning where scents were previously
encountered is likely to be another important mechanism to relo-
cate scents without any need to detect airborne scent molecules.
Through associative learning, darcin induces not only attraction to
individual male odours but also attraction to spatial cues associated
with the pheromone (Roberts, Davidson, McLean, Beynon, & Hurst,
2012). Repeated contact with male soiled bedding in the same
location (Martinez-Ricos, Agustin-Pavon, Lanuza, & Martinez-
Garcia, 2007), or more specifically with male urinary scent marks
(Roberts et al., 2012), induces a conditioned preference for that
location in adult females, suggesting that male scent is rewarding
(Martinez-Garcia et al., 2009). Repeated scent exposure is not
required to induce this remembered preference though; females
given a single learning trial with either male urine containing
darcin or darcin alone show as strong a remembered location
preference as those given two or three learning sessions with scent
in the same site. This remembered preference for a site is as strong

(a) (b) P = 0.014

30 30
Time under stimulus (s)

P = 0.008
**
P = 0.006 P = 0.013
20 20 NS NS ***
*
*** NS NS
10 NS 10

M F M F M F M F M F M F M F M F M F
0 0
Same Different Same Different Same Different Lab male urine without darcin
Naïve females Experienced females Natural darcin +Buffer +rdarcin +rMUP7

Figure 2. Female learned attraction to the individual-specific volatile signature of male house mice stimulated by darcin. Wild-stock female house mice were given prior contact
with urine test stimuli in a different arena before being tested with airborne odours from male versus control female urine that could not be contacted during the test (see Fig. 3 for
illustration of test set-up). (a) Females spent more time near male airborne urinary odour when this was from the same individual wild-stock male donor as urine contacted prior to
the test but showed no attraction when airborne odour was from a different but equivalent individual. Females showed the same response whether previously naïve to adult males
and their odours or were socially experienced adults that had been captured from seminatural enclosure populations (data redrawn from Ramm et al., 2008, Figure 2 and 3). (b)
Females learned attraction to the individual-specific airborne odours of normal wild-stock males that expressed natural levels of darcin after contact with their urine but failed to
learn attraction to associated airborne urinary odour after contact with laboratory male urine that did not contain darcin unless recombinant darcin was added artificially. Addition
of other recombinant MUPs (here the male-specific rMUP7) failed to stimulate such learned attraction (data redrawn from Roberts et al., 2010, Figure 10). Data are means ± SEM.
Asterisks show matched-pair t test comparisons between time near male and female airborne odour within each test (*P < 0.05; **P < 0.01; ***P < 0.005). P values compare the bias
in response between tests (a: matched-pair t tests; b: independent-samples t tests). Control female urine was from BALB/c laboratory mice.
 S. A. Roberts et al. / Animal Behaviour 97 (2014) 313e321 317

0.4
Male airborne odour

Conditioned preference
Prior contact with scent
0.2 M F

M Test F
0

Female control –0.2

7 14 21 28
Retention (days)

Figure 3. Retention of female conditioned preference for male airborne urinary odour (mean ± SEM for each retention interval). Female (F) wild-stock house mice were given prior
contact with 10 ml urine samples from wild-stock male (M) house mice versus control urine from BALB/c laboratory females for 30 min in a neutral arena then returned to their
home cage or tested immediately. After differing periods of time (0, 1, 7, 14, 28 days, N ¼ 8e14 females per time point), females were given a choice between airborne odour from
the same male and female urine stimulus they had previously contacted (urine samples presented above the perforated arena lid where they could not be contacted, following the
methods and housing conditions detailed in Ramm et al., 2008; Roberts et al., 2010). Conditioned preference is shown as time spent near male odour as a proportion of total time
spent near male or female odour  0.5. Conditioned preference for the male odour did not differ significantly across the five retention intervals (ANOVA: F4,53 ¼ 2.20, P ¼ 0.081),
with a highly significant preference for male odour across all trials (t57 ¼ 9.54, P < 0.0001), although planned contrasts with response immediately after contact suggested a reduced
preference after 14 days (P ¼ 0.03). This was due to an elevated response to female odour after this time interval rather than a reduced response to male odour and thus might have
been a chance effect. Separate tests confirmed that the expected conditioned preference for airborne male odour was evident at each time point (immediately after contact:
t7 ¼ 5.92, P ¼ 0.0005; 1 day: t11 ¼ 3.97, P ¼ 0.001; 7 days: t13 ¼ 7.56, P < 0.0001; 14 days: t11 ¼ 3.99, P ¼ 0.001; 28 days: t11 ¼ 2.73, P ¼ 0.01; one-tailed tests).

as their initial preference when scent was detected there and can
last for at least 14 days, although extinction of this response occurs
rapidly if scent is no longer present when females return to the site
(Roberts et al., 2012). This suggests that females rapidly update
their memory of remembered spatial associations. Airborne urinary
volatiles from male scent marks fail to condition any preference for
scent location, even if females have already learned an attraction to
these volatiles through association with darcin (i.e. there is no
secondary conditioning, Roberts et al., 2012). Contact with the
darcin pheromone is thus crucial for learning a spatial preference
for male scent-marked sites.
