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Female gestation and lactation as zeitgebers for circannual rhythmicity in the hibernating ground squirrel, Citellus lateralis
Camp. Biochem. Physiol., 1915, Vol. %A, pp. 621 to 625. Pergamon Press. Printed in Great Bntain
FEMALE GESTATION AND LACTATION AS ZEITGEBERS FOR CIRCANNUAL RHYTHMICITY IN THE HIBERNATING GROUND SQUIRREL,
CITELLUS
LATERALLY
E.T. PJJNGELLEYAND SALLY J. ASMUND~~N Department of Biology, University of California, Riverside, California 92502, U.S.A. (Received 16 November 1973) Abstrati-1. Free-running circannual rhythms in females of the hibernator, Citellus lateralis, have been studied in relation to breeding. 2. It has been demonstrated that, in comparison to controls, females which go through gestation and lactation in the spring, and are subsequently kept in constant environmental conditions, hibernate with a more accurate phase relationship to the presumptive seasons. 3. Breeding as a zcitgebcr for the circammal rhythm is discussed, and the mechanisms involved in spring arousal are speculated upon.
INTRODUCTION THE PROBLEM of determining
entraining agents or zeitgebers for endogenous circannual rhythms have been discussed by Pengelley & Fisher (1963, 1966), Pengelley (1967, 1969), Pengelley & Asmtmdson (1969, 197Oa, b, 1972a, b), Heller & Poulson (1970) and Pengelley et al. (1972). It has been suggested as a result of experimentation that in ground squirrels temperature (Pengelley & Fisher, 1963), light (Pengelley & Asmundson, 197Oa) and locomotor activity (Pengelley & Fisher, 1966) may all be involved, but the results have not been conclusive when compared to experiments involving circadian rhythms. In birds, however, photoperiod is probably the primary zeitgeber (Gwirmer, 1967, 1968, 1969) as might be expected. On the other hand, Heller & Poulson (1970) have suggested that in the ground squirrel, Citellus lateralis, the free-running period of the rhythm may be so close to 365 days that no zeitgeber is necessary. This seems unlikely, for after much experimentation it seems probable that under controlled and constant conditions, and after the initial period, the free-running circannual rhythm is nearly always less than a year and it seems far more likely that as Pengelley et al. (1972) have pointed out, there are many and complex interacting zeitgebers involved. In the case of circadian rhythms, light in one form or another is almost invariably a zeitgeber (Pittendrigh, 1958, 1960; Halberg et al., 1959; Aschoff, 1960, 1963, 1965) which keeps the endogenous period of oscillation in phase with the environmental 24hr oscillation. Researchers have somewhat inevitably assumed this would be the case in circannual rhythms also. However, with the greatly increased complexity of
exogenous factors influencing a circarmual period, it seems highly probable that no simple solution can be expected. Furthermore, in ground squirrels light would not be expected to play such a dominant role as in birds, since the animals spend so much of their time underground, and perhaps half or more of the year in a state of hibernation where such a stimulus is likely to have a minimal effect. Thus it seems necessary to look more closely for various zeitgebers which may interact with the endogenous circannual rhythm keeping it in phase with the oscillation of the seasons. Since pilot experiments (Pengelley & Asmundson, 1971) showed that males and females of C. lateralis seemed to behave differently so far as hibernation was concerned, and since the one major event which females in the wild undergo which does not occur in males, is gestation and lactation, it occured to us that the latter two events might be involved as circamual zeitgebers in the wild. Since C. lateralis females successfully raise litters in the laboratory (Pengelley, 1966) an experiment was designed utilizing this information to test the effect of breeding on circannual rhythmicity. MATEFUALS AND METHODS From previous studies (Pengelley, 1966) it was known approximately at what time we could expect the females of the golden-manteled ground squirrel, Cite&s lateralis, to be pregnant in the wild state. Accordingly between 4 and 15 May 1970, some twenty females were trapped, at an altitude of about 7500 ft. near Bia Bear Lake in the San Bernardino Mountains of Calif&nia. They were brought to the laboratory and housed in individual cages in a quiet room with a photoperiod of 12 hr and an ambient temperature of 23+ 1°C. Purina lab chow 621
622
E. T. PENGELLEYANDSALLYJ. ASMUNDSON
and water were supplied ad lib. and diet was supplemented by carrots for pregnant and lactating squirrels. Of the twenty animals nine produced and successfully raised litters. Young were born between 14 May and 4 June after a gestation period of about 35 days. The young ground squirrels can be weaned at 32 days after birth with 100 per cent survival, and they were in fact weaned at about 35 days. Under these conditions and at this time of year ground squirrels rapidly gain weight and on 31 July, which is long before any hibernation would normally be expected, they were divided into two groups of nine each, i.e. those which had had litters and those which had not, and both groups were placed in environmental chambers with a constant 12 hr artificial photoperiod and a constant 35 1°C ambient temperature. Throughout the subsequent experiment food and water were available ad lib. The animals were carefully observed every day, though disturbances were kept to a minimum, and hibernation was determined by the method of Pengelley & Fisher (1961). It is important to note that these ground squirrels have distinct periods (months) of hibernation during which they are heterothermic, and these alternate with periods of activity during which the animals are active and homothermic. While an animal is in the heterothermic state, it has a body temperature close to the ambient, but within these long periods of heterothermy there are short arousals during which the animal becomes fully active
with a homothermic temperature of 37”C, maintains this for a few hours and then enters hibernation again. These are technically referred to as “periodic arousals” but for the purpose of this experiment they are not considered, and reference is only made to the entire heterothermic period of several months. Accurate weight records of each animal were kept by methods which do not disturb their hibernating behavior (Pengelley & Asmundson, 1969).
