Ultrasonic courtship vocalizations in wild house mice: spectrographic analyses
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House mice emit ultrasonic vocalizations (USVs) during courtship, which are sexually dimorphic and function to attract mates. Spectrographic analyses of laboratory mice show that USVs are surprisingly complex and have features of song. In this study, we conducted the first spectral and temporal analyses of recordings from wild house mice (F1 from wild-caught Mus musculus musculus). Inspection of the spectral shape of syllables shows that the USVs from wild mice can be classified by both frequency and duration, and the most apparent distinction is between low- versus high-frequency calls. High-frequency calls of wild mice seem to be emitted at a much higher frequency range than previously found in some laboratory mice. Interestingly, we found that 20% of males do not vocalize at all, though the reason for their behaviour is unclear. Future studies are needed to determine what kind of information is conveyed in these complex vocalizations, and why some males appear to be non-vocalizers.
KeywordsMus musculus musculus Ultrasonic vocalization Spectrographic analyses Wild house mouse
Like many rodents, house mice (Mus musculus) communicate using ultrasonic vocalizations (USVs) (Nyby 1983; White et al. 1998; Holy and Guo 2005). USVs are produced by pups as distress calls, and by adults, though especially males during courtship in response to encountering females or their scent (laboratory mice: Nyby et al. 1977, 1979; Whitney and Nyby 1979; wild mice: Hoffmann et al. 2009; Musolf et al. 2010). The evolutionary functions of males’ courtship USVs are not entirely clear, and it is not known what kind of information is communicated in their vocalizations. Spectrographic analyses show that males’ courtship USVs are surprisingly complex, and even have features of birdsong (Holy and Guo 2005). As spectrographic analyses have only been conducted on domesticated, laboratory mice, our aim here is to conduct spectral and temporal analyses of recordings from wild male house mice (F1 from wild-caught Mus musculus musculus).
Studies on domesticated, laboratory strains of house mice have provided many interesting findings about USVs (Portfors 2007). For example, there are differences in the USVs among strains, suggesting that these differences are genetic (Bell et al. 1972; Sales 1979; Scattoni et al. 2008; Kikusui et al. 2011). The USVs of laboratory mice are likely to differ significantly from their wild counterparts, as with their morphology, physiology, behaviour (Crawley et al. 1997), reproduction and other life history traits (Miller et al. 2002), and in particular, they are likely to have reduced inter-individual variation due to having been inbred for about two centuries (van Zuphten et al. 1995). Indeed, a recent study comparing USVs of wild California mice (Peromyscus californicus) versus a domesticated, laboratory strain revealed that wild mice showed higher variability of calls (Kalcounis-Rueppell et al. 2010). We recently began the first studies on the USVs of wild house mice (Hoffmann et al. 2009; Musolf et al. 2010), and found that females are attracted to playbacks of male USVs, especially unfamiliar and unrelated males versus siblings (Musolf et al. 2010). Our findings indicate that males’ USVs contain signals of individuality and kinship, which we recently confirmed with spectrographic analysis (Hoffmann et al. 2011). In this study, we provide the first spectrographic analyses of the USVs of wild house mice.
Subjects and housing
Fifteen adult male (>8 weeks; mean body mass ± SD = 20.9 ± 1.4 g), F1 progeny of wild-caught house mice (Mus musculus musculus), were used in this study. Mice were trapped at three different locations (<1 km apart) in Gänserndorf, Austria. Mice were trapped with wooden live traps with a metal lid (Mäuse-Wippfalle, DeuFa GmbH) stocked with bedding, peanut butter bait and apple as a water supply. Traps were placed inside different buildings (stable, farms, enclosures) for 10 days. All traps were set in the late afternoon, and checked that evening and early the following morning. At all other times traps were left in place but unset. Caught mice (except visibly lactating females which were released) were transferred to normal colony cages (see below) and moved to the laboratory by car. We are not aware of any study on the impact of maternal confinement on wild litters, but as house mice often breed communally with several mothers attending a nest, we assume that there was no adverse effect of trapping on litters. In total 99 wild mice were caught and maintained in the colony through outbreeding (e.g., no matings between relatives by crossing mice from different trapping locations). Breeding pairs were kept for several litters with parents and offspring housed together (in mixed sex groups) up to weaning of the young at 21 days of age. Thereafter, males (subjects) were housed individually (to prevent aggressive and otherwise often fatal interactions) whereas females (urine donors) were housed as sister pairs in type II cages (size 26.5 × 20.5 × 18 cm, plus high stainless-steel covers, mesh width 1 cm) with bedding (Abedd: aspen wood chips) and enrichment material consisting of nesting material (Abedd: aspen wood shavings), paper rolls and nest boxes (Tecniplast). Mice were kept at mean temperature of 20 ± 1°C and light:dark cycle of 12:12 h. Water and mouse chow (Altromin, 1314 Forti; Lage, Germany) was provided ad libitum. There was no acoustic separation between animals in the colony room before or after testing periods. To standardize social experience for the adult male subjects, all individuals were exposed to adult conspecifics of both sexes before the experiment took place (procedure described in Hoffmann et al. 2009 and Musolf et al. 2010). At the end of the study, experimental animals were reintegrated into the breeding population. The study was approved by the Austrian Federal Ministry of Science and Research.
