Daily torpor in Peromyscus leucopus on an adequate diet

Comp. Biochem. Physiol., 1975, Vol. 51A, pp. 413 to 423. Pergamon Press. Printed in Great Britain

DAILY TORPOR IN P E R O M Y S C U S LEUCOPUS ON AN ADEQUATE DIET

RICHARD W. HILL

The Museum and Department of Zoology, Michigan State University, East Lansing, Michigan 48824, U.S.A.

(Received 12 March 1974)

Abstract--1. Seventeen mice were tested for the occurrence of torpor in the presence of excess food over periods of 6-30 days at ambient temperatures of 13-15°C.

2. Seven animals displayed regular daily torpor, and four displayed sporadic episodes of torpor. Body temperatures in mice showing regular daily torpor ranged from 16.8 to 27-7°C, and metabolic rates remained below 2'0 cm a O2/g per hr for an average of 4.3-12.7 hr/day in various individuals.

3. It is estimated that episodes of torpor in the laboratory environment resulted in energetic savings of 44(I-1900 cal in animals showing regular daily torpor.

INTRODUCTION

IN 1951 HOWARD reported the occurrence of torpor in free-ranging Peromyscus leucopus noveboracensis and P. maniculatus bairdi occupying artificial nest boxes in the field during winter. He induced torpor in three P. maniculatus in an outdoor enclosure by limiting their food supply but raised the possibility that some of the free-ranging animals were undergoing torpor in the presence of adequate food inasmuch as they were found in torpor in nest boxes containing stored food. Morhardt & Hudson (1966) and Morhard t (1970) studied torpor in the laboratory in several species of Peromyscus, including P. leucopus texanus. Whereas one P. maniculatus and one P. crinitus entered torpor in the presence of excess food, most animals, including all of the P. leucopus, did not enter torpor unless starved to some extent. The authors concluded that starvation sufficient to cause a significant drop in body weight is generally a necessary stimulus to torpor.

In the present study, eleven P. leucopus noveboracensis were found to enter torpor in the presence of excess food. Seven of these did so in a regular daily pattern. This indicates that, at least in some individuals, torpor is a routine physiological process, not requiring deficiency of food as a stimulus. A similar conclusion was reached by Gaertner et al. (1973) in studies on P. leucopus which were carried out contemporaneously with those reported here, and Fuller et al. (1969) have recently reported spontaneous torpor in P. maniculatus. These results add a significant new dimension to our understanding of this important genus.

In this study the occurrence of torpor was monitored over continuous periods of 6-30 days by determination of oxygen consumption. This method

x4 4 1 3

has the advantage of leaving the animals completely unencumbered by implanted telemeters or thermocouple leads. It also permits estimation of the energetic savings realized by torpor.

MATERIALS AND METHODS

Peromyscus leucopus noveboracensis were captured around Ann Arbor, Michigan, and Newark, Delaware. Of the seventeen animals studied, six (three from Dela- ware, three from Michigan) were wild-caught individuals, eight (all from Michigan) were first-generation, laboratory-reared offspring of wild-caught individuals and three (all from Michigan) were second-generation laboratory offspring. The animals were maintained in pairs in plastic mouse cages ( 2 9 x 1 9 x 1 3 c m deep). Cedar or pine shavings were used as bedding, and cotton was provided as nesting material. Food (Purina Mouse Chow) and water were supplied adlib.

Prior to experimentation animals were maintained under controlled conditions in a Sherer-Gillett Model CEL 37-14 environmental chamber for at least a month and generally for many months. The temperature was 13-15°C. Lights (four 25-W incandescent bulbs) were on daily from 0500 to 1900 E.S.T. or from 0600 to 2100 E.S.T., depending on the experiment. The remainder of each day was completely dark.

For studies of torpor, animals were transferred individually to metabolism chambers fashioned from new 1-gal paint cans. The lid of each chamber was fitted with a Plexiglas window (6 cm dia) and with inflow and outflow tubes for air. A port on the side of the chamber (9 cm above the bottom) provided for introduction of a water sipper tube from the outside. The seals between the Plexiglas window and lid, between the lid and bottom and between the sipper tube and wall of the chamber were air-tight. The environment in the metabolism chamber during experimentation was very similar to that to which the animals had been previously acclimated. Cedar or pine shavings were again used as bedding, and cotton

414 R I ~ W. HILL

nesting material was provided. All animals built nests approximating 12-15 cm dia. The chamber was placed in a controlled temperature cabinet, again at 13-15°C. Light from a 25-W incandescent bulb entered the chamber through the Plexiglas window, and the photoperiod was the same as the acclimation photoperiod. Experiments ran continuously for 6-30 days. Sufficient food for this period was placed in the chamber at the start of the experiment. When the chamber was opened at the termination of the experiment, a minimum of 25 g of food pellets remained. Water was provided from a small vial on the outside of the chamber through the sipper tube. Water was added to the vial once daily, and this necessitated brief removal of the sipper tube from the port into the chamber. This was the only overt disturbance during the entire experimental period and always occurred around the time that the lights went off at night. The time of the disturbance was adjusted as necessary so as not to coincide with episodes of torpor.

