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Aspects of temperature adaptation in Peromyscus Hayward, John Stanley 1964

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ASPECTS OF TEMPERATURE ADAPTATION IN PEROMYSCUS by JOHN STANLEY HAYWARD B.Sc„, University of B r i t i s h Columbia, 1958 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1964 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of • B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that, copying or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission* Department of Zoology  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada Date August 28, 1964 Chairman: Professor I. McT. Cowan i ABSTRACT Six races of Peromyscus, from a v a r i e t y of habitats, have been studied with respect to factors involved i n adaptation to environmental temperature. The central theme of the study was to assess the extent to which metabolic rate i s involved i n the processes of d i s t r i b u t i o n and speciation in t h i s genus. A unique, e l e c t r o l y t i c respirometer for the accurate measurement of oxygen consumption was constructed and reported i n the l i t e r a t u r e . With t h i s apparatus, the metabolic rate c h a r a c t e r i s t i c s of the six races were measured over the temperature range 0° to 35°C. After acclimation to standardized laboratory conditions, c r i t i c a l temperatures and metabolic responses to temperatures below thermoneutrality were pr i m a r i l y related to body s i z e . They show, therefore, no evidence of r a c i a l metabolic rate adaptation or s i g n i f i c a n t i n s u l a t i v e differences. Body weight per se i s not correlated with the climate of the respective habitats. A single equation which predicts the metabolic rate of these races at any temperature between 0° and 27°C, from a knowledge of body weight and body temperature, i s derived. When considered as a single group, the basal oxygen consumption of a l l races varied with body weight^*^^ and was i n s i g n i f i c a n t l y d i f f e r e n t from the accepted interspecies approximation. The basal metabolic rates of each race showed no temperature-adaptive differences, e s p e c i a l l y when considered i n r e l a t i o n to body composition. The body composition i n terms of water, f a t and protein was determined for a l l individuals. The importance of consider-ing body composition, e s p e c i a l l y fatness, in comparative studies of metabolic rate i s emphasized. It i s concluded that metabolic rate i s inadaptive to climate i n these races of Peromyscus and consequently has played no important r o l e i n t h e i r d i s t r i b u t i o n and speciation. I t i s shown that the major temperature-adaptive feature of these small mammals i s the use of a suitable microclimate. Measurements of the ambient temperatures p r e v a i l i n g i n the microhabitats of the six races during winter and summer are presented. These data indicate that there i s no s i g n i f i c a n t d i f f e r e n t i a l s e l e c t i v e pressure for temperature adaptation among the six races, and are complementary to the finding that metabolic rate i s inadapt-ive to gross climate i n Peromyscus. i i i TABLE OF CONTENTS PAGE ABSTRACT i TABLE OF CONTENTS i i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGMENTS X FOREWORD x i I. THE GROSS BODY COMPOSITION OF SIX GEOGRAPHIC RACES OF PEROMYSCUS 1 Abstract 1 Introduction 2 Experimental 5 Animals 5 Care and Maintenance of Mice 5 Chemical Methods 8 Results 10 The Body Composition of Laboratory-acclimated Deer Mice 10 Body Composition of Mice in t h e i r Natural Habitat 18 The Changes in Body Composition Associated with Laboratory Acclimation 20 Discussion 22 References 27 II . METABOLIC RATE AND ITS TEMPERATURE-ADAPTIVE SIGNIFICANCE IN SIX GEOGRAPHIC RACES OF PEROMYSCUS 31 Abstract 31 Introduction 32 Animals 34 iv PAGE Methods 35 Metabolic Rate 35 Body Temperature 37 Results 37 A. Metabolic rate in Relation to Ambient Temperature 37 (1) . Laboratory-acclimated mice 37 (2) . Seasonal comparisons of mice from th e i r natural habitat 48 B. Basal Metabolism and Body Weight 50 C„ Basal Metabolism and Body Composition 55 Discussion 59 References 66 II I . MICROCLIMATE TEMPERATURE AND ITS ADAPTIVE SIGNIFICANCE IN SIX GEOGRAPHIC RACES OF PEROMYSCUS 70 Abstract 70 Introdu ct ion 71 Temperature Measuring Technique 73 Habitat Descriptions 77 Results 79 Discussion 84 References 91 APPENDIX. A SIMPLE ELECTROLYTIC RESPIROMETER FOR ... SMALL ANIMALS 95 Abstract 95 Introduction 95 P r i n c i p l e 96 Description of the Respirometer 97 Operation of the Respirometer 99 V PAGE Cal i b r a t i o n and Calculation 102 Results and Discussion 103 References 3/08 EPILOGUE 109 LIST OF TABLES v i TABLE PAGE I. THE GROSS BODY COMPOSITION OF SIX GEOGRAPHIC RACES OF PEROMYSCUS I The i d e n t i f i c a t i o n of the experimental mice (genus Peromyscus) along with the l o c a l i t y where they were trapped. A b r i e f catagorization of t h e i r habitat types i s included. 6 II The gross body composition of the six races of Peromyscus acclimated to. si m i l a r laboratory conditions for more than s i x months. Comparable data for white mice are from Bailey est a l . (4) l l III An example of the i n t r a r a c i a l v a r i a b i l i t y of fa t content (P. m. nebrascensis) 13 IV The average seasonal body composition of the six races of Peromyscus l i v i n g i n t h e i r natural habitat (summer and winter) 19 II. METABOLIC RATE AND ITS TEMPERATURE-ADAPTIVE SIGNIFICANCE IN SIX GEOGRAPHIC RACES OF PEROMYSCUS I Regression equations for the r e l a t i o n s h i p of metabolic rate (MR, cc02/gm/hr) and ambient temperature (T A, °C) between 0° and 27°C 40 II A comparison of the calculated and measured body temperatures of the six races of Peromyscus 44 III Calculated lower c r i t i c a l temperatures and c r i t i c a l gradients for the six races of Peromyscus 47 v i i LIST OF FIGURES FIGURE PAGE I. THE GROSS BODY COMPOSITION OF SIX GEOGRAPHIC RACES OF PEROMYSCUS A map of western North America showing the locations from which the mice i d e n t i f i e d i n Table I were sampled. 7 The decrease in the hydration of the f a t - f r e e body at high f a t l e v e l s . 15 The r e l a t i o n s h i p of protein to water i n Peromyscus. 16 A comparison of the f a t le v e l s of mice caught in the summer with those acclimated to laboratory conditions. 21 II . METABOLIC RATE AND ITS TEMPERATURE-ADAPTIVE SIGNIFICANCE IN SIX GEOGRAPHIC RACES OF PEROMYSCUS The r e l a t i o n s h i p between rate of oxygen consumption and ambient temperature for the six races of Peromyscus. The curves extrapolate to an average body temperature ( T B ) . To change to energetic units, 1 cc of oxygen i s equivalent to 4.73 x 10~ 3 Cal. 38 The re l a t i o n s h i p between the mean body weights of the races and the slopes of t h e i r metabolic responses to cold. 42 A comparison of the metabolic rate character-i s t i c s of summer and winter-caught individuals of J?. m. oreas in r e l a t i o n to the curve representing the average of the laboratory-acclimated representatives of t h i s race. 49 v i i i FIGURE PAGE Basal metabolic rate i n r e l a t i o n to body weight for Peromyscus and comparison with Brody's interspecies approximation. Standard errors of regression are for the Peromyscus data. 51 Comparison of the basal metabolic rate (BMR): body weight relationships of the ind i v i d u a l races of Peromyscus used i n t h i s study. 53 Basal metabolic rate considered in r e l a t i o n to t o t a l body water content for the six races of Peromyscus. 56 II I . MICROCLIMATE TEMPERATURE AND ITS ADAPTIVE SIGNIFICANCE IN SIX GEOGRAPHIC RACES OF PEROMYSCUS 1 A drawing of the connector-thermistor device in i t s two operating pos i t i o n s . The hinged bar at the posterior end of the connector i s made to f i t t i g h t l y enough that a gentle tug on the nylon l i n e i s s u f f i c i e n t to release the mouse. 75 2 This photograph shows a deer mouse with the connector and thermistor attached. The connector device i s e a s i l y made by a jewellery mechanic who has the small hinges necessary. The thermistor i s a Fenwall GB32P8. 76 3 A t y p i c a l summer recording of outside and burrow temperatures over a 24-hour period (subalpine h a b i t a t ) . 81 4 Comparison of outside (solid lines) and burrow (broken lines) temperatures in summer and winter and in a l l six habitats. The shaded areas are temperatures warmer than the burrows in summer and colder than the burrows in winter. 82 5 A comparison of the seasonal outside-temperature extremes i n each habitat with the average burrow temperatures at the same times. The broken l i n e s indicate how the burrow temperatures are moderated in comparison to the seasonal extremes. This diagram emphasizes the po t e n t i a l a v o i d a b i l i t y of temperatures capable of inducing thermoregulatory stress i n deer mice. 86 ix FIGURE PAGE APPENDIX. A SIMPLE ELECTROLYTIC RESPIROMETER FOR SMALL ANIMALS A diagrammatic representation of the respirometer. The spirometer i s shown in two posit i o n s . 98 A general view of the respirometer. 100 An example of the results obtained with the respirometer showing the re l a t i o n s h i p between environmental temperature and the res t i n g rate of oxygen consumption of a 25-gm Swiss albino mouse. 104 A record of the 24-hour oxygen consumption of a deer mouse (Peromyscus maniculatus) at 25°C. Points are the average of 1-hour i n t e r v a l s . 106 X ACKNOWLEDGMENTS The author wishes to express sincere thanks to Dr. I. McT. Cowan, whose advice, support and encouragement were always forthcoming during the course of t h i s study. The many ideas and timely encouragements of Dr. A. J. Wood have been a continual source of i n s p i r a t i o n and are due a l a s t i n g appreciation. Thanks are also due to Dr. R. D. Taber, who arranged the i n i t i a l sampling of mice from the Wyoming alpine. I would l i k e to express my gratitude to the National Research Council of Canada for the bursary and studentships held during my graduate t r a i n i n g . F i n a l l y , the author i s sincerely g r a t e f u l to h i s wife, Mary, whose s a c r i f i c e s and encouragement have made these years of graduate study possible. x i FOREWORD The format of t h i s thesis i s somewhat unusual. The study can be divided into three major aspects; body composition, metabolic rate, and microclimate. Accordingly, i t was decided to write these three sections as independent papers suitable for p u b l i c a t i o n . The s t y l e i s that of the Canadian Journal of Zoology. The three papers follow a l o g i c a l sequence and the ideas therein are c a r e f u l l y cross-referenced. I t i s hoped to submit these as a series, with some revision, in the above-mentioned journal. A description of the respirometer which was developed for t h i s study has already been published i n the Canadian Journal of Zoology, and i s included herein as an appendix. 1 I. THE GROSS BODY COMPOSITION OF SIX GEOGRAPHIC RACES OF PEROMYSCUS Abstract The body composition i n terms of fat, water and protein has been determined for 115 deer mice (genus Peromyscus) of six r a c i a l stocks. The changes i n composition that are c h a r a c t e r i s t i c of seasonal extremes and that accompany laboratory acclimation are presented. The composition of the f a t - f r e e body exhibits the constancy which has been found i n other mammals. Body protein averaged 22.97 percent and body water 69.71 percent of the f a t - f r e e body weight. Body f a t l e v e l s are shown to vary considerably among individuals and races. The highest f a t le v e l s occurred i n the desert-adapted race (P. m. sonoriensis). Apparent genetic differences between the races include the proportion of hide and pelage i n the t o t a l body weight, the water content of the hide, the protein:water r a t i o and the capacity to store f a t . The importance of considering body composition i n comparative studies of metabolic rate i s discussed. 