No litters
Litters
1970 Julv-i
-
1971 Jan 1971
July
1972 1972 Jan
‘11
x
11111 I
July
x
I
1973 Jan 1973
to cold / July
t
1974faaJ
RESULTS
Fig. 1. Graphical representation of free-running circannual neriods of eighteen female animals (C. lateralis) for over 3 years. upper group of nine had a litter in spring 1970, lower group had no litters. Black bars indicate heterothermic period, clear space homothermic period and the beginning to beginning of each black bar x= indicates one free-running circannual period. death; “to cold” indicates transfer of animals from 22 to 3°C. Artificial photoperiod throughout of 12 hr.
The data for this experiment which have been collected over three complete free-running circannual periods are summarized and expressed graphically in Fig. 1. The symbols are explained in the caption, but it is important to note that the horizontal time scale is over 3 years. Prior to the words “to cold” (31 July 1970) all animals were under constant environmental conditions of 22°C and an artificial photoperiod of 12 hr, and after that time were constantly at 3 + 1°C with the same artificial photoperiod. The free-running circannual periods are measured from the beginning of each heterothermic period to the beginning of the next. Figure 1 clearly indicates several things. The group which had litters in the spring of 1970 hibernated for the first time considerably later than the no litter group. In this respect there is no overlap between the two groups. Analysis using a twotailed t-test for small samples indicates that for each group the number of days of activity before the initial hibernation is significant beyond the 0.001 level. By 3 September all animals but one in the no litter group had entered hibernation, while
in the group with litters the onset of hibernation was spread over the months of September and October. From field observations it is known that the latter situation is the one that is much closer to that in the wild state, i.e. the group with litters hibernated more closely in synchrony with the presumptive autumn. The time of onset of the second period of hibernation, i.e. the beginning of the second free-running circannual period, is also significantly different, beyond the 0.001 level, between the two groups. The group which had had a litter in the spring of 1970 continued to hibernate later in the fall of 1971 and more synchronously with the presumptive autumn. Thus the difference in the phase relationship of the two groups is quite clear for at least two free-running circannual periods. By the beginning of the third circannual period the difference, while still apparent, is not statistically significant. Table 1 summarizes the actual number of days in each free-running circannual period for all animals in the experiment and from these data it is possible
Circammal rhythmicity in the hibernating Table 1. Comparison of tirst, second and third freerunning circammal periods (onset to onset of hibernation) in days of C. lateralis under constant environmental conditions of 3°C and 12 hr photoperiod Free-running
circannual
periods
(days) Animals with litters in Spring 1970 1 2 3 4 2 7 8 9 Animals with no litters 10 11 12 13 14 15 16 17 18
First (1970-71)
Second (1971-72)
Third (1972-73)
353 397 392 392 346 361 356 317 301
337
310
306 295 274 314 350 349 320
334 311 313 277
348
337
304 297 323 302 337
318 345 336 341
355 345 363 362 376 343 340 383 281
336
300
Animals 1-9 had litters in the Spring of 1970, and animals IO-18 had no litters.
to draw certain inferences. There does not appear to be any significant difference between the two groups in the length of the lirst or subsequent freerunning periods and the individual variations are in keeping with previous observations. Thus the free-running period of the female animals was not altered by their having a litter. However, in both groups the second free-running period is significantly less than the iirst, but the third, while less than the first, is not less than the second. It is also true that the 6rst free-running period is closer to the presumptive annual period of 365f days than either the second or third periods. It may also be noted here that ah animals in the experiment exhibited an endogenous body weight rhythm synchronous with the circannual rhythm of homothermy-heterothermy and there were no apparent differences between the two groups. The body weight is at a maximum at the beginning of the heterothermic phase and at a minimum shortly after the beginning of the homothermic phase (Pengelley & Fisher, 1963; Pengelley & Asmundson, 1969). DISCUSSION The French physiologist Dubois (1896) and later Kayser (1940) and Lyman (1954) were all aware of
ground squirrel
623
the temporal nature of hibernation in a variety of animals and the fact that this was not a simple response of an animal to some environmental stimulus. However, it is only in the last 10 years or so that some of the problems have been seriously studied. As Pengelley & Asmundson (1972a) have pointed out, the basic problem can be stated as follows: how do animals repeatedly perform the correct behavioral or physiological act at the specific time during the year when presumably it is most beneficial for the biological fitness of the species? The answer to this question seems to be that there are only three possible ways that an animal can synchronize its behavioral physiology to a changing environment. These are (a) a direct response to some changing geophysical stimuli, and there are more of these than is commonly realized (Weihaupt, 1964); (b) an endogenous rhythm which programs the animal’s behavior to the exogenous temporal period, i.e. 24 hr or 365$ days; or (c) a combination of both. So far as the temporal period of 24 hr is concerned, there seems very little doubt that animals and plants, from single-celled protists to the most highly developed animals, have evolved a combination of both mechanisms by which various zeitgebers entrain an endogenous circadian rhythm. Aschoff (1964) has explained the survival value of such mechanisms and these mechanisms are even more likely to have great survival value during a circammal period. Periodic reproduction in all animals is an essential for survival; and it is nearly always the case that, in order for the young to survive, reproductive activities must take place at rather specific times during the annual cycle of the seasons. The mechansisms by which this is ensured depend on the species and are of great variety, but it was the work of Rowan (1926, 1938) that clearly linked photoperiodism, reproductive periodism and the annual migrations of birds, thus elucidating a mechanism for the accurate timing of reproductive activities. Later, Bissonette (1935) showed that modification of some mammalian sexual cycles was possible by manipulation of the photoperiod. From both field observations, previous laboratory work and the results of these experiments, it appears quite certain that in these ground squirrels mating takes place immediately or very soon after emergence of the females from hibernation and that the young are born and quickly raised in the early spring. This not only has survival value for the young but, as these experiments show, production of a litter in the spring also helps to program the mother to enter hibernation in the fall at the most advantageous time. Thus gestation and lactation in the females are in fact acting as zeitgebers; for, as previously explained, the group with no litters hibernated much too early for the presumptive fall and, in consequence, aroused too early from the heterothermic