This pheromone-induced learned attraction to spend time in the
specific scent-marked locations where they have encountered
darcin, and wherever females then perceive the airborne odour
signature that they learned on contact with a male's scent marks, is
likely to underpin female attraction to spend time within particular
male territories, as well as their attraction to the owners of those
territories when females are ready to mate. Notably, though, the
pheromone that induces this targeted learned attraction is identical
across all adult males. However, because it is involatile and
apparently perceived only when females actively deliver male scent
to the vomeronasal organ on nasal contact, exposure to this key
pheromone is under the female's own control. This means that
females may use other information in a male's scent to determine
whether or not they deliver scent likely to contain darcin to the
necessary receptor system. So far, the mechanism underlying such
female attraction to male scents has been worked out only for
house mice. None the less, similar pheromone-induced learning
mechanisms are likely to be important in other mammalian species
that advertise their location, identity and quality through scent
marks. Prior contact with a particular male's scent (with no other
prior interaction) has been shown in a range of species to strongly
increase preference for the individual owner of that scent over
equivalent males of similar high status and health but with unfa-
miliar scent (e.g. harvest mice, Micromys minutes: Brandt &
Macdonald, 2011; Roberts & Gosling, 2004; golden hamsters,
Mesocricetus auratus: Johnston, Munver, & Tung, 1995; meadow
voles, Microtus pennsylvanicus: Johnston, Sorokin, & Ferkin, 1997;
pygmy loris, Nycticebus pygmaeus: Fisher, Swaisgood, & Fitch-
Snyder, 2003). Such prior exposure may be important because it
allows females the opportunity to fully investigate and process all
information in the male's scent to assess his suitability as a po-
tential mate. Pheromone-induced social learning may be wide-
spread in this and other social contexts, but the considerable
complexity of mammalian scents coupled with the difficulties of
isolating and testing specific candidates while controlling prior
experience of subjects makes identification and understanding
these pheromonal mechanisms particularly challenging. Selection
on communication signals leads to rapid evolution and diversifi-
cation (e.g. Emes, Beatson, Ponting, & Goodstadt, 2004), further
increasing the difficulty of identifying signals in other species that
may play similar roles. Lipocalin proteins with similar structures to
darcin are present in a wide range of rodent scent sources (Beynon
et al., 2008; Stopka, Stopkov a, & Janotova, 2012), including the
aphrodisin protein in hamster vaginal secretions that has been
implicated as an aphrodisiac signal for males (Briand, Trotier, &
Pernollet, 2004). However, while other lipocalins provide initial
candidates for investigation in other species, it should be recog-
nized that even very similar MUPs in house mice do not have the
same pheromonal role as darcin and there are many other candi-
date molecules in complex mammalian scents. The first and only
other clear example of pheromone-induced learning reported to
date occurs in the very different social context of mothereoffspring
communication, where the volatile rabbit mammary pheromone
(2-methyl-but-2-enal) produced by lactating European rabbits,
Oryctolagus cuniculus, induces rapid learning of other associated
odorants to improve the efficiency of nipple location and grasping
during a short sensitive postnatal period (see Schaal, 2014; for a
detailed review).
MALE INVESTMENT AND FEMALE CHOOSINESS
Although darcin is a key pheromone that underpins the tar-
geting of female attraction to specific males, investment by males in
other associated components of their scent signals is likely to be
particularly important in determining female contact with this
pheromone under competitive conditions, and thus female learned
attraction to particular males. In seminatural populations, female
house mice mate almost exclusively with male territory owners
(e.g. Hurst, 1987; Meagher, Penn, & Potts, 2000; Potts, Manning, &
318 S. A. Roberts et al. / Animal Behaviour 97 (2014) 313e321
Wakeland, 1991; Wolff, 1985). Territory owners make a substantial
investment in scent marking, in terms of both their behaviour (time
and energy spent patrolling the territory, refreshing scents, coun-
termarking any competitor scents and attempting to exclude such
competitors) and their investment in scent components that are
added to urinary scent marks (Hurst & Beynon, 2004). In the
presence of a neighbouring competitor, breeding territory owners
substantially increase their rate of urinary scent marking compared
with males in isolated breeding pairs; the concentration of MUPs
excreted in their scent marks also doubles (Garratt et al., 2012). In
seminatural populations, where territory owners experience mul-
tiple competitors, the MUP investment of breeding adult males
increases approximately five-fold compared with those in isolated
breeding pairs (mean ± SEM concentration of urinary pro-
tein ¼ 71.9 ± 3.5 mg/mg creatinine to control for urine dilution,
data for 26 breeding males living in 250 m2 enclosures at a popu-
lation density of 0.64 adult mice/m2, sex ratio 1.2:1; subset of data
in Hurst & Beynon, 2013, Figure 9.2, but for known breeding males
only).
This elevated investment in MUPs under competitive conditions,
together with the increased rate of scent refreshment, influences
not only the amount of darcin deposited around a male's territory
but also the strength of volatile odours. Darcin itself is highly
resistant to degradation and remains in the environment for a
substantial period after deposition, but females are initially
attracted to contact darcin by airborne volatiles. After contact, fe-
males are also subsequently attracted to spend time near a male's
associated airborne volatile signature. However, these volatile sig-
nals are gradually lost from urinary scents as they age, such that
there is much higher emission from freshly deposited scents. When
presented with scents from two unfamiliar adult male territory
owners, females consistently prefer the owner of the freshest, most
recently deposited scent or countermark, as long as there is a clear
difference in the age of the scents (Rich & Hurst, 1999) and each
male has a distinctive individual identity signature encoded by
different MUP patterns that allows females to discriminate between
the owners (Cheetham et al., 2007). It is likely that this preference
for the owner of the fresher scent is because females preferentially
approach and contact scent marks that emit much stronger volatile
signals; thus, they learn an attraction to the airborne odour signa-
ture of the owner of the fresher (stronger) scent. However, in the
absence of competing fresh scent, females are still more attracted to
the owner of familiar 24 h aged scent marks than to a male whose
scent they have not met. This indicates that females are still able to
recognize a male's identity from 24 h aged scent despite the gradual
loss of volatile signatures (Cheetham, 2006).