2 Introduction This i s the f i r s t i n a series of communications dealing with the question of adaptation to environmental temperature in a sel e c t i o n of closely-related, but geographically and c l i m a t i c a l l y d i s t i n c t , races of the North American genus Peromyscus (deer mice). The central theme of the study involves comparisons of the metabolic rates of these mice when acclimated to sim i l a r laboratory conditions. At an early stage in these investigations, i t became obvious that a more r e l i a b l e reference base for metabolic rate comparisons than t o t a l body weight or surface area would be desirable. The l i t e r a t u r e i s well documented with evidence (1, 10, 15, 19, 20, 25, 35, 36, 37) that physio-l o g i c a l functions such as basal metabolic rate are best expressed i n r e l a t i o n to some single or multiple component of body composition, e s p e c i a l l y when comparisons are to be made on an i n t r a s p e c i f i c l e v e l . Despite t h i s evidence, metabolic rate i s s t i l l commonly related to surface area or to some function of t o t a l body weight. The use of such baselines assumes a homogeneity of body composition, which may not be the case. This problem was extensively discussed by Benedict (6). In a following report (16), the basal metabolic rates of these races of Peromyscus w i l l be presented i n r e l a t i o n to t h e i r body composition. The present report attempts to describe the differences that occur i n the 3 body composition of these mice as r e f l e c t i o n s of genetic va r i a t i o n , n u t r i t i v e condition, seasonal changes, and as mechanisms of adaptive s i g n i f i c a n c e . The pronounced compositional changes that take place when mice are brought from t h e i r natural habitat and housed and fed i n the laboratory are stressed. The study of body composition i s one of the fo c a l points of contemporary e f f o r t s i n human and animal biology (9^). Evidence of t h i s i s provided by the extensive recent symposia devoted to t h i s subject (11, 38). Investigators i n the f i e l d of animal husbandry have structured body composition into an a n a l y t i c a l d i s c i p l i n e in t h e i r e f f o r t s to understand the process of growth and development and the n u t r i t i o n of domestic animals. Also, the human b i o l o g i s t , investigating the fundamentals of health and disease, has contributed many advances i n t h i s f i e l d . There has been no u t i l i z a t i o n of these concepts i n the study of comparative physiology of wild mammals. Where ph y s i o l o g i c a l adaptation of cl o s e l y - r e l a t e d populations i s in question, differences are l i k e l y to be small, and measurement of body composition as a reference c r i t e r i o n permits more meaningful interpretations. The only extensive study of the gross body composition of small wild mammals i s that of P i t t s (31). He studied a large number of Alaskan and temperate zone mammals i n order to determine whether constancy of composition of the f a t - f r e e body i s obtained on an i n t e r s p e c i f i c basis among mammals. 4 The body composition i n terms of water, f a t and protein has been determined for 115 deer mice representing six geographic races. The samples include animals kept under laboratory conditions for more than six months and animals caught i n t h e i r natural habitats during the summer and winter seasons. In addition to i t s function as part of a larger study of Peromyscus, the data w i l l add to the fund of knowledge of body composition based upon d i r e c t chemical analysis; such knowledge provides the fundamental reference point for the i n d i r e c t analysis of l i v e i n d i v i d u a l s . 5 Experimental Animals Representatives of six geographic populations of t h i s genus were established i n a laboratory colony. The populations were chosen so as to sample as wide a v a r i e t y of gross c l i m a t i c environments as possible. This would maximize the p o s s i b i l i t y of discerning temperature-adaptive character-i s t i c s , i f any e x i s t . The i d e n t i t y of these mice according to Osgood (26) and Cowan and Guiguet (12), along with a description of the trapping l o c a l i t i e s , i s given i n Table I. The s i t u a t i o n of these l o c a l i t i e s on a map of western North America i s shown by F i g . 1. A more det a i l e d outline of the habitats of these mice w i l l be given in a l a t e r report which describes the microclimate encountered by these mice (17). Five of the experimental populations are taxonomically d i f f e r e n t i a t e d at the subspecies or r a c i a l level? the si x t h (P. s i t k e n s i s prevostensis) at the species l e v e l . However, recent evidence (13) indicates that P. si t k e n s i s i s a c t u a l l y conspecific with P. maniculatus. The older c l a s s i f i c a t i o n w i l l be used i n t h i s study. In the interests of brevity, a l l groups w i l l be referred to hereafter as "races". Care and Maintenance of Mice The mice were housed in a temperature and humidity-controlled animal unit. The v e n t i l a t i o n system provided an TABLE I The i d e n t i f i c a t i o n of the experimental mice (genus Peromyscus) along with the l o c a l i t y where they were trapped. A b r i e f catagprization of t h e i r h a b i t a t types i s included Race Trapping l o c a l i t y Habitat type P^ . maniculatus nebrascensis (Coues) Beartooth Pass, Wyoming B. P. m. austerus (Baird) C. j?. m. sonoriehsis Le Conte D. j?. m. artemisiae (Rhoads) E. P. ro. oreas Bangs F. P. sitkensis prevostensis Osgood Point Grey, B. C. S i l v e r Peak, Nevada Oliver, B. C. A l l i s o n Pass, B. C. Hotspring Island, Queen Charlotte Islands treeless alpine, 10,500 f t a l t i t u d e Puget Sound lowland forest high-altitude desert, 4,800 f t ari d , i n t e r i o r v a l l e y subalpine forest, 4,500 f t a l t i t u d e wet, coastal i s l a n d 7 F i g . 1. A map of western North America showing the locations from which the mice i d e n t i f i e d i n Table I were sampled. 8 a i r temperature of 21° to 23°C (dry bulb) and a r e l a t i v e humidity of 50 to 55 percent. A r t i f i c i a l illumination was regulated to a 12-hour d a i l y photoperiod. The d e t a i l s of caging and management for t h i s type of colony have been described (21). A p e l l e t e d r a t i o n deemed n u t r i t i o n a l l y adequate on the basis of the performance of laboratory mice (21) was fed ad libitum. The r a t i o n has a metabolizable energy value of approximately 2900 Calories per kilogram, and a protein to Calorie r a t i o of 80 mgm/Cal derived from a mixture of fishmeal and cereal proteins of good b i o l o g i c a l value. The mice were caged in groups of 2 to 4 individuals and the pans and bedding were changed weekly. The incidence of intermittent i n f e c t i o n i n the colony was n e g l i g i b l e . Under these management conditions, a healthy colony of deer mice has been maintained for three years. Chemical Methods Mice for body composition analysis were k i l l e d immediately subsequent to metabolic rate determinations. o They were weighed and stored at -20 C u n t i l ready for ana l y s i s . The analysis of the major body constituents followed standard procedures (2). Body water was determined by desiccation to constant weight in an oven maintained at 105°C (24 hours). The dry carcass was q u a n t i t a t i v e l y transferred to a tared soxhlet extraction 9 thimble. The bodies were extracted with petroleum ether (b.p. 30° - 60°C) i n a soxhlet apparatus for 16 hours and the decrease i n weight of the dry carcass was recorded as the weight of the f a t or ether-extractable l i p i d . The f a t - f r e e , dry body i n the nitrogen-free extraction thimble was then transferred to a macro-kjeldahl digestion flask and the t o t a l contents hydrolyzed using copper sulphate as the c a t a l y s t . Duplicate samples of the digestion mixture were d i s t i l l e d i n a m i c r o d i s t i l l a t i o n apparatus using a b o r i c acid trap. I t was assumed that a l l of the nitrogen present was of protein o r i g i n and that the mixed body proteins contained 16 percent nitrogen. The remainder of the t o t a l body weight that was unaccounted for i n the analyses averaged about 7 to 8 percent of the t o t a l body weight and w i l l be referred to hereafter as "not analysed". This component presumably represents the body ash (not determined) plus the sum of errors in the water, f a t and protein determinations. 10 Results I t i s a good f i r s t - o r d e r approximation to consider the animal body as consisting e s s e n t i a l l y of two major components; a r e l a t i v e l y constant f a t - f r e e body and a variable amount of fa t (5). The expression of body constituents as percentages of the f a t - f r e e body weight (FFBW) f a c i l i t a t e s i n t e r i n d i v i d u a l comparisons and enables s i g n i f i c a n t deviations from constancy to be discerned. Accordingly, compositional comparisons of individuals and races of mice in t h i s study are on a f a t - f r e e body weight basis. The Body Composition of Laboratory-acclimated Deer Mice A summary of the gross body composition of 50 laboratory-acclimated mice that had previously undergone metabolic rate t r i a l s i s presented i n Table I I . The most s t r i k i n g feature of the data i s the large range of f a t content among the races. The Nevada race (P. m. sonoriensis) has a s i g n i f i c a n t l y higher mean f a t content than any of the other races (t = 0.05), but additional s i g n i f i c a n t differences i n f a t content were not found due to the large i n t r a r a c i a l v a r i a b i l i t y i n fatness. The minimum f a t l e v e l observed was 6.1 percent of the FFBW. Four mice of 3 r a c i a l stocks shared the highest values for f a t content: 63.5 to 64.5 percent of the FFBW (approximately 39 percent of the t o t a l body weight). This apparent consistency of TABLE II The gross body composition of the six races of Peromyscus acclimated to s i m i l a r laboratory conditions for more than six months. Comparable data for white mice are from B a i l e y et. a l . (4) Mean body weight Race n (gm) P.m. nebrascensis 5 18.36 14.60 3. 77 10. 23 3. 19 1. 18 25.82 70. 07 21.85 8.08 P.m. austerus 10 19.4.8 16.24 3. 24 11. 50 3. 61 1. 12 19.95 70. 81 22.22 6.90 P.m. sonoriensis 5 20.64 13.56 7. 08 9. 22 3. 12 1. 21 52.21 68. 00 23.01 8.92 P.m. artemisiae 10 23.34 20.43 2. 91 14. 32 4. 66 1. 45 14. 24 70. 09 22.81 7.10 P.m. oreas 10 24.47 19.91 4. 55 13. 83 4. 59 1. 49 22.85 69. 46 23.06 7.48 P. si t k e n s i s 10 28.55 25.59 2. 96 17. 72 6. 09 1. 78 11.57 69. 25 23.80 6.96 white mice 111 26.3 23.9 2. 4 17. 4 5. 2 10.0 72. 8 21.8 Mean weight of Mean constituent (gm) Mean percent of FFBW FFBW • — (gm) f a t water pro- not f a t water pro- not t e i n anal- t e i n anal-yzed yzed maximum f a t l e v e l may indicate the maximum fattening p o t e n t i a l of these mice. Although there were indications that females have a higher f a t l e v e l than males, the difference i s not s i g n i f i c a n t due to the large i n d i v i d u a l v a r i a b i l i t y already mentioned. To further i l l u s t r a t e t h i s v a r i a b i l i t y , the actual f a t le v e l s i n the f i v e Wyoming mice (P. m. nebrascensis) are presented in Table I I I . The r e l a t i v e constancy of the f a t - f r e e body weight of mammals i s evident again in these Peromyscus data .'