621
E. T. PENC;ELLEY AND
phase for the presumptive spring. In fact, most aroused during the presumptive winter which in the wild would probably be fatal. If it had been possible to get the group which had had a litter in the spring of 1970 to reproduce again in the spring of 1971, it appears highly probable that the onset of hibernation in the fall of 1971 would have been delayed so that they would have remained better in phase with the presumptive seasons and the difference with the no litter group would have been even more marked. The fact that there is no difference in the free-running circannual period for the first year between the two groups indicates that the endogenous circannual rhythm can run close to the period of the exogenous annual rhythm for at least a year, even under very artificial conditions. However thereafter, the evidence is clearly that one or more zeitgebers are necessary for entrainment, and from these experiments it is clear that for females one of these is in fact the production of a litter in the spring. This of course has the dual function of ensuring the survival of the species, reproductively, and ensuring the survival of the mother in her complex adaptations to a harsh environment. It has already been noted that an animal, such as C. lateralis, which spends most of the winter underground and in a state of hibernation, is most unlikely to have its physiology influenced at this time of year by photoperiod, and one may justly ask the question: what in fact causes it to arouse in the spring so that reproduction can be assured? It has been suggested by earlier workers that possibly this was due to the fact that the stored fat which the animals consume during hibernation became depleted by the spring and that this caused arousal. However, Pengelley & Fisher (1963) clearly demonstrated that this was not the case, for when they sacrificed animals which had just aroused from the heterothermic state, they found that the animals still had ample fat available; in fact, it was estimated that there was enough for another 6 months of hibernation. It is probable also that any marked increase in burrow temperature can also be ruled out as a mechanism of arousal in the spring; thus we are left with some kind of endogenous clock mechanism which is programmed in such a way that during the months of hibernation it brings the animals’ physiology to a state where mating and reproduction are possible. On the other hand, it is important to note that reproductive competence per se is not necessary either for spring arousal or the maintenance of a circannual rhythm under constant conditions. Pengelley & Kelly (1966) have shown that castrated animals do not behave differently in these respects from normal animals. This does not negate light and ambient temperature as zeitgebers, for these, as pointed out previously have been clearly implicated (Pengelley & Fisher, 1963; Pengelley & Asmundson,
SALLYJ. ASMUNDSON
1970a) as possible zeitgebers in the long run of circannual rhythmicity, but simply that they are unlikely to cause actual arousal in the spring which is so important for reproduction. It is not known whether spring breeding has a similar effect in males in terms of programming the time of fall hibernation. However, there are some indications that the male ground squirrels rhythm is not as precise as that of the female. In conclusion it may be noted that in addition to gestation and lactation, it follows inevitably that for females of this species, a sexually active male at the right place and the right time is in fact a circannual zeitgeber. Acknowledgements-This study was supported by Grant No. GB-40827 from the National Science Foundation, and secondarily by an Intramural Grant from the University of California. We wish to thank Mr. Brian Barnes for his able technical assistance. REFERENCES ASCHOFF J. (1960) Exogenous and endogenous components in circadian rhythms. Cold. Spring Harb. Symp. quart. Biol. 25, 1 l-28. ASCHOFF J. (1963) Comparative physiology: diurnal rhythms. Ann. Rev. Phyiiol. 25, 581400. ASCHOFF J. (19641 Survival value of diurnal rhythms. Symp. Zooi So;. Lond. 13, 79-98. AXHOFF J. (1965) Circadian Clocks. North Holland, Amsterdam. BI~~ONETTET. H. (1935) Modification of mammalian sexual cycles. J. exp. Zoo/. 71, 341-373. DUBOISR. (1896) J%ude sur le mkhanisme de la thermogenese et du sommeil chez les mammifhres. Physiologie cornpar&. de la marmotte. Ann. Univ. Lyon 25, l-268. GWINNER E. (1967) Circannuale Periodik der Mauser und der Zugunruhe hei einem Vogel. Naturwiss. 54,447. GRINNER E. (1968) Circannuale Periodik als grundlage des jahreszeitlichen Funktionswandels bei ZugvGgeln. Untersuchungen am Fitis (PhylIoscopus trochilus) und am Waldlaubslnger (P. sibilatrix). J. Ornithoi. 109, 70-95. GWINNER E. (1969) Untersuchungen zur Jahresperiodik von Laubstingem. J. Omithol. 110, 1-21. HALBERGE., HALBERGF., BARNUM C. P. & BITTNER J. J. (1959) Physiologic 2-hr periodicity in human beings and mice, the lighting regimen and daily routine. In Photoperiodism and Related Phenomena in Plants and Animals, pp.803-878. Amer. Assoc. Adv. Sci., Washington, Pub. 55. HELLER H. C. & POUL~~N T. L. (1970) Circannian Endogenous and exogenous factors rhythms-II. controlling reproduction and hibernation in chipmunks (Eitamias) and ground squirrels (Spermophilus). Comu. Biochem. Physiol. 33. 357-383. KAYSE; C. (1940) &sai d’ahalyse du m&anisme du sommeil hibemal. Ann. Physiol. 16, 313-372. LYMANC. P. (1954) Activity, food consumption and hoarding in hibernators. i Mammal. 35, 545-552. PENGELLEYE. T. (1966) Differential developmental patterns and their adaptive value in various species of the genus Citellus. Growth 30, 137-142.
