Ontogeny of Sexually Dimorphic Ultrasonic Vocalizations in Mongolian Gerbils c
S. D. HOLMAN W. T. C. SEALE MRC Neuroendocrine Development and Behaviour Group Institute of Animal Physiology and Genetics Research, Babraham Cambridge, CB2 4AT, U K
Sexual differentiation of emission rates (Experiment 1) and physical structure of ultrasonic vocalizations (Experiment 2) were investigated in Mongolian gerbils between 17 and 85 days of age. Animals from different litters were allowed to interact in iso- and heterosexual pairs. Vocalization rates increased in all groups, reaching a plateau at approximately day 56. Female pups had significantly higher rates of all vocalizations than male pairs before the plateau stage. Five stereotypic categories of physical structure were easily distinguished and confirmed by sound spectrographic analysis. Only mid-range frequencies of vocalizations appeared to decline throughout the juveniles’ lives: this change may communicate the animal’s age. Two male and one female categories were sexually dimorphic and similar in structure to those made during courtship interactions. Rates of male-type vocalizations appear to increase as testicular androgens start rising.
Ultrasonic vocalizations in rodents have proved to be an ideal model system for studying neuroendocrine processes underlying behavior (gerbils, Holman, 198 1; Holman & Hutchison, 1985; rats, Barfield & Geyer, 1972;mice, Warburton, Sales, & Milligan, 1989; hamsters, Floody, Walsh, & Flanagan, 1979). Although ultrasonic emissions show a close relationship with other sexual behavioral patterns in rodents, their communicatory function is not fully understood. Authors have suggested that vocalizations by female hamsters in estrus have an “advertising” function (Floody & F’faff, 1977) in sexual interactions, whereas the post-ejaculatory call of male rats communicates a period of “non-availability” for further sexual interactions (Anisko, Suer, McClintock, & Adler, 1978). In Mongolian gerbils Reprint requests should be sent to S. D. Holman, MRC Neuroendocrine Development and Behaviour Group, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, CB2 4AT, United Kingdom. Received for publication 8 May 1990 Revised for publication 14 September 1990 Accepted at Wiley 9 November 1990 Developmental Psychobiology 24(2):103-1 15 (1991) 0 1 9 9 1 by John Wiley & Sons, Inc.
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(Meriotzes rrn#rric.rrlcrzrrs), ultrasonic vocalizations are also thought to have communicatory functions (Holman, 1980; Thiessen, Graham, & Davenport, 1978); for example. the masculine upsweep, described below, may stimulate female movement sequences ready for male mounting attempts (Burley, 1980). Recent work has attempted to establish the role of steroid hormones on the activation of courtship ultrasonic vocal behavior, which is independent of genital feedback (mice, Nunez & Tan, 1984; deermice, Clemens & Pomerantz, 1982; hamsters, Floody & Bauer, 1987). During sexual interactions, adult male Mongolian gerbils fhrfciiones tinguicuiatus) emit ultrasonic vocalizations that can be classified into three very distinct structural types: the upsweep, unmodulated, and modulated vocalizations (Holman, 1980). Upsweep vocalizations are produced at high rates, particularly in male-female chasing sequences, and high-intensity, unmodulated vocalizations are emitted for a short period after each ejaculation. Similar to the rat, there are a number of ejaculations in each mating session (Agren, Zhou, & Zhong, 1989; Davis, Estep, & Dewsbury, 1974; Kuehn & Zucker, 1968). Modulated vocalizations are produced in fewer mating encounters than the upsweep or unmodulated vocalization, and are recorded most often when the female is not in complete behavioral estrus. In adult castrates, the upsweep call is sensitive to testosterone (Holman and Hutchison, 1982). Testosterone is also involved during a neonatal sensitive period in the sexual differentiation of this vocalization (Holman, 1981) by local effects on the medial hypothalamic area of the brain (Holman & Hutchison, 1985). Production of the masculine upsweep during courtship is associated with a region, the Sexually Dimorphic Area (SDA), in the hypothalamus (Holman & Hutchison, in press). A group of cells within the SDA, the pars compacta (SDApc, identified and described by Cornmins & Yahr, 1984), is sexually differentiated by the presence of androgens in the neonate (Yahr, 1988) which are of testicular origin in normal males (Holman & Hutchison, 1990).The volume of SDApc is positively correlated with the courtship upsweep (Holman, Hutchison, Wozniak, & Hutchison, 1989). Little is known about the sequence and structure of ultrasonic vocalizations in the period between weaning and adulthood. We know of no study that characterizes the development of adult ultrasounds. In this study we describe the development of sexually dimorphic vocalizations in juvenile gerbils. Experiment I identifies sex differences in the rates of vocalization produced at different ages in heteroand isosexual pair encounters. Experiment 2 (a) identifies and categorizes juvenile vocalizations, and (b) relates the development of juvenile sounds to those of the adult.
General Methods
Animals and Maintenance Mongolian gerbils (Mcviones ~ ~ n g i i i c ~ l uwere t u ~ )reared in our laboratory and housed in 22.5 x 38.0 x 35.5 cm high opaque, polycarbonate cages. Sawdust and paper strips were provided for bedding material. SBS hamster breeding diet and water were continuously available and the temperatures of the rooms were maintained at 20°C ( *1.6"C). The rooms were illuminated between 0430 h to 1830 h.
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Parents were removed when their offspring reached the age of 28 days. Animals remained in their litter group until the end of the experiment. Individual experimental animals (n = 77) in each litter (n = 18) were marked at the start of these investigations when they were aged 17 days and at subsequent intervals when necessary.
Procedure In Experiments 1 and 2 , pairs of litters born on the same day and containing 6-7 young, with approximately half being of one sex, were matched to be identical in numbers of offspring. Individuals from each matched-litter were placed together in a semi-anechoic chamber, the second animal following the first within 10-15 s. After approximately 10-30 s sound monitoring began and continued for 30 s. Selection of animals was carried out using a latin-square pairing design so that each animal in one of the litters was paired with each animal in the other matchedlitter during the course of one testing session. Tests were carried out twice weekly.
Ultrasound Recording To minimize unwanted extraneous noise created by pairs digging or scratching, sound recordings were made in a semi-anechoic chamber (internal dimensions 37 x 32 x 30 cm high). The internal surface of the chamber was insulated with a layer of cotton wool held in place with fine plastic netting (Harrison & Holman, 1978). The only uninsulated internal surface was the front wall which consisted of a perspex observation window. The floor of the chamber was covered with a layer (approximately 5 cm thick) of slightly moistened sphagnum peat. Sounds were detected using a QMC solid dielectric capacitance microphone (type CM1: Experiment 1) or a Bruel and Kjzr (B & K) calibrated air dielectric capacitance microphone (type 4135: Experiment 2 only). Both microphones were placed in the lid of the semi-anechoic chamber, slightly off-set from the central point and approximately 3 I cm above the floor. The QMC microphone was connected to a QMC S200 encoder in “countdown 16” mode. In this mode, the frequency of the strongest component present is reduced by a factor of 16, producing an audible analogue of the ultrasounds which can be monitored through headphones (Experiment 1). In Experiment 2, the B & K microphone was connected to a measuring amplifier (type 2610) and a Krohn-Hite variable filter (type 3550) set in band pass mode (5-100 kHz). A Racal Thermionic instrumentation recorder (4DL) with direct channel facilities (running at a tape speed of 38 cm/s) enabled permanent sound recordings to be made. Sound analysis was carried out on first generation copies made from the original Racal recordings played back at 9.5 cmis tape speed via the B & K measuring amplifier and Krohn-Hite filter (set at band-pass 1-20 kHz) and rerecorded onto an Akai GX-370 D tape recorder running at 19 cm/s. These first generation copies were then replayed on the Akai at a tape speed of 9.5 cm/s which was an eighth of the recording speed. Signals, after being amplified by a B & K measuring amplifier and filtered (Krohn-Hite set at band pass 1-8 kHz), were monitored using headphones. Representative vocalizations were further analysed using a Kay 6061B Sona-graph Spectrum Analyzer.