Greater investment in MUPs increases the concentration of
bound volatile ligands in the scent and thus helps to extend the
duration over which scent marks continue to emit detectable levels
of volatiles. Investment in MUPs and associated volatile ligands
declines as adult males start to age; urinary protein levels start to
decline when wild-stock males (housed singly in the laboratory)
are >18 months old, declining to approximately one-third of the
amount excreted by younger adult males once males are aged
25e32 months (Garratt, Stockley, Armstrong, Beynon, & Hurst,
2011). This decline in urinary protein correlates strongly with the
decline in epididymal sperm counts as males start to senesce. The
abundance of urinary volatile ligands as adult males age mirrors the
three-fold decline in MUPs, consistent with the sequestration of
volatile ligands that bind to MUPs with a 1:1 stoichiometry. From a
functional perspective, the impact of this decline becomes most
apparent not when urine is freshly deposited but as scent marks
age. Although volatile abundance differs in fresh urine, the level of
volatiles released from scent marks freshly deposited by older
males is still relatively strong. Females given a choice between fresh
urine scents from an unfamiliar young adult versus a senesced
adult male approach and investigate both equally. However, in
most cases scent marks are not likely to be encountered very soon
after deposition. As scent marks age and the abundance of volatiles
declines, the amount of 2-sec-butyl-4,5-dihydrothiazole (an
androgen-dependent volatile that binds largely to darcin) in sen-
esced male urine after 4 h is equivalent to that in younger adult
male urine after 8e12 h. Given a choice between urine marks 12 h
after deposition, females are significantly less attracted to investi-
gate those from a senesced adult male where the emission of vol-
atiles is now extremely weak (Garratt et al., 2011). As yet, we have
limited understanding of the temporal dynamics of scent marks
(e.g. Cavaggioni, Mucignat-Caretta, & Redaelli, 2008; see also
Mucignat & Caretta, 2014), as molecular analyses usually focus only
on levels of signalling components in freshly collected scents. We
do not know, for example, whether individuals that refresh their
scents at a higher rate invest less in the molecular strength of those
signals or whether investment in all components of signalling
correlate positively with the quality of a male.
The amount of darcin expressed by individual males correlates
with the total concentration of other urinary MUPs, with both
showing a similar decline in output as males age (Garratt et al.,
2011). Little is yet known about how different aspects of male
quality might influence expression specifically of darcin, although
initial observations suggest that expression may not be particularly
sensitive to issues of current status such as infection (see Lanuza
et al., 2014). However, the proportion of total urinary MUP output
that is due to darcin varies across individual males, in part because
of genetically determined differences in individual MUP patterns
(e.g. see Figure 9.4 in Hurst & Beynon, 2013). Experiments that
manipulate the amount of darcin independently of other urinary
components including other MUPs will be needed to understand
how variation in investment in this specific urinary pheromone
influences male attractiveness to females.
Additional information in other components of a male's scent
about a male's potential quality as a mate may be used to modulate
female contact or response to the darcin pheromone. Male house
mice that are not able to defend their own territories (low quality or
young males) often attempt to live as subordinates within another
male's territory, where they are forced to reduce production of
androgen-dependent scents that otherwise induce aggression from
the resident territory owner (Hurst, Payne, et al., 2001). In addition
to largely suppressed scent-marking behaviour to avoid competi-
tion with the owner, the preputial gland development of subordi-
nate males is much reduced (Bronson & Marsden, 1973; Homady &
Brain, 1983) leading to much lower concentrations of two
androgen-dependent sesquiterpenes, E,E-a-farnesene and E-b-far-
nesene, added to urine (Novotny, Harvey, & Jemiolo, 1990). Another
androgen-dependent volatile constituent of preputial glands has
recently been identified, an unsaturated aliphatic alcohol Z5-14:OH,
which is also secreted in male urine (Yoshikawa, Nakagawa, Mori,
Watanabe, & Touhara, 2013). Although it is not yet known
whether this component is reduced in subordinate males, the much
smaller size of their preputial glands makes this likely. Each of these
preputial secretions stimulates female investigation of unfamiliar
adult male urine (Jemiolo, Xie, & Novotny, 1991; Yoshikawa et al.,
2013), while females show little investigatory interest in urine
from subordinate males, castrated males or preputialectomized
males without addition of synthetic analogues of these components
(Bronson & Caroom, 1971; Jones & Nowell, 1974; Ninomiya &
Kimura, 1988). By contrast, there appears to be little quantitative
difference in urinary protein output between dominant and sub-
ordinate males (Malone, Payne, Beynon, & Hurst, 2001; Payne,
2001), although further studies are required to establish whether
darcin production may decrease. However, females appear able to
 S. A. Roberts et al. / Animal Behaviour 97 (2014) 313e321
recognize the status of subordinate scent owners from airborne
volatiles without the need for close contact investigation, and show
no interest in investigating subordinate male scents. Thus, they are
unlikely to be exposed to the darcin pheromone in subordinate
male urine even if this is expressed.
Females also discriminate against urinary odours from males
subclinically infected with parasites or viruses, in many cases
showing active avoidance of odours from infected males (reviewed
by Beltran-Bech & Richard, 2014; Choleris, Clipperton-Allen, Phan,
Valsecchi, & Kavaliers, 2012; Kavaliers, Choleris, & Pfaff, 2005). As
yet, the urinary components used to identify infection are un-
known; it is unclear whether infection results in reduced expres-
sion of male sexual signals, detection of cues from the parasite
itself, detection of cues that reflect activation of the host immune
system or a combination of these. However, as subject females are
usually prevented from direct contact with male odours during
these tests, it appears that they are able to detect infection on the
basis of airborne volatiles alone (e.g. Ehman & Scott, 2002;
Kavaliers & Colwell, 1995a, 1995b; Kavaliers, Colwell, Braun, &
Choleris, 2003; Kavaliers, Fudge, Colwell, & Choleris, 2003). Thus,
females may detect and avoid urine scents from infected males
without delivering these scents to the vomeronasal organ, which
would avoid contact with darcin, but this needs to be examined
further (see also Lanuza et al., 2014). Females can also show
reduced attraction to scents from males that are less genetically
compatible to themselves, such as very close relatives (reviewed by
Barnard et al., 1991; D'Udine & Alleva 1983; Pusey & Wolf, 1996),
regardless of the male's investment in signals that are highly
attractive to other females. As yet, we do not understand the
mechanisms that alter female attraction to male scents according to
additional information gained on genetic compatibility, or whether
this influences associative learning, memory and/or response to
individual airborne scent signatures.