Oxygen consumption was monitored continuously over the experimental period using an open-circuit system conforming to condition B of Hill (1972). The oxygen content of air flowing into and out of the animal chamber was measured with a Beckman G-2 paramagnetic oxygen analyzer and recorded on a Honeywell Class 15 potentiometer. The rate of flow of air into the animal chamber was determined with a Brooks Model 1110 rotameter. Ascarite (A. H. Thomas Co.) and Drierite (W. A. Hammond Co.) were used to absorb carbon dioxide and water vapor, respectively, from the air stream at appropriate parts of the system. Oxygen consumption was calculated by the method of Depocas & Hart (1957) and Hill (1972). The rate of air flow into the animal chamber was relatively stable in any given experiment. Daily variation was generally less than 10 cma/min from the mean daily flow rate. The average flow rate varied in different experiments from 235 to 420 cma/min of dry air at STP. At these flow rates, the concentration of oxygen in dry, CO2-free effluent air did not fall below 20.45 per cent during long periods when the animals were at rest and only rarely fell below 20.0 per cent when the animals were active (rest and activity being assessed from the record of oxygen consumption).

Air temperature in the animal chamber was monitored continuously by a copper-constantan thermocouple positioned in the outlet port of the chamber and connected to a Honeywell Class 15 recording potentiometer.

The body temperature of the animal was determined at the end of the experiment using a thermocouple probe inserted rectally to a depth of 2-3 cm. The probe was constructed of 40-gauge copper and constantan wires welded at the tip and threaded into Clay-Adams Intra- medic PES0 polyethylene tubing (o.d. 0.97mm). Removal of the animal from the metabolic chamber and determination of body temperature required 45 sec or less. Animals which were undergoing regular daily episodes of torpor were removed during a period of torpor. Other animals were removed at a time when the metabolic record indicated that they were at rest.

Animals were weighed before and after experimental periods on a Torsion Model RX-1 balance. The average of the initial and terminal weights was utilized in calculating weight-specific oxygen consumption.

All gas volumes reported in the results have been corrected to standard conditions of temperature and pressure (STP).

RESULTS

All animals, regardless of whether they exhibited torpor, spent long periods during the light period of each day at relatively stable metabolic rates averag- ing 2.6-3.6 cm 8 O2/g per hr. These are interpreted to be the resting metabolic rates of animals maintaining high body temperatures under the conditions of these experiments. This interpretation receives strong support from the fact that nine animals removed from the metabolic chamber when showing such rates had body temperatures of 36"3-37-4°C. Resting metabolic rates in the present experiments were lower than those indicated by Hart 's (1953) data at similar ambient temperatures, 4-6-6.2 cm a Ol/g per hr. Hart's mice were not provided with a nest and were acclimated to their test chambers for only 50-400 min, as compared to many days in the present experiments. Both factors could be important in explaining the differences in results. Metabo- lic rates of the mice in the present experiments tended to be higher and more variable during at least parts of their dark period than during the light period. This is indicative of nocturnal activity and corrobo- rates the findings of Pearson (1947) and Morrison (1948).

Some animals, as illustrated in Fig. 1, underwent protracted periods of depressed metabolism during which oxygen consumption fell below 2.0 cm a O2/g per hr and, commonly, below 1.0 cm a O2/g per hr. These periods are interpreted as episodes of torpor. Hypothermia was confirmed by measurements of body temperature on seven animals removed from the test chamber during episodes of depressed metabolism (Table 1). It is noteworthy that several animals had body temperatures only a few degrees higher than ambient temperature.

Table 1. Body temperatures of animals removed from apparatus during torpor and metabolic rates immediately

before removal

Body Oxygen Animal temperature consumption No. (°C) (cm a O2/g per hr)

1001 18.1 0.68 1034 17.6 0"50 2001 23.4 0.74 2002 17.9 0-46 2037 20-6 0.83 2051 16.8 0.47 2082 27.7 1.32

Of the seventeen animals studied, the seven listed in Table 1 displayed regular daily torpor. Two of these (numbers 1001 and 1034) were wild-caught from Michigan, and five (numbers 2001, 2002, 2037, 2051 and 2082) were first-generation, laboratory- reared offspring of Michigan mice. Animals 2051 and 2037 were studied twice, in June 1970 and April

Daily torpor in Peromyscus leucopus 415

1972, and displayed daily torpor on both occasions. Data presented for these individuals in Fig. 1, Table 1, and subsequently are from the experiments in April 1972, which lasted 19 days as compared to only 8 days in the earlier experiments.

Four additional animals displayed episodes of torpor according to the metabolic records but did not do so in a consistent daily pattern. These mice were all laboratory-reared from Michigan stock. Three (numbers 2033, 2046 and 2050) were first- generation offspring, and one (number 3001) was a second-generation offspring. Hypothermia was not confirmed by measurement of body temperature in these individuals.

Six of the seventeen animals studied failed to exhibit any evidence of torpor in experiments lasting 8-22 days. These included the three mice from Delaware, one wild-caught animal from Michigan and two second-generation, laboratory-reared animals from Michigan.

A wild-caught individual from Michigan (number 1047) was observed in torpor on a single occasion in the animal colony but was not included in the present experiments.

Animals displaying regular daily torpor

Figures 1-3 provide a comparison of the metabolic records for three animals undergoing daily torpor. It must be noted that the method used to determine oxygen consumption provides an accurate measure of the oxygen consumption of the animal only under steady-state conditions. Thus, when the recorded

O

.z

O ; • . . . . . . DO 03 06 09 12 1S

noon EASTERN STANDARD TIME (hr)

Fig. 1. Oxygen consumption in animal 2051 on days 15, 16 and 17 (from top to bottom) during a 19-day experiment. The average resting metabolic rate of this animal when maintaining high body temperatures was estimated from many records to be about 3-0cm 30~/g per hr.