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Experiment 1: Rates of Vocalization In this experiment, we investigate (1) the development of vocalization emissions between sexes and (2) whether rates depend on the sex of the partner during behavioral interactions.
Met hods Ultrasonic vocalization rates of individuals housed in litter-mate groups (n = 7; 7 : 7; 7 : 6; 6) were recorded in a latin-square experimental design (see General Method). Rates were recorded from all possible female-female, female-male, and male-male pairs. Each pair was placed together in a semi-anechoic chamber and allowed to interact freely. Pairs were tested twice each week between the ages of 17 and 85 days. No attempt was made in Experiment 1 to distinguish between the structural types of ultrasound produced. Sounds below 20 kHz were not included in the analysis.
Analysis Vocalization rates were submitted to logarithmic transformation (Sokal & Rohlf, 1981) in order to normalize distribution before carrying out analyses. The rates from all pairs within a testing group, that is, composed of female-female, female-male, and male-male individuals, were averaged over 10 day blocks (age) apart from the first block which contained nine days. In total there were seven age blocks. A two-way analysis of variance evaluated the effects of group composition and age on vocalization rates. A subsequent post hoc multiple comparison test (Fisher’s Protected Least Significance Difference (Winer, 1971)) detected differences between groups at each age.
Results and Discussion Rarely did the first animal placed in the chamber vocalize: Vocalizations began when the second animal was introduced. Recording of ultrasounds started 10-30 s later, after the first bout of calling had subsided. Calling was emitted in bursts usually following investigation of the anechoic chamber’s floor or the partner’s head or anogenital region. As animals aged, vocalizations were emitted immediately before or after behavioral interactions which included olfactory investigations and sexual and aggressive play interactions. Emission rates of ultrasonic vocalizations increased significantly as the pups became older, F = 19.46, p = 0.0001, df = 6 (Fig. 1). As the animals approached puberty, rates reached a plateau in all three pairing types and remained at this level until the end of the experiment. A significant difference in vocalization rates between the three groups was found F = 22.13, p = 0.0001, df = 2 (Fig. 1). It was established on the first age of testing, (17-25 days, Fisher PLSD = 0.1 1, p 5 0.05) and disappeared as the various pairing types reached their maximum rates between 36 and 75 days of age (Fig. 1). At the youngest ages (days 17-36), the female-female group produced the greatest rate of calling. The female-female group reached maximum rates of calling sooner than the male-male group (36-45 days);
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I I I I I I 26-35 36-45 46-55 56-65 66-75 76-85
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Age (days)
Fig. I . Maturational increases in ultrasonic vocalization rates (mean per min 2 sem) of Mongolian gerbils in paired encounters. Thirty sec of each encounter was recorded. Individuals of each pair came from different litters (n = 6). Unfilled squares represent female-female pairs (n = 34), filled squares represent male-male pairs (n = 34) and female-male pairs (n = 66) are represented by filled diamond symbols. * Different from male-female and male-male groups or t Male-female group only (Fisher PLSD, p 5 0.05).
male vocalizing increased very rapidly between days 36 and 75 (Fig. 1). Analysis of the male-female group revealed that its mean vocalizing rate tended to be midrange between female-female and male-male mean rates. Thus, young gerbils seem to vocalize independently of the sex of their partners, with a greater rate occurred in young females before the ages of between 56-65 days. The communicatory function of the total rate of vocalizations emitted by juveniles is uncertain: a function would also need to incorporate a reason for the sex difference seen. Vocalizations may have a communicatory “contact” function, similar to some of those produced by birds (Hinde, 1952; Thielcke, 1976; Messmer & Messmer, 1956; Gompertz, 1961). If it is assumed that the function of a contact vocalization is to help the development of cohesion within a group of animals, would sex differences be shown? Relationships between the maturing sexes, dependent on the socio-ecological environment, would need to be investigated to answer this question in gerbils. No attempt was made in this experiment to distinguish the frequency of occurrence of different structural types of vocalization (see Experiment 2). Production of a specific type of vocalization may be related to a special context, Calling by males increased rapidly between the ages of 56 to 75 days. During this period testosterone levels begin approaching adult levels (Probst, 1987). This suggests that vocalizations, as well as serving to maintain contact
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between individuals, may indeed also have a significant sexual function. Data from Experiment 2 support this hypothesis.
Experiment 2: Vocalization Structure Vocalizations with different structures emitted by gerbils during mating encounters depend on the stage reached in the sexual interaction (see Introduction). In Experiment 2, we examined in sexually immature gerbils whether (a) ultrasonic vocalizations could be identified and categorized, and (b) the development of juvenile sounds are related to those of the adult.
Method Permanent sound recordings were made during encounters of gerbils (see General Method). Forty-four pairs of animals (female-female, n = 15: femalemale, n = 14: male-male, n = 15) produced vocalizations during three age ranges, 17-25, 26-70, and 71-85 days.
Analysis Criteria by which vocalizations can be classified have been partially determined in preliminary experiments (Holman, Seale, & Hutchison, 1989).Categories of vocalization were distinguished by ear from tape recordings replayed at reduced speed (see Methods). A spectrographic trace in 15-20% of the vocalizations made by pairs verified the structural category. Spectrograms were also made when the category of vocalization was not evident. On average, I0 traces were made from each recording of a particular pair. A G-test of independence (Sokal & Rohlf, 1981) was carried out between numbers of vocalizations of each category, summed in the female-female or male-male groups.
Results and Discussion Description of Vocalizations Vocalizations were very stereotypic (Fig. 2) and therefore easily classified when heard on replayed recordings. The five simple definitions outlined were able to accommodate the majority of vocalizations. Approximately 7% did not fit into one of the five categories [Miscellaneous group (Fig. 211. Inspection of spectrograms revealed no evidence that the ultrasonic vocalizations comprised second harmonics, or formants, above the single fundamental frequency (first harmonic) trace (Fig. 2). In a few (less than 1%) spectrographic traces, both animals emitted sounds almost simultaneously. In all cases, it was obvious that two vocalizations were being produced. Two broad super categories of calls could be distinguished: rectilinear or curvilinear vocalizations.
DEVELOPMENT OF SEXUALLY DIMORPHIC ULTRASOUNDS a. Short rectilinear
d. Tailed curvilinear
b. Long rectilinear
e. Warble
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c. Steep curvilinear
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Fig. 2. Spectrogram traces representing ultrasonic vocalizations made by juvenile Mongolian gerbils. Six classes were emitted and vocalizations appeared to be stereotypic to each class. Vocalizations b and c were made primarily by males whereas sound e was made by females aged under 25 days.
Rectilinear In our definition, the drift from horizontal was no more than kO.8 kHz from the mid-range frequency. (Fig. 2a,b). Rectilinear calls were subdivided into: Short rectilinear. Calls under 15.1 ms duration. Some short calls were on the borderline between being defined as Long rectilinear or Tailed curvilinear (Fig. 2a). Long rectilinear. Calls over 15 ms duration (Fig. 2b). The mean duration was 58 ms (sem k 9 . 0 ) but some calls were up to 550 ms in length.