MATING ATTRACTION AND STIMULATION
A number of volatile pheromones that bind to MUPs in male
scent marks have priming effects on a female's reproductive
physiology when she delivers male scent to her vomeronasal organ
during close contact investigation. These priming pheromones
accelerate puberty in young females (Novotny et al., 1999;
Vandenbergh, 1969) and promote ovulation in adult females
(Jemiolo, Andreolini, Xie, Wiesler, & Novotny, 1989; Jemiolo,
Harvey, & Novotny, 1986; Whitten, Bronson, & Greenstein, 1968)
so that they are ready to mate. However, by the time that females
are ready to mate under natural conditions, it is likely that decisions
about preferred mates are already made. Indeed, the choice of fe-
males to settle and nest within a particular male's territory is a
major factor in mate choice, as most females will mate with the
owner of their resident territory (Hurst, 1987; Potts et al., 1991;
Wolff, 1985). This may be an important factor in reducing the risk
of infanticide (Hrdy, 1979), as males reduce infanticidal behaviour
towards their mate's offspring after cohabitation and copulation
(Elwood & Ostermeyer, 1984; McCarthy & Saal, 1986; Soroker &
Terkel, 1988). However, female house mice typically range across
several male territories and frequently mate multiply with neigh-
bouring territory owners; in a survey of wild-caught mice, 23% of
litters were sired by multiple males (Dean, Ardlie, & Nachman,
2006) while multiple mating can be even higher in high-density
captive populations (Stockley, Bottell, & Hurst, 2013; Thonhauser,
Raveh, Hettyey, Beissmann, & Penn, 2013). Thonhauser et al.
(2013) found more multiple paternity of litters when scent mark
investment among males in the local population was more even,
suggesting that females showed less discrimination to mate with
one particular territory owner.
319
Under free-ranging conditions, mate choice is due to oestrous
females approaching males when ready to mate; males do not mate
outside their defended territories (Hurst, 1987; Potts et al., 1991;
Wolff, 1985) and females return repeatedly to the same mating
site for copulation, typically moving away between successive
copulations (e.g. Hurst, 1986). How the specific site for mating is
chosen is not yet known and may simply depend on where the
female encounters one of her preferred males when in an appro-
priate state to mate. Prior to copulation, both female and male
spend time sniffing the facial and anogenital regions of the po-
tential mate. During these interactions, additional signals may
facilitate copulation itself. The tear fluid of male mice contains an
exocrine gland secreting the protein ESP1, which is a 7 kDa peptide
that activates a specific vomeronasal receptor and enhances
lordosis behaviour in females (Haga, Kimoto, & Touhara, 2007;
Kimoto, Haga, Sato, & Touhara, 2005). This is expressed in the
extraorbital lacrimal gland of wild-derived inbred strains and some
domesticated laboratory strains (BALB/c, DBA) under androgen
control. Interestingly though, like darcin, expression of this peptide
pheromone is absent in many domesticated strains (Haga et al.,
2010; Kimoto et al., 2007). As yet, nothing is known about differ-
ential male investment in this priming pheromone among wild
house mice but, unlike darcin, ESP1 is produced at very low levels
and plays a role in coordinating mating behaviour when mice are
already in close contact and mate attraction has already occurred.
FUTURE DIRECTIONS
Our recent discovery of darcin, an involatile sex pheromone in
male mouse urine, has provided new insight into the control of
female attraction to male scents and the importance of both
inherent attraction to a male-specific pheromone and associative
learning to target attraction flexibly to specific males. Notably, as
this pheromone can be detected through contact investigation only,
attraction is stimulated only once females have chosen to investi-
gate a male's scent closely including activation of the accessory
olfactory system. Scent deposition patterns and male investment in
other components of their scents both appear to be important in
attracting such contact under competitive conditions, while fe-
males may detect additional cues such as infection status that
inhibit scent attraction. However, as yet, we do not fully understand
how different information in male scents is integrated in making
these decisions. Do females fully investigate all information in male
scents and modify their inherent response to darcin according to
information gained about the scent owner through higher-level
processing? Or is attraction to closely investigate male scents
adjusted according to the relative strength of airborne volatiles and
other information gained through the main olfactory system, thus
altering contact with darcin and learned attraction to that male?
Progress in understanding this would be greatly aided by better
tools to identify when animals actively deliver scents to the vom-
eronasal organ under normal free-ranging conditions, a process
that is currently invisible in species that do not show a flehmen
response and requires invasive and often blunt approaches such as
entire removal or functional blocking of the vomeronasal system.
Comparative studies are needed to establish the extent to which
pheromones like darcin are present in other mammalian scents and
whether this is a widespread mechanism used in social learning.
Many other rodents produce sex-specific scent proteins (Beynon
et al., 2008) but the presence of pheromones that induce
individual-specific learning has yet to be tested. In addition to un-
derstanding the importance of differential investment in darcin and
what influences this, further work is needed to understand the wide
range of androgen-specific volatiles in male scents and whether
such complexity is important for providing individual-specific
320 S. A. Roberts et al. / Animal Behaviour 97 (2014) 313e321
signatures and/or additional information about male quality. It
should also be noted that scent advertisement is not restricted only
to males; females also advertise through scent marks, which may
not only indicate oestrous state and readiness to mate but also
appear to be involved in competitive signalling for breeding op-
portunities (Stockley et al., 2013). A major future challenge will be to
understand scent components produced by females and how these
are also used for sexual and competitive communication under
more natural social conditions than laboratory cages.
Acknowledgments
We are grateful to members of both the Mammalian Behaviour
and Evolution Group and the Protein Function Group for essential
support for this work, particularly to Lynn McLean, Dr Stuart
Armstrong, Dr Richard Humphries, John Waters, Rachel Spencer
and Joshua Beeston, and to many other members for discussion of
ideas and approaches. The work was funded by research grants to
J.L.H. and R.J.B. from the Biotechnology and Biological Sciences
Research Council (BB/J002631/1) and the Natural Environment
Research Council (NE/G018650).
References
Armstrong, S. D., Robertson, D. H. L., Cheetham, S. A., Hurst, J. L., & Beynon, R. J.