Recordings took place in an isolated recording room with no other animals present. Recordings were performed in an isolated wooden recording chamber (see Hoffmann et al. 2009; Musolf et al. 2010). One subject at a time was placed in a type II cage containing clean bedding and a water bottle on the cage lid. Food was removed during recordings to reduce sound interference. The cage was then positioned in the centre of the recording chamber, and after a 5 min habituation period, the recording session was initiated by introducing a freshly voided female urine stimulus, as we previously found that males are more responsive to fresh versus frozen urine (Hoffmann et al. 2009). Five females, which were all unfamiliar and unrelated to the subjects, served as donors of fresh urine. Urine of each of the five females was collected on clean aluminium foil as described by Nyby and colleagues (1979) and mixed. The oestrus stage of females was not controlled, as it was previously shown to have no influence on male USV response (Nyby et al. 1979). Immediately after pooling the urine, 60 μl was pipetted directly from the aluminium surface onto a clean cotton swab and put into the test cage (storage time ≤5 min). To record only direct responses to the stimulus, the acclimatization period was further prolonged if a male produced any USVs during that time until 2 min without USVs elapsed. Each of the 15 individual males was recorded for one 90-min session. Between trials, the recording chamber was cleaned with a handheld vacuum cleaner. Temperature in the testing room was 20.4 ± 0.9°C. During recording, no experimenter or other person was present in the recording room.
USV structure and terminology
As the terminology used to describe animal vocalizations is diverse, the following definitions will be used in this study: calls or syllables are defined as units of sound separated by silence from other sound units (Doupe and Kuhl 1999). Syllables or calls consist of one or more elements, continuous markings on a sonogram. Here, syllables consisting of one element are termed general syllables, whereas syllables consisting of two elements including a major sudden frequency jump (≥10 kHz) are termed 1-frequency step syllables (Grimsley et al. 2011). There is no separation in time between the elements. Syllables that are composed of three elements without separation in time and that include two major sudden frequency jumps (each jump ≥10 kHz) are termed 2-frequency step syllables (Grimsley et al. 2011). In this paper, we use the term frequency step syllables for what we previously called complex syllables (Musolf et al. 2010) to improve clarity and consistency in terminology among studies. A syllable type or call type is defined as a category of syllables, observed regularly in the animal’s vocalization, distinct from other syllable types (Holy and Guo 2005).
Prior to feature analyses performed with Sound Analysis Pro (SAP, version 1.04) (Tchernichovski et al. 2001), the duration of the exceptionally short USV syllables of the mice was extended using the time-warp feature of the software Goldwave (50% via rate, Goldwave v5.14, http://www.goldwave.com), and the sampling rate was adjusted from 250 kHz (sampling rate when recorded) to 22.05 kHz (SAP sampling rate). The sound files were then segmented into sounds in the feature batch mode of SAP. Here, the USVs are separated from non-vocalization background noise. The thresholds were adjusted separately for each individual recording session by performing 30 fake trials. Parameters ranged between 22 ± 3 dB for amplitude and 4.5 ± 0.1 for Wiener entropy across all sessions. Segmentation was validated by visual inspection of the segments. Afterwards, all USV segments were analyzed using SAP feature batches. Resulting data included the features duration, pitch, mean frequency, Wiener entropy, frequency modulation (FM) and goodness of pitch, which is a measure of sounds’ periodicity. After feature batch analyses via SAP, values for durations and frequencies were calculated back to their real, recorded sampling rate and time for further parameter analyses. In addition to these spectrographical features the absolute number of syllables was determined.