Circannual rhythmicity in the hibernating PENGELLEY
E. T. (1967) The relation of the external conditions to the onset and termination of hibernation and estivation. In Proc. III Intl. Symp. Mammal. Hi6 (Edited by FISHERK. C., DAWE A. R., LYMAN C. P., SCH~NBAUM E. & SOUTHF. E., JR.), pp. l-29. Oliver & Boyd, Edinburgh. PENGELLEY E. T. (1969) Influence of light on hibernation in the mojave ground squirrel, Citellus mohavensis. In Physioiogicaf Systems in Semiarid Environments (Edited bv HOFF C. C. & RIEDESELM. L.). nv. 11-16. University of New Mexico Press, Albuquerque. PENGELLEYE. T. & ASMUNDSONSALLYJ. (1969) Freerunning periods of endogenous circamrian rhythms in the golden-mantled ground squirrel, Citellus lateralis.
Comp. Biochem. Physiol. 30, 177-183.
PENGELLEYE. T. & ASMUNDSONSALLY J. (1970a) The effect of light on the freerunning circannual rhythm of the golden-mantled ground squirrel, Citellus lateralis. Comp. Biochem. Physiol. 32, 155-160. PENGELLEYE. T. & ASMUND~~N SALLY J. (1970b) Circannual rhythmicity-evidence and theory. Life Sciences and Space Research VIII (Edited by LAVOR~TE F. G. & VISCHNIACW.), pp. 235-239. Proc. XII Plenary Meeting, COSPAR, Prague, Czechoslovakia. North-Holland, Amsterdam. PENGELLEYE. T. & ASMUND~~NSALLYJ. (1971) Circannual rhythmicity in hibernating ground squirrels, Citellus lateralis, in relation to sex and opportunity to breed. Vol. IX. Proc. XXV Intl. Union Physiol. Sci. Munich, Germany. PENGELLEYE. T. & ASMUND~~NSALLY J. (1972a) An analysis of the mechanisms by which mammalian hibernators synchronize their behavioral physiology with the environment. In Hibernation and Hypothermia, Perspectives and Challenges, pp. 637-661. Elsevier, Amsterdam. PENGELLEYE. T. & ASMUNDSONSALLY J. (1972b) Variations in hibernating behavior of the ground squirrel, Citellus lateralis, in response to day length and intensity of light. Cryobiology 9, 308-309.
ground squirrel
625
PENGELLEYE. T., BARTHOU~MEWG. A. & LIGHT P. (1972) Problems in the chronobiology of hibernating mammals. In Hibernation and Hypothermia, Perspectives and Challenges, pp. 713-716. Elsevier, Amsterdam. PENGELLEYE. T. & FISHER K. C. (1961) Rhythmical arousal from hibernation in the golden-mantled ground squirrel, Citellus lateralis tescorum. Can. J. Zool. 39, 105-120.
PENGELLEYE. T. & FISHER K. C. (1963) The effect of temperature and photoperiod on the yearly hibernating behavior of captive golden-mantled ground squirrels. Citellus lateralis tescorum. Can. J. Zoo/. 41,1103-l 120. PENGELLEYE. T. & FISHER K. C. (1966) Locomotor activity patterns and their relation to hibernation in the golden-mantled ground squirrel, Citellus lateralis. J. Mammal. 41, 63-73.
PENGELLEYE. T. KELLY K. H. (1966) A “circannian” rhythm in hibernating species of the genus Citellus with observations on their physiological evolution. Comp. Biochem. Physiol. 19,603-617. P~ITENDRIGHC. S. (1958) Perspectives in the study of biological clocks. In Perspectives in Marine Biology, pp. 239-268. University of California Press, Berkeley, 621. P~T~ENDRIGHC. S. (1960) Circadian rhythms and the circadian organization of living systems. Cold Spr. Harb. Symp. quart. Biol. 25, 159-184.