(2005). Structural and functional differences in isoforms of mouse major uri-
nary proteins: a male-specific protein that preferentially binds a male phero-
mone. Biochemical Journal, 391, 343e350.
Bacchini, A., Gaetani, E., & Cavaggioni, A. (1992). Pheromone binding proteins of the
mouse, Mus musculus. Experientia, 48, 419e421.
Barnard, C. J., Hurst, J. L., & Aldhous, P. (1991). Of mice and kin: the functional
significance of kin bias in social behaviour. Biological Reviews, 66, 379e430.
Baum, M. J. (2012). Contribution of pheromones processed by the main olfactory
system to mate recognition in female mammals. Frontiers in Neuroanatomy, 6.
Beltran-Bech, S., & Richard, F. J. (2014). Impact of infection on mate choice. Animal
Behaviour, 90, 159e170.
Berry, R. J. (1981). Biology of the house mouse. London: Academic Press.
Beynon, R. J., & Hurst, J. L. (2004). Urinary proteins and the modulation of chemical
scents in mice and rats. Peptides, 25, 1553e1563.
Beynon, R. J., Hurst, J. L., Mirton, M. J., Robertson, D. H. L., Armstrong, S. D.,
Cheetham, S. A., et al. (2008). Urinary lipocalins in rodenta: is there a generic
model? Chemical Signals in Vertebrates, 11(11), 37e49.
Brandt, R., & Macdonald, D. W. (2011). To know him is to love him? Familiarity and
female preference in the harvest mouse, Micromys minutus. Animal Behaviour,
82, 353e358.
Breer, H., Fleischer, J., & Strotmann, J. (2006). The sense of smell: multiple olfactory
subsystems. Cellular and Molecular Life Sciences, 63, 1465e1475.
Brennan, P. A., & Kendrick, K. M. (2006). Mammalian social odours: attraction and
individual recognition. Philosophical Transactions of the Royal Society B-Biolog-
ical Sciences, 361, 2061e2078.
Briand, L., Trotier, D., & Pernollet, J. C. (2004). Aphrodisin, an aphrodisiac lipocalin
secreted in hamster vaginal secretions. Peptides, 25(9), 1545e1552.
Bronson, F. H. (1979). The reproductive ecology of the house mouse. Quarterly
Review of Biology, 54, 265e299.
Bronson, F. H., & Caroom, D. (1971). Preputial gland of male mouseeattractant
function. Journal of Reproduction and Fertility, 25, 279e282.
Bronson, F. H., & Marsden, H. M. (1973). Preputial gland as an indicator of social
dominance in male mice. Behavioral Biology, 9, 625e628.
Butenandt, A., Beckmann, R., Stamm, D., & Hecker, E. (1959). Uber Den sexual-
Lockstoff Des Seidenspinners Bombyx morieReindarstellung Und Konstitution.
Zeitschrift Fur Naturforschung Part B-Chemie Biochemie Biophysik Biologie Und
Verwandten Gebiete, 14, 283e284.
Cavaggioni, A., Mucignat-Caretta, C., & Redaelli, M. (2008). Mice recognize recent
urine scent marks by the molecular composition. Chemical Senses, 33, 655e663.
Cheetham, S. A. (2006). Chemical communication in the house mouse: Linking
biochemistry and behaviour (Doctoral thesis). Liverpool, U.K.: University of
Liverpool.
Cheetham, S. A., Smith, A. L., Armstrong, S. D., Beynon, R. J., & Hurst, J. L. (2009).
Limited variation in the major urinary proteins of laboratory mice. Physiology
and Behavior, 96, 253e261.
Cheetham, S. A., Thom, M. D., Jury, F., Ollier, W. E., Beynon, R. J., & Hurst, J. L. (2007).
The genetic basis of individual-recognition signals in the mouse. Current
Biology, 17, 1771e1777.
Choleris, E., Clipperton-Allen, A. E., Phan, A., Valsecchi, P., & Kavaliers, M. (2012).
Estrogenic involvement in social learning, social recognition and pathogen
avoidance. Frontiers in Neuroendocrinology, 33, 140e159.
Dean, M. D., Ardlie, K. G., & Nachman, M. W. (2006). The frequency of multiple
paternity suggests that sperm competition is common in house mice (Mus
domesticus). Molecular Ecology, 15, 4141e4151.
Drea, C. M., Vignieri, S. N., Kim, S., Weldele, M. L., & Glickman, S. E. (2002). Re-
sponses to olfactory stimuli in spotted hyenas (Crocuta crocuta): II. Discrimi-
nation of conspecific scent. Journal of Comparative Psychology, 116, 342e349.
Drickamer, L. C. (1989). Odor preferences of wild-stock female house mice (Mus
domesticus) tested at 3 ages using urine and other cues from conspecific males
and females. Journal of Chemical Ecology, 15, 1971e1987.
D'Udine, B. A. A. E., & Alleva, E. N. R. I. C. O. (1983). Early experience and sexual
preferences in rodents. In P. Bateson (Ed.), Mate choice (pp. 311e327). Cam-
bridge, U.K.: Cambridge University Press.
Dulac, C., & Axel, R. (1995). A novel family of genes encoding putative pheromone
receptors in mammals. Cell, 83, 195e206.
Ehman, K. D., & Scott, M. E. (2002). Female mice mate preferentially with non-
parasitized males. Parasitology, 125, 461e466.
Elwood, R. W., & Ostermeyer, M. C. (1984). Does copulation inhibit infanticide in
male rodents. Animal Behaviour, 32, 293e294.
Emes, R. D., Beatson, S. A., Ponting, C. P., & Goodstadt, L. (2004). Evolution and
comparative genomics of odorant-and pheromone-associated genes in rodents.
Genome research, 14, 591e602.
Ferrero, D. M., Moeller, L. M., Osakada, T., Horio, N., Li, Q., Roy, D. S., et al. (2013).
A juvenile mouse pheromone inhibits sexual behaviour through the vomer-
onasal system. Nature, 502, 368e371.