(Table I I ) . The average body water content of mammals i s variously stated to be between 71.0 and 73.2 percent of the FPBW (3, 27, 28), and as low as 67 to 69 percent i n healthy humans (23). This percentage i s f a i r l y constant aft e r the age of "chemical maturity" (24), but does continue to decrease slowly with further aging. The o v e r a l l percentage of body water in the FFBW for these deer mice (69.71) i s lower than the i n t e r -species averages c i t e d . One possible reason for t h i s lower value i s that the mice were at l e a s t 2 years old when, analyzed; well past the i n i t i a l stage of chemical maturity (55-60 days i n white mice (4)). The more important explanation i s that these mice have a r e l a t i v e l y large f r a c t i o n of t h e i r f a t - f r e e dry matter i n the form of skin and hair, which i s associated with much less water than the res t of the carcass. Separate analysis of the hides and hide-free carcasses of a sample of a l l races showed that the hides accounted for an 13 TABLE III t An example of the i n t r a r a c i a l v a r i a b i l i t y of f a t content (P. m. nebrascensis) Total body Sex weight (gm) f a t ^ m 14.95 2.58 m 22.00 4.69 f 16.75 1.06 f 17.40 2.40 f 20.72 8.11 14 average of 27.8 percent of the t o t a l body protein and that the hide has only 50.90 percent water on a f a t - f r e e basis as against 72.26 percent for the res t of the carcass. It has been emphasized frequently that the t o t a l body water content i s independent of the amount of f a t present (3, 28, 32). The data for Peromyscus suggest that t h i s may not be so i n cases of high f a t content or obesity. When the fa t content i s above approximately 30 percent of the FFBW, there i s an apparent decrease in the hydration of the f a t -free body from 70-71 percent down to about 65 percent at the highest f a t l e v e l s (Fig. 2). The most probable explanation for t h i s lowering would be a p a r t i a l replacement of some function of body water by body f a t . The reduction of body water at high f a t l e v e l s has been noted by others (29, 33). A few of the many records of body protein content (N x 6.25) i n d i f f e r e n t animals are summarized by M i t c h e l l (22). The interspecies average protein percentage of the FFBW i s 22.2. The i n t e r r a c i a l Peromyscus average i s only s l i g h t l y higher (22.97 percent) and i s exactly the same as the value c i t e d for the r a t by Pace and Rathbun (29). There are no s i g n i f i c a n t differences in t o t a l body water or protein percentages of the FFBW when comparing the s i x races of deer mice studied (Table I I ) . However, when the r e l a t i o n s h i p of protein to water i s plotted, one race i s d i f f e r e n t and poses an in t e r e s t i n g interpretation. F i g . 3 shows that the largest race (P. sitkensis) i s a 15 F i g . 2. The decrease in the hydration of the f a t - f r e e body at high f a t l e v e l s . 0-74 0-72 0 7 0 WATER 0 6 8 L FFBW 0-66 k 0-64 H o o o o o o o o o o o O O O O o 0 0 0-1 0 2 0-3 0-4 0-5 0-6 0-7 FAT FFBW 16 F i g . 3. The relationship of protein to water i n Peromyscus. GRAMS OF BODY PROTEIN (Y) 17 r e l a t i v e l y " d r i e r " mouse as judged by i t s high protein to water r a t i o . The l i n e of best f i t of the P. s i t k e n s i s data using the method of l e a s t squares i s s i g n i f i c a n t l y d i f f e r e n t from the l i n e f i t t e d to the data of the other f i v e races (t = 0.05). Recalling that compositional analyses were performed on hides and hide-free carcasses separately i n a sample of mice, these data showed that the r a t i o of hide protein to carcass protein i n P. s i t k e n s i s (mean = 0.394) was higher than that of the other races combined (mean = 0.343). P. s i t k e n s i s has, therefore, more of i t s protein i n the form of skin and h a i r than the other races, and, as has been pointed out, such protein i s associated with less water. This example serves to i l l u s t r a t e an apparent, genetic, compositional difference between P. s i t k e n s i s and the other races. Figure 3 also v e r i f i e s that the mice used i n t h i s study were a l l mature as judged chemically. The protein to water r a t i o has been suggested as an index of ph y s i o l o g i c a l age (4). It increases exponentially u n t i l chemical maturity and there-a f t e r maintains a f a i r l y constant slope; so that a series of mature animals plotted i n such a manner w i l l form a l i n e a r r e l a t i o n on an arithmetic scale. The Peromyscus data presented form such a r e l a t i o n . 18 Body Composition of Mice i n Their Natural Habitat It was expected that deer mice l i v i n g in t h e i r natural habitat might have a d i f f e r e n t gross body composition than those acclimated to the laboratory, e s p e c i a l l y with regard to fa t content. Perhaps, also, t h e i r protein l e v e l s might be d i f f e r e n t due to diet a r y protein quality, a c t i v i t y l e v e l , changes i n the amount of pelage, or some combination of these. Furthermore, i t was of int e r e s t to see i f there were compositional differences at seasonal c l i m a t i c extremes. The body composition of mice caught i n t h e i r natural habitat and k i l l e d immediately are presented for the summer and winter seasons i n Table IV. Several features of the seasonal values may be noted. F i r s t l y , the average body weights of the winter mice were less than those i n the summer sample. The winter mice may have been younger, yet t h e i r compositions indicate that they were chemically mature. Secondly, the average f a t content in r e l a t i o n to the FFBW was nearly twice as great i n winter in a l l races except P_. m. sorroriensis from Nevada. The higher winter f a t values may be attributed to the functions of increased tiss u e i n s u l a t i o n (7) and energy reserve. Although i t i s extremely cold i n Nevada in the winter season, the higher summer f a t l e v e l i n ^ . m. sonoriensis may be related to the increased emphasis on f a t storage i n desert mammals during hot, a r i d conditions (8, 41). TABLE IV The average seasonal body composition of the s i x races of Peromyscus l i v i n g in the i r natural habitat (summer and winter) Mean Mean weight of body Mean constituent (gm) Mean percent of FFBW Race and weight FFBW season n (gm) (gm) f a t water pro- not f a t water pro- not te i n anal- t e i n anal-yzed yzed P_.m. nebrascensis summer winter P_.m. austerus summer winter P_.m. sonoriensis summer winter P..m. artemisiae summer winter P_.m. oreas summer winter P. si t k e n s i s 7 19.80 19.01 0.79 13.32 1* 17.83 16.35 1.48 11.10 4 6 5 8 summer winter #nil 3 9 3 11 8 21.52 20.95 0.57 15.19 14.19 13.51 0.68 9.38 15.97 14.64 1.33 10.38 12.24 11.68 0.56 7.94 4.12 1.58 4.16 70.07 21.67 8.31 3.95 1.30 9.05 67.89 24.16 7.95 4.08 1.68 2.72 72.50 19.47 8.02 2.93 1.21 5.03 69.43 21.69 8.96 2.99 1.27 9.08 70.90 20.42 8.68 2.85 0.89 4.79 67.98 24.40 7.62 20.22 18.85 1.37 13.23 4.27 1.36 7.27 70.19 22.65 7.21 23.47 22.33 1.14 16.13 16.48 14.97 1.51 10.45 22.97 21.87 1.10 15.61 23.51 22.07 1.44 15.26 4.47 1.73 5.11 72.23 20.02. 7.75 3.28 1.23 10.09 69.81 21.91 8.22 4.33 1.95 5.03 71.38 19.80 8.92 4.85 2.08 6.52 69.14 21.98 9.42 * rigorous winter conditions made trapping extremely d i f f i c u l t . # the mouse population i n t h i s area was such that trapping was unsuccessful. 20 Inspection of the average protein l e v e l s in Table IV shows that there i s a uniformly higher percentage of body protein i n winter than i n summer. Combining t h i s f a c t with the observation that percentage body water i n the FFBW i s simultaneously decreased, suggests that the additional winter protein may be due to a r e l a t i v e l y larger amount of pelage. If th i s i s the case, i t provides i n d i r e c t evidence that pelage in s u l a t i o n i s increased to some extent i n winter. In t h i s context, i t i s also noteworthy that the highest protein l e v e l s occur i n P_. m. nebrascensis (alpine) and P_. m. sonoriensis (desert), from the two areas having the greatest extremes of temperature and solar r a d i a t i o n . The Changes i n Body Composition Associated with Laboratory  Acclimation The combined influences of high plane of n u t r i t i o n , moderate temperature, and sedentary existence i n the laboratory colony produced mice that are su b s t a n t i a l l y d i f f e r e n t in body composition from t h e i r wild counterparts, e s p e c i a l l y with respect to body f a t (comparing Tables II and IV). The large increase i n body f a t in the laboratory mice can best be v i s u a l i z e d from a bar chart (Fig. 4). The average increase i s 440 percent of the wild l e v e l i n summer. There i s also some i n d i c a t i o n that the protein l e v e l s i n the laboratory colony are greater in most of the races studied. 21 F i g . 4 . A comparison of the f a t l e v e l s of mice caught i n the summer with those acclimated to laboratory conditions. FAT FFBW P m. nebrascensis R m. sonoriensis R m. oreas P m. austerus p m. artemisiae P. sitkensis 22 Discussion The gross body composition of six races of deer mice under various conditions has been described. The r e s u l t s show that the amount of body f a t i n i n d i v i d u a l mice i s considerably variable, and one may speculate on the reasons for t h i s v a r i a b i l i t y . Of considerable influence i s the previous n u t r i t -ional h i s t o r y of the mice. For example, Peckham et a l . (30) have shown that the a b i l i t y to accrete body f a t in mature rats i s i n large part dependent upon the number of adipose c e l l s formed p r i o r to weaning. Simi l a r l y , Heggeness (18) demonstrated that weanling rats subjected to periods of c a l o r i c r e s t r i c t i o n a l t e rnating with periods of ad l i b i t u m intake showed, during unrestricted feeding, rates of gain and f a t accumulation greater than those of animals fed ad l i b i t u m continuously. The l i k e l i h o o d of v a r i a b i l i t y i n the previous n u t r i t i o n a l regimens of individuals and races of deer mice in t h e i r natural habitat may, therefore, account for the observed differences i n fatness under laboratory conditions. Another consideration may also be important in t h i s regard. I t has been shown by several investigators (39, 40) that c e r t a i n animals demonstrate seasonal c y c l i n g of f a t reserves when t h e i r n u t r i t i o n a l require-ments are s a t i s f i e d . In view of the f a c t that the laboratory deer mice were k i l l e d over an extended period of time in conjunction with metabolism t r i a l s , there i s the p o s s i b i l i t y that i n d i v i d u a l mice may have been sampled at various stages in 23 t h e i r f a t cycle; i f such a cycle exists i n t h i s genus. It may be pointed out that the f a t content of these mice under natural conditions i s s u f f i c i e n t as an energy reserve for only a very short period of f a s t i n g . For example, consider the one mouse (17.83 gm t o t a l weight) that was caught i n the Wyoming alpine during winter. From metabolic rate t r i a l s (16), i t s d a i l y energy production at 10°C may be estimated at approx-imately 13 Cal. At a metabolizable energy figure of 9.0 Cal per gram of fat, the 1.48 grams of body f a t would be s u f f i c i e n t for only about one day, assuming f a t as the sole contributor to the energy requirements. The s i g n i f i c a n t l y higher f a t l e v e l s found i n JP. m. sonoriensis when under laboratory conditions suggest that t h i s race has a g e n e t i c a l l y determined fattening p o t e n t i a l which i s of adaptive s i g n i f i c a n c e . An emphasis on f a t storage i s one of a series of c h a r a c t e r i s t i c s common to desert-adapted mammals. Another noteworthy c h a r a c t e r i s t i c of the body composition of P. m. sonoriensis i s that the hide has a s i g n i f i c a n t l y lower percentage of water on a f a t - f r e e basis (mean = 46.75) than that of the other races (mean = 52.06) (t = 0.05). I t can be reasoned that t h i s dryness has adaptive value for the purpose of water conservation by the mechanism of reduced external insensible water-loss i n an environment where water conservat-ion i s as important, or more so, than heat regulation. These examples serve to i l l u s t r a t e the way