ROWAN W. (1926) On photoperiodism, reproductive periodicity, and the annual migrations of birds and certain fishes. Proc. Boston Sot. Nat. Hist. 38,147-189. ROWAN W. (1938) Light and seasonal reproduction in animals. Biol. Rev. 13, 374402. WEIHAUPTJ. G. (1964) Geophysical biology. Bioscience 14,18-24. Key Word Index-Circamrual rhythms; Citellus lateralis; gestation; zeitgeber.
hibernation;
FEMALE GESTATION AND LACTATION AS ZEITGEBERS FOR CIRCANNUAL RHYTHMICITY IN THE HIBERNATING GROUND SQUIRREL,
CITELLUS
LATERALLY
E.T. PJJNGELLEYAND SALLY J. ASMUND~~N Department of Biology, University of California, Riverside, California 92502, U.S.A. (Received 16 November 1973) Abstrati-1. Free-running circannual rhythms in females of the hibernator, Citellus lateralis, have been studied in relation to breeding. 2. It has been demonstrated that, in comparison to controls, females which go through gestation and lactation in the spring, and are subsequently kept in constant environmental conditions, hibernate with a more accurate phase relationship to the presumptive seasons. 3. Breeding as a zcitgebcr for the circammal rhythm is discussed, and the mechanisms involved in spring arousal are speculated upon.
INTRODUCTION THE PROBLEM of determining
entraining agents or zeitgebers for endogenous circannual rhythms have been discussed by Pengelley & Fisher (1963, 1966), Pengelley (1967, 1969), Pengelley & Asmtmdson (1969, 197Oa, b, 1972a, b), Heller & Poulson (1970) and Pengelley et al. (1972). It has been suggested as a result of experimentation that in ground squirrels temperature (Pengelley & Fisher, 1963), light (Pengelley & Asmundson, 197Oa) and locomotor activity (Pengelley & Fisher, 1966) may all be involved, but the results have not been conclusive when compared to experiments involving circadian rhythms. In birds, however, photoperiod is probably the primary zeitgeber (Gwirmer, 1967, 1968, 1969) as might be expected. On the other hand, Heller & Poulson (1970) have suggested that in the ground squirrel, Citellus lateralis, the free-running period of the rhythm may be so close to 365 days that no zeitgeber is necessary. This seems unlikely, for after much experimentation it seems probable that under controlled and constant conditions, and after the initial period, the free-running circannual rhythm is nearly always less than a year and it seems far more likely that as Pengelley et al. (1972) have pointed out, there are many and complex interacting zeitgebers involved. In the case of circadian rhythms, light in one form or another is almost invariably a zeitgeber (Pittendrigh, 1958, 1960; Halberg et al., 1959; Aschoff, 1960, 1963, 1965) which keeps the endogenous period of oscillation in phase with the environmental 24hr oscillation. Researchers have somewhat inevitably assumed this would be the case in circannual rhythms also. However, with the greatly increased complexity of
exogenous factors influencing a circarmual period, it seems highly probable that no simple solution can be expected. Furthermore, in ground squirrels light would not be expected to play such a dominant role as in birds, since the animals spend so much of their time underground, and perhaps half or more of the year in a state of hibernation where such a stimulus is likely to have a minimal effect. Thus it seems necessary to look more closely for various zeitgebers which may interact with the endogenous circannual rhythm keeping it in phase with the oscillation of the seasons. Since pilot experiments (Pengelley & Asmundson, 1971) showed that males and females of C. lateralis seemed to behave differently so far as hibernation was concerned, and since the one major event which females in the wild undergo which does not occur in males, is gestation and lactation, it occured to us that the latter two events might be involved as circamual zeitgebers in the wild. Since C. lateralis females successfully raise litters in the laboratory (Pengelley, 1966) an experiment was designed utilizing this information to test the effect of breeding on circannual rhythmicity. MATEFUALS AND METHODS From previous studies (Pengelley, 1966) it was known approximately at what time we could expect the females of the golden-manteled ground squirrel, Cite&s lateralis, to be pregnant in the wild state. Accordingly between 4 and 15 May 1970, some twenty females were trapped, at an altitude of about 7500 ft. near Bia Bear Lake in the San Bernardino Mountains of Calif&nia. They were brought to the laboratory and housed in individual cages in a quiet room with a photoperiod of 12 hr and an ambient temperature of 23+ 1°C. Purina lab chow 621
622
E. T. PENGELLEYANDSALLYJ. ASMUNDSON
and water were supplied ad lib. and diet was supplemented by carrots for pregnant and lactating squirrels. Of the twenty animals nine produced and successfully raised litters. Young were born between 14 May and 4 June after a gestation period of about 35 days. The young ground squirrels can be weaned at 32 days after birth with 100 per cent survival, and they were in fact weaned at about 35 days. Under these conditions and at this time of year ground squirrels rapidly gain weight and on 31 July, which is long before any hibernation would normally be expected, they were divided into two groups of nine each, i.e. those which had had litters and those which had not, and both groups were placed in environmental chambers with a constant 12 hr artificial photoperiod and a constant 35 1°C ambient temperature. Throughout the subsequent experiment food and water were available ad lib. The animals were carefully observed every day, though disturbances were kept to a minimum, and hibernation was determined by the method of Pengelley & Fisher (1961). It is important to note that these ground squirrels have distinct periods (months) of hibernation during which they are heterothermic, and these alternate with periods of activity during which the animals are active and homothermic. While an animal is in the heterothermic state, it has a body temperature close to the ambient, but within these long periods of heterothermy there are short arousals during which the animal becomes fully active
with a homothermic temperature of 37”C, maintains this for a few hours and then enters hibernation again. These are technically referred to as “periodic arousals” but for the purpose of this experiment they are not considered, and reference is only made to the entire heterothermic period of several months. Accurate weight records of each animal were kept by methods which do not disturb their hibernating behavior (Pengelley & Asmundson, 1969).
