A maternal chemosignal maintains paternal behaviour in the biparental california mouse, Peromyscus californicus

Anita. Behav., 1990,39, 936-942

A maternal chemosignal maintains paternal behaviour in the biparental California mouse, Peromyscus californicus D A V I D J. G U B E R N I C K

Psychology Department, University of Wisconsin, Madison, Wisconsin 53706, U.S.A. Abstract. Relatively little is known about the proximate mechanisms underlying paternal behaviour in

mammals. Paternal behaviour in the biparental California mouse, Peromyscus californicus, is maintained during the first 3 days postpartum by the male's mate and by maternal excreta. Here, it is reported that (1) maintenance of paternal responsiveness is specific to the male's mate. Fathers exposed to excreta from their mates were significantly more likely to exhibit parental behaviour than fathers exposed to virgin or lactating female excreta, or no excreta. (2) Mere familiarity with a female was not sufficient to maintain paternal behaviour. (3) Maternal urine was sufficient to maintain paternal solicitude. Fathers given 100/A of maternal urine on their nares twice a day were parental compared to fathers similarly exposed to distilled water. (4) The maternal urinary chemosignal(s) was contained in the volatile fraction of maternal urine. The existence of paternal behaviour poses an interesting theoretical problem for evolutionary analysis. Males normally maximize reproductive success by mating with several females, whereas female fitness is ordinarily limited by time and energy constraints (Trivers 1972). Females are likely to benefit from increased male parental assistance (Wittenberger & Tilson 1980). However, any male investment in one female's offspring necessarily reduces the male's chances of inseminating other females. Thus, if a male shifts his reproductive effort from mating to parental care, he forfeits some of his potential reproductive success. This situation appears to create an insurmountable evolutionary barrier to male parental care (Kurland & Gaulin 1984). Paternal behaviour is uncommon in mammals and occurs primarily among rodents, carnivores and primates (Kleiman & Malcolm 1981). Species in which males display parental care are of particular importance for understanding conditions favouring the evolution of paternal behaviour in mammals. Assuming that paternal behaviour confers some adaptive advantage to males, then natural selection should operate to provide the proximate machinery (mechanisms) necessary to ensure the emergence of male care (Kurland & Gaulin 1984). Relatively little is known about proximate mechanisms underlying paternal behaviour in mammals (Daly & Wilson 1978), whereas much is known about the proximate causation of maternal behaviour, particularly in rodents (Rosenblatt & 0003-3472/90/050936 + 07 $03.00/0

Siegel 1981; Elwood 1983a). Studies of male parental care in rodents are largely descriptive (Elwood 1983b) and, with few exceptions, provide little information about underlying mechanisms. To understand paternal behaviour, we must ultimately integrate proximate and ultimate explanations and different levels of analysis. It is within this context that I have been exploring the proximate causes of male parental care through investigation of the biparental care system of the California mouse, Peromyscus californicus (Gubernick & Alberts 1987). Peromyscus californicus offers a model system for analysis of mammalian paternal care because it is a social species characterized by persistent, if not monogamous, association between males and females (Dewsbury 1981; Ribble & Salvioni, in press). Furthermore, and most importantly, males invest extensively in the care of their young and exhibit all the components of parental behaviour displayed by mothers and to the same extent, except lactation (Dudley 1974; Gubernick & Alberts 1987). Different proximate mechanisms underlie the postpartum maintenance of paternal and maternal behaviour in P. californicus (Gubernick & Alberts 1989). During the first 3 days postpartum, pup stimulation is indispensable for the maintenance of maternal behaviour. In marked contrast, pup stimulation is not essential for maintenance of paternal care. Rather, it is the presence of the mother that maintains parental responsiveness in male P. californicus. In the absence of pup stimulation, fathers were more likely to exhibit parental

9 1990The Association for the Study of Animal Behaviour 936

Gubernick: Maternal chemosignal behaviour if the mother was present than if she was absent. Maintenance of maternal behaviour was unaffected by presence of the male (Gubernick & Alberts 1989). An olfactory cue(s) from the female was implicated in the maintenance of paternal responsiveness. Fathers exposed to maternal excreta (urine, faeces and other olfactory substances) via a modified drip metabolism cage were parental compared to unexposed fathers (cf. Gubernick & Alberts 1989 for details). In experiments 1-3 below, I examined whether maintenance of paternal behaviour is: (1) specific to the male's mate, (2) due to mere familiarity with a female, and (3) mediated by a chemosignal(s) in maternal urine. In experiment 4, I examined whether the maternal chemosignal(s) is contained in the volatile fraction of maternal urine. EXPERIMENT1