Fisher, H. S., Swaisgood, R. R., & Fitch-Snyder, H. (2003). Odor familiarity and female
preferences for males in a threatened primate, the pygmy loris Nycticebus
pygmaeus: applications for genetic management of small populations. Natur-
wissenschaften, 90, 509e512.
Garratt, M., Stockley, P., Armstrong, S. D., Beynon, R. J., & Hurst, J. L. (2011). The scent
of senescence: sexual signalling and female preference in house mice. Journal of
Evolutionary Biology, 24, 2398e2409.
Garratt, M., McArdle, F., Stockley, P., Vasilaki, A., Beynon, R. J., Jackson, M. J., et al.
(2012). Tissue-dependent changes in oxidative damage with male reproductive
effort in house mice. Functional Ecology, 26, 423e433.
Gosling, L. M., & Roberts, S. C. (2001). Testing ideas about the function of scent
marks in territories from spatial patterns. Animal Behaviour, 62, 7e10.
Haga, S., Kimoto, H., & Touhara, K. (2007). Molecular characterization of vomer-
onasal sensory neurons responding to a male-specific peptide in tear fluid:
sexual communication in mice. Pure and applied chemistry, 79(4), 775e783.
Haga, S., Hattori, T., Sato, T., Sato, K., Matsuda, S., Kobayakawa, R., et al. (2010). The
male mouse pheromone ESP1 enhances female sexual receptive behaviour
through a specific vomeronasal receptor. Nature, 466, 118e122.
Halpern, M., & Martinez-Marcos, A. (2003). Structure and function of the vomer-
onasal system: an update. Progress in Neurobiology, 70, 245e318.
Herrada, G., & Dulac, C. (1997). A novel family of putative pheromone receptors in
mammals with a topographically organized and sexually dimorphic distribu-
tion. Cell, 90, 763e773.
Homady, M., & Brain, P. F. (1983). A comparison of preputial glands in individually-
housed, dominant and subordinate laboratory mice. Aggressive Behavior, 9,
109e110.
Howard, R. W., & Blomquist, G. J. (2005). Ecological, behavioral, and biochemical
aspects of insect hydrocarbons. Annual Review of Entomology, 50, 371e393.
Hrdy, S. B. (1979). Infanticide among animalsereview, classification, and exami-
nation of the implications for the reproductive strategies of females. Ethology
and Sociobiology, 1, 13e40.
Humphries, R. E., Robertson, D. H. L., Beynon, R. J., & Hurst, J. L. (1999). Unravelling
the chemical basis of competitive scent marking in house mice. Animal
Behaviour, 58, 1177e1190.
Hurst, J. L. (1986). Mating in free-living wild house mice (Mus domesticus). Journal of
Zoology, 210, 623e628.
Hurst, J. L. (1987). Behavioral variation in wild house mice Mus domesticus rutty: a
quantitative assessment of female social-organization. Animal Behaviour, 35,
1846e1857.
Hurst, J. L., Beynon, R. J., Humphries, R. E., Malone, N., Nevison, C. M., Payne, C. E., et al.
(2001). Information in scent signals of competitive social status: the interface
between behaviour and chemistry. Chemical Signals in Vertebrates, 9(9), 43e52.
Hurst, J. L., Payne, C. E., Nevison, C. M., Marie, A. D., Humphries, R. E.,
Robertson, D. H. L., et al. (2001). Individual recognition in mice mediated by
major urinary proteins. Nature, 414, 631e634.
Hurst, J. L., Robertson, D. H. L., Tolladay, U., & Beynon, R. J. (1998). Proteins in urine
scent marks of male house mice extend the longevity of olfactory signals. An-
imal Behaviour, 55, 1289e1297.
Hurst, J. L., & Beynon, R. J. (2004). Scent wars: the chemobiology of competitive
signalling in mice. Bioessays, 26, 1288e1298.
Hurst, J. L., & Beynon, R. J. (2013). Rodent urinary proteins: genetic identity signals
and pheromones. Chemical Signals in Vertebrates, 12, 117e133.
Jemiolo, B., Andreolini, F., Xie, T. M., Wiesler, D., & Novotny, M. (1989). Puberty-
affecting synthetic analogs of urinary chemosignals in the house mouse, Mus
domesticus. Physiology and Behavior, 46, 293e298.
Jemiolo, B., Harvey, S., & Novotny, M. (1986). Promotion of the whitten effect in
female mice by synthetic analogs of male urinary constituents. Proceedings of
the National Academy of Sciences of the United States of America, 83, 4576e4579.
Jemiolo, B., Xie, T. M., & Novotny, M. (1991). Sociosexual olfactory preference in
female miceeattractiveness of synthetic chemosignals. Physiology and Behavior,
50, 1119e1122.
 S. A. Roberts et al. / Animal Behaviour 97 (2014) 313e321
Johansson, B. G., & Jones, T. M. (2007). The role of chemical communication in mate
choice. Biological Reviews, 82, 265e289.
Johnson, R. P. (1973). Scent marking in mammals. Animal Behaviour, 21, 521e535.
Johnston, R. E., Munver, R., & Tung, C. (1995). Scent counter markseselective
memory for the top scent by golden-hamsters. Animal Behaviour, 49,
1435e1442.
Johnston, R. E., Sorokin, E. S., & Ferkin, M. H. (1997). Female voles discriminate
males' over-marks and prefer top-scent males. Animal Behaviour, 54, 679e690.
Jones, R. B., & Nowell, N. W. (1974). Comparison of aversive and female attractant
properties of urine from dominant and subordinate male mice. Animal Learning
& Behavior, 2, 141e144.
Karlson, P., & Butenandt, A. (1959). Pheromones (Ectohormones) in insects. Annual
Review of Entomology, 4, 39e58.
Kaur, A. W., Ackels, T., Kuo, T. H., Cichy, A., Dey, S., Hays, C., et al. (2014). Murine
pheromone proteins constitute a context-dependent combinatorial code gov-
erning multiple social behaviors. Cell, 157(3), 676e688.