Curvilinear This category contained vocalizations which tended to have an ascending sigmoid-shaped spectrographic trace. The end frequency was greater than the starting frequency in most cases (but see Warble curvilinear). In proportion to
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Table I Structural Categories c$ Ultrasonic Vocalizations Emitted b y Mongolian Gerbils of Various Ages during Paired Encounters. Ultrasorinds Were Categorized by Ear. Spectrograms, (Fig. 21, Conjrmed Each Vocalization Category (Method Experiment 2 ) . 5% vocalization Category (mid-range frequency kHz)*
Group
17-25
Age (days) 26-70
71-85
~~
Female-female
Short rectilinear Long rectilineart Steep curvilinear? Tailed curvilinear W arblet Miscellaneous
0.0 0.0 0.0 25.0 (37 kHz) 62.5 (41 kHz) 12.5 (42 kHz)
4.35 (31 kHz) 0.0 4.35 (33 kHz) 82.6 (32 kHz) 0.0 8.7 (32 kHz)
0.0 0.0 16.9 (30 kHz) 76.2 (32 kHz) 0.0 3.9 (32 kHz)
Female-male
Short rectilinear Long rectilinear Steep curvilinear Tailed curvilinear Warble Miscellaneous
5.9 (32 kHz) 5.9 (32 kHz) 0.0 70.5 (36 kHz) 0.0 17.6 (35 kHz)
3.3 13.3 16.7 56.7 0.0 10.0
3.6 (30 kHz) 28.6 (30 kHz) 38.9 (31 kHz) 20.0 (30 kHz) 0.0 8.9 (32 kHz)
Short rectilinear Long rectilinear Steep curvilinear Tailed curvilinear Warble Miscellaneous
0.0 9.0 (34 kHz) 0.0 82.0 (37 kHz) 0.0 9.0 (37 kHz)
0.0 5.3 36.8 47.4 0.0 10.5
(30 kHz) (29 kHz) (34 kHz) (32 kHz) (34 kHz)
~~
Male-male
(29 kHz) (32 kHz) (31 kHz)
(32 kHz)
1.3 (33 kHz) 2.7 (32 kHz) 29.3 (32 kHz) 61.3 (31 kHz) 0.0 5.4 (32 kHz)
* t
Mid-range frequencies calculated from spectrograms. Difference in numbers of vocalizations of each category produced by female-female or malefemale groups (p s 0.05, G-test, 2-tailed. Sokal and Rohlf, (1981)). The total number of spectrograms was summed over all ages.
other calls, Steep and Tailed curvilinear calls were emitted at high rates by paired animals (Table 1). Both types of call were emitted in bouts of 3-5 vocalizations. The mid-range frequency of the Steep curvilinear was similar to the Tailed curvilinear at the various ages (Fig. 2c,d, Table 1). Curvilinear vocalizations were classified into: Steep curvilinear. Vocalizations in which the frequency rise of 7.8 kHz per 25 ms was always exceeded within the first 5 ms. Calls had a duration of less than 1 ms (Fig. 2c). Tailed curvilinear. Vocalizations in which the frequency change within the first 5 ms was always less than or equal to a rate of 7.8 kHz per 25 ms. The slow increase in slope gave the calls a “tailed” appearance (Fig. 2d). Warbled curvilinear. Spectrographic traces showed a series of marked frequency modulations. The vocalization sounded like twittering or warbling sounds at reduced playback speed (Fig. 2e). The maximum durations of calls in this category extended up to approximately 160 ms. In a few examples the lowest frequency of the modulations was less than the starting frequency.
Miscellaneous vocalizutions This category represented vocalizations which could not be included in those above, including dome and basin shaped traces and traces showing an end fre-
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quency considerably less than the starting frequency. They often appeared to be similar to short phases of the Warble category, that is, comprised of one or less than one cycle of modulation (Fig. 2). Vocalizations in the Miscellaneous category may be curvilinear calls which were interrupted or altered by, for example, positions of the animal’s body causing laryngeal or air-way deformations. They were not formed by the superimposition of two vocalizations being made simultaneously as each separate call was distinguishable during such circumstances.
Age and Sex Differences One general feature found was that the mid-range frequencies of a particular type of vocalization decreased as animals became older in all categories of vocalization. Thus, for example, the. mid-range frequency for the Tailed curvilinear category produced by isosexual female pairs fell from 37 kHz to 32 kHz between the youngest and oldest groups (Table 1). Similar decreases were seen in other vocalizations. Theoretically, it could be argued that smaller laryngeal structures, which seem to be the source of ultrasound production in rodents (Thiessen, Kittrell, & Graham, 1980; Roberts, 1975), should produce higher mid-range frequencies than larger structures. We infer that this simple change may communicate the emitter’s age to receivers of the signal. Two structural types of vocalizations were affected by the age of the animal. One vocalization, the Warble, was only made by preweanling (17-25 days) female pups (difference between the numbers of vocalization produced by female-female or male-male groups, G = 57.8, p < 0.01) (Table 1). We have unpublished data, however, that this type of vocalization is also made by preweanling (10-16 days) males, particularly in situations where the animal has just been handled. A vocalization which was recorded only in male-male and female-male pairs was the Long rectilinear call (Fig. 2d). It was not produced by females at any age (G = 5.0, p = 0.05, between female-female and male-male groups, Table 1). The third age-related ultrasound, the Steep curvilinear, was made from day 26 onwards at high rates by male-male and female-male pairs. A significant difference was found between the numbers of vocalizations made by male-male pairs compared to female-female pairs (G = 8.2, p < 0.01) (Table I).
General Discussion Between the ages of 17 (preweaning) and 85 days, Mongolian gerbils emit a varied range of ultrasonic vocalizations during paired encounters. The rate of emissions for the total number of vocalizations also increases as they become older. Gerbils isolated during sound monitoring, however, show a decrease in the rate of ultrasonic calling up to 20 days of age, after this age vocalizations are no longer detectable (De Ghett, 1974). Thus, behavioral interactions induce calling in animals 20 days and older. Categories of vocalizations were stereotypical, although within a category there was some variation in call structure. Therefore, classification of calls was a simple procedure. The total number of vocalizations produced was also different between the sexes in paired encounters, with female-female groups emitting more vocalizations than male-male groups before the age of 56 days. A methodological point which is pertinent to other studies on vocal behavior is that only interacting
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pairs of gerbils produced vocalizations in this study (c.f., DeGhett, 1974). Thus, a vocalization in which the rate is sexually dimorphic is made in a context that is difficult to match between the two sexes (Gyger, Pontet, & Schenk, 1986; Warburton et al., 1989). For example, an ultrasound produced at high rates in male-male and female-male interactions but significantly less in female-female interactions may not be produced by the male because the important variable determining the rate of the vocalization may not be the sex of the emitter but the sex of the partner. That is, the context in which a call is made is critical for the call’s production. Parallel studies using devocalized animals and correlations between emission and other behavioral components need to be performed before this question may be resolved. The three sexually dimorphic categories of ultrasound identified in this study, the Warble, Long rectilinear, and Steep curvilinear may be precursors of sounds made during sexual interactions. Only females were heard emitting the Warble call in this study. It appears that very young animals in discomfort make this category of sound. Mongolian gerbil pups reach their full endothermic potential by about 21 days of age (McManus, 1971) and our first two tests were carried out before this age. Therefore, females, which have generally lower body weight than males, may have been undergoing the effects of falls in body temperature during testing as they were not allowed to huddle with their sibs or parents and thus reduce heatloss or gain heat, respectively. A similar call to the Warble is produced during sexual interactions particularly when the female is not fully in behavioral estrus (the modulated vocalization, Holman, 1980). Aggression often occurs when a female is not in complete estrus during mating (Holman, Hutchison, & Snelson, 1982). Thus, this confirms our suggestion made in Experiment 2 that the Warble call has a distress function. In avian species, it is often the case that during periods of conflict in courtship, birds will use immature patterns of behavior (“displacement activities”) (Hinde, 1970). Long rectilinear and Steep curvilinear made by juvenile males are similar to those made by adult males in sexual interactions (Holman, 1980). During sexual interactions in adults, a vocalization, striking because of its high sound intensity (75 db recorded 12 cm from its source, Holman, 1980), is made by males within 10 s after ejaculation: bursts of this sound continue for about 45 s in the postejaculatory interval. The spectrograph representing the post-ejaculatory ultrasound is similar to the unmodulated vocalization made by immature gerbils: However, the central frequency of the post-ejaculatory ultrasound was lower by approximately 5 kHz than the Long rectilinear call and, although it is not presented in this report, the intensity of the Long rectilinear ultrasound was less intense than the post-ejaculatory call. The Long rectilinear call may be a developmental precursor of post-ejaculatory sounds because of its structural similarity. The structure of the Steep curvilinear resembles that of the masculine vocalization made during courtship sexual interactions, the upsweep (Holman, 1980).Male courtship upsweep vocalizations are similar to those made byjuveniles, in that a greater rate (approximately 96%) is emitted compared with other types of vocalizations. Courtship upsweeps have a bandwidth frequency of 28-32 kHz (mid-range approximately 30 kHz) but tend to be longer (median duration, 19 ms) than the Steep curvilinear call (Holman, 1980). In the present study, we suggest that the Steep curvilinear is produced at greater rates as males mature and that this vocalization, in proportion to others,