i n which body composition data can lead to inferences regarding adaptation to p a r t i c u l a r 24 environments. In t h i s regard, Gam (14) emphasizes that " i t i s not enough to describe variations i n body composition: i t i s ultimately necessary to ascertain the reasons for such v a r i a t i o n s " . The importance of the ro l e of gross body composition in the scheme of temperature adaptation must be viewed i n conjunction with other temperature-adaptive mechanisms at the disposal of small mammals, such as the use of a micro-climate (17). The composition of the f a t - f r e e body of deer mice has exhibited the r e l a t i v e constancy that t h i s component i s known to have from a l l other work on body composition (31). There are, however, s l i g h t corrections to t h i s generalization, such as the influence of high f a t l e v e l s on body hydration, and the v a r i a b i l i t y of the protein to water r a t i o due to differences in the amount of hide or pelage. The primary purpose of th i s description of the body composition of Peromyscus has been to point out the large in d i v i d u a l and i n t e r r a c i a l v a r i a b i l i t y that i s t y p i c a l of the body f a t compartment. I t also serves to emphasize that t h i s f a c t must be taken into account when there i s an a p r i o r i reason to believe that the amount of f a t has a bearing on the ph y s i o l o g i c a l function in question. For such p h y s i o l o g i c a l functions as basal metabolic rate, t h i s i s p a r t i c u l a r l y important i n comparative studies. P i t t s (34) has commented that i n t e r s p e c i f i c studies generally deal with mean values, a procedure which s t a t i s t i c a l l y minimizes in d i v i d u a l 25 va r i a t i o n s i n body f a t . On the other hand, in i n t r a s p e c i f i c studies and comparison of si m i l a r forms such as i s the topic of the following report i n t h i s series (16), in d i v i d u a l differences i n body f a t contribute heavily to the o v e r - a l l v a r i a b i l i t y . There i s a basic need, therefore, for a dependable reference c r i t e r i o n for i n t e r i n d i v i d u a l comparisons of such functions as basal metabolic rate. Pace and Rathbun (28) suggest that data should be expressed i n terms of the t o t a l body water present, which would be independent of f a t content. Although body water i s not the d i r e c t causative factor of p h y s i o l o g i c a l a c t i v i t i e s , i t i s probably the gross body component most h i g h l y correlated with the energy-exchanging mass (23). In the Peromyscus studied, body water was the most consistent component of gross body composition and suggests i t s e l f as the reference base of choice. Body protein of deer mice would be an i n f e r i o r reference due to the r e l a t i v e amount deposited as metabolically inactive hair, and the r a c i a l v a r i a b i l i t y i n that amount. Water i s also the most suitable reference base from a pragmatic viewpoint, being the most e a s i l y measured of body constituents. In a l a t e r report (16) the basal metabolic rate of these races of deer mice, expressed i n r e l a t i o n to t h e i r body water content, w i l l be presented. F i n a l l y , i t i s necessary to stress the importance of recognizing the changes which occur i n gross body composition, 26 especially fat, when Peromyscus, and probably most small mammals, are brought from their natural environment and acclimated to laboratory conditions such as described in this experiment. Aside from many behavioural changes, very tangible proof has been presented of a fundamental change in body structure; a change that is l i k e l y to influence a l l subsequent parameters measured, whatever they may be. 27 References Ashworth, U. S. and Cowgill, G. R. 1938. Body composition as a factor governing the basal heat production and the endogenous nitrogen excretion. J. N u t r i t i o n 15, 73-81. — Association of O f f i c i a l A g r i c u l t u r a l Chemists. 1955. O f f i c i a l methods of analysis. 8th edi t i o n . Association of O f f i c i a l A g r i c u l t u r a l Chemists, Washington, D. C. Babineau, L. and Page, E. 1955. On body f a t and body water i n r a t s . Can. J. Biochem. Physiol. 33, 970-979. : Bailey, C. B., K i t t s , W. D. and Wood, A. J . 1960. Changes in the gross chemical composition of the mouse during growth i n r e l a t i o n to the assessment of p h y s i o l o g i c a l age. Can. J. Animal S c i . ^ 0, 143-155. Behnke, A. R. 1961. Comment on the determination of whole body density and a resume of body composition data. In Techniques for measuring body composition. Edited by J. Brozek and A. Henschel. Nat. Acad. S c i . - National Research Council, Washington, D. C. pp. 118-133. Benedict, F. G. 1938. V i t a l energetics, a study i n comparative basal metabolism. Carnegie Inst, of Washington. Publ. No. 503. Blaxter, K. L. 1962. The energy metabolism of ruminants. Hutchinson Co., London. Bodenheimer, F. S. 1952. Problems of animal ecology and physiology i n deserts. _In Desert research. Research Council of I s r a e l s p e c i a l publ. No. 2, pp. 205-229. Brozek, J . 1961. Body composition. Science 134, 920-930. 1961. Methods for the study of body composition- addenda, comments, recommendations: an epilogue. _In Techniques for measuring body composition. Edited by J. Brozek and A. Henschel. Nat. Acad. S c i . - National Research Council, Washington, D. C. pp. 267-270. 28 11. and Hen sch e l , A., Eds. 1961. Techniques for measuring body composition. Nat. Acad. S c i . -National Research Gouncil, Washington, D. C. 12. Cowan, I. McT. and Guiguet, C. J. 1960. The mammals of B r i t i s h Columbia. 2nd edi t i o n . B r i t i s h Columbia P r o v i n c i a l Museum Handbook No. 11. 13. Foster, J . B. 1963. The evolution of the native land mammals of the Queen Charlotte islands and the problem of i n s u l a r i t y . Ph.D. thesis, Dept. of Zoology, Univ e r s i t y of B r i t i s h Columbia. 14. Garn, S. M. 1963. Human biology and research i n body composition. Annals New York Acad. S c i . 110, 429-446. 15. Grande, F. 1961. N u t r i t i o n and energy balance i n body composition studies. In Techniques for measuring body composition. Edited by J. Brozek and A. Henschel. Nat. Acad. S c i . - National Research Council, Washington, D. C. 16. Hayward, J . S. 19 . Metabolic rate and i t s temperature-adaptive s i g n i f i c a n c e i n six geographic races of Peromyscus. Submitted for publication, (included in t h i s thesis) . 17. 19 . Microclimate temperature and i t s adaptive s i g n i f i c a n c e i n six geographic races of Peromyscus. Submitted for publication, (included in t h i s t h e s i s ) . 18. Heggeness, F. W. 1961. Weight gains and f a t accumulation in rats subjected to periods of c a l o r i c r e s t r i c t i o n . Amer. J. Physiol. 201, 1044-1048. 19. Kinney, J . M., L i s t e r , J . and Moore, F. D. 1963. Relationship of energy expenditure to t o t a l exchangeable potassium. Annals New York Acad. S c i . 110, 711-722. 20. M i l l e r , A. T. J r . 1954. Energy metabolism and metabolic reference standards. Methods i n Medical Res. 6, 74-84. ™ 21. M i l l e r , J . R. and Wood, A. J. 1961. An economical laboratory mouse colony. Can. J. Animal S c i . 41, 143-149. — 29 22. M i t c h e l l , H. H. 1962. Comparative n u t r i t i o n of man and domestic animals. Vol. 1. Academic Press, New York. p. 194. 23. Moore, F. D. and Boyden, C. M. 1963. Body c e l l mass and l i m i t s of hydration of the f a t - f r e e body: t h e i r r e l a t i o n to estimated s k e l e t a l weight. Annals New York Acad. S c i . 110, 62-71. 24. Moulton, C. R. 1923. Age and chemical development i n mammals. J . B i o l . Chem. 57, 79-97. 25. Muldowney, F. P. 1961. Lean body mass as a metabolic reference standard. In Techniques for measuring body composition. Edited by J . Brozek and A. Henschel. Nat. Acad. S c i . - National Research Council, Washington, D. C. 26. Osgood, W. H. 1909. Revision of the mice of the American genus Peromyscus. North American Fauna No. 28. 27. Osserman, E. F., P i t t s , G. C , Welham, W. C. and Behnke, A. R. 1950. In vivo measurement of body f a t and body water i n a group of normal men. J. Appl. Physiol. 2, 633-639. 28. Pace, N. and Rathbun, E. N. 1945. Studies on body composition. 3. The body water and chemically combined nitrogen content i n r e l a t i o n to f a t content. J. B i o l . Chem. 158, 685-691. 29. Passmore, R. 1961. The r e l a t i o n between the metabolic mixture and the water content of the body i n man. Nutr. Dieta. _3, 1-16. 30. Peckham, S. C , Entenman, C. and Carrol, H. W. 1962. The influence of a hypercaloric d i e t on gross body and adipose tiss u e composition i n the rat. J. N u t r i t i o n 77, 187-197. 31. P i t t s , G. C. 1960. A study of gross body composition of small Alaskan mammals as compared with those from the temperate zone. A r c t i c Aeromedical Laboratory Technical Report 59-3. 32. 1962. Density and composition of the lean body compartment and i t s re l a t i o n s h i p to fatness. Amer. J. Physiol. 202, 445-452. 30 33. S i r i , W. E. 1956. The gross composition of the body. Advances i n B i o l o g i c a l and Medical Physics 4^, 239-280. ~ 34. Steele, J . M., Ed. 1954. Methods in medical research, Vol. 6. Comment on paper by A. T. M i l l e r , p. 83. 35. Tanner, J . M. 1949. Fa l l a c y of per-weight and per-surface area standards and th e i r r e l a t i o n to spurious c o r r e l a t i o n s . J . Appl. Physiol. 2, 1-15. "~ 36. Von Dobeln, W. 1956. Human standard and maximal metabolic rate i n r e l a t i o n to f a t - f r e e body mass. Acta. Physiol. Scand. _37, Suppl. 126. 79 pp. 37. Wedgwood, R. J., Bass, D. E., Klimas, J. A., Kleeman, C. R., and Quinn, M. 1953. Relationship of body composition to basal metabolic rate i n normal man. J. Appl. Physiol. 6, 327-334. 38. Wipple, H. E., Siverzweig, S. and Brozek, J., Eds. 1963. Body composition. Annals New York Acad. S c i . 110, 1018 pp. 39. Wilber, C. G. and Musacchia, X. J. 1950. Fat metabolism in the a r c t i c ground s q u i r r e l . J. Mammal. 31, 304-309. 40. Wood, A. J., Cowan, I. McT„ and Nordan, H. C. 1962. P e r i o d i c i t y of growth in ungulates as shown by deer of the genus Odocoileus. Can. J. Zool. 40, 593-603. 41. Wright, N. C. 1954. Domesticated animals inhabiting desert areas. _In Biology of deserts. Edited by J.•L. Cloudsley-Thompson. In s t i t u t e of Biology, Tavistock Square, London. 