OIN 0 I I I I I I I I I I I I I I

g , ~7

8 Z

~ 4 o

2

o O0 03 06 09 12 15

noon

EASTERN STANDARD TIME (hr)

Fig. 2. Oxygen consumption in animal 2037 on days 15, 16 and 17 (from top to bottom) during a 19-day experiment. The average resting metabolic rate of this animal when maintaining high body temperatures was estimated from many records to be about 2-6cm 3 O2/g per hr.

2

4 z 0

:~ 2

Z 0

u 0

Z u.l

× 4 0

0 i i i i I i i i i i , ,

OO 03 06 0 9 12 noon


Fig. 3. Oxygen consumption in animal 2082 on days 20, 21 and 22 (from top to bottom) during a 23-day experiment. The average resting metabolic rate of this animal when maintaining high body temperatures was estimated from many records to be about 2-7 cm ~ Oz/g per hr.

416 R[CI.-mRD W. HmL

oxygen consumption is steady over a period of time, it corresponds to the actual oxygen consumption of the animal. However, when the recorded oxygen consumption is changing appreciably over time, qualitatively reflecting a change in the rate of oxygen consumption of the animal, the recorded rate does not correspond quantitatively to the oxygen consumption of the animal.

The pattern of entry into torpor shown for animals 2051 and 2037 in Figs. 1 and 2 was characteristic of animals 1001, 1034, 2001 and 2002 as well. Oxygen consumption typically declined smoothly from levels compatible with maintenance of high body temperatures to levels below 1 cm a Oz/g per hr. In animals 2051 (Fig. 1), 1001, 1034 and 2002, oxygen consumption then usually remained relatively stable at levels below 1 cm s O2/g per hr until arousal occurred. The oxygen consumption of animal 2037 (Fig. 2) varied to a considerably greater extent during torpor. Lesser variability was seen in animal 2001. Arousal was characterized by a rapid increase in the rate of oxygen consumption. Generally, oxygen consumption rose transiently to high levels and then fell to levels characteristic of the resting animal maintaining high body temperature. The magnitude of the overshoot of resting metabolism is underestimated in the metabolic records since the experimental system did not have time to achieve steady-state. All animals consistently displayed a strong overshoot of resting metabolism during arousal except for animal 2002, which in some instances showed a strong overshoot but in others displayed little or no overshoot.

Animal 2082 (Fig. 3) displayed distinctly more shallow episodes of torpor than any other animal undergoing regular daily torpor. Recorded oxygen consumption generally fell close to or below 1 cm a Oz/g per hr during the initial entry into torpor but then usually rose well above I cm a O~/g per hr in less than an hour and remained above that level for most or all of the remainder of the episode. Recorded oxygen consumption not uncommonly rose transiently above 2 cm 30~/g per hr. The final exit from torpor was always well marked by a dramatic rise in recorded oxygen consumption above 4.5 cm 8 Oz/g per hr, followed by resumption of the oxygen consumption characteristic of rest at high body temperatures. Note that animal 2082 had a relatively high body temperature when removed from the apparatus at a metabolic rate of 1.32 cm 30~/g per hr, a rate which was reasonably representative of this animal when in torpor (Table 1). Considering the high measured body temperature and the metabolic records, it seems likely that this animal, unlike at least some of the others, did not allow its body temperature to fall close to ambient temperature when torpid.

The complete metabolic records of all animals which showed regular daily torpor are summarized in Figs. 4 and 5. It is assumed in these Figures that

rates of oxygen consumption below 2.0 cm s OJg per hr are generally indicative of torpor. This assumption is supported by the following observations. First, an oxygen consumption of 2.0 cm 8 O2/g per hr or less is well below the levels believed to be characteristic of resting animals maintaining high body temperatures. Second, when the oxygen

ANIMAL DAY b

2001 ~ ~ - =

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b 1034

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Fig. 4. Summary of the metabolic records of animals 2001, 1034, 2002 and 1001. In the record for each animal, each horizontal line corresponds to a single day of the experiment, as indicated on the left. The line remains blank whenever the recorded oxygen consumption was greater than or equal to 2.0 cm 30~/g per hr. The line is filled with a thin dark line whenever the recorded oxygen consumption was less than 2"0cm302/gperhr but greater than or equal to 1"0 cm 3 O2/g per hr. The line is filled with a thick dark line whenever recorded oxygen consumption was less than 1.0 cm a O~/g per hr. The experiment began at "b" and was terminated at "x". Body temperatures recorded in Table 1 were measured immediately after removal of the animal at time "x". For each animal the dark line below the abscissa indicates

the dark period of the daily light-dark cycle.

consumption fell below 2.0 cm a O2/g per hr, it generally continued to fall to lower levels and remained at low levels for an hour or more. That is, oxygen consumption generally did not fall below 2-0 cm s O2/g per hr unless part of a patterned reduction in metabolism lasting an appreciable period of time. In some records (such as those for animals 1001 and 2002), there are occasional drops of


oxygen consumption below 2.0 cm 80~/g per hr lasting only 20 min or less. These are difficult to interpret. They may or may not represent transient tendencies toward hypothermia.