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increases as the animals age (Table 1). If the emission rate of the Steep curvilinear sound increases from 17 to 56 days, the difference between emission rates of the sexes would eventually disappear (Fig. 1). As animals mature, therefore, the ratio of the different categories of calls made by the sexes also changes. This would suggest that vocalizations might be selected from a range of categories, described in this study, and subsequently used by adults in the specific contexts where the vocalization may have a clear meaning. For example, the masculine juvenile Steep curvilinear call becomes the male-type upsweep (Holman, 1980). Other variables, such as the effect of gonadal secretions, may also play a role in determining the rate and type of juvenile vocalization. This study provides a model by which the development of vocal behavior may be investigated in terms of relationships between the sexes and their dependency on gonadal secretions. In such an observational study as that reported here, the suggestion of communicatory functions for vocalizations are imprecise and only extensive experimental research would remedy this deficiency. It has been suggested that vocalizations are artifacts of lung compression in body movements in gerbils, such as in locomotion perhaps: air is expelled through laryngeal structures causing ultrasound (Thiessen et al., 1980). We do not support this hypothesis for a number of major reasons. First, few calls were made in the first moments when an animal was alone in the testing chamber even though it was moving while investigating its surroundings. Second, many categories of vocalizations last longer than the time it would take for a normal expulsion of breath. Furthermore, single peaks of pressure intensity are not seen within calls which occur during the lung deflation cycle. Third, calls are of different structural stereotypes, where one would assume that artifacts would tend to vary considerably in physical structure. Also, intensity modulations are seen which are associated with specific types of call. Fourth, sexual dimorphisms exist between the various categories of vocalizations, whereas there was no obvious difference in locomotory activity between the pairs. However, the reasons stated here do not rule out that some vocalizations may be associated with locomotion and have no communicatory value. Nevertheless, the major question that remains unanswered is whether a communication function can be assigned to a precursor of a sexual vocalization in juveniles. We can only suggest that as play sexual motor patterns are seen in gerbils and rats (Meaney & Stewart, 1981) so will sexual play vocalizations be seen. Experiments statistically correlating the various categories of ultrasound to sexual “practice” components in juveniles would provide the basis for this hypothesis.
Notes We wish to thank Drs. John and Rosemary Hutchison and Chris and Magdalena Jams for commenting on, and Miss Helen Potter for typing this manuscript.
References Agren, G . , Zhou, Q., & Zhong, W. (1989). Ecology and social behaviourof Mongolian gerbils, Meriones unguiculafus, at Xininhot, Inner Mongolia, China. Animal Behauiour, 37, 11-27. Anisko, J. J., Suer, S . F., McClintock, M. T., & Adler, N. T. (1978). Relation between 22-kHz ultrasonic signals and sociosexual behavior in rats. Physiology & Behavior 92, 821-829.
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Barfield, R. J.. & Geyer. L. A. (1972). Sexual behavior: Ultrasonic post-ejaculatory song of the male rat. Science, 176, 1349-1350. Burley, R. A. (1980). Pre-copulatory and copulatory behaviour in relation to stages of the oeslrous cycle in the female Mongolian gerbil. Behauiour. 72, 211-241. Clemens, L. G., & Pomerantz, S. M. (1982). Testosterone acts as a pro-hormone to stimulate copulatory behavior in male deer mice (Peromyscus manicirluius bairdi). Journal of Comparative and Physiological Psychology, 96, 114-122. Commins, D., & Yahr, P. (1984). Adult testosterone levels influence the morphology of a sexually dimorphic area in the Mongolian gerbil brain. Journnl of Cotnparatiue Neurology, 224, 132-140. Davis, H. N., Estep, D. Q . , & Dewsbury, D. A. (1974). Copulatory behavior of Mongolian gerbils (Meriones unguiculutus). Animal Learning and Behuuior, 2 , 69-73. De Ghett, V. J . (1974). Developmental changes in the rate of ultrasonic vocalization in the Mongolian gerbil. Developrnenral Psychobiology, 7, 267-272. Floody, 0. K., & Bauer, G . B. (1987). Selectivity in the responses of hamsters to conspecific vocalizations. Hormone3 and Behavior, 21, 522-527. Floody, 0 . R., & Pfaff, D. W. (1977). Communication among hamsters by high-frequency acoustic signals: 1. Physical characteristics of hamster calls. Journol of Compurative and Physiological Psychology, 91, 794-806. Floody, 0 . R., Walsh, C., & Flanagan, M. T. (1979). Testosterone stimulates ultrasound production by male hamsters. Hormones and Behauior, 12, 164-171. Gompertr, T. (1961). The vocabulary of the Great Tit. British Birds, 54, 369-418. Gyger, M., Pontet, A . , & Schenk, F. (1986). Le canal acoustique chez les rongeurs: Les emissions A haute frkquence. Revue SuiJse Zoologie, 93, 623-640. Harrison, G . E., & Holman, S. D. (1978). A device to enable the automatic recording of rodent ultrasonic vocalizations. Behuuior Research Method.s und In.strumenfation, 10, 689-692. Hinde, R. A. (1952). The behaviour of the Great Tit (faru,v m u j o r ) and some other related species. Behaviour, Suppl. 2, 1-201. Hinde, R. A. (1970). Animal Behaviovr: A Synthesis o f Etiiology uttd Cnmparariue Psychology. Tokyo: McGraw-Hill Kogakusha, Ltd. Holman, S . D. (1980). Sexually dimorphic, ultrasonic vocalizations of Mongolian gerbils. Behavioral and Neural Biology, 28, 183-192. Holman, S. D. (1981). Neonatal androgenic influences on masculine ultrasonic vocalizations of Mongolian gerbils. Physiology & Behavior, 26, S83-586. Holman, S . D., & Hutchison, J. B. (1982). Pre-copulatory behaviour in the male Mongolian gerbil: I . Differences in dependence on androgen of component patterns. Animal Behauiour, 30, 221 -230. Holman, S. D., & Hutchison, J . B . (1985). Effects of intracranial androgen on the development of masculine ultrasonic vocalisations in the Mongolian gerbil (Mvriones ungiric:ulu/us).Journul qf Endocrinology, 107, 355-363. Holman, S . D., & Hutchison, J . B. (1990). Lateralisation of hormone-dependent brain mechanisms during sexual differentiation of behaviour. Journal of Endocrinology, 124, 266. Holman, S. D., & Hutchison, J . B. (in press). lntracranial steroid micro-implants: Their preparation and subsequent use in the developing brain. In B. Greenstein (Ed). Neuroendocrine Reseurch Methods. London: Harwood Academic Publishers. Holman, S. D., Hutchison, J. B., & Snelson, D. (1982). Pre-copulatory behaviourin the maleMongolian gerbil: 11. Effects of post-castration sexual and aggressive interactions on responsiveness to androgen. Animal Behaviour. 30, 231-239. Holman, S . D., Hutchison, R., Wozniak. A. & Hutchison, J . (1989). Is asymmetric brain development related to sexual behavior? Socie/y for Neuro~cirnc.rAbstruc/s, 15, 379. Holman, S . D., Seale. W., & Hutchison, J. B . (1989). Sexual differentiation ofjuvenile ultrasounds: Their relationship to oestrogen-action in the sexually dimorphic area of the Mongolian gerbil. In J. Balthazart (Ed.), Int~~rnu~ionalconferewc~e on hormones, bruin und hehauiour, Abstracts. Liege: University Press. (pp. 87-88). Kuehn, R. W., & Zucker, I. (1968). Reproductive behavior of the Mongolian gerbil (Meriones ungrricula!us). Journul of Cotnpiiru~ivecind Physiological Psychology, 66, 747-752. Meaney, M. J., & Stewart, J. (1981). A descriptive study of social development in the rat (Rartus norvegicus). Animal Behaviour, 29, 34-45. McManus, J . J. (1971). Early postnatal growth and the development of temperature regulation in the Mongolian gerbil (Meriones unguiculatus). Journal of Mummalogy, 42, 782-792.