No litters
Litters
1970 Julv-i
-
1971 Jan 1971
July
1972 1972 Jan
‘11
x
11111 I
July
x
I
1973 Jan 1973
to cold / July
t
1974faaJ
RESULTS
Fig. 1. Graphical representation of free-running circannual neriods of eighteen female animals (C. lateralis) for over 3 years. upper group of nine had a litter in spring 1970, lower group had no litters. Black bars indicate heterothermic period, clear space homothermic period and the beginning to beginning of each black bar x= indicates one free-running circannual period. death; “to cold” indicates transfer of animals from 22 to 3°C. Artificial photoperiod throughout of 12 hr.
The data for this experiment which have been collected over three complete free-running circannual periods are summarized and expressed graphically in Fig. 1. The symbols are explained in the caption, but it is important to note that the horizontal time scale is over 3 years. Prior to the words “to cold” (31 July 1970) all animals were under constant environmental conditions of 22°C and an artificial photoperiod of 12 hr, and after that time were constantly at 3 + 1°C with the same artificial photoperiod. The free-running circannual periods are measured from the beginning of each heterothermic period to the beginning of the next. Figure 1 clearly indicates several things. The group which had litters in the spring of 1970 hibernated for the first time considerably later than the no litter group. In this respect there is no overlap between the two groups. Analysis using a twotailed t-test for small samples indicates that for each group the number of days of activity before the initial hibernation is significant beyond the 0.001 level. By 3 September all animals but one in the no litter group had entered hibernation, while
in the group with litters the onset of hibernation was spread over the months of September and October. From field observations it is known that the latter situation is the one that is much closer to that in the wild state, i.e. the group with litters hibernated more closely in synchrony with the presumptive autumn. The time of onset of the second period of hibernation, i.e. the beginning of the second free-running circannual period, is also significantly different, beyond the 0.001 level, between the two groups. The group which had had a litter in the spring of 1970 continued to hibernate later in the fall of 1971 and more synchronously with the presumptive autumn. Thus the difference in the phase relationship of the two groups is quite clear for at least two free-running circannual periods. By the beginning of the third circannual period the difference, while still apparent, is not statistically significant. Table 1 summarizes the actual number of days in each free-running circannual period for all animals in the experiment and from these data it is possible
Circammal rhythmicity in the hibernating Table 1. Comparison of tirst, second and third freerunning circammal periods (onset to onset of hibernation) in days of C. lateralis under constant environmental conditions of 3°C and 12 hr photoperiod Free-running
circannual
periods
(days) Animals with litters in Spring 1970 1 2 3 4 2 7 8 9 Animals with no litters 10 11 12 13 14 15 16 17 18
First (1970-71)
Second (1971-72)
Third (1972-73)
353 397 392 392 346 361 356 317 301
337
310
306 295 274 314 350 349 320
334 311 313 277
348
337
304 297 323 302 337
318 345 336 341
355 345 363 362 376 343 340 383 281
336
300
Animals 1-9 had litters in the Spring of 1970, and animals IO-18 had no litters.
to draw certain inferences. There does not appear to be any significant difference between the two groups in the length of the lirst or subsequent freerunning periods and the individual variations are in keeping with previous observations. Thus the free-running period of the female animals was not altered by their having a litter. However, in both groups the second free-running period is significantly less than the iirst, but the third, while less than the first, is not less than the second. It is also true that the 6rst free-running period is closer to the presumptive annual period of 365f days than either the second or third periods. It may also be noted here that ah animals in the experiment exhibited an endogenous body weight rhythm synchronous with the circannual rhythm of homothermy-heterothermy and there were no apparent differences between the two groups. The body weight is at a maximum at the beginning of the heterothermic phase and at a minimum shortly after the beginning of the homothermic phase (Pengelley & Fisher, 1963; Pengelley & Asmundson, 1969). DISCUSSION The French physiologist Dubois (1896) and later Kayser (1940) and Lyman (1954) were all aware of
ground squirrel
623
the temporal nature of hibernation in a variety of animals and the fact that this was not a simple response of an animal to some environmental stimulus. However, it is only in the last 10 years or so that some of the problems have been seriously studied. As Pengelley & Asmundson (1972a) have pointed out, the basic problem can be stated as follows: how do animals repeatedly perform the correct behavioral or physiological act at the specific time during the year when presumably it is most beneficial for the biological fitness of the species? The answer to this question seems to be that there are only three possible ways that an animal can synchronize its behavioral physiology to a changing environment. These are (a) a direct response to some changing geophysical stimuli, and there are more of these than is commonly realized (Weihaupt, 1964); (b) an endogenous rhythm which programs the animal’s behavior to the exogenous temporal period, i.e. 24 hr or 365$ days; or (c) a combination of both. So far as the temporal period of 24 hr is concerned, there seems very little doubt that animals and plants, from single-celled protists to the most highly developed animals, have evolved a combination of both mechanisms by which various zeitgebers entrain an endogenous circadian rhythm. Aschoff (1964) has explained the survival value of such mechanisms and these mechanisms are even more likely to have great survival value during a circammal period. Periodic reproduction in all animals is an essential for survival; and it is nearly always the case that, in order for the young to survive, reproductive activities must take place at rather specific times during the annual cycle of the seasons. The mechansisms by which this is ensured depend on the species and are of great variety, but it was the work of Rowan (1926, 1938) that clearly linked photoperiodism, reproductive periodism and the annual migrations of birds, thus elucidating a mechanism for the accurate timing of reproductive activities. Later, Bissonette (1935) showed that modification of some mammalian sexual cycles was possible by manipulation of the photoperiod. From both field observations, previous laboratory work and the results of these experiments, it appears quite certain that in these ground squirrels mating takes place immediately or very soon after emergence of the females from hibernation and that the young are born and quickly raised in the early spring. This not only has survival value for the young but, as these experiments show, production of a litter in the spring also helps to program the mother to enter hibernation in the fall at the most advantageous time. Thus gestation and lactation in the females are in fact acting as zeitgebers; for, as previously explained, the group with no litters hibernated much too early for the presumptive fall and, in consequence, aroused too early from the heterothermic