A N I M A L DAY b 2051 - - - - .

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2082

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Fig. 5. Summary of the metabolic records of animals 2051, 2037 and 2082. See legend of Fig. 4 for explanation.

All animals showed evidence of torpor within 36hr after their introduction to the metabolic apparatus. Three showed torpor on the first day after their introduction. In two cases (1001 and2001) the pattern of torpor remained similar from day to day, from the time torpor made its first appearance to the end of the experiment. More usually the pattern of torpor showed some adjustments in the early days of the experiment. For example, episodes of torpor became progressively longer in animal 1034, and the first episodes displayed by animal 2051 were short and shallow by comparison to later episodes. The record for animal 2082 is of some interest. Recall that this animal displayed the most shallow torpor of all animals undergoing regular daily torpor. As seen in Fig. 5, it also showed the most inconsistent incidence of torpor in the early days of the experiment. Torpor occurred on some days but not others, and on two days it occurred unusually in the evening.

In all animals, once the initial period of adjustment was over, torpor occurred consistently each day. For each animal, the principal episode of torpor

tended to occur at about the same time each day. Some basic similarities among individuals in the timing of torpor are evident and noteworthy. The principal episode of torpor began in the dark, early morning hours or shortly after the lights came on in the morning. It ended in the morning or afternoon, always well in advance of the time that the lights went off again. Some individuals underwent relatively short, and usually relatively shallow, secondary episodes of torpor. These generally occurred late in the afternoon or shortly after the lights went off at night. Animal 1001 is particularly noteworthy in this regard. It regularly underwent two episodes of torpor each day, a long, principal episode starting between 0230 and 0640 E.S.T. and a short, secondary episode starting between 1520 and 1740 E.S.T. The incidence of secondary episodes in other animals was more sporadic.

Table 2 summarizes the amount of time that each animal spent in torpor per day during the final days of its experimental run. Three animals (1034, 2001 and 2051) averaged 11.4-12-7 hr/day in torpor, the

Table 2. Total time at depressed metabolic rates per day: range and mean*

Time at metabolic Time at metabolic rate under rate under

Animal 2.0 cm 3 O2]g per hr 1.0 cm 3 O2/g per hr No. (hr) (hr)

1001 7"3-10"1 (9"0) 5.0-7.0 (6'2) 1034 9"3-13"3 (11.7) 7'0-11.3 (9"6) 2001 9"8-14"3 (12.7) 7.1-9.9 (8"6) 2002 6.7-11 "0 (8"2) 4"1-8.0 (5.7) 2037 6.7-9.3 (7"6) 2.0-6.3 (4'3) 2 0 5 1 10"8-12-0 (l 1 "4) 7"1-10'5 (9"4) 2082 1 "74"0 (4"3) 0'0-1 '3 (0"4)

* Means given in parentheses. Data are summarized for the last six full days of the test period for all animals except 2001. Data are summarized for the last three full days of the test period for animal 2001.

metabolic rate being below 1.0 cm 30~/g per hr for an average of 8.6-9.6 of these hours. Three other animals (1001, 2002 and 2037) averaged 7-6-9-0 fir/day in torpor (4-3-6"2hr/day below l ' 0 c m 8 O~/gperhr) . Animal 2082 exhibited the least amount of torpor per day and averaged a particularly short time at metabolic rates below 1.0 cm 3 O2/g per hr.

Animals which displayed torpor but not in a regular daily pattern

The episodes of depressed metabolism displayed by these animals were, in general, shallow and short by comparison to those of the animals which

418 RICHARD W. HILL

underwent regular daily torpor. Episodes during which oxygen consumption remained at or below 2.0 cm s O2/g per hr for at least 30 min are interpreted as periods of torpor. These episodes generally began with a gradual decline of oxygen consumption from the resting level compatible with maintenance of high body temperatures. In some instances the period of torpor terminated with a clear metabolic overshoot, but in other cases no overshoot was apparent. There was a general correlation between the length of the episode of torpor and the occurrence of a terminal overshoot. An overshoot was observed in all of six episodes lasting 2"5 hr or more but was observed in only one of seven episodes lasting 1.5 hr or less.

The episodes of torpor displayed by these animals are summarized below. The length of each episode was determined as the period during which recorded oxygen consumption was at or below 2.0 cm a O2/g per hr. Oxygen consumption fell below 1.0cm s O2/g per hr during only a single episode (the last episode of animal 2046) and then did so for only 0.4 hr. All animals received light between 0500 and 1900 E.S.T.

Animal 2033 was studied for 30 days and underwent three episodes of torpor on days 16 (1.5 hr), 23 (0-7 hr), and 25 (1.1 hr). Episodes began between 0730 and 0825 E.S.T., and the minimal recorded oxygen consumption during the three episodes ranged from 1.4 to 1.8 cm s O2/g per hr.

Animal 2050 was studied for 12 days and underwent two episodes of torpor on days 4 (1.3 hr) and 11 (3-5 hr). Episodes began at 0759 and 0647 E.S.T., and minimal recorded oxygen consumption during the episodes was 1.3 and 1.4 cm 80~/g per hr.

Animal 3001 was studied for 29 days and underwent three episodes of torpor on consecutive days: day 24 (0.7 hr), day 25 (3.3 hr), and day 26 (2"9 hr). Episodes began between 0317 and 0623 E.S.T., and the minimal recorded oxygen consumption ranged from 1.0 to 1.3 cm 3 O2/g per hr.