Kavaliers, M., Choleris, E., & Pfaff, D. W. (2005). Genes, odours and the recognition of
parasitized individuals by rodents. Trends in Parasitology, 21, 423e429.
Kavaliers, M., & Colwell, D. D. (1995a). Discrimination by female mice between the
odors of parasitized and non-parasitized males. Proceedings of the Royal Society
B: Biological Sciences, 261, 31e35.
Kavaliers, M., & Colwell, D. D. (1995b). Odors of parasitized males induce aversive
responses in female mice. Animal Behaviour, 50, 1161e1169.
Kavaliers, M., Colwell, D. D., Braun, W. J., & Choleris, E. (2003). Brief exposure to the
odour of a parasitized male alters the subsequent mate odour responses of
female mice. Animal Behaviour, 65, 59e68.
Kavaliers, M., Fudge, M. A., Colwell, D. D., & Choleris, E. (2003). Aversive and
avoidance responses of female mice to the odors of males infected with an
ectoparasite and the effects of prior familiarity. Behavioral Ecology and Socio-
biology, 54, 423e430.
Kimoto, H., Haga, S., Sato, K., & Touhara, K. (2005). Sex-specific peptides from
exocrine glands stimulate mouse vomeronasal sensory neurons. Nature, 437,
898e901.
Kimoto, H., Sato, K., Nodari, F., Haga, S., Holy, T. E., & Touhara, K. (2007). Sex- and
strain-specific expression and vomeronasal activity of mouse ESP family pep-
tides. Current Biology, 17, 1879e1884.
Kwak, J., Grigsby, C. C., Rizki, M. M., Preti, G., Koksal, M., Josue, J., et al. (2012).
Differential binding between volatile ligands and major urinary proteins due to
genetic variation in mice. Physiology and Behavior, 107, 112e120.
Lanuza, E., Martín-Sa nchez, A., Marco-Manclús, P., Bernardita Ca diz-Moretti, B.,
Lluís Fortes-Marco, L., Hern andez-Martínez, A., et al. (2014). Sex pheromones
are not always attractive: changes induced by learning and illness in mice.
Animal Behaviour, 97, 265e272.
Leinders-Zufall, T., Lane, A. P., Puche, A. C., Ma, W. D., Novotny, M. V., Shipley, M. T.,
et al. (2000). Ultrasensitive pheromone detection by mammalian vomeronasal
neurons. Nature, 405, 792e796.
Liberles, S. D., & Buck, L. B. (2006). A second class of chemosensory receptors in the
olfactory epithelium. Nature, 442, 645e650.
Luo, M., Fee, M. S., & Katz, L. C. (2003). Encoding pheromonal signals in the
accessory olfactory bulb of behaving mice. Science, 299(5610), 1196e1201.
Malone, N., Payne, C. E., Beynon, R. J., & Hurst, J. L. (2001). Social status, odour
communication and mate choice in wild house mice. Chemical Signals in Ver-
tebrates, 9(9), 217e224.
Martel, K. L., & Baum, M. J. (2009). Adult testosterone treatment but not surgical
disruption of vomeronasal function augments male-typical sexual behavior in
female mice. Journal of Neuroscience, 29, 7658e7666.
Martinez-Garcia, F., Martinez-Ricos, J., Agustin-Pavon, C., Martinez-Hernandez, J.,
Novejarque, A., & Lanuza, E. (2009). Refining the dual olfactory hypothesis:
pheromone reward and odour experience. Behavioural Brain Research, 200,
277e286.
Martinez-Ricos, J., Agustin-Pavon, C., Lanuza, E., & Martinez-Garcia, F. (2007).
Intraspecific communication through chemical signals in female mice: rein-
forcing properties of involatile male sexual pheromones. Chemical Senses, 32,
139e148.
Martinez-Ricos, J., Agustin-Pavnon, C., Lanuza, E., & Martinez-Garcia, F. (2008). Role
of the vomeronasal system in intersexual attraction in female mice. Neurosci-
ence, 153, 383e395.
Matsunami, H., & Buck, L. B. (1997). A multigene family encoding a diverse array of
putative pheromone receptors in mammals. Cell, 90, 775e784.
Mccarthy, M. M., & Saal, F. S. V. (1986). Inhibition of infanticide after mating by wild
male house mice. Physiology and Behavior, 36, 203e209.
Meagher, S., Penn, D. J., & Potts, W. K. (2000). Male-male competition magnifies
inbreeding depression in wild house mice. Proceedings of the National Academy
of Sciences of the United States of America, 97, 3324e3329.
Moncho-Bogani, J., Lanuza, E., Hernandez, A., Novejarque, A., & Martinez-Garcia, F.
(2002). Attractive properties of sexual pheromones in mice: innate or learned?
Physiology and Behavior, 77, 167e176.
Mucignat, C., & Caretta, A. (2014). Message in a bottle: major urinary proteins and
their multiple roles in mice intraspecific chemical communication. Animal
Behaviour, 97, 255e263.
Mucignat-Caretta, C., Caretta, A., & Cavaggioni, A. (1995). Acceleration of puberty
onset in female mice by male urinary proteins. Journal of Physiology-London,
486, 517e522.
Mudge, J. M., Armstrong, S. D., McLaren, K., Beynon, R. J., Hurst, J. L., Nicholson, C.,
et al. (2008). Dynamic instability of the major urinary protein gene family
321
revealed by genomic and phenotypic comparisons between C57 and 129 strain
mice. Genome Biology, 9.
Ninomiya, K., & Kimura, T. (1988). Male odors that influence the preference of female
miceeroles of urinary and preputial factors. Physiology and Behavior, 44, 791e795.
Novotny, M., Harvey, S., & Jemiolo, B. (1990). Chemistry of male-dominance in the
house mouse, Mus domesticus. Experientia, 46, 109e113.
Novotny, M. V., Ma, W. D., Wiesler, D., & Zidek, L. (1999). Positive identification of
the puberty-accelerating pheromone of the house mouse: the volatile ligands
associating with the major urinary protein. Proceedings of the Royal Society B:
Biological Sciences, 266, 2017e2022.