A cue(s) in maternal excreta is sufficient to maintain male parental care in P. ealifornieus for at least the first 3 days postpartum (Gubernick & Alberts 1989). In species that form persistent pair bonds between males and females, such as P. ealifornieus (Eisenberg 1962, 1963), it is possible that fathers are more responsive to maternal cues than to cues from other females (Mendoza & Mason 1986). Disruption of the pair bond reduced paternal responsiveness, whereas maternal excreta reinstated paternal solicitude (Gubernick & Alberts 1989). In experiment 1, I determined whether exposure to excreta from the male's mate, a virgin female or another lactating female would equally maintain paternal behaviour during the first 3 days postpartum. Methods

Subjects Eighty adult male P. ealifornieus 120-150 days old at the start of the study served as subjects. Animals in all studies were laboratory reared and were descendants of mice originally captured in the Santa Monica mountains, northeast of Los Angeles, California. A virgin male and female were paired and housed together continuously in a standard mouse maternity cage. Purina Mouse Chow and water were provided ad libitum and cages were cleaned each week. The colony room was maintained at 2 3 _ I~ with a 16:8 h light:dark cycle initiated at 0700 hours.

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Procedure On the day of birth (day 0), fathers were separated from the mothers and their young. Males were placed into a standard mouse cage measuring 27-9 • 19 • 12.5 cm that formed the lower level of a specially constructed two-storey cage. The upper level consisted of a standard mouse cage with a metal nestbox attached to one end. The floor was removed and replaced with 0.64-cm hardware cloth that allowed urine and faeces to fall into a metal trough measuring 27-3 • 17.8 • 0-16 cm. The sides of the trough were attached to the upper level and were angled inward to form a slit (0.3 cm wide) that ran the length of the trough. The trough fitted into the male's cage and acted as a cover to prevent the male's escape. The slit permitted urine and faeces to drop into the male's cage below and prevented the male from having visual access to the upper level. For one group of fathers (N= 20), their mate and young were placed in the upper level on the day of birth (within 4 h after parturition). For another group of fathers ( N = 20), an unfamiliar lactating female and her young were placed in the upper level on the day of birth. The female was at the same stage of lactation as the male's mate. For a third group of fathers (N=20), a virgin female was placed in the upper level. Fathers were thus exposed to female excreta until the time of testing. The final group of fathers (N = 20) lived in the lower level of the cage until testing but were unexposed to female excreta. On day 3 postpartum, the upper level and trough were removed and replaced with a wire lid and the male remained undisturbed in the lower level for 60 min prior to testing. Males were given a standard 10-min test with a single alien pup, 1-3 days old. The pup was placed in the centre of the cage. Animals in this and subsequent experiments were tested only once and were used in only one experiment. A male was considered parental if it spent 1 min or more either licking the pup or crouched over it in a nursing posture. Parental mice typically spent 2-6 min licking a pup or huddled over it. Nonparental males either ignored or attacked the test pup. When attacks occurred, we immediately hit the top of the cage to disrupt the aggression. The test was ended, the pup removed and returned to its parents and reared normally. This was a fairly successful procedure, but about 10% of attacked pups still were killed. Each pup was used only once. The 10-min test was sufficiently long to elicit parental behaviour reliably. Attacks occurred typically

Animal Behaviour, 39, 5

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Figure 1. The percentageof fathers exhibitingparental ( 9 or non-parental (attack: []; ignore: []) behaviour towards a test pup on day 3 postpartum. Fathers were removed from their mate and pups and were either exposed to excreta from their mate (a) or no excreta (b), or excreta from a virgin female(c) or another lactating female(d). N= 20 males/group. within 1 min of pup presentation. Animals that ignored the test pup in the first 10 rain continued to ignore the pup 30 min later.

Data analysis For statistical analyses, I used the two-tailed chisquared test at 0-05 level of significance. All tests were in the form of either 2 x 4 (experiment 1) or 2 x 2 contingency tables which compared the number of fathers that were parental versus those that were non-parental (attack + ignore) under the different experimental conditions. Because sample sizes differed between experiments, data are presented in the Figures as the percentage of mice exhibiting parental or non-parental behaviour, whereas frequencies were used for statistical analyses.

Results and Discussion Significantly more fathers exposed to their own mate's excreta were parental (95%) compared with father's exposed to excreta from another lactating female (50%), a virgin female (45%), or unexposed fathers (40%; Z2= 15.8, df=3, P<0.01). The percentage of fathers that were parental towards, attacked or ignored the test pup as a function of exposure to female excreta is shown in Fig. 1. These results replicate my previous findings (Gubernick & Alberts 1989) and extend them to indicate that paternal responsiveness is maintained specifically by excreta from the male's mate.