Animal 2046 was observed in torpor on a single occasion in the animal colony. It showed no evidence of torpor during a 7-day test period in June 1970. When retested for 12 days in July 1971, it displayed five episodes of torpor on days 5 (0.6 hr), 6 (2.5 hr), 9 (1.0hr), 10 (3.0hr) and 12 (4"8 hr). The first episode began at 1201 E.S.T.; the others began between 0710 and 0931 E.S.T. Minimal recorded oxygen consumption ranged from 0.9 to 1.5 cm 3 O~/g per hr.

The episodes of torpor displayed by animals 2033, 2050 and 3001 were sporadic in occurrence. It is possible that animal 2046, during the experiments in July 1971, was undergoing a long adjustment period similar to that described earlier for animal 2082 and would have settled into a consistent daily pattern of torpor had the experiment been continued for a longer time.

Energetics

Daily torpor, in common with other forms of hypothermia, is expected to have energetic advan- tages. The data gathered in this study permit approximation of the energetic savings realized by torpor in the laboratory environment. The types of calculations performed may be illustrated with respect to animal 2051 on day 15 of its experimental period (see upper graph in Fig. 1). The average resting oxygen consumption of this animal when maintaining high body temperatures was estimated at 3-0 cm 3 O2/g per hr. For purposes of this discussion, this rate will be termed the simple resting metabolic rate. On day 15, oxygen consumption fell below the simple resting rate at about 0100 E.S.T. The ensuing episode of torpor terminated with a metabolic overshoot, following which oxygen consumption fell to within 10 per cent of the simple resting rate at 1506 E.S.T. The 14.1-hr period between 0100 and 1506 E.S.T. is taken to be the period over which torpor and arousal from torpor caused displacement of oxygen consumption from the simple resting rate. Had the animal not undergone torpor and remained at the simple resting rate over this period, its total oxygen consumption would have been 42.3 cm ~ O2/g or, given a body weight of 16"86 g, 713 cm 3 02. Integration of the recorded oxygen consumption over this period indicates, instead, that the animal actually expended 18.0 cm 3 O2/g, or 303 cm 3 02. The animal thus realized an energetic saving equivalent to 410cm 3 02. This saving is equivalent to 57 per cent of the metabolic cost that would have been incurred over the 14-1-hr period had the animal not undergone torpor but instead remained at the simple resting metabolic rate.

It is of interest to determine the metabolic savings due to torpor as a percentage of the total daily energy expenditure in the absence of torpor. The latter quantity was estimated by integrating recorded oxygen consumption over the entire day (from midnight to the next midnight) except that the simple resting metabolic rate was substituted for the actual oxygen consumption over the 14.1-hr period of torpor and arousal. The total daily energy expenditure in the absence of torpor estimated for animal 2051 on day 15 was 78.0 cm a Oz/g, or 1315 cm a 02. The savings realized by torpor amount to 31 per cent of this total.

Calculations like the above are summarized in Table 3 for the metabolic records depicted in Figs. 1-3. Energetic savings over the period of torpor and arousal averaged 399cm302 in animal 2051, 232 cm 8 O2 in animal 2037 and 93 cm 3 02 in animal 2082. Assuming 1 cm 3 02 to be equivalent to 4.75 cal, these savings correspond to 1895, 1102 and 442 cal, respectively. The heats of combustion of various plant and animal foods that might be used by Peromyscus range around 5 cal/mg dry weight (e.g.

Daily torpor in Peromyscus leucopus

Table 3. Estimated energetic savings realized by torpor in the laboratory environment

419

Animal No. 2051 2037 2082

Day 15 16 17 15 16 17 20 21 22

Average weight (g) 16-86 19.93 22-58 Simple resting metabolic rate 3-0 2.6 2.7

(cm 30~/g per hr)

Length (hr) Actual oxygen consumption (cm a O2/g) Estimated oxygen consumption in the

absence of torpor (cm s O2/g) Savings (cm a O~) Savings as per cent of estimated oxygen

consumption in the absence of torpor

Period of torpor and overshoot 14"1 13"7 13"5 8"5 9"6 12"4 6"3 6"8 8-9 18"0 18"1 16"9 13.5 13"0 17"9 12"9 15.2 19"0 42'3 41.1 40.5 22.1 25"0 32"2 17.0 18.4 24"0

410 388 398 171 238 286 93 71 114 57 56 58 39 48 44 24 17 21

Estimated total daily oxygen consumption 78.0 78.3 78.0 in the absence of torpor (cm 3 O2/g)

Savings as per cent of estimated total daily 31 29 30 oxygen consumption in the absence of torpor

71"0 69"1 68"5 76-2 77"0 76"2

12 17 21 5"4 4'1 6"6

Golley, 1961; Wiegert, 1965). The average daily savings of animals 2051, 2037 and 2082 thus correspond, respectively, to about 380, 220 and 88 mg of dry food assuming (unrealistically) that the food is assimilated completely and catabolized to the same end-products as are produced in bomb calori- metry. The reduction in actual food intake permitted by torpor will, of course, be greater insofar as the food is incompletely utilized in energy metabolism and will be greater when expressed in terms of wet weight than in terms of dry weight. In animals 2051, 2037 and 2082, respectively, the energetic savings realized by torpor average 30, 17 and 5 per cent of the estimated total daily energy expenditure in the absence of torpor (Table 3).