Payne, C. E. (2001). Urinary proteins and their ligands in wild house mice: Modulation,
heterogeneity and response (Doctoral thesis). Liverpool, U.K.: University of Liverpool.
Potts, W. K., Manning, C. J., & Wakeland, E. K. (1991). Mating patterns in seminatural
populations of mice influenced by mhc genotype. Nature, 352, 619e621.
Pusey, A., & Wolf, M. (1996). Inbreeding avoidance in animals. Trends in Ecology &
Evolution, 11(5), 201e206.
Ramm, S. A., Cheetham, S. A., & Hurst, J. L. (2008). Encoding choosiness: female
attraction requires prior physical contact with individual male scents in mice.
Proceedings of the Royal Society B: Biological Sciences, 275, 1727e1735.
Restrepo, D., Arellano, J., Oliva, A. M., Schaefer, M. L., & Lin, W. H. (2004). Emerging
views on the distinct but related roles of the main and accessory olfactory
systems in responsiveness to chemosensory signals in mice. Hormones and
Behavior, 46, 247e256.
Rich, T. J., & Hurst, J. L. (1999). The competing countermarks hypothesis: reliable
assessment of competitive ability by potential mates. Animal Behaviour, 58,
1027e1037.
Rich, T. J., & Hurst, J. L. (1998). Scent marks as reliable signals of the competitive
ability of mates. Animal Behaviour, 56, 727e735.
Riviere, S., Challet, L., Fluegge, D., Spehr, M., & Rodriguez, I. (2009). Formyl peptide
receptor-like proteins are a novel family of vomeronasal chemosensors. Nature,
459, 574e577.
Roberts, S. A., Davidson, A. J., McLean, L., Beynon, R. J., & Hurst, J. L. (2012). Pher-
omonal induction of spatial learning in mice. Science, 338, 1462e1465.
Roberts, S. A., Simpson, D. M., Armstrong, S. D., Davidson, A. J., Robertson, D. H.,
McLean, L., et al. (2010). Darcin: a male pheromone that stimulates female
memory and sexual attraction to an individual male's odour. BMC Biology, 8.
Roberts, S. C., & Gosling, L. M. (2004). Manipulation of olfactory signalling and mate
choice for conservation breeding: a case study of harvest mice. Conservation
Biology, 18, 548e556.
Robertson, D. H. L., Marie, A. D., Veggerby, C., Hurst, J. L., & Beynon, R. J. (2001).
Characteristics of ligand binding and release by major urinary proteins.
Chemical Signals in Vertebrates, 9, 169e176.
Ryba, N. J. P., & Tirindelli, R. (1997). A new multigene family of putative pheromone
receptors. Neuron, 19, 371e379.
Schaal, B. (2014). Chemical signals 'designed for' mammalian newborns. Animal
Behaviour, 97, 289e299.
Singer, A. G., Tsuchiya, H., Wellington, J. L., Beauchamp, G. K., & Yamazaki, K. (1993).
Chemistry of odortypes in mice: fractionation and bioassay. Journal of chemical
ecology, 19(3), 569e579.
Soroker, V., & Terkel, J. (1988). Changes in incidence of infanticidal and parental
responses during the reproductive-cycle in male and female wild mice Mus
Musculus. Animal Behaviour, 36, 1275e1281.
Spehr, M., Spehr, J., Ukhanov, K., Kelliher, K. R., Leinders-Zufall, T., & Zufall, F. (2006).
Parallel processing of social signals by the mammalian main and accessory
olfactory systems. Cellular and Molecular Life Sciences, 63, 1476e1484.
Stockley, P., Bottell, L., & Hurst, J. L. (2013). Wake up and smell the conflict: odour
signals in female competition. Philosophical Transactions of the Royal Society of
London. Series B: Biological Sciences, 368, 20130082.
Stopka, P., Stopkova , R., & Janotova , K. (2012). Mechanisms of chemical communica-
tion. Cambridge, U.K.: Cambridge University Press.
Thonhauser, K. E., Raveh, S., Hettyey, A., Beissmann, H., & Penn, D. J. (2013). Why do
female mice mate with multiple males? Behavioral Ecology and Sociobiology, 67,
1961e1970.
Todrank, J., & Heth, G. (2003). Odor-genes covariance and genetic relatedness
assessments: rethinking odor-based “recognition” mechanisms in rodents.
Advances in the Study of Behavior, 32, 77e130.
Vandenbergh, J. G. (1969). Male odor accelerates female sexual maturation in mice.
Endocrinology, 84, 658e660.
Vogt, K., Zimmermann, F., Ko € lliker, M., & Breitenmoser, U. (2014). Scent-marking
behaviour and social dynamics in a wild population of Eurasian lynx Lynx lynx.
Behavioural Processes, 106, 98e106.
Whitten, W. K., Bronson, F. H., & Greenstein, J. A. (1968). Estrus-inducing phero-
mone of male miceetransport by movement of air. Science, 161, 584e585.
Witzgall, P., Kirsch, P., & Cork, A. (2010). Sex pheromones and their impact on pest
management. Journal of Chemical Ecology, 36, 80e100.
Wolff, R. J. (1985). Mating-behavior and female choiceetheir relation to social-
structure in wild caught house mice (Mus Musculus) housed in a semi-natural
environment. Journal of Zoology, 207, 43e51.
Wyatt, T. D. (2014). Pheromones and animal behaviour (2nd ed.). Cambridge, U.K.:
Cambridge University Press.
Yoshikawa, K., Nakagawa, H., Mori, N., Watanabe, H., & Touhara, K. (2013). An un-
saturated aliphatic alcohol as a natural ligand for a mouse odorant receptor.
Nature Chemical Biology, 9, 160e162.
Zala, S. M., Potts, W. K., & Penn, D. J. (2004). Scent-marking displays provide honest
signals of health and infection. Behavioral Ecology, 15, 338e344.