621
E. T. PENC;ELLEY AND
phase for the presumptive spring. In fact, most aroused during the presumptive winter which in the wild would probably be fatal. If it had been possible to get the group which had had a litter in the spring of 1970 to reproduce again in the spring of 1971, it appears highly probable that the onset of hibernation in the fall of 1971 would have been delayed so that they would have remained better in phase with the presumptive seasons and the difference with the no litter group would have been even more marked. The fact that there is no difference in the free-running circannual period for the first year between the two groups indicates that the endogenous circannual rhythm can run close to the period of the exogenous annual rhythm for at least a year, even under very artificial conditions. However thereafter, the evidence is clearly that one or more zeitgebers are necessary for entrainment, and from these experiments it is clear that for females one of these is in fact the production of a litter in the spring. This of course has the dual function of ensuring the survival of the species, reproductively, and ensuring the survival of the mother in her complex adaptations to a harsh environment. It has already been noted that an animal, such as C. lateralis, which spends most of the winter underground and in a state of hibernation, is most unlikely to have its physiology influenced at this time of year by photoperiod, and one may justly ask the question: what in fact causes it to arouse in the spring so that reproduction can be assured? It has been suggested by earlier workers that possibly this was due to the fact that the stored fat which the animals consume during hibernation became depleted by the spring and that this caused arousal. However, Pengelley & Fisher (1963) clearly demonstrated that this was not the case, for when they sacrificed animals which had just aroused from the heterothermic state, they found that the animals still had ample fat available; in fact, it was estimated that there was enough for another 6 months of hibernation. It is probable also that any marked increase in burrow temperature can also be ruled out as a mechanism of arousal in the spring; thus we are left with some kind of endogenous clock mechanism which is programmed in such a way that during the months of hibernation it brings the animals’ physiology to a state where mating and reproduction are possible. On the other hand, it is important to note that reproductive competence per se is not necessary either for spring arousal or the maintenance of a circannual rhythm under constant conditions. Pengelley & Kelly (1966) have shown that castrated animals do not behave differently in these respects from normal animals. This does not negate light and ambient temperature as zeitgebers, for these, as pointed out previously have been clearly implicated (Pengelley & Fisher, 1963; Pengelley & Asmundson,
SALLYJ. ASMUNDSON
1970a) as possible zeitgebers in the long run of circannual rhythmicity, but simply that they are unlikely to cause actual arousal in the spring which is so important for reproduction. It is not known whether spring breeding has a similar effect in males in terms of programming the time of fall hibernation. However, there are some indications that the male ground squirrels rhythm is not as precise as that of the female. In conclusion it may be noted that in addition to gestation and lactation, it follows inevitably that for females of this species, a sexually active male at the right place and the right time is in fact a circannual zeitgeber. Acknowledgements-This study was supported by Grant No. GB-40827 from the National Science Foundation, and secondarily by an Intramural Grant from the University of California. We wish to thank Mr. Brian Barnes for his able technical assistance. REFERENCES ASCHOFF J. (1960) Exogenous and endogenous components in circadian rhythms. Cold. Spring Harb. Symp. quart. Biol. 25, 1 l-28. ASCHOFF J. (1963) Comparative physiology: diurnal rhythms. Ann. Rev. Phyiiol. 25, 581400. ASCHOFF J. (19641 Survival value of diurnal rhythms. Symp. Zooi So;. Lond. 13, 79-98. AXHOFF J. (1965) Circadian Clocks. North Holland, Amsterdam. BI~~ONETTET. H. (1935) Modification of mammalian sexual cycles. J. exp. Zoo/. 71, 341-373. DUBOISR. (1896) J%ude sur le mkhanisme de la thermogenese et du sommeil chez les mammifhres. Physiologie cornpar&. de la marmotte. Ann. Univ. Lyon 25, l-268. GWINNER E. (1967) Circannuale Periodik der Mauser und der Zugunruhe hei einem Vogel. Naturwiss. 54,447. GRINNER E. (1968) Circannuale Periodik als grundlage des jahreszeitlichen Funktionswandels bei ZugvGgeln. Untersuchungen am Fitis (PhylIoscopus trochilus) und am Waldlaubslnger (P. sibilatrix). J. Ornithoi. 109, 70-95. GWINNER E. (1969) Untersuchungen zur Jahresperiodik von Laubstingem. J. Omithol. 110, 1-21. HALBERGE., HALBERGF., BARNUM C. P. & BITTNER J. J. (1959) Physiologic 2-hr periodicity in human beings and mice, the lighting regimen and daily routine. In Photoperiodism and Related Phenomena in Plants and Animals, pp.803-878. Amer. Assoc. Adv. Sci., Washington, Pub. 55. HELLER H. C. & POUL~~N T. L. (1970) Circannian Endogenous and exogenous factors rhythms-II. controlling reproduction and hibernation in chipmunks (Eitamias) and ground squirrels (Spermophilus). Comu. Biochem. Physiol. 33. 357-383. KAYSE; C. (1940) &sai d’ahalyse du m&anisme du sommeil hibemal. Ann. Physiol. 16, 313-372. LYMANC. P. (1954) Activity, food consumption and hoarding in hibernators. i Mammal. 35, 545-552. PENGELLEYE. T. (1966) Differential developmental patterns and their adaptive value in various species of the genus Citellus. Growth 30, 137-142.