The values obtained for these three animals should be reasonably representative of the range of values for animals which underwent regular daily torpor in this study. Animal 2051 was one of the three animals which exhibited especially long episodes of torpor (Table 2). Three animals, including 2037, displayed episodes of intermediate length. Again, animal 2082 exhibited distinctly shorter and more shallow episodes than the other animals. Its energetic savings were accordingly relatively small.

Effects o f food deprivation

The effect of food deprivation was appraised on two animals, 2033 and 3001, which displayed only sporadic episodes of torpor on an adequate diet.

Animal 2033 displayed three very short episodes of torpor over a period of 30 days in the presence of adequate food. The maximal duration of these episodes was 1.5hr, and the minimal oxygen consumption recorded was 1.4 cm 3 O2/g per hr. At

the end of the 30-day experiment, all but 4 g of food was removed from the animal chamber. The mouse did not evidence torpor on the first day following the restriction of food supply but did so on the second. Oxygen consumption fell below 2.0 cm a O~/g per hr for 3"8 hr and reached a minimum of 1.2 cm 3 O2/g per hr. The episode terminated with a large metabolic overshoot. This episode was longer and deeper than any observed during the period on adequate food. On the third day after the restriction of the food supply, the animal displayed two episodes of torpor, one lasting 2.4 hr, the other, 5.7 hr. The latter episode was profound. Oxygen consumption remained near 1'1 cm a O~/g per hr for 2 hr. Torpor continued to occur on days 4, 5 and 6. The animal was removed on day 6 at an oxygen consumption of 1.5 cm a O2/g per hr. Its body temperature was 24.6°C, and its weight was 29 per cent lower than when food was initially restricted.

Animal 3001 was studied for 29 days in the presence of adequate food. It displayed torpor on days 24, 25 and 26 but not on any preceding day or on days 27, 28 and 29. The maximal duration of the episodes displayed on an adequate diet was 3-3 hr, and the minimal oxygen consumption recorded was 1.0 cm a O~/g per hr. On the evening of the twenty- ninth day, all but 4 g of food was removed from the animal chamber. There was no evidence of torpor over the two succeeding days, but on the third day following restriction of the food supply the animal underwent a 3-6 hr episode of torpor during which oxygen consumption fell to 1.1 cm ~ OJg per hr. The episode on the fourth day lasted 6.1 hr, and oxygen consumption remained in the range of 0-7-1.1 cm 30~/g per hr for 3 hr. This episode was clearly longer and deeper than any exhibited when the food

420 RICHARD W. HILL

supply was adequate. On the fifth day the animal underwent a 2-9 hr episode in the morning in which oxygen consumption fell to 0.7 cm 30~/g per hr and then entered torpor again in the late afternoon. It was removed during this latter episode at a metabolic rate of 0.3 cm 3 O2/g per hr. Its body temperature was 15-3°C, and its weight was 22 per cent lower than when food was initially restricted. After a 14 hr period of recovery the animal was returned to the metabolic apparatus with an abundance of food. It then failed to give evidence of torpor for 7 additional days of study.

These experiments, while limited in scope, corroborate the conclusion of Morhardt & Hudson (1966) and Morhardt (1970) that inadequacy of food is a stimulus to torpor in P. lettcopus.

DISCUSSION

The past decade has witnessed increasing knowledge of widespread ability to undergo torpor among North American cricetine rodents. Spontaneous torpor (that is, torpor on an adequate diet) has previously been reported in Peromyscus maniculatus (Fuller et aL, 1969) and Reithrodontomys raviventris (Fisler, 1965). This report and the recent study of Gaertner et al. (1973) extend knowledge of spontaneous torpor to P. leucopus. Fasting-induced torpor has been found in Baiomys taylori (Hudson, 1965) and several species ofPeromyscus (MacMillen, 1965; Morhardt & Hudson, 1966; Morhardt, 1970). It is now apparent that controlled hypothermia occurs quite commonly among cricetines, and it seems likely that future work will reveal the pheno- menon in additional species. This knowledge adds a new dimension to our understanding of the species concerned and of their place in the energy dynamics of the ecological communities which they occupy.

The timing of torpor in P. leucopus is of interest. With few exceptions, animals entered torpor in the dark hours of early morning or shortly after the lights came on in the morning and had emerged from torpor by the onset of dark in the evening. Gaertner et aL (1973) report that mice undergoing spontaneous torpor in their study usually entered torpor shortly after returning to their nest at the end of nightly activities. This pattern of torpor would appear to be well integrated with demands for feeding and other activities in a nocturnal animal. The mouse is prepared to commence feeding at the onset of darkness and thus can potentially utilize the entire night if necessary to find adequate food. We can hypothesize that the animal enters torpor only after demands for feeding and other nocturnal activities have been met.

An overshoot of the simple resting metabolic rate during arousal is generally observed in P. leucopus and has been reported in some other small rodents (e.g. Perognathus californicus; Tucker, 1965a). The

overshoot in effect diminishes the energetic savings of an episode of torpor, but, on the other hand, the stimulation of metabolism reflected by the overshoot should act to augment the rapidity of rewarming. The rate of rewarming will be increased insofar as the arousing animal minimizes thermal conductance and maintains a maximal metabolic heat production (aerobic and anaerobic) for its body temperature until the body temperature reaches non-torpid levels (see Tucker, 1965b). The extent to which P. leucopus brings this entire physiological complex into play cannot be appraised from the data available, but it is clear that aerobic metabolism is generally stimulated well above minimal levels during at least the terminal phases of arousal. A decline in metabolic rate to the simple resting rate, as often observed in P. leucoptts, can be expected once rewarming is complete.