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Messmer, V. E., & Messmer, I. (1956). Die Entwicklung der LautauSerungen und einiger Verhaltensweisen der Amsel (Turdus merula merula L.). Zeitschr$t fur Tierpsychologie, 13, 341-441, Nunez, A. A., & Tan, D. T. (1984). Courtship ultrasonic vocalizations in male Swiss-Webster mice: Effects of hormones and sexual experience. Physiology & Behavior, 32, 717-721. Probst, B. (1987). Developmental changes in the pituitary-gonadal-axisin male Mongolian gerbils from birth to adulthood. Experimental and Clinical Endocrinology, 90, 157-166. Roberts, L. H. (1975). Evidence for the laryngeal source of ultrasonic and audible cries of rodents. Journal of Zoology (London), 175, 243-257. Sokal, R. R., & Rohlf, B. J. (1981). Biometry. The Principles and Practice of Statistics in Biological Research. San Francisco: Freeman and Company. Thielcke, G. A. (1976). Bird sounds (English Translation). Ann Arbor: University of Michigan Press. (Original work published as G. Thielcke (1970). Vogelstimmen. Berlin: Springer-Verlag). Thiessen, D. D., Graham, M., & Davenport, R. (1978). Ultrasonic signalling in the gerbil (Meriones unguiculatus): Social interaction and olfaction. Journal of Comparative and Physiological Psychology, 92, 1041-1049. Thiessen, D. D., Kittrell, M. W., & Graham, J. M. (1980). Biomechanics of ultrasonic emissions in Mongolian gerbils, Meriones unguiculatus. Behavioral and Neural Biology, 29, 415-429. Warburton, V. L., Sales, G. D., & Milligan, S. R. (1989). The emission and elicitation of mouse ultrasonic vocalizations: The effects of age, sex and gonadal status. Physiology &Behavior, 45, 41-47.
Winer, B. J. (1971). Statistical Principles in Statistical Design. New York: McGraw-Hill. Yahr, P. (1988). Pars compacta of the Sexually Dimorphic Area of the gerbil hypothalamus: Postnatal ages at which development responds to testosterone. Behavioral and Neural Biology, 49, 118-124.
S. D. HOLMAN W. T. C. SEALE MRC Neuroendocrine Development and Behaviour Group Institute of Animal Physiology and Genetics Research, Babraham Cambridge, CB2 4AT, U K
Sexual differentiation of emission rates (Experiment 1) and physical structure of ultrasonic vocalizations (Experiment 2) were investigated in Mongolian gerbils between 17 and 85 days of age. Animals from different litters were allowed to interact in iso- and heterosexual pairs. Vocalization rates increased in all groups, reaching a plateau at approximately day 56. Female pups had significantly higher rates of all vocalizations than male pairs before the plateau stage. Five stereotypic categories of physical structure were easily distinguished and confirmed by sound spectrographic analysis. Only mid-range frequencies of vocalizations appeared to decline throughout the juveniles’ lives: this change may communicate the animal’s age. Two male and one female categories were sexually dimorphic and similar in structure to those made during courtship interactions. Rates of male-type vocalizations appear to increase as testicular androgens start rising.
Ultrasonic vocalizations in rodents have proved to be an ideal model system for studying neuroendocrine processes underlying behavior (gerbils, Holman, 198 1; Holman & Hutchison, 1985; rats, Barfield & Geyer, 1972;mice, Warburton, Sales, & Milligan, 1989; hamsters, Floody, Walsh, & Flanagan, 1979). Although ultrasonic emissions show a close relationship with other sexual behavioral patterns in rodents, their communicatory function is not fully understood. Authors have suggested that vocalizations by female hamsters in estrus have an “advertising” function (Floody & F’faff, 1977) in sexual interactions, whereas the post-ejaculatory call of male rats communicates a period of “non-availability” for further sexual interactions (Anisko, Suer, McClintock, & Adler, 1978). In Mongolian gerbils Reprint requests should be sent to S. D. Holman, MRC Neuroendocrine Development and Behaviour Group, Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, CB2 4AT, United Kingdom. Received for publication 8 May 1990 Revised for publication 14 September 1990 Accepted at Wiley 9 November 1990 Developmental Psychobiology 24(2):103-1 15 (1991) 0 1 9 9 1 by John Wiley & Sons, Inc.
CCC 0012- I630/91/020103-13$04.O0
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(Meriotzes rrn#rric.rrlcrzrrs), ultrasonic vocalizations are also thought to have communicatory functions (Holman, 1980; Thiessen, Graham, & Davenport, 1978); for example. the masculine upsweep, described below, may stimulate female movement sequences ready for male mounting attempts (Burley, 1980). Recent work has attempted to establish the role of steroid hormones on the activation of courtship ultrasonic vocal behavior, which is independent of genital feedback (mice, Nunez & Tan, 1984; deermice, Clemens & Pomerantz, 1982; hamsters, Floody & Bauer, 1987). During sexual interactions, adult male Mongolian gerbils fhrfciiones tinguicuiatus) emit ultrasonic vocalizations that can be classified into three very distinct structural types: the upsweep, unmodulated, and modulated vocalizations (Holman, 1980). Upsweep vocalizations are produced at high rates, particularly in male-female chasing sequences, and high-intensity, unmodulated vocalizations are emitted for a short period after each ejaculation. Similar to the rat, there are a number of ejaculations in each mating session (Agren, Zhou, & Zhong, 1989; Davis, Estep, & Dewsbury, 1974; Kuehn & Zucker, 1968). Modulated vocalizations are produced in fewer mating encounters than the upsweep or unmodulated vocalization, and are recorded most often when the female is not in complete behavioral estrus. In adult castrates, the upsweep call is sensitive to testosterone (Holman and Hutchison, 1982). Testosterone is also involved during a neonatal sensitive period in the sexual differentiation of this vocalization (Holman, 1981) by local effects on the medial hypothalamic area of the brain (Holman & Hutchison, 1985). Production of the masculine upsweep during courtship is associated with a region, the Sexually Dimorphic Area (SDA), in the hypothalamus (Holman & Hutchison, in press). A group of cells within the SDA, the pars compacta (SDApc, identified and described by Cornmins & Yahr, 1984), is sexually differentiated by the presence of androgens in the neonate (Yahr, 1988) which are of testicular origin in normal males (Holman & Hutchison, 1990).The volume of SDApc is positively correlated with the courtship upsweep (Holman, Hutchison, Wozniak, & Hutchison, 1989). Little is known about the sequence and structure of ultrasonic vocalizations in the period between weaning and adulthood. We know of no study that characterizes the development of adult ultrasounds. In this study we describe the development of sexually dimorphic vocalizations in juvenile gerbils. Experiment I identifies sex differences in the rates of vocalization produced at different ages in heteroand isosexual pair encounters. Experiment 2 (a) identifies and categorizes juvenile vocalizations, and (b) relates the development of juvenile sounds to those of the adult.