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E. T. (1967) The relation of the external conditions to the onset and termination of hibernation and estivation. In Proc. III Intl. Symp. Mammal. Hi6 (Edited by FISHERK. C., DAWE A. R., LYMAN C. P., SCH~NBAUM E. & SOUTHF. E., JR.), pp. l-29. Oliver & Boyd, Edinburgh. PENGELLEY E. T. (1969) Influence of light on hibernation in the mojave ground squirrel, Citellus mohavensis. In Physioiogicaf Systems in Semiarid Environments (Edited bv HOFF C. C. & RIEDESELM. L.). nv. 11-16. University of New Mexico Press, Albuquerque. PENGELLEYE. T. & ASMUNDSONSALLYJ. (1969) Freerunning periods of endogenous circamrian rhythms in the golden-mantled ground squirrel, Citellus lateralis.
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PENGELLEYE. T. & ASMUNDSONSALLY J. (1970a) The effect of light on the freerunning circannual rhythm of the golden-mantled ground squirrel, Citellus lateralis. Comp. Biochem. Physiol. 32, 155-160. PENGELLEYE. T. & ASMUND~~N SALLY J. (1970b) Circannual rhythmicity-evidence and theory. Life Sciences and Space Research VIII (Edited by LAVOR~TE F. G. & VISCHNIACW.), pp. 235-239. Proc. XII Plenary Meeting, COSPAR, Prague, Czechoslovakia. North-Holland, Amsterdam. PENGELLEYE. T. & ASMUND~~NSALLYJ. (1971) Circannual rhythmicity in hibernating ground squirrels, Citellus lateralis, in relation to sex and opportunity to breed. Vol. IX. Proc. XXV Intl. Union Physiol. Sci. Munich, Germany. PENGELLEYE. T. & ASMUND~~NSALLY J. (1972a) An analysis of the mechanisms by which mammalian hibernators synchronize their behavioral physiology with the environment. In Hibernation and Hypothermia, Perspectives and Challenges, pp. 637-661. Elsevier, Amsterdam. PENGELLEYE. T. & ASMUNDSONSALLY J. (1972b) Variations in hibernating behavior of the ground squirrel, Citellus lateralis, in response to day length and intensity of light. Cryobiology 9, 308-309.
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PENGELLEYE. T., BARTHOU~MEWG. A. & LIGHT P. (1972) Problems in the chronobiology of hibernating mammals. In Hibernation and Hypothermia, Perspectives and Challenges, pp. 713-716. Elsevier, Amsterdam. PENGELLEYE. T. & FISHER K. C. (1961) Rhythmical arousal from hibernation in the golden-mantled ground squirrel, Citellus lateralis tescorum. Can. J. Zool. 39, 105-120.
PENGELLEYE. T. & FISHER K. C. (1963) The effect of temperature and photoperiod on the yearly hibernating behavior of captive golden-mantled ground squirrels. Citellus lateralis tescorum. Can. J. Zoo/. 41,1103-l 120. PENGELLEYE. T. & FISHER K. C. (1966) Locomotor activity patterns and their relation to hibernation in the golden-mantled ground squirrel, Citellus lateralis. J. Mammal. 41, 63-73.
PENGELLEYE. T. KELLY K. H. (1966) A “circannian” rhythm in hibernating species of the genus Citellus with observations on their physiological evolution. Comp. Biochem. Physiol. 19,603-617. P~ITENDRIGHC. S. (1958) Perspectives in the study of biological clocks. In Perspectives in Marine Biology, pp. 239-268. University of California Press, Berkeley, 621. P~T~ENDRIGHC. S. (1960) Circadian rhythms and the circadian organization of living systems. Cold Spr. Harb. Symp. quart. Biol. 25, 159-184.
ROWAN W. (1926) On photoperiodism, reproductive periodicity, and the annual migrations of birds and certain fishes. Proc. Boston Sot. Nat. Hist. 38,147-189. ROWAN W. (1938) Light and seasonal reproduction in animals. Biol. Rev. 13, 374402. WEIHAUPTJ. G. (1964) Geophysical biology. Bioscience 14,18-24. Key Word Index-Circamrual rhythms; Citellus lateralis; gestation; zeitgeber.
hibernation;