31 II . METABOLIC RATE AND ITS TEMPERATURE-ADAPTIVE SIGNIFICANCE IN SIX GEOGRAPHIC RACES OF PEROMYSCUS Abstract The metabolic rate c h a r a c t e r i s t i c s of s ix races of Peromyscus, selected from a wide range of habitats, have been determined over the temperature range 0° to 35°C. After acclimation to standardized laboratory conditions, c r i t i c a l temperatures and metabolic responses to temperatures below thermoneutrality were p r i m a r i l y a function of body weight. They show, therefore, no evidence of r a c i a l metabolic rate adaptation or s i g n i f i c a n t i n s u l a t i v e differences. Body weight per se i s not correlated with the climate of the respective habitats. A single equation which predicts the metabolic rate of these races at any temperature between 0° and 27°C, from a knowledge of body weight and body temperature, i s derived. Data on the metabolic rates of four races during winter and summer indicate no metabolic acclimatization over the range of temperatures which these mice are known to encounter i n t h e i r microhabitat (21). When considered as a single group, the basal oxygen consumption of a l l races varied with body weight^*69 over the body weight range of 14.7 to 36.0 gm and was i n s i g n i f i c a n t l y d i f f e r e n t from the accepted interspecies approximation. The 32 basal metabolic rates of each race showed no temperature-adaptive differences, e s p e c i a l l y when considered i n r e l a t i o n to body composition. I t i s concluded that metabolic rate i s nonadaptive to climate in these races of Peromyscus and consequently has played no important part in t h e i r d i s t r i b u t i o n and speciation. Introduction Studies of temperature adaptation in homoeotherms necessarily begin with reference to the findings of Scholander et a l . (41). These investigators showed, i n a comparison of a r c t i c and t r o p i c a l mammals and birds, that basal metabolic rate and body temperature were nonadaptive to climate. They concluded that "the phylogenetic adaptation to cold or hot climate therefore has taken place only through factors that regulate the heat d i s s i p a t i o n , notably the fur and skin insulation" (p269). However, i t was found in the above study and more recently by Hart (17) that the smaller mammals (approximately 2 kg and below) have much less fur ins u l a t i o n and are capable of only li m i t e d seasonal i n s u l a t i v e adjust-ment in comparison to larger mammals. This knowledge has stimulated renewed e f f o r t s to f i n d evidences of metabolic rate adaptation and acclimatization i n small mammals. In the present study, s i x geographic races of the deer mouse, genus Peromyscus, have been selected from a wide range of c l i m a t i c habitats i n an attempt to ascertain the importance, i f any, of metabolic rate adaptation to environmental 'temper-ature. Such findings would further the understanding of the d i s t r i b u t i o n and evolution of t h i s genus. Because of t h e i r widespread d i s t r i b u t i o n in North America and th e i r demonstrated genotypic p l a s t i c i t y , deer mice have been used i n many morphological studies of mammalian speciation (6). More recently, they have been used i n phys i o l o g i c a l approaches to adaptation and speciation. Three such studies have been concerned with e s s e n t i a l l y the same question as i s the topic of t h i s report. In two cases, the interpretation o'f res u l t s has been confused by considerations of hypoxia (10) or "nervous temperament" (33). In-an extensive investigation, McNab and Morrison (29) found a s i g n i f i c a n t l y lower basal metabolic rate in desert subspecies of Peromyscus as evidence of c l i m a t i c adaptation i n these mammals. There i s s t i l l need, however, for a more d e f i n i t i v e generalization on the importance of metabolic adaptation to temperature i n t h i s genus as a representative small mammal. Measurements of metabolic rate i n r e l a t i o n to environ-mental temperature, body weight, and body water are presented and compared for the six races acclimated to sim i l a r laboratory conditions. In addition, a comparison of the metabolic c h a r a c t e r i s t i c s of summer and winter-caught mice allows some evaluation of the magnitude of seasonal acclimatization. 34 The r e s u l t s of t h i s study show that, i n terms of r e l a t i v e importance, metabolic rate plays an i n s i g n i f i c a n t r o l e in the temperature adaptation of Peromyscus. In a l a t e r report (21), the temperature-adaptive feature considered to be of major importance i s described. Animals The s i x geographic subspecies (races) of Peromyscus, representing two species, have been described i n d e t a i l , along with trapping l o c a l i t i e s , i n a previous communication (20). They were selected from the following wide range of c l i m a t i c habitats in B r i t i s h Columbia and the western United States. A. _P. maniculatus nebrascensis- alpine, Wyoming. B. P_. m. austerus- mesic coast, B. C. C. P. m. sonoriensis- high-altitude desert, Nevada. D. P^ . m. artemisiae- a r i d v a lley, B. C. E. P. m. oreas- subalpine, B. C. F. P_. s i t k e n s i s prevostensis- mesic island, B. C. Approximately twenty mature individuals of each race were brought to the laboratory and maintained under s i m i l a r conditions (20) for at l e a s t s i x months before commencement of metabolic rate determinations. This period was consider-ed to be of s u f f i c i e n t duration for any seasonal acclimatiz-ation differences to be l o s t (16). Presumably, any demonstrable differences i n energy metabolism would be a 3 5 r e f l e c t i o n of genetic v a r i a t i o n ; to be judged i n terms of temperature-adaptive s i g n i f i c a n c e . Methods Metabolic Rate Conditions for the measurement of metabolic rate for comparative purposes must be well-defined ( 5 ) . A l l metabolism t r i a l s were conducted between 9 A.M. and 4 P.M. These mice are usually r e s t i n g during t h i s period and hence display a minimal metabolic rate ( 2 2 ) . This s i m p l i f i e d the standard-i z a t i o n of a c t i v i t y l e v e l s , which i s a major problem i n metabolic rate studies ( 5 , 3 3 ) . In preparation for a metabolic rate determination, a mouse was removed from i t s normal housing and placed i n a metabolism cage 1 2 hours before commencement of oxygen consumption measurements. Food was not provided. Information gathered from a representative sample of these mice indicated that the time of passage of t h e i r p e l l e t e d r a t i o n was approximately 6 hours. Consequently, the mice were considered to be i n a post-absorptive condition during the metabolism determinations. The apparatus used for metabolic rate measurements was an i n d i r e c t , volumetric respirometer with oxygen supplied e l e c t r o l y t i c a l l y . A description of the respirometer and i t s operation appears elsewhere ( 2 2 ) (see Appendix). The 36 metabolic rate of each mouse was determined i n two phases: f i r s t l y , from 35°C to 0°C at 5-degree interva l s , and, several days l a t e r , i n the region of thermoneutrality at 1-degree i n t e r v a l s . Subsequent to the thermoneutral zone measurements, the mice were k i l l e d for body composition analysis (20). One-half hour was allowed for adjustment to each new temperature and oxygen consumption was recorded during periods when the mouse was at complete r e s t . At low temperatures, periods of i n a c t i v i t y were of shorter duration. The resp i r a t o r y quotient (RQ) determined under these described conditions averaged about 0.75. Accordingly, an energy equivalent of 4.73 Calories per l i t e r of oxygen (STP) consumed was used for estimation of heat production (7). The experimental mice comprised equal numbers of each sex and were selected only i f judged to be mature on the basis of pelage c h a r a c t e r i s t i c s and body weight. Subsequent data on body composition tended to substantiate the maturity of the indivi d u a l s selected (20). For the measurement of seasonal (winter and summer) metabolic rate c h a r a c t e r i s t i c s of mice i n t h e i r natural habitat, individuals from the four areas i n B r i t i s h Columbia were brought to the laboratory as quickly as possible and metabolic rate t r i a l s conducted within 1-3 days of capture. The distances and time involved i n bringing mice from Wyoming and Nevada precluded t h e i r p a r t i c i p a t i o n i n t h i s aspect of the study. 37 Body Temperature Body temperature was measured by a method si m i l a r to that of Morrison and Ryser (31). An iron-constantan I thermocouple was inserted r e c t a l l y to a depth of 3 cm and temperature was recorded continuously on a Brown potentiometer (Minneapolis-Honeywell). The body temperature of these mice fluctuates as much as 4°C i n response to the excitement and a c t i v i t y of handling. In order to obtain a measure of the res t i n g body temperature that could be related to the metabolic rate data, the following procedure was followed. Mice were housed i n d i v i d u a l l y and when each was sleeping, i t was quickly removed and the thermocouple inserted. The time between i n i t i a l disturbance and in s e r t i o n was noted (usually 25-30 sec). The recorder p l o t of increasing body temperature could then be extrapolated back, i n time, to the temperature of the sleeping or re s t i n g state. Results A. Metabolic Rate in Relation to Ambient Temperature (1). Laboratory-acclimated mice. The relationships between rate of oxygen consumption and ambient temperature for the s i x races in t h i s study are summarized i n F i g . 1. Individual curves represent the average of ten individuals of each race, except P. m. nebrascensis and P. m. sonoriensis which are each the average of 5 in d i v i d u a l s . 38 F i g . 1. The rela t i o n s h i p between rate of oxygen consumption and ambient temperature for the six races of Peromyscus. The curves extrapolate to an average body temperature (T_). To change to energetic _ 3 units, 1 cc of oxygen i s equivalent to 4.73 x 10 Cal. 39 These data are presented i n a grouped manner (with some loss of d e t a i l ) i n order to f a c i l i t a t e important comparisons. There are several trends of difference between these curves which can be explained l a r g e l y on the basis of body weight. Associated with increasing body weight are: (a) decreased metabolic response to temperatures below thermoneutrality, (b) decrease i n minimum metabolic rate, (c) a general s h i f t i n g of the thermoneutral zone to a lower temperature range, and (d) a greater metabolic response to temperatures above thermoneutrality, with one exception (P. m. nebrascensis). To evaluate these r a c i a l metabolic rate c h a r a c t e r i s t i c s for evidence of metabolic rate adaptation per se, they must be compared i n a manner that eliminates differences associated with body weight alone. The important r e l a t i o n s h i p between minimal or basal metabolic rate and body weight w i l l be considered i n a l a t e r section. Consider now the r e l a t i o n s h i p between the slopes of the curves (Fig. 1) below thermo-n e u t r a l i t y and the average body weights of the s i x races. I t i s f i r s t necessary to calculate these slopes. Using the average metabolic rates at temperature^ below 27°C, the equations of these l i n e s (Table I) were calculated using the method of l e a s t squares. They i l l u s t r a t e , using _P. m. nebrascensis as an example, that oxygen consumption increases Table I Regression equations f o r the relationship of metabolic rate (MR, cc0 2/gni/hr) and ambient temperature (T A, C) between 0° and 27°G. Race Body weight, S„ of regression n mean and Regression equation , e . . & v range (gm) (cc02/gmAr/uC) A. P. m. nebrascensis B. P. m. austerus 10 C. P. m. sonoriensis D. P. m. artemisiae 10 E. P. m. oreas .10 F. P. sitkensis 10 18.93 (14.95-22.00) 19.53 (14.70-23.82) 20.38 (17.22-24.75) 23.19 (18.08-29.91) 24.58 (20.56-36.00) 28.33 (21.75-33.94) MR = 9.401 MR = 8.758 MR = 8.911 MR - 8.250 MR = 7.515 MR = 6.470 - 0.254T A - 0.239TA - 0.236TA - 0.234T A - 0.206T A - 0.184TA 0.16 0.17 0.16 0.13 0.12 0.11 o 0.254 cc/gm/hr for every 1 C decrease in ambient temperature (T A) and at 0°C the metabolic rate i s 9.401 cc02/gm/hr. Comparative values for the metabolic increment per °C decrease in T for Peromyscus (29, 30) are s l i g h t l y lower at equal body weight than those reported here. The rel a t i o n s h i p between the mean body weights of the races and the slopes of t h e i r metabolic response to cold i s presented i n F i g . 2. The high c o r r e l a t i o n (0.96) between these two variables i s s i g n i f i c a n t l y d i f f e r e n t from p= 0 at the 1 percent l e v e l . This r e l a t i o n s h i p emphasizes that the observed differences in metabolic response to cold are pr i m a r i l y a function of size; the heavier mice expending less energy for thermoregulation at cold temperatures. A sim i l a r relationship, with the exception of three desert subspecies, i s described for Peromyscus by McNab and Morrison (29). In view of the above res u l t , the question i s immediately asked whether body s i z e (weight) i t s e l f exhibits an adaptive r e l a t i o n to climate i n these races. I t does not. The smallest race (P. m. nebrascensis), with the greatest metabolic response to cold, was sampled from an alpine habitat. Conversely, the largest race (P. s i t k e n s i s ) , with the lowest metabolic response to cold, was sampled from a mesic habitat. A discussion of these observations as related to behavioural temperature regulation i s presented i n a following report (21). A further c h a r a c t e r i s t i c of the curves shown in F i g . 