General Methods
Animals and Maintenance Mongolian gerbils (Mcviones ~ ~ n g i i i c ~ l uwere t u ~ )reared in our laboratory and housed in 22.5 x 38.0 x 35.5 cm high opaque, polycarbonate cages. Sawdust and paper strips were provided for bedding material. SBS hamster breeding diet and water were continuously available and the temperatures of the rooms were maintained at 20°C ( *1.6"C). The rooms were illuminated between 0430 h to 1830 h.
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Parents were removed when their offspring reached the age of 28 days. Animals remained in their litter group until the end of the experiment. Individual experimental animals (n = 77) in each litter (n = 18) were marked at the start of these investigations when they were aged 17 days and at subsequent intervals when necessary.
Procedure In Experiments 1 and 2 , pairs of litters born on the same day and containing 6-7 young, with approximately half being of one sex, were matched to be identical in numbers of offspring. Individuals from each matched-litter were placed together in a semi-anechoic chamber, the second animal following the first within 10-15 s. After approximately 10-30 s sound monitoring began and continued for 30 s. Selection of animals was carried out using a latin-square pairing design so that each animal in one of the litters was paired with each animal in the other matchedlitter during the course of one testing session. Tests were carried out twice weekly.
Ultrasound Recording To minimize unwanted extraneous noise created by pairs digging or scratching, sound recordings were made in a semi-anechoic chamber (internal dimensions 37 x 32 x 30 cm high). The internal surface of the chamber was insulated with a layer of cotton wool held in place with fine plastic netting (Harrison & Holman, 1978). The only uninsulated internal surface was the front wall which consisted of a perspex observation window. The floor of the chamber was covered with a layer (approximately 5 cm thick) of slightly moistened sphagnum peat. Sounds were detected using a QMC solid dielectric capacitance microphone (type CM1: Experiment 1) or a Bruel and Kjzr (B & K) calibrated air dielectric capacitance microphone (type 4135: Experiment 2 only). Both microphones were placed in the lid of the semi-anechoic chamber, slightly off-set from the central point and approximately 3 I cm above the floor. The QMC microphone was connected to a QMC S200 encoder in “countdown 16” mode. In this mode, the frequency of the strongest component present is reduced by a factor of 16, producing an audible analogue of the ultrasounds which can be monitored through headphones (Experiment 1). In Experiment 2, the B & K microphone was connected to a measuring amplifier (type 2610) and a Krohn-Hite variable filter (type 3550) set in band pass mode (5-100 kHz). A Racal Thermionic instrumentation recorder (4DL) with direct channel facilities (running at a tape speed of 38 cm/s) enabled permanent sound recordings to be made. Sound analysis was carried out on first generation copies made from the original Racal recordings played back at 9.5 cmis tape speed via the B & K measuring amplifier and Krohn-Hite filter (set at band-pass 1-20 kHz) and rerecorded onto an Akai GX-370 D tape recorder running at 19 cm/s. These first generation copies were then replayed on the Akai at a tape speed of 9.5 cm/s which was an eighth of the recording speed. Signals, after being amplified by a B & K measuring amplifier and filtered (Krohn-Hite set at band pass 1-8 kHz), were monitored using headphones. Representative vocalizations were further analysed using a Kay 6061B Sona-graph Spectrum Analyzer.
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Experiment 1: Rates of Vocalization In this experiment, we investigate (1) the development of vocalization emissions between sexes and (2) whether rates depend on the sex of the partner during behavioral interactions.
Met hods Ultrasonic vocalization rates of individuals housed in litter-mate groups (n = 7; 7 : 7; 7 : 6; 6) were recorded in a latin-square experimental design (see General Method). Rates were recorded from all possible female-female, female-male, and male-male pairs. Each pair was placed together in a semi-anechoic chamber and allowed to interact freely. Pairs were tested twice each week between the ages of 17 and 85 days. No attempt was made in Experiment 1 to distinguish between the structural types of ultrasound produced. Sounds below 20 kHz were not included in the analysis.
Analysis Vocalization rates were submitted to logarithmic transformation (Sokal & Rohlf, 1981) in order to normalize distribution before carrying out analyses. The rates from all pairs within a testing group, that is, composed of female-female, female-male, and male-male individuals, were averaged over 10 day blocks (age) apart from the first block which contained nine days. In total there were seven age blocks. A two-way analysis of variance evaluated the effects of group composition and age on vocalization rates. A subsequent post hoc multiple comparison test (Fisher’s Protected Least Significance Difference (Winer, 1971)) detected differences between groups at each age.
Results and Discussion Rarely did the first animal placed in the chamber vocalize: Vocalizations began when the second animal was introduced. Recording of ultrasounds started 10-30 s later, after the first bout of calling had subsided. Calling was emitted in bursts usually following investigation of the anechoic chamber’s floor or the partner’s head or anogenital region. As animals aged, vocalizations were emitted immediately before or after behavioral interactions which included olfactory investigations and sexual and aggressive play interactions. Emission rates of ultrasonic vocalizations increased significantly as the pups became older, F = 19.46, p = 0.0001, df = 6 (Fig. 1). As the animals approached puberty, rates reached a plateau in all three pairing types and remained at this level until the end of the experiment. A significant difference in vocalization rates between the three groups was found F = 22.13, p = 0.0001, df = 2 (Fig. 1). It was established on the first age of testing, (17-25 days, Fisher PLSD = 0.1 1, p 5 0.05) and disappeared as the various pairing types reached their maximum rates between 36 and 75 days of age (Fig. 1). At the youngest ages (days 17-36), the female-female group produced the greatest rate of calling. The female-female group reached maximum rates of calling sooner than the male-male group (36-45 days);
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r
0
1
*
I
I 17-25
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T
I I I I I I 26-35 36-45 46-55 56-65 66-75 76-85
I
Age (days)
Fig. I . Maturational increases in ultrasonic vocalization rates (mean per min 2 sem) of Mongolian gerbils in paired encounters. Thirty sec of each encounter was recorded. Individuals of each pair came from different litters (n = 6). Unfilled squares represent female-female pairs (n = 34), filled squares represent male-male pairs (n = 34) and female-male pairs (n = 66) are represented by filled diamond symbols. * Different from male-female and male-male groups or t Male-female group only (Fisher PLSD, p 5 0.05).
male vocalizing increased very rapidly between days 36 and 75 (Fig. 1). Analysis of the male-female group revealed that its mean vocalizing rate tended to be midrange between female-female and male-male mean rates. Thus, young gerbils seem to vocalize independently of the sex of their partners, with a greater rate occurred in young females before the ages of between 56-65 days. The communicatory function of the total rate of vocalizations emitted by juveniles is uncertain: a function would also need to incorporate a reason for the sex difference seen. Vocalizations may have a communicatory “contact” function, similar to some of those produced by birds (Hinde, 1952; Thielcke, 1976; Messmer & Messmer, 1956; Gompertz, 1961). If it is assumed that the function of a contact vocalization is to help the development of cohesion within a group of animals, would sex differences be shown? Relationships between the maturing sexes, dependent on the socio-ecological environment, would need to be investigated to answer this question in gerbils. No attempt was made in this experiment to distinguish the frequency of occurrence of different structural types of vocalization (see Experiment 2). Production of a specific type of vocalization may be related to a special context, Calling by males increased rapidly between the ages of 56 to 75 days. During this period testosterone levels begin approaching adult levels (Probst, 1987). This suggests that vocalizations, as well as serving to maintain contact
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between individuals, may indeed also have a significant sexual function. Data from Experiment 2 support this hypothesis.