Because 1- and 2-frequency step syllables are visually distinct from general calls, they were treated as individual call categories. They were extracted from all other calls and analyzed separately. To reveal potential syllable type categories of general calls, histograms and scatter plots of all measured parameters and parameter pairs were computed. Log transformations were performed to meet requirements for parametric tests. Multivariate analysis of variance (MANOVA, Hotelling’s trace, two-tailed) including all measured parameters was conducted, to test for overall differences between two potential call type categories. After confirmation of two distinguishable call type categories, Dunnett’s T3 post hoc tests were used to detect significant differences of the single parameters. The distinct frequency step syllables differ in the number of their sudden frequency jumps (1 and 2 jumps), therefore a first classification was made on this basis. Ranges and mean values of features obtained by SAP feature batches were calculated for each of the two call types. Further, histograms and scatter plots of all measured parameters and parameter pairs were computed to reveal more potential syllable type categories. For parametrical statistical tests (MANOVA, Hotelling’s trace), log-transformed values were used.
Males emitted significantly higher numbers of low- (N = 13664, 94.19%) than high-frequency calls (N = 843, 5.81%) to female urinary cues (binomial probability distribution: N = 14670, P ≤ 0.001). Interestingly, of the 843 high-frequency syllables, 621 had energy exclusively above 100 kHz (73.67% of high-frequency syllables; 4.28% of all syllables).
Comparison of low- and high-frequency syllables (Nlow-frequency calls = 13664, Nhigh-frequency calls = 843) emitted by 12 different male mice in response to female urinary cues
Dunnett T3 post hoc test
45.60 to 89.95
91.95 to 109.79
Basis of classification
7.85 to 194.02
7.86 to 45.50
20.10 to 89.22
22.01 to 107.26
Goodness of pitch
11.2 to 115.1
25.6 to 87.6
0.07 to 0.79
0.04 to 0.57
−6.47 to −4.77
−5.90 to −5.01
Comparison of 1-frequency step (N = 121) versus 2-frequency step (N = 155) ultrasonic syllables emitted by 12 different male house mice in response to female urinary cues
1-frequency step syllables
2-frequency step syllables
Dunnett T3 post hoc test
50.68 to 81.25
50.68 to 85.01
11.26 to 66.69
9.31 to 49.04
21.27 to 81.41
39.02 to 82.88
Goodness of pitch
11.1 to 111.2
18.0 to 89.3
0.007 to 0.415
0.036 to 0.415
−6.08 to −5.96
−6.22 to −5.01
Most males readily emitted USVs upon exposure to female urine, as previously described in laboratory mice (Nyby et al. 1977, 1979; Whitney and Nyby 1979) and wild house mice (Hoffmann et al. 2009; Musolf et al. 2010), as expected. Surprisingly, however, 20% (3/15) of the males emitted no vocalizations during the 90-min session. If males’ USVs function as courtship to attract females, as is generally assumed, then it is puzzling that some males do not vocalize. These non-vocalizing males may have been more distressed in the recording apparatus than vocalizing males. However, non-vocalizing males are also found with laboratory mice and especially with some strains (D’Amato and Moles 2001; J. Nyby, person. comm.), which suggests that non-calling is not a result of distress in captivity. Future studies are needed to determine the proximate causes of this variation in USV production (genetic, developmental, such as intra-uterine position, or social status or condition) and adaptive consequences. If non-vocalizing is due to distress, then such a finding might suggest that males’ courtship USV production provides a reliable indicator of a male’s quality, condition or resistance to stressful circumstances.
Inspection of the spectral shape of the USVs of wild males indicates that syllables consisting of one element can be classified by both frequency and duration, and the most apparent distinction is between low- and high-frequency calls, as previously found with BALB/c laboratory mice (Barthelemy et al. 2004). In addition to the distinction between high- versus low-frequency calls, male USVs could also be classified based on the appearance and number of sudden frequency jumps (frequency step syllables). Previous studies on USVs of adult laboratory male mice also found several distinct call categories, mostly classified by their pitch jumps (Holy and Guo 2005; Grimsley et al. 2011; Kikusui et al. 2011). We found that 5% of the USVs of wild male mice are frequency step syllables. Studies with a laboratory strain (CBA/CaJ) found similar proportions of these complex call types in their repertoire (Grimsley et al. 2011). Moreover, the overall shape of USVs from wild and laboratory mice is very similar, which demonstrates that production of USVs is not the result of domestication.
Although it is difficult to compare acoustic studies in mice because most of the studies differ in methodology, it appears that wild male mice produce a higher proportion of syllables within high frequency ranges than laboratory mice investigated in previous studies (BALB/c: Barthelemy et al. 2004; B6D2F1: Holy and Guo 2005; CBA/CaJ: Grimsley et al. 2011; C57BL/6 and BALB/c: Kikusui et al. 2011). Of all syllables uttered by wild males, 4.28% had energy exclusively above 100 kHz. Previous studies on USVs of adult laboratory male mice, however, showed that laboratory mice did not produce any USVs with energy exclusively above 100 kHz (BALB/c: Barthelemy et al. 2004; CBA/CaJ: Grimsley et al. 2011). Nevertheless, Grimsley and colleagues (2011) could show that a small proportion of CBA/CaJ males produced calls with dominant frequencies above 100 kHz (0.6% of all syllables). Certainly, these differences could be due to differences in recording and analyzing methods. However, interestingly, this difference is consistent with comparisons of wild versus laboratory California mice (Peromyscus californicus) (Kalcounis-Rueppell et al. 2010): USVs of wild P. californicus were also higher in frequency than USVs of their domestic counterparts.