1 i s that, i n accordance with Fourier's law of heat flow (26), 42 F i g . 2. The relat i o n s h i p between the mean body weights of the races and the slopes of th e i r metabolic responses to cold. 43 the regressions of metabolic response to cold should extra-polate to body temperature (T B) at a the o r e t i c a l , zero metabolic rate. In Table II, the body temperatures of each race, calculated from the equations given i n Table I, are compared with actual measurements of body temperature. The body temperatures of mice kept at an ambient temperature of 2° .- 3°c for 24 hours with food available are also included. The calculated body temperatures are very close to those a c t u a l l y measured and to those reported by others (29, 33) for Peromyscus. The agreement/of the predicted and measured values of body temperature provides experimental v a l i d i t y of the temperature-metabolism r e l a t i o n s h i p s . Prom Table II i t i s also evident that body temperature of these mice i s r e l a t i v e l y independent of ambient temperature down to at l e a s t 2°C. I t has been shown that the slope of the metabolic response to temperatures below thermoneutrality i s l a r g e l y a function of body weight (Fig. 2) and also that the l i n e must extrapolate to body temperature. Consequently, an equation which predicts metabolic rate at any temperature below thermoneutrality (assuming body temperature i s constant) on the basis of body weight (W i n grams) and body temperature (T B i n °C) can be formulated. Thus, the metabolic rate (MR i n cc02/gm/hr) at a temperature T x (°C) i s as follows: MRT = m(T x - T B) (1) Table II A comparison of the calculated arid measured body temperatures of the six races of Peromyscus. Race Body temperature from regression (°C) Mean measured body temperature (°C) T A = 25°C T = 2-3°C A A. P. m. nebrascensis 36.94 35.9 34.8 B. P. m. austerus 36.65 36.3 36.6 C. P. m. sonoriensis 37.80 36.7 35.7 D. P. m. artemisiae 35.20 37.2 36.9 E. £• m. oreas 36.52 36.2 36.6 F. £• sitkensis 35.09 36.0 36.7 i n t e r r a c i a l average 36.37 36.3 36.1 45 where m i s the weight-dependent slope. Substituting for m from the regression equation of F i g . 2, MR = (-0.378 +:, 0.0068W) (T - T ) (2) T x X B y i e l d s the equation r e l a t i n g body weight and body temperature to metabolic rate at any desired environmental temperature between 0° and 27°C. The p r e d i c t i v e value of t h i s equation for Peromyscus may be useful i n estimating the energy requirements of wild deer mice exposed to known environmental temperatures without having to conduct metabolic rate t r i a l s . Examples of recent studies where energy budgets of small mammals have been determined are those of Buckner (8) for shrews and McNab (28) for deer mice. Assuming a value of 4.73 C a r per l i t e r of oxygen consumed, metabolic rate i n equation 2 can be converted to energy units (Cal/day) by multiplying by body weight (gm) and a factor of 0.1135 Cal«hr/cc0 2/day. The " c r i t i c a l temperature" of a homoeotherm i s defined as that temperature at which physical heat regulation i s i n s u f f i c i e n t to maintain body temperature and chemical heat production i s induced. The lower c r i t i c a l temperature i s determined by the minimal thermal conductance and the basal or minimal metabolic rate. The difference between the c r i t i c a l temperature and the body temperature i s termed the " c r i t i c a l gradient" (40) and i s a measure of the i n s u l a t i v e 46 capacity of the animal. The c r i t i c a l temperatures (T ) of c these races of deer mice can be calculated by substituting the average basal metabolic rate (BMR) of each race into t h e i r respective equations (Table I) r e l a t i n g metabolic rate to ambient temperature. The calculated lower c r i t i c a l temperatures and c r i t i c a l gradients are presented i n Table I I I . The range of c r i t i c a l temperatures i s less than 4°C and the v a r i a t i o n within t h i s narrow range tends to be weight-dependent, with one notable exception. P. m. sonoriensis (Nevada) has the highest T c (30.00°C) although i t i s not the l i g h t e s t race. This deviation from the observed rela t i o n s h i p may be inte r p r e t -ed as an indicati o n of s l i g h t l y i n f e r i o r i n s u l a t i o n i n thi s race; l i k e l y due to the noticeable clumping and o i l i n e s s of the fur when these p a r t i c u l a r mice are kept i n the laboratory. I t could also be an a r t i f a c t associated with the lower-than-expected BMR i n thi s race, which i s a topic of l a t e r discussion. The c r i t i c a l gradients i n Table III are very low compared to larger a r c t i c mammals (40). This means that Peromyscus i s sen s i t i v e metabolically to even moderately-low environmental temperatures. The upper c r i t i c a l temperatures of these mice are more d i f f i c u l t to estimate accurately from the present data. Consequently, the widths of the thermoneutral zones can only be estimated as approximately 3° - 5°C. Table III Calculated lower c r i t i c a l temperatures and c r i t i c a l gradients for the six races of Peromyscus Mean body Mean BMR Lower c r i t i c a l C r i t i c a l weight (gm) (cc02/gm/hr) temperature (°C) gradient (°C) A. P.m. nebrascensis 18.93 2.08 28.77 8.17 B. P.m. austerus 19.53 2.04 28.12 8.53 C. P.m. sonoriensis 20.38 1.84 30.00 7.80 D. P.m. artemisiae 23.19 1.99 26.71 8.49 E- P.m. oreas 24.58 1.77 27.91 8.61 F. P. sitkensis 28.33 1.65 26.14 8.95 48 (2). Seasonal comparisons of mice from t h e i r natural  habitat. I t has been shown above that laboratory-acclimated individuals of the s i x races of Peromyscus show no major differences i n t h e i r metabolic responses to cold that can be considered temperature-adaptive. This would suggest that seasonal acclimatization of metabolic rate within a single race i s an u n l i k e l y phenomenon. Evidence to t h i s e f f e c t i s presented i n F i g . 3. The metabolic rates of summer and winter-caught individuals of P. m. oreas (subalpine) are compared with the curve for the laboratory-acclimated i n d i v i d u a l s . There i s no s i g n i f i c a n t difference between the average metabolic rates of winter indiv i d u a l s and those acclimated to the laboratory in t h i s representative race. S i m i l a r l y , the metabolic rates of 3 summer^caught individuals, measured only at 15°C, are i n accord with the laboratory and winter values. The three other B r i t i s h Columbia races that were tested seasonally gave si m i l a r r e s u l t s except that, at equal body weight, winter individuals tended to have a very s l i g h t l y lower metabolic response at low temperatures. This difference was not s i g n i f i c a n t using the few individuals that could be caught i n winter. In t o t a l , these few seasonal data suggest that there i s no metabolic rate acclimatization per se over the range of temperatures tested. There i s an indica t i o n of s l i g h t i n s u l a t i v e improvement i n winter as was found by Hart (17) for 49 F i g . 3. A comparison of the metabolic rate c h a r a c t e r i s t i c s of summer and winter-caught individuals of _P. m. oreas in r e l a t i o n to the curve representing the average of the laboratory-acclimated representatives of t h i s race. P m. oreas s o \ O N © N N laboratory-acclimated mice N \ p ° o ° o / I A ir-summer • winter O 10 20 25 30 35 TEMPERATURE, °C 50 P_. maniculatus. B. Basal Metabolism and Body Weight The best genetic measure of metabolic rate i s the basal l e v e l . This e n t i t y i s uncomplicated by i n s u l a t i v e differences and i s meant to represent the minimum rate of energy expendit-ure under c a r e f u l l y defined conditions of a c t i v i t y , diet, etc. (5). I t i s well known that basal metabolic rate does not vary d i r e c t l y with body weight and i s usually expressed in r e l a t i o n to some f r a c t i o n a l power of body weight. On the basis of interspecies comparisons, Brody (7) suggested the use of the power function:-BMR (Cal/day) = 70.5W0'7 (w = body weight i n kg) as a reference generalization for making i n t e r - and i n t r a s p e c i f i c comparisons of basal metabolic rate. In t h i s way, individuals of d i f f e r e n t body weight can be compared. If, i n the Peromyscus data, r a c i a l differences from t h i s r e l a t i o n s h i p are observed, speculations as to temperature-adaptive s i g n i f i c a n c e are i n order. In F i g . 4, the double-log regression l i n e that relates the basal metabolic rate and body weight of a l l 50 individuals of the s i x races of Peromyscus i s presented. The basal metabolism of each i n d i v i d u a l was taken as the lowest metabolic rate value i n the thermoneutral zone. This r e l a t i o n s h i p i s compared with the Brody approximation regression, which i s seen to l i e within the r e l a t i v e l y low standard error of 51 F i g . 4 . Basal metabolic rate i n r e l a t i o n to body weight for Peromyscus and comparison with Brody's interspecies approximation. Standard errors of regression are for the Peromyscus data. 52 regression of the Peromyscus data. There are no s i g n i f i c a n t differences i n slope or p o s i t i o n of these two regressions. I t i s concluded that, as a group, these representatives of the genus Peromyscus do not d i f f e r i n t h i s r e l a t i o n s h i p from Brody's interspecies approximation. In comparison to Kleiber's (25) suggested use of the three-fourth (0.75) power of body weight as the metabolic u n i t of body size, the Peromyscus data are approximately 10 percent higher than the p r e d i c t i o n on t h i s b a s i s . For the present purposes, i t i s of greater i n t e r e s t to compare the BMR - body weight relationships of the i n d i v i d u a l races. These data are presented i n F i g . 5 and compared with the t o t a l Peromyscus regression. The equation of each l i n e i s included. I t i s seen that within races the weight exponents are quite d i f f e r e n t . The low exponents of P_. m. nebrascensis (0.43) and P. m. sonoriensis (0.25) are each based on only 5 individuals of low weight range. Consequently, these exponents are l e s s meaningful than the other four which range from 0.54 to 0.94. I t i s d i f f i c u l t to assign reasons for these differences i n slope among in d i v i d u a l populations. I t i s a worthy problem i n i t s e l f . The l i t e r a t u r e i s well documented with evidence of species weight-exponents that d i f f e r from the interspecies value (5, 7, 24). For example, in a study of the energetics of the meadow vole (Microtus  pennsylvanicus pennsylvanicus), Wiegert (44) found an exponent of 0.52 for non-fasted and 0.64 for fasted 53 F i g . 5. Comparison of the basal weight relationships of Peromyscus used i n t h i s metabolic rate (BMR) : body the i n d i v i d u a l races of study. LOG- BODY WEIGHT, kg -1-5 CQ 4 -A. P m. nebroscensis- B M R • 0-43 2 5 0 W V •5-7% -5« r = 0 8 2 a P m. ousterus— B M R = 38-1 W 0 8 4 •7-4 % -6-9 h r« 0-72 c. P m. sonoriensis— B M R * 11.5 W 0 2 S SR' + 6-0 ej -5-7 / o r • 0-63 a P m. artemlsiae — B M R = I 7 8 . 9 W 0 9 4 V r = 0.88 E. P m. ore as— B M R = 4 8 . 0 W 0 6 ' V r • 0.76 F P sitkensis— B M R = 6 0 . 4 W 0 - 6 8 V r • 0-88 T O T A L - B M R .0.69 6 6 - 8 V T " S = ! i i % r .o-92 3 -15 2 0 2 5 3 0 BODY WEIGHT, kg * io3 3 5 4 0 54 ind i v i d u a l s . Hart and Heroux (19) have found an exponent of 0.83 for summer-caught wild r a t s . Using the values of BMR at the mean body weights of each race (given in Table I I I ) , there are no s i g n i f i c a n t differences from the i n t e r r a c i a l average regression of F i g . 