The body temperatures of the seven mice which displayed daily spontaneous torpor ranged from 16-8 to 27.7°C. Temperatures of 24.6 and 15.3°C, respectively, were observed in the two mice which were deprived of food. It is possible that temperatures of 15-18°C were representative of a passive thermoregulatory state during torpor. Such temperatures were only modestly above the ambient temperature, and the insulation of the cotton nest would probably lead to higher body temperatures in a passive state than would be observed in mice without a nest. It seems likely that mice with body temperatures above 20°C were actively thermoregu- lating while in torpor. Animal 2082 provides the extreme example. It never allowed its metabolic rate to fall to the low levels observed in other mice and had a body temperature of 27.7°C when removed from the apparatus. The observed response of this mouse is compatible with the hypothesis of thermoregulation according to a lowered thermoregulatory set-point during torpor. In mice which were induced to undergo torpor through fasting, Morhardt (1970) observed body temperatures of 17-25°C in tests at ambient temperatures of 13-15°C. The range of body temperatures is similar to that observed here.

According to calculations presented in the Results section, the energetic savings realized by an episode of torpor in animals undergoing stabilized spontaneous daily torpor ranged from a mean of 442 cal in animal 2082 to a mean of almost 1900cal in animal 2051. Expressed as a percentage of total daily energy expenditure in the absence of torpor, the savings ranged from 5 to 30 per cent. These figures depend on the assumptions made in the calculations, and it is difficult to appraise the extent to which the assumptions are at variance with conditions in nature.

One significant assumption is that the metabolic rate over the period of torpor would have remained at the simple resting rate had the animal not entered torpor. First it should be recognized that this assumption is basically conservative, for it minimizes the calculated energetic savings. Were the


savings to be computed by comparing the actual metabolic expenditure during torpor with a non- torpid expenditure greater than the simple resting expenditure, the calculated savings would be greater. The ideal in calculating savings would be to know what the actual metabolic expenditure would have been had torpor not occurred. Since this can never be known with certainty, some assumption will always be necessary and, at the present time, the most conservative assumption has merit. There are biological grounds for making this assumption in P. leucopus since torpor generally occupies pre- dominantly daylight hours, when the animal would probably be resting in its nest whether torpid or not.

In calculating the per cent daily energy savings realized through torpor, the energy expenditure during the non-torpid part of the day must enter in. This can be measured directly, and measured values were utilized in the calculations performed here. An assumption implicit in the calculation is that metabolism during the non-torpid part of the day would have been the same even if the animal had not entered torpor. There are no compelling reasons for rejecting this assumption at present, but it should be recognized. An additional question is whether the energy expenditure during the non-torpid period as measured in these experiments is a reasonable approximation of that incurred by mice in nature. There is good reason to think that the expenditure in nature would be significantly greater (see, for example, Mullen, 1970). Mice in nature move around over a vastly greater area than that provided in the experiments, and they must find and some- times catch their food rather than having it immediately available. The computed per cent daily energy savings realized by torpor decreases with increasing energy expenditure during the non-torpid period. In all likelihood, use of the laboratory data on this expenditure therefore has the effect of over- estimating the per cent daily energy savings due to torpor in nature. Note that this error is in the opposite direction of any error encountered in using the simple resting metabolic rate as a basis for comparison in computing the energy savings during the period of torpor itself.

Additional problems of interpretation also arise, among them the question of whether the length and depth of torpor observed experimentally are representative of the length and depth in nature. Other authors (e.g. Hudson, 1973) have discussed the difficulty of estimating energy savings in natural situations.

It seems clear from the data obtained that daily energy demands were reduced by torpor in the experimental environment utilized. An important point is that individual mice varied considerably in the savings realized.

One of the striking aspects of the present results is the individual variation. Among seventeen animals

tested for spontaneous torpor under identical experimental conditions, some underwent relatively deep and prolonged episodes in a regular daily pattern; one in particular (2082) underwent distinctly shallow and short episodes on a daily basis; some underwent only sporadic and relatively shallow episodes; and some failed altogether to enter torpor. Fisler (1965) reported considerable individual variability in his study of spontaneous torpor in R. raviventris. Some mice entered torpor on over 80 per cent of test days; some never entered torpor; and some displayed an intermediate incidence of torpor. Other workers on P. leucopus have not dealt so explicitly with individual variation but indicate, at least indirectly, that it was observed. In his studies of fasting-induced torpor, Morhardt (1970) found that the percentage of weight lost before the onset of torpor varied significantly among individuals. Gaertner et aL (1973) report their data on spontaneous torpor as the percentage of test days on which torpor occurred, irregardless of the individual displaying torpor. The incidence of torpor varied from as high as 89 per cent of test days to as low as 0 per cent in at least a quasi-seasonal pattern. The authors do not give the percentage of test animals which displayed torpor, but the implication is that this varied with season. It is explicitly noted that one out of seven groups of mice failed to show torpor when tested in winter, the season when torpor was most likely. Gaertner et aL induced torpor through starvation in some mice and found that some individuals entered deep torpor after only a single day of inadequate food, whereas one individual had to be starved to the brink of death before showing torpor.