If laboratory strains vocalize less frequently at higher frequencies than wild house mice, then this difference might be due to genetic, environmental or G × E effects. If genetic, there are several possible explanations. First, it could be due to domesticated laboratory mice being largely derived from another subspecies (Mus musculus domesticus), or because laboratory mice are hybrids of both subspecies (Mus musculus domesticus, Mus musculus musculus and others) (Wade and Daly 2005). Second, lower frequencies may have been selectively favoured in the laboratory. The acoustic environment is surely different in cages than in the wild, and the selective advantage for calling at high frequencies (in terms of attracting mates versus avoiding predators) may be lost in captivity. Third, some laboratory strains may have evolved lower frequencies due to inadvertent selection, a non-adaptive by-product of other traits that evolved during domestication, such as increased body mass (Garland et al. 1995). In other mammals, body mass is correlated with several vocalization parameters, including fundamental frequency, formant dispersion (Pfefferle et al. 2006) and formant frequencies (Frey and Riede 2003). However, we found no correlation between body weight and USV frequency among the wild males, which also indicates that USVs do not provide honest indicators of a male’s size, contrary to the handicap principle (Zahavi and Zahavi 1999). Whichever hypothesis proves correct, the differences between wild versus domesticated mice is consistent with other evidence that USVs in pups and adults are influenced by genetics (Bell et al. 1972; Sales 1979; Maggio and Whitney 1986; Scattoni et al. 2008; Kikusui et al. 2011). Interestingly, the vocal repertoire of domesticated silver foxes (Vulpes vulpes) differs from vocalizations of their wild counterparts (Gogoleva et al. 2008a, 2008b, 2009).
Frequency step USVs of wild male mice are composed of 2 elements with one frequency jump or 3 elements with two frequency jumps. Thus, these calls are distinguishable into two call type categories, and different individuals uttered a distinct number of the two frequency step syllable types. Birds also emit multi-element syllables (Vallet et al. 1997), which are more difficult to perform than other syllables (Janicke et al. 2008). Those calls elicit high levels of copulation solicitation displays, and are predominantly produced by successful breeders (Leitner and Catchpole 2002; Janicke et al. 2008). As these syllables are used in mating, courtship and other sexual contexts, they were called ‘sexy syllables’ (Leitner and Catchpole 2002). In our previous study on the influence of different olfactory stimuli on male courtship USVs in wild mice (Musolf et al. 2010) we found that male mice produced frequency step USV syllables in different ratios depending upon the stimulus. The proportion of frequency step syllables was greatest when responding to the urine of unfamiliar females versus male urine or water. It remains to be seen whether the rate of production of frequency step syllables in mice carries different information from that of general ones, or perhaps reflects males’ quality or condition. It would be interesting to determine whether some males cannot afford the physiological costs of producing USVs or specific syllable types, but there are no studies on this question to our knowledge. Further studies are needed to determine if multi-element syllable types of male mice USVs are similar to those ‘sexy syllables’ in birds.
In summary, our spectral analysis confirms previous work showing that wild house mice, as well as laboratory strains, emit complex ultrasonic vocalizations (Holy and Guo 2005). Most males readily respond to female urine, but curiously, we found that some males do not produce any USVs whatsoever, though it is unclear why not. Among vocalizers, males produce two general call types, a high- and a low-frequency call, as with domesticated mice. However, wild male mice seem to produce a higher proportion of syllables within high frequency ranges compared with laboratory mice investigated in previous studies (Barthelemy et al. 2004; Grimsley et al. 2011). Additionally, wild mice produce certain specific call categories termed frequency step syllables, which were found to be emitted in comparable proportions in laboratory strains. We found similarities and differences in the USVs emitted by wild mice compared with previous studies with laboratory mice, and we predict that laboratory strains will show reduced variability and complexity in USVs due to domestication.
We thank Prof. H. Winkler for helpful advice. We are grateful to C. Kohl and H. Sasse for their help in taking care of the mice. F. Galler built the recording box. M. Kalcounis-Rueppell made very constructive comments on an earlier version of this manuscript.
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