Experiment 2: Vocalization Structure Vocalizations with different structures emitted by gerbils during mating encounters depend on the stage reached in the sexual interaction (see Introduction). In Experiment 2, we examined in sexually immature gerbils whether (a) ultrasonic vocalizations could be identified and categorized, and (b) the development of juvenile sounds are related to those of the adult.
Method Permanent sound recordings were made during encounters of gerbils (see General Method). Forty-four pairs of animals (female-female, n = 15: femalemale, n = 14: male-male, n = 15) produced vocalizations during three age ranges, 17-25, 26-70, and 71-85 days.
Analysis Criteria by which vocalizations can be classified have been partially determined in preliminary experiments (Holman, Seale, & Hutchison, 1989).Categories of vocalization were distinguished by ear from tape recordings replayed at reduced speed (see Methods). A spectrographic trace in 15-20% of the vocalizations made by pairs verified the structural category. Spectrograms were also made when the category of vocalization was not evident. On average, I0 traces were made from each recording of a particular pair. A G-test of independence (Sokal & Rohlf, 1981) was carried out between numbers of vocalizations of each category, summed in the female-female or male-male groups.
Results and Discussion Description of Vocalizations Vocalizations were very stereotypic (Fig. 2) and therefore easily classified when heard on replayed recordings. The five simple definitions outlined were able to accommodate the majority of vocalizations. Approximately 7% did not fit into one of the five categories [Miscellaneous group (Fig. 211. Inspection of spectrograms revealed no evidence that the ultrasonic vocalizations comprised second harmonics, or formants, above the single fundamental frequency (first harmonic) trace (Fig. 2). In a few (less than 1%) spectrographic traces, both animals emitted sounds almost simultaneously. In all cases, it was obvious that two vocalizations were being produced. Two broad super categories of calls could be distinguished: rectilinear or curvilinear vocalizations.
DEVELOPMENT OF SEXUALLY DIMORPHIC ULTRASOUNDS a. Short rectilinear
d. Tailed curvilinear
b. Long rectilinear
e. Warble
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f. Miscellaneous
c. Steep curvilinear
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6 0-l
1 n
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200
u
0
100
200
ms
Fig. 2. Spectrogram traces representing ultrasonic vocalizations made by juvenile Mongolian gerbils. Six classes were emitted and vocalizations appeared to be stereotypic to each class. Vocalizations b and c were made primarily by males whereas sound e was made by females aged under 25 days.
Rectilinear In our definition, the drift from horizontal was no more than kO.8 kHz from the mid-range frequency. (Fig. 2a,b). Rectilinear calls were subdivided into: Short rectilinear. Calls under 15.1 ms duration. Some short calls were on the borderline between being defined as Long rectilinear or Tailed curvilinear (Fig. 2a). Long rectilinear. Calls over 15 ms duration (Fig. 2b). The mean duration was 58 ms (sem k 9 . 0 ) but some calls were up to 550 ms in length.
Curvilinear This category contained vocalizations which tended to have an ascending sigmoid-shaped spectrographic trace. The end frequency was greater than the starting frequency in most cases (but see Warble curvilinear). In proportion to
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Table I Structural Categories c$ Ultrasonic Vocalizations Emitted b y Mongolian Gerbils of Various Ages during Paired Encounters. Ultrasorinds Were Categorized by Ear. Spectrograms, (Fig. 21, Conjrmed Each Vocalization Category (Method Experiment 2 ) . 5% vocalization Category (mid-range frequency kHz)*
Group
17-25
Age (days) 26-70
71-85
~~
Female-female
Short rectilinear Long rectilineart Steep curvilinear? Tailed curvilinear W arblet Miscellaneous
0.0 0.0 0.0 25.0 (37 kHz) 62.5 (41 kHz) 12.5 (42 kHz)
4.35 (31 kHz) 0.0 4.35 (33 kHz) 82.6 (32 kHz) 0.0 8.7 (32 kHz)
0.0 0.0 16.9 (30 kHz) 76.2 (32 kHz) 0.0 3.9 (32 kHz)
Female-male
Short rectilinear Long rectilinear Steep curvilinear Tailed curvilinear Warble Miscellaneous
5.9 (32 kHz) 5.9 (32 kHz) 0.0 70.5 (36 kHz) 0.0 17.6 (35 kHz)
3.3 13.3 16.7 56.7 0.0 10.0
3.6 (30 kHz) 28.6 (30 kHz) 38.9 (31 kHz) 20.0 (30 kHz) 0.0 8.9 (32 kHz)
Short rectilinear Long rectilinear Steep curvilinear Tailed curvilinear Warble Miscellaneous
0.0 9.0 (34 kHz) 0.0 82.0 (37 kHz) 0.0 9.0 (37 kHz)
0.0 5.3 36.8 47.4 0.0 10.5
(30 kHz) (29 kHz) (34 kHz) (32 kHz) (34 kHz)
~~
Male-male
(29 kHz) (32 kHz) (31 kHz)
(32 kHz)
1.3 (33 kHz) 2.7 (32 kHz) 29.3 (32 kHz) 61.3 (31 kHz) 0.0 5.4 (32 kHz)
* t
Mid-range frequencies calculated from spectrograms. Difference in numbers of vocalizations of each category produced by female-female or malefemale groups (p s 0.05, G-test, 2-tailed. Sokal and Rohlf, (1981)). The total number of spectrograms was summed over all ages.
other calls, Steep and Tailed curvilinear calls were emitted at high rates by paired animals (Table 1). Both types of call were emitted in bouts of 3-5 vocalizations. The mid-range frequency of the Steep curvilinear was similar to the Tailed curvilinear at the various ages (Fig. 2c,d, Table 1). Curvilinear vocalizations were classified into: Steep curvilinear. Vocalizations in which the frequency rise of 7.8 kHz per 25 ms was always exceeded within the first 5 ms. Calls had a duration of less than 1 ms (Fig. 2c). Tailed curvilinear. Vocalizations in which the frequency change within the first 5 ms was always less than or equal to a rate of 7.8 kHz per 25 ms. The slow increase in slope gave the calls a “tailed” appearance (Fig. 2d). Warbled curvilinear. Spectrographic traces showed a series of marked frequency modulations. The vocalization sounded like twittering or warbling sounds at reduced playback speed (Fig. 2e). The maximum durations of calls in this category extended up to approximately 160 ms. In a few examples the lowest frequency of the modulations was less than the starting frequency.
Miscellaneous vocalizutions This category represented vocalizations which could not be included in those above, including dome and basin shaped traces and traces showing an end fre-
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quency considerably less than the starting frequency. They often appeared to be similar to short phases of the Warble category, that is, comprised of one or less than one cycle of modulation (Fig. 2). Vocalizations in the Miscellaneous category may be curvilinear calls which were interrupted or altered by, for example, positions of the animal’s body causing laryngeal or air-way deformations. They were not formed by the superimposition of two vocalizations being made simultaneously as each separate call was distinguishable during such circumstances.