EXPERIMENT

2

In experiment 1, males were familiar with their mate and presumably with her excreta. Fathers

exposed to their mates' excreta in the two-storey cage were thereby exposed to familiar olfactory cues, whereas the other fathers were exposed to excreta from unfamiliar females. In experiment 2, I examined whether mere familiarity with a female would be sufficient to maintain paternal responsiveness.

Methods

Subjects A total of 30 males served as subjects. Maintenance conditions were the same as described earlier.

Procedure An adult male was paired with a virgin female and placed into a cage measuring 45.7x 25.4 x 15"2 cm made of galvanized metal equipped with a hardware cloth lid. Another virgin female (hereafter 'familiar' female) was placed into the same cage and separated from the pair by a double wire-mesh barrier (the familiar-female compartment measured 15.2 x 25.4 • 15.2 cm). The wiremesh barrier prevented physical contact between the pair and the familiar female but allowed for olfactory, auditory and visual communication. The animals were housed in this way until the male's mate gave birth. On the day of birth, the male was removed and placed into the lower level of the two-storey cage described earlier. For one group of fathers (N = 15), the male's mate and young were placed in the upper level on the day of birth (within 4 h of parturition)

Guberniek: Maternal ehemosignal 90'

939

Methods

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Subjects 7o

A total of 30 adult males served as subjects. Housing and maintenance conditions were the same as those in experiment 1.

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Figure 2. The percentage of fathers exhibiting parental or non-parental behaviour towards a test pup on day 3 postpartum. Fathers were either exposed to excreta from their mate (a) or excreta from a familiar virgin female (b). N= 15 males/group. See Fig. 1 for details.

and males were thus exposed to maternal excreta until the time of testing. For a second group of fathers (N=15), the familiar virgin female was placed in the upper level on the day of birth and fathers were thus exposed to excreta from the familiar female. Males were given our standard 10min test on day 3 postpartum with a single alien pup, 1-3 days old, as described earlier.

On the day of birth, a father was removed from his mate and young within 4 h after parturition and was placed into a clean mouse maternity cage for the remainder of the experiment. One group of fathers (N= 15) was exposed daily to their mates' urine. To ensure contact with the urine, 100/A of maternal urine was expressed twice a day via a micropipette directly onto the male's nares. Males were thus exposed for 3 days to maternal urine. We collected urine daily from mothers by holding a female over a petri dish and gently rubbing her flanks. A second group of fathers (N= 15) was similarly treated with 100 pl of distilled water twice a day for 3 days. Males were given our standard 10-min test on day 3 postpartum with a single alien pup, 1-3 days old, as described earlier. Results and Discussion

Results and Discussion Significantly more fathers exposed to their mates' excreta were parental (66.7%) compared with fathers exposed to excreta from a familiar female (26.7 %;Z 2 = 4.8, df= 1, P < 0-05), indicating that mere familiarity alone is not sufficient to maintain paternal responsiveness (Fig. 2).

Significantly more fathers exposed to maternal urine on their nares were parental (80%) compared with fathers exposed to distilled water (26.7%; Z2 = 8.6, dr= 1, P < 0.01), indicating that maternal urine is the critical stimulus sufficient to maintain paternal responsiveness (Fig. 3).

EXPERIMENT4 EXPERIMENT 3 The results of the previous two experiments indicate that paternal behaviour is maintained postpartum specifically by the male's mate and that mere familiarity with a female is not sufficient to maintain paternal responsiveness. The relevant cue(s) that maintains paternal behaviour is contained in maternal excreta (urine, faeces, etc.). Urinary chemosignals are potent regulators of reproduction and social behaviour in rodents (Vandenbergh 1983). In experiment 3, I examined whether a chemosignal(s) in maternal urine was sufficient to maintain paternal responsiveness.

Experiment 3 demonstrated that a chemosignal(s) in maternal urine was sufficient to maintain paternal responsiveness postpartum. Because urine was placed directly onto the nares, males were exposed to both the volatile and non-volatile fraction of maternal urine. The non-volatile fraction of urine is of a higher molecular weight than the volatile fraction and must generally be contacted directly to be detected. The volatile, lighter weight, fraction of urine is air borne and detectable at a distance (Vandenbergh 1983). If the volatile fraction of maternal urine is involved in maintenance of paternal behaviour, then males allowed to sff~ff maternal urine, but prevented direct physical contact with the urine, should still be parental relative

Animal Behaviour, 39, 5

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Figure 3. The percentage of fathers exhibiting parental or non-parental behaviour towards a test pup on day 3 postpartum. Fathers were either exposed to maternal urine (a) or distilledwater (b) on their nares (experiment3, N= 15 males/group) or were exposed to, but prevented contact with, maternal urine (c) or distilled water (d) (experiment 4, N= 20 males/group). See Fig. 1 and text for details. to unexposed males. In experiment 4, I examined whether the volatile fraction of maternal urine was involved in maintaining paternal behaviour.

standard 10-min test on day 3 postpartum with a single alien pup, 1-3 days old, as described earlier.