5. This conformity of r a c i a l basal metabolic rate c h a r a c t e r i s t i c s i s strong evidence that the exploitation of a va r i e t y of cl i m a t i c habitats by Peromyscus i s not contingent upon any fundamental change in metabolic rate. There i s some evidence worthy of mention pertaining to some of the s l i g h t differences that do ex i s t in the r a c i a l regressions of F i g . 5. There i s no doubt that there are small differences i n the behavioural responses of these races to c a p t i v i t y and experimental manipulations (12, 15). Despite the most stringent s p e c i f i c a t i o n s regarding a c t i v i t y during measurement of basal metabolism, i t i s hard to imagine that these behavioural differences w i l l not be manifested to a small extent i n the BMR data. The Peromyscus r e s u l t s of Murie (33) are interpreted on t h i s b a s i s . In t h i s regard, i t was noticed in the present study that P_. m. artemisiae was the most d i f f i c u l t , and j?. s i t k e n s i s the easiest race to " s e t t l e down" i n the metabolism chamber. Perhaps t h e i r respective positions i n F i g . 5 are a r e f l e c t i o n of t h i s s l i g h t influence. I t i s also noted in F i g . 5 that the regression of P. m. sonoriensis (Nevada) i s , at an average body weight, 55 7.5 percent lower than the t o t a l Peromyscus regression. The same trend to lower BMR i n desert inhabiting subspecies of Peromyscus has been found by McNab and Morrison (29) who noted a 13 percent reduction below the interspecies prediction for P. m. sonoriensis and even greater reductions in other desert subspecies. In the following section, a possible interpretat-ion of these reductions i s offered. In the r e l a t i o n s h i p of BMR to body weight, there were no s i g n i f i c a n t d i f f e r e n c e s . a t t r i b u t a b l e to sex. C. Basal Metabolism and Body Composition I t was pointed out i n a previous report (20) that there i s a great deal of evidence supporting the use of some component of body composition as the most r e l i a b l e reference base for basal metabolic rate comparisons. At thermoneutral-i t y , metabolic rate i s determined by the lowest l e v e l of integrated (25) tissue metabolism. At t h i s point, i t i s not determined by heat-loss phenomena. Body f a t i s the most variable component of body composition i n mature mammals as has been shown for these races of Peromyscus, e s p e c i a l l y under laboratory conditions (20). Since body f a t contributes r e l a t i v e l y l i t t l e to the basal metabolic rate (32), the elimination of t h i s v a r i a ble weight component from the reference base should enable more dis c r e t e BMR comparisons. From the previous determination of the body composition of the experimental mice (20), body water was suggested as the reference base of choice. In F i g . 6, the relationships 56 F i g . 6. Basal metabolic rate considered in r e l a t i o n to t o t a l body water for the six races of Peromyscus. LOG. BODY WATER, kg -z.i - 2 0 — I — -1.7 -1.6 C D. P m. nebrascensis— BMR = 129 X 0 23 • . 7 . ^ % r - 0-59 B. P m. austerus-0-61 P m. sonoriensis— BMR = 23-7X 0-37 c . - 6 - 0 % S D " - 5 - 7 r • P m. artemisiae — E. P m. o reo s— F /? sitkensis— 0-99 BMR = 2187 X .0 -89 S _ • D " - 8 - 6 0 - 6 0 BMR = 601 X Sc •5-2. , 0-63 BMR = 366-5X" " S = !'§3% r = 0 9 0 r * 0-74 " 5 . 0 7. r = 0-90 4 -0-8 _ o o o ft: CD O 0-6 3 -T 0 T A L — BMR = 47-6 X 0 5 3 S - ^ S K . R " 7 - 4 r • 0 - 8 6 0-5 10 12 15 BODY WATER, kg x ,03 (X) 20 25 57 between t o t a l body water and basal metabolic rate of each race are presented. Here again, the regressions of each race are cl o s e l y grouped and are not s i g n i f i c a n t l y d i f f e r e n t in p o s i t i o n r e l a t i v e to the t o t a l regression. The co r r e l a t i o n c o e f f i c i e n t s are not better than those r e l a t i n g BMR to t o t a l body weight. However, there are two important differences between these body water and the body weight regressions. One i s the observation that the slope of the regression for P_. m. oreas i s much higher (0.89) when related to body water than when related to t o t a l body weight (0.61). This i s due, primarily, to the fact that one in d i v i d u a l of t h i s race was excessively f a t (14.1 gm f a t in a t o t a l body weight of 36.0 gm). On the basis of t o t a l body weight, therefore, the metabolic rate of th i s heavy mouse was misleadingly low and reduced the slope accordingly. When the BMR of t h i s i n d i v i d u a l was related to body water, i t was s l i g h t l y higher than the average for t h i s race. The second, and most i n t e r e s t i n g difference observed when BMR i s related to body water i s the change i n p o s i t i o n of the P. m. sonoriensis regression. When BMR was related to body weight, t h i s race f e l l below the t o t a l regression, which can lead to interpretation as an indicati o n of desert adapt-ation. I t has been shown previously (20) that t h i s race, under laboratory conditions, was the f a t t e s t of the s i x races. When t h i s influence i s removed by r e l a t i n g BMR to body water, the regression of t h i s race i s no longer low r e l a t i v e to the others but i s a c t u a l l y s l i g h t l y higher than the t o t a l regression. 58 One can conclude from these two examples that correction for s i g n i f i c a n t body f a t differences in individuals or races can eliminate, in some cases, what appear to be deviations from the "normal" r e l a t i o n s h i p . In general, i t was noted that in excessively f a t individuals or races, correction by r e l a t i o n to body water seemed to overcompensate, r e s u l t i n g i n somewhat higher than normal BMRs. This suggests that the body f a t i s "demanding" (5) a certain metabolic contribution even under basal conditions. The importance of measuring body composition in comparative studies of basal metabolic rate i s obvious from these examples. Before an adaptive difference in BMR i s proposed, such a difference should be checked for c o r r e l a t i o n with body composition, e s p e c i a l l y fatness. Words from the c l a s s i c a l work of Benedict (5) are pertinent here - "When the f i n a l comparisons are drawn, i t i s of special moment not only in comparisons within one species, but esp e c i a l l y i n i n t e r s p e c i f i c comparisons to bear i n mind those animals having a large proportion of f a t i n the body, p a r t i c u l a r l y i f there are any seemingly anomalous energy values associated with the type of very f a t animal." ( i t a l i c s mine). 5 9 Discussion The main purpose of t h i s investigation has been to attempt to answer the question- are there inherent or genetic metabolic rate differences between races of the genus Peromyscus that can be considered temperature-adaptive? I t i s an attempt to explore one possible case of phy s i o l o g i c a l speciation i n t h i s diverse and widespread genus. At the same time, the r e s u l t s for t h i s group may have implications that would further understanding of the processes of temperature regulation and t h e i r r o l e i n the d i s t r i b u t i o n of small mammals in general. In t h i s study, i t has been found that the basal metabolic rates of the races concerned are not d i f f e r e n t from a predictable, weight-dependent r e l a t i o n s h i p . Furthermore, evidence concerning c r i t i c a l temperatures and metabolic response to temperatures below thermoneutrality indicate that metabolic rate other than the basal l e v e l i s s i m i l a r l y inadaptive. The same data are i n d i r e c t evidence that i n s u l a t i v e differences are i n s i g n i f i c a n t . In t o t a l , the data render the v e r d i c t that metabolic rate, as a basic p h y s i o l o g i c a l function, does not p a r t i c i p a t e in the adaptive processes of speciation i n these representatives of the genus Peromyscus. This i s contingent upon the maintenance of constant and si m i l a r body temperatures. The close r e l a t i o n s h i p between heat production and body weight suggests that body weight may be related to climate. 60 I t has already been pointed out that t h i s i s not the case in the races used in t h i s study. This evidence adds support to Scholander's c r i t i c i s m (38) of Bergmann's Rule. In the l i t e r a t u r e on small mammals, there i s i n s u f f i c i e n t evidence to warrant the conclusion that, i n genetic terms, metabolic rate i s temperature-adaptive. Cook and Hannon (30) compared the metabolic rates of three geographic races of Peromyscus maniculatus and found a s i g n i f i c a n t l y lower standard metabolism i n the high- a l t i t u d e form. They postulate that t h i s may be related to the lower oxygen tension of t h e i r environment. Murie (33) accounted for observed differences in "basal" metabolism of P_. maniculatus and P. eremicus as r e f l e c t i o n s of differences i n "nervous temperament". In a study of two species of voles (Clethrionomys) i n Finland, A. M. Pearson (34) showed that, on a weight-specific basis, basal metabolism was equal in the two species. A survey of the metabolic patterns of several species of small mammals was conducted by 0. P. Pearson (35) who found no adaptive differences or even good c o r r e l a t i o n with body weight. Enger (14) was unable to conclude that metabolic rate i s adaptive to climate i n a sel e c t i o n of t r o p i c a l mammals and b i r d s . In opposition to the above evidence of nonadaptive basal metabolic rate, several authors have observed a generally lower BMR than the standard curve p r e d i c t i o n i n small, desert-adapted rodents. These include subspecies of Peromyscus (29) and three members of the family Heteromyidae (4, 9, 11). To what extent 61 these reductions are due to lowered body temperature, in conjunction with the p o s s i b i l i t y of high body f a t le v e l s such as found in the P_. m. sonoriensis of t h i s study i s a question that requires more consideration before f i n a l interpretation of these observations. For example, the body temperatures of the kangaroo mouse (Microdipodops pallidus) (4) and the pocket mouse (Perognathus longimembris) (2) have been shown to be p a r t i c u l a r l y l a b i l e , even at room temperature. The f a c u l t a t -ive hypothermia of such desert mammals has been interpreted, as an adaptive response to summer heat and a r i d i t y (4). With regard to the p o s s i b i l i t y of high f a t l e v e l s accounting for the apparent lower metabolic rates, Bartholomew and MacMillan (4) noted that t h e i r kangaroo mice had conspicuous deposits of adipose tissue, p a r t i c u l a r l y when in the laboratory. Similarly, in a study of the r e l a t i o n of metabolism to climate and d i s t r i b u t i o n in finches (Carpodacus), Sa l t (37) describes the misleadingly lower metabolic rates of fat, winter in d i v i d u a l s . The foregoing discussion of the importance of metabolic rate in temperature adaptation involves comparison of gen e t i c a l l y d i f f e r e n t populations and species. Another approach to evaluation of the r e l a t i o n between metabolic c h a r a c t e r i s t i c s and environmental temperature 1 involves studies within populations. Included in these are the studies of seasonal acclimatization and the voluminous works on laboratory acclimation (36). In a review and comparison of these two processes, Hart (18) showed that i n the deer mouse acclimation and acclimatization are e s s e n t i a l l y the same, metabolically. In acclimatization to seasonal cold in these mammals, there i s no change in the c r i t i c a l temperature, which suggests that i n s u l a t -ive changes are i n s i g n i f i c a n t . As i n the present study, the metabolic rate at any temperature below thermoneutrality i s similar in summer and winter mice, with the exception that in winter the low temperature l i m i