Age and Sex Differences One general feature found was that the mid-range frequencies of a particular type of vocalization decreased as animals became older in all categories of vocalization. Thus, for example, the. mid-range frequency for the Tailed curvilinear category produced by isosexual female pairs fell from 37 kHz to 32 kHz between the youngest and oldest groups (Table 1). Similar decreases were seen in other vocalizations. Theoretically, it could be argued that smaller laryngeal structures, which seem to be the source of ultrasound production in rodents (Thiessen, Kittrell, & Graham, 1980; Roberts, 1975), should produce higher mid-range frequencies than larger structures. We infer that this simple change may communicate the emitter’s age to receivers of the signal. Two structural types of vocalizations were affected by the age of the animal. One vocalization, the Warble, was only made by preweanling (17-25 days) female pups (difference between the numbers of vocalization produced by female-female or male-male groups, G = 57.8, p < 0.01) (Table 1). We have unpublished data, however, that this type of vocalization is also made by preweanling (10-16 days) males, particularly in situations where the animal has just been handled. A vocalization which was recorded only in male-male and female-male pairs was the Long rectilinear call (Fig. 2d). It was not produced by females at any age (G = 5.0, p = 0.05, between female-female and male-male groups, Table 1). The third age-related ultrasound, the Steep curvilinear, was made from day 26 onwards at high rates by male-male and female-male pairs. A significant difference was found between the numbers of vocalizations made by male-male pairs compared to female-female pairs (G = 8.2, p < 0.01) (Table I).
General Discussion Between the ages of 17 (preweaning) and 85 days, Mongolian gerbils emit a varied range of ultrasonic vocalizations during paired encounters. The rate of emissions for the total number of vocalizations also increases as they become older. Gerbils isolated during sound monitoring, however, show a decrease in the rate of ultrasonic calling up to 20 days of age, after this age vocalizations are no longer detectable (De Ghett, 1974). Thus, behavioral interactions induce calling in animals 20 days and older. Categories of vocalizations were stereotypical, although within a category there was some variation in call structure. Therefore, classification of calls was a simple procedure. The total number of vocalizations produced was also different between the sexes in paired encounters, with female-female groups emitting more vocalizations than male-male groups before the age of 56 days. A methodological point which is pertinent to other studies on vocal behavior is that only interacting
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pairs of gerbils produced vocalizations in this study (c.f., DeGhett, 1974). Thus, a vocalization in which the rate is sexually dimorphic is made in a context that is difficult to match between the two sexes (Gyger, Pontet, & Schenk, 1986; Warburton et al., 1989). For example, an ultrasound produced at high rates in male-male and female-male interactions but significantly less in female-female interactions may not be produced by the male because the important variable determining the rate of the vocalization may not be the sex of the emitter but the sex of the partner. That is, the context in which a call is made is critical for the call’s production. Parallel studies using devocalized animals and correlations between emission and other behavioral components need to be performed before this question may be resolved. The three sexually dimorphic categories of ultrasound identified in this study, the Warble, Long rectilinear, and Steep curvilinear may be precursors of sounds made during sexual interactions. Only females were heard emitting the Warble call in this study. It appears that very young animals in discomfort make this category of sound. Mongolian gerbil pups reach their full endothermic potential by about 21 days of age (McManus, 1971) and our first two tests were carried out before this age. Therefore, females, which have generally lower body weight than males, may have been undergoing the effects of falls in body temperature during testing as they were not allowed to huddle with their sibs or parents and thus reduce heatloss or gain heat, respectively. A similar call to the Warble is produced during sexual interactions particularly when the female is not fully in behavioral estrus (the modulated vocalization, Holman, 1980). Aggression often occurs when a female is not in complete estrus during mating (Holman, Hutchison, & Snelson, 1982). Thus, this confirms our suggestion made in Experiment 2 that the Warble call has a distress function. In avian species, it is often the case that during periods of conflict in courtship, birds will use immature patterns of behavior (“displacement activities”) (Hinde, 1970). Long rectilinear and Steep curvilinear made by juvenile males are similar to those made by adult males in sexual interactions (Holman, 1980). During sexual interactions in adults, a vocalization, striking because of its high sound intensity (75 db recorded 12 cm from its source, Holman, 1980), is made by males within 10 s after ejaculation: bursts of this sound continue for about 45 s in the postejaculatory interval. The spectrograph representing the post-ejaculatory ultrasound is similar to the unmodulated vocalization made by immature gerbils: However, the central frequency of the post-ejaculatory ultrasound was lower by approximately 5 kHz than the Long rectilinear call and, although it is not presented in this report, the intensity of the Long rectilinear ultrasound was less intense than the post-ejaculatory call. The Long rectilinear call may be a developmental precursor of post-ejaculatory sounds because of its structural similarity. The structure of the Steep curvilinear resembles that of the masculine vocalization made during courtship sexual interactions, the upsweep (Holman, 1980).Male courtship upsweep vocalizations are similar to those made byjuveniles, in that a greater rate (approximately 96%) is emitted compared with other types of vocalizations. Courtship upsweeps have a bandwidth frequency of 28-32 kHz (mid-range approximately 30 kHz) but tend to be longer (median duration, 19 ms) than the Steep curvilinear call (Holman, 1980). In the present study, we suggest that the Steep curvilinear is produced at greater rates as males mature and that this vocalization, in proportion to others,
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increases as the animals age (Table 1). If the emission rate of the Steep curvilinear sound increases from 17 to 56 days, the difference between emission rates of the sexes would eventually disappear (Fig. 1). As animals mature, therefore, the ratio of the different categories of calls made by the sexes also changes. This would suggest that vocalizations might be selected from a range of categories, described in this study, and subsequently used by adults in the specific contexts where the vocalization may have a clear meaning. For example, the masculine juvenile Steep curvilinear call becomes the male-type upsweep (Holman, 1980). Other variables, such as the effect of gonadal secretions, may also play a role in determining the rate and type of juvenile vocalization. This study provides a model by which the development of vocal behavior may be investigated in terms of relationships between the sexes and their dependency on gonadal secretions. In such an observational study as that reported here, the suggestion of communicatory functions for vocalizations are imprecise and only extensive experimental research would remedy this deficiency. It has been suggested that vocalizations are artifacts of lung compression in body movements in gerbils, such as in locomotion perhaps: air is expelled through laryngeal structures causing ultrasound (Thiessen et al., 1980). We do not support this hypothesis for a number of major reasons. First, few calls were made in the first moments when an animal was alone in the testing chamber even though it was moving while investigating its surroundings. Second, many categories of vocalizations last longer than the time it would take for a normal expulsion of breath. Furthermore, single peaks of pressure intensity are not seen within calls which occur during the lung deflation cycle. Third, calls are of different structural stereotypes, where one would assume that artifacts would tend to vary considerably in physical structure. Also, intensity modulations are seen which are associated with specific types of call. Fourth, sexual dimorphisms exist between the various categories of vocalizations, whereas there was no obvious difference in locomotory activity between the pairs. However, the reasons stated here do not rule out that some vocalizations may be associated with locomotion and have no communicatory value. Nevertheless, the major question that remains unanswered is whether a communication function can be assigned to a precursor of a sexual vocalization in juveniles. We can only suggest that as play sexual motor patterns are seen in gerbils and rats (Meaney & Stewart, 1981) so will sexual play vocalizations be seen. Experiments statistically correlating the various categories of ultrasound to sexual “practice” components in juveniles would provide the basis for this hypothesis.
Notes We wish to thank Drs. John and Rosemary Hutchison and Chris and Magdalena Jams for commenting on, and Miss Helen Potter for typing this manuscript.
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