Results and Discussion Methods

Subjects A total of 40 adult males served as subjects. Housing and maintenance conditions were the same as those in experiment 1.

Procedure The procedures were the same as those in experiment 3, except that males were exposed to 100/~1of urine twice a day in a plastic vial instead of on their nares. The vial was a Monojet 1-cm3 tuberculin syringe case and removable top measuring 9.2 cm long x 1.3 cm in diameter. Two holes measuring 0-64cm in diameter were drilled opposite each other approximately 2-86 cm from the bottom of the vial. The holes allowed the mouse to insert his nose into the vial but prevented contact with the cotton pellet at the bottom of the vial. The urine was expressed into the cotton pellet with a micropipette through the top of the vial. The urine did not touch the inner surfaces of the vial or the holes. We removed and replaced any males that chewed through the vials. The vial was suspended through the stainless steel lid of the male's cage within 2 cm of the male's nest. One group of fathers ( N = 20) was exposed to a vial containing their mate's urine for 3 days and another group of fathers (N=20) was similarly exposed to distilled water, Males were given our

Significantly more fathers were parental when exposed to the volatile fraction of their mate's urine (65%) than fathers similarly exposed to distilled water (30%; X2 = 4.9, dr= 1, P < 0,05; Fig. 3). These results indicate that the volatile fraction of maternal urine helps maintain paternal responsiveness. We cannot from these results rule out the possible involvement of the non-volatile fraction. GENERAL DISCUSSION The results of the present series of experiments indicate that postpartum paternal behaviour in P. californicus is maintained'specificallyby the male's mate and that the effect is not due to mere familiarity with a female. These results suggest that males individually recognize, and are differentially affected by, their own mates. In species that form persistent pair bonds between males and females, such as P. ealifornicus, males may be more responsive to cues from their mate than from other females. It is possible, however, that a familiar pregnant and subsequently lactating female might also maintain paternal responsiveness. This possibility remains to be explored. The vast majority of males exhibit parental behaviour only when exposed to maternal chemosignals, whereas approximately 26-45% of males in the various control groups continued to exhibit parental behaviour even in the absence of a mater-

941

Gubernick: Maternal chemosignal nal signal. I cannot account for this variation between males. Perhaps for some males a chemosignal or experience that occurs during the female's pregnancy is sufficient to maintain male care postpartum. However, for most males a postpartum maternal chemosignal appears essential. Perhaps the most interesting finding of the present study is that paternal solicitude in male P. californicus is mediated by a chemosignal(s) in the mother's urine. Mammalian chemosignals, or pheromones, affect a wide variety of behaviours including acceleration (Vandenbergh 1967) and delay (Drickamer 1977) of female sexual maturation, pregnancy block (Bellringer et al. 1980), induced ovulation (Johns et al. 1978) and mating behaviour (Winans et al. 1982). The maternal urinary chemosignal(s) that maintains paternal responsiveness in P. californicus represents another functional class of chemical signals known to influence social behaviour. In P. californicus, maternal urine also delays sexual maturation of the mother's daughters (unpublished data). It remains to be explored whether this chemosignal(s) is the same as the maternal urinary chemosignal(s) that maintains paternal behaviour. The chemosignal(s) maintaining paternal responsiveness is contained in the volatile fraction of maternal urine. Some success has been achieved in identifying the sexual attractant in hamster vaginal secretion (Singer et al. 1976; Macrides et al. 1977), puberty acceleration chemosignal (Vandenbergh et al. 1976) and chemosignals promoting inter-male aggression in mice (Novotny et al. 1985). We are currently working on the isolation, identification and synthesis of critical compounds in the volatile fraction of maternal urine that maintain the male's parental behaviour. Recent field data indicate that in P. californicus a male's home range overlaps with that of just one female and that the male and female spend time together in the nest (Ribble & Salvioni, in press), although they may also maintain separate nests. We found, with the aid of fiber optics, that a male will also be in the nest with the female and her young (Gubernick & Ribble, personal observations). Thus, males are likely to be exposed to their mate's chemosignals prior to and following birth of their young. More field research is needed to elucidate further why the proximate mechanisms underlying paternal behaviour in P. californicus are designed to operate the way they do. The biparental care

system of P. caliJbrnicus affords the opportunity to test and to integrate proximate and ultimate explanations and different levels of analysis into a broader, more complete view of parent-offspring relations.

ACKNOWLEDGMENTS This research was supported by the National Institute of Child Health and Human Development (Grant HD-21233).

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(Received 5 June 1989; initial acceptance 20 July 1989; final acceptance 14 August 1989; MS. number: A5583)