Individual variation in t". leucopus and perhaps other cricetines may well be so great that it demands study in its own right (that is, cannot be treated as "noise"). This conclusion is indicated by the results reported here. It may be that all individuals are basically alike but show short-term variations in their proclivity toward torpor, thus giving rise to the range of behaviour observed within any given test period. On the other hand, individuals may show basic and persistent differences in their proclivity toward torpor, perhaps due to genetic causes. Should heritable differences be present, we would have opportunity to study an example of mammalian torpor in a potential state of contemporary evolu- tionary flux.

In this context the relationships of the animals in this study deserve note. All animals which displayed spontaneous torpor were from Michigan and were drawn from a colony of 119 individuals, including 22 pairs of wild-caught animals and their offspring. The colony was established in connection with other research, and the occurrence of torpor was dis- covered during routine cage cleaning. Seven animals had been observed in torpor in this manner before the organized studies on torpor were undertaken:

422 RJCHAm~ W. HILL

animals 1034, 1047, 2001, 2002, 2037, 2046 and 2051. The genetic relationships of all animals were available in colony records, but the author was unaware of these relationships at the time the original observations of torpor were made. When the records were consulted at the beginning of the studies on torpor, it was found that the following pairs of animals were known relatives: 1047 and 2051 (mother--daughter), 2001 and 2002 (siblings) and 2037 and 2046 (siblings). Since the routine cage cleaning provided for numerous, randomized observations of 119 animals distributed in 22 genetic lines, it seemed remarkable that 6 of the 7 animals found in torpor should prove to be from three genetic lines. To assess whether this result is unusual enough to warrant interest, it is necessary to compute the a priori probability of obtaining a similar result, or one indicating even more genetic relationship, under the assumption that torpor occurred randomly among the individuals in the colony. The twenty- two genetic lines had from three to ten adult individuals per line, giving the situation sufficient com- plexity to make computation of this probability by formal methods most arduous. The problem was thus approached by having a computer repeatedly select seven individuals at random from the 119 individuals and assess the degree of relatedness displayed by the individuals in each set of seven. In 517 iterations, seven of the sets (1.4 per cent) dear ly displayed as much, or more, relatedness as actually observed (for example, two iterations placed two individuals in each of three genetic lines; three iterations placed three individuals in one line and two individuals in another; and one iteration placed four individuals in one line and two in another). Twenty-one (4.1 per cen0 of the 517 iterations placed 3 individuals in one genetic line and the remaining 4 individuals in four different lines. It is uncertain whether this result should be considered to represent as great a degree of relatedness as the result actually observed; but, even if it is so considered, the total estimated probability of obtaining a result showing as much, or more, relatedness as observed is still only 5.5 per cent. The apparently low probability of obtaining a result like the one observed under the assumption that torpor is randomly distributed indicates that it might well be worth while to explore the potential of a heritable influence on torpor in P. leucopus.

Once the relationships of the original animals found in torpor had been elucidated, six additional known relatives were tested. Four displayed torpor: 1001 (father of 2001 and 2002), 3001 (the father of which was a sibling of 2037 and 2046) and 2033 and 2050 (siblings of 2051). Two known relatives failed to show torpor: 1026 (father of 2037 and 2046) and 3003 (sibling of 3001). Two animals which displayed torpor, 1034 and 2082, and four animals which failed to show torpor had no known relatives included in this study.

The three studies now available on torpor in P. leucopus have all indicated that fasting is a stimulus to torpor (see Morhardt, 1970 and Gaertner et aL, 1973). Gaertner et al. have also reported spontaneous torpor, but Morhardt & Hudson (1966) and Morhardt (1970) failed to see torpor in the absence of fasting and, indeed, concluded that fasting was probably necessary to induce torpor, at least in laboratory animals. Morhardt worked on the sub- species texanus rather than noveboracensis and made use of implanted thermocouples, which he recognized might disturb the animals sufficiently to interfere with the full manifestation of torpor. His failure to observe spontaneous torpor when it has been observed in both of the other studies could be attributable to differences in the origins of his animals or in his experimental techniques. Gaertner et al. have presented evidence, however, that the incidence of spontaneous torpor increases with decreasing temperature of acclimation. Should this be true, it may provide an explanation for Morhardt 's different results, for his animals were acclimated and initially tested at a relatively high temperature, 23-26°C. It is noteworthy that the present results are compatible with the hypothesis of Gaertner et al., for spontaneous torpor occurred in mice acclimated at a lower temperature than that utilized by Mor- hardt but apparently did not occur at as high an incidence as observed by Gaertner et al. during some months of the late fall and winter, when temperatures were still lower.

If mice differ in their proclivity toward torpor, it is possible that what we have observed in the studies so far is a recruitment of torpor which starts with the most torpor-prone individuals and ends with the least torpor-prone and depends in its extent on the strength of torpor-provoking environmental stimuli. Fasting is clearly a suitable stimulus, and lowered temperature of acclimation may well be another.

Acknowledgements--Much of this work was performed at the University of Delaware, and I express my gratitude to the Department of Biological Sciences and University of Delaware Research Foundation for their financial support.

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Key Word Index--Torpor; hypothermia; thermoregulation; metabolism; energetics; rodents; Peromyscus leucopus.

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Daily torpor in Peromyscus leucopus on an adequate diet