t for survival i s extended by approximately 20°C. This extension i s due to an enhanced ') "capacity" for more intensive and sustained metabolic a c t i v i t y . I t i s l i k e l y a function of the a v a i l a b i l i t y of metabolic nutrients (42) and development of the mechanisms for t h e i r oxidation, such as nonshivering thermogenesis (19). Support-Y ing evidence of such development, involving the thr^oid gland (13) and blood c h a r a c t e r i s t i c s (43) has recently been presented for deer mice. It should be emphasized at t h i s point that the phenomenon of seasonal metabolic acclimatization as described above by Hart (18) does not allow continuous exposure to low temperatures. Its function i s l i k e l y that of permitting short periods of intensive energy production associated with occasional exposure to very low winter temperatures. Such exposure may occur when i t i s necessary to forage for food. A t h e o r e t i c a l c a l c u l a t i o n may help to c l a r i f y t h i s point. From equation 2 above, the energy requirement for thermo-regulation alone at a continuous exposure to -15°C would be 63 approximately 25 Cal per day for a 20 gm mouse. Can such a mouse eat s u f f i c i e n t to balance t h i s c a l o r i c requirement? Data on the feed intake of these mice (1) indicate that a 20 gm in d i v i d u a l eats approximately 1.95 gm of food (2.9 metabolizable Calories per gram) a day at 21° - 23°C, which i s equivalent to about 5.7 Cal. Therefore, i t would have to more than quadruple i t s food intake to meet energy requirements at —15°C. Evidence for rats (27) showed that the food intake of a rat i o n of 2.9 Cal/gm i s approximately 60 percent of the maximum intake determined by digestive capacity and time of passage. Using these estimates, the maximum energy intake of the 20 gm mouse would be 9.5 Cal/day; a value far short of that necessary for continuous exposure to -15°C. Since natural foods are u n l i k e l y to have a higher c a l o r i c density than the rat i o n used i n t h i s c a l c u l a t i o n , i t i s safe to say that a mouse in i t s natural winter environment i s forced, i n terms of i t s energy balance, to seek a moderated microclimate. Further evidence on the subject of metabolic acclimatization i s a v a i l a b l e . In a seasonal comparison,.of metabolic rates of Alaskan mammals of various sizes, Irving et a l . (23) showed that the seasons did not modify the basal metabolic rates: "Insulation was the only general factor i n the regulation of animal heat which showed seasonal modification, and that appeared only in the two larger mammals". In the present metabolic rate study.of Peromyscus, i n d i r e c t evidence for s i g n i f i c a n t seasonal change i n 64 i n s u l a t i o n i s lacking, as has been found for wild rats (19). In actual measurements of fur ins u l a t i o n of Peromyscus (17, 29, 33), seasonal and taxonomic differences are small. The evidence for the lack of metabolic rate adaptation or seasonal acclimatization i n the s i x races of deer mice used in t h i s study (over the temperature range 0° to 35°C) can be assessed i n the following t h e o r e t i c a l scheme. I f a small, non-hibernating mammal such as Peromyscus i s exposed to a stressing environmental temperature, i t s f i r s t response would be to avoid such a temperature. If t h i s response i s not completely e f f e c t i v e , gradual changes i n body insula t i o n would be expected. I f there s t i l l remains a temperature stress, a sel e c t i o n for suitable metabolic c h a r a c t e r i s t i c s may occur. However, such a metabolic rate s e l e c t i o n i s improbable due to the fa c t that when there i s an opposing temperature stress, the previously selected metabolic rate would then have negative s u r v i v a l value. For example, a low metabolic rate during the hot summers i n Nevada may have survival value, but during the low temperatures of winter i n the same area, i t would be a disadvantage. Therefore, any selection for factors c o n t r o l l i n g metabolism must be for those that are re v e r s i b l e i n nature. In t h i s regard, the comparative endocrinology of a r c t i c and t r o p i c a l or desert mammals deserves more intensive study. From another viewpoint, continuous exposure to cold would require high energy intake and i s therefore u n l i k e l y under natural circumstances (hence 65 hibernation). On the other hand, continuous exposure to hot conditions requires very l i t t l e energy intake to maintain body temperature such that the p o s s i b i l i t y of a genetic p o t e n t i a l for low metabolic rate i n desert adaptation cannot be eliminated. The very low basal metabolic rate of the Poor-will (Phalaenoptilus n u t t a l l i i ) (3) seems to be of t h i s nature. In view of the findings of the present study, metabolic rate and ins u l a t i o n responses to temperature appear to play an i n s i g n i f i c a n t r o l e i n the cl i m a t i c adaptation, and hence in the d i s t r i b u t i o n and speciation of the genus Peromyscus. This i s strong i n d i r e c t evidence of the paramount importance i n these small mammals of the behavioural response to temperature; the seeking and modification of a suitable microclimate. This i s the topic of the next report (21). On the basis of these i n f r a s p e c i f i c comparisons of Peromyscus, and evidence i n the l i t e r a t u r e , there i s no good reason to beli e v e that small, non-hibernating mammals are any exception to the generalization of Scholander et a l . (41) that basal metabolic rate i s "phylogenetically nonadaptive to external temperature conditions." 66 References 1. Addison, R. Personal communication. Dept. of Zoology, University of B r i t i s h Columbia. 2. Bartholomew, G. A. and Cade, T. J. 1957. Temperature regulation, hibernation, and aestivation i n the l i t t l e pocket mouse, Perognathus longimembris. J . Mammal. 38, 60-72. 3. , Hudson, J . W. and Howell, T. R. 1962. Body temperature, oxygen consumption, evaporative water loss, and heart rate i n the Poor-will. Condor 64, 117-125. 4. and MacMillan, R. E. 1961. Oxygen consumption, estivation, and hibernation i n the kangaroo mouse, Microdipodops p a l l i d u s . Physiol. Zool. 34, 177-183. 5. Benedict, F. G. 1938. V i t a l energetics, a study i n comparative basal metabolism. Carnegie Inst, of Washington. Publ. No. 503. 6. B l a i r , W. F. 1950. Ecological factors in speciation of Peromyscus. Evolution 4, 253-275. 7. Brody, S. 1945. Bioenergetics and growth. Reinhold Publ. Corp., New York. 8. Buckner, C. H. 1964. Metabolism, food capacity and feeding behaviour in four species of shrews. Can. J. Zool. 42, 259-279. 9. Carpenter, R. E. 1963. A comparison of thermoregulation and water metabolism i n the kangaroo rats, Dipodomys a g i l i s and Dipodomys merriami. Dissert. Abstracts 24, 899-900. 10. Cook, S. F. and Hannon, J. P. 1954. Metabolic differences between three strains of P_. maniculatus. J . Mammal. 35, 553-560. 11. Dawson, W. R. 1955. The r e l a t i o n of oxygen consumption to temperature i n desert rodents. J . Mammal. 36, 543-553. — 67 12. Eisenberg, J . F. 1963. The i n t r a s p e c i f i c s o c i a l behaviour of some c r i c e t i n e rodents of the genus Peromyscus. Amer. Midland N a t u r a l i s t 69, 240-246. 13. Eleftheriou, B. E, and Zarrow, M„ X. 1962. Seasonal v a r i a t i o n i n thyroid gland a c t i v i t y i n deermice. Proc. Soc. Exp. B i o l . Med. 110, 128-131. 14. Enger, P. S. 1957. Heat regulation and metabolism i n some t r o p i c a l mammals and b i r d s . Acta. Physiol. Scand. 40, 161-166. 15. Foster, D. D. 1959. Differences i n behaviour and temperament between two races of the deer mouse. J . Mammal. 40, 496-513. 16. Hart, J. S. 1953. Rate of gain and loss of cold resistance in mice. Can. J . Zool. _31, 112-116. 17. 1956. Seasonal changes i n i n s u l a t i o n of the fur. Can. J . Zool. 34, 53-57. 18. 1957. Climatic and temperature induced changes i n the energetics of homeotherms. Rev. Can. B i o l . 16, 133-174. 19. and Heroux, 0. 1963. Seasonal acclimatization in wild rats (Rattus norvegicus). Can. J. Zool. 41, 711-716. = 20. Hayward, J . S. 196_. The gross body composition of s i x geographic races;of Peromyscus. Submitted for p u b l i c a t i o n . (Included i n t h i s t h e s i s ) . 21. ' 7-96—. Microclimate temperature and i t s adaptive s i g n i f i c a n c e i n six geographic races of Peromyscus. Submitted for p u b l i c a t i o n . (Included i n t h i s t h e s i s ) . 22. , Nordan, H. C. and Wood, A. J. 1963. A simple e l e c t r o l y t i c respirometer for small animals. Can. J . Zool. 4JL, 63-68. 23. Irving, L.,Krog, H. and Monson, M. 1955. The metabolism of some Alaskan animals i n winter and summer. Physiol. Zool. 28, 173-185. 24. Kleiber, M. 1947. Body s i z e and metabolic rate. Physiol. Rev. 27, 555-541. 68 25. 1961. The f i r e of l i f e . Wiley and Sons, Inc., New York. 26. . 1963. Trophic responses to cold. In Proceedings of the international symposium on temperature acclimation. Fed. Proc. _22, 112-11 A. 27. McKenzie, R. M. 1964. The response of the laboratory r a t to changes i n the c a l o r i c density/and protein: c a l o r i e r a t i o of i t s r a t i o n . Unpublished Master's Thesis, Dept. of Animal S c i . , U n i v e r s i t y of B r i t i s h Columbia. i 28. McNab, B. K. 1963. A model of the energy budget of a wild mouse. Ecol..44, 521-532. 29. and Morrison, P. 1963. Body temperature and metabolism i n subspecies of Peromyscus from a r i d and mesic environments. Ecol. Monog. 33, 63-82. ~ 30. Morrison, P. R. and1 Ryser, F. A. 1951. Temperature and metabolism i n some Wisconsin mammals. Fed. Proc. 10, 93-94. 31. 1959. Body temperature i n the white-footed mouse, Peromyscus leucopus  noveborascensis. Physiol. Zool. 32, 90-103. 32. Muldowney, F. P. 1961. Lean body mass as a metabolic reference standard. JEn Techniques for measuring body composition. Edited by J . Brozek and A. Henschel. Nat. Acad. S c i . - Nat. Res. Council. Washington, D. C. 33. Murie, M. 1961. Metabolic c h a r a c t e r i s t i c s of mountain, desert and coastal populations of Peromyscus. Ecol. 42, 723-740. 34. Pearson, A. M. 1962. A c t i v i t y patterns, energy metabolism, and growth rate of the voles Clethrionomys rufocanus and C_. glareolus i n Finland. Ann. Zool. Soc, "Vanamo" _24, 1-58. 35. Pearson, 0. P. 1947. The rate of metabolism of some small mammals. Ecol. _28, 127-145. 36. Proceedings of the international symposium on temperature acclimation. 1963. R. E. Smith ( e d i t o r - i n - c h i e f ) . Fed. Proc. 22, 687-960. 69 37. Salt, G„ W„ 1952. The r e l a t i o n of metabolism to climate and d i s t r i b u t i o n i n three finches of the genus Carpodacus. Ecol. Monog. 22, 121-152. 38. Scholander, P. F. 1955. Evolution of cl i m a t i c adaptation in homeotherms. Evolution _9, ' 15-26. 39. , Walters, V., Hock, R. and Irving, L. 1950a. Body ins u l a t i o n of some a r c t i c and t r o p i c a l mammals and b i r d s . B i o l . B u l l . 5>9, 225-236. 40. __, Hock, R., Walters, V., Johnson, F. and Irving, L. 1950b. Heat regulation i n some a r c t i c and t r o p i c a l mammals and b i r d s . B i o l . B u l l . 99, 237-258. — 41. , Hock, R., Walters, V.. and Irving, L, 1950c. Adaptation to cold i n a r c t i c and t r o p i c a l mammals and bir d s i n r e l a t i o n to body temperature, in s u l a t i o n and basal metabolic rate. B i o l . B u l l . .99, 259-271. 42. Sealander, J . A. 1951. Survival of Peromyscus i n r e l a t i o n to environmental temperature and acclimation at high and low temperatures. Amer. Midland N a t u r a l i s t 46, 257-311. 43. 1962. Seasonal changes i n blood values of deer mice and other small mammals. Ecol. 43, 107-119. — 44. Wiegert, R. G. 1961. Respiratory energy loss and a c t i v i t y patterns i n the meadow vole, Microtus  pennsylvanicus pennsylvanicus. Ecol. 4_2, 245-253. 70 I I I . MICROCLIMATE TEMPERATURE AND ITS ADAPTIVE SIGNIFICANCE IN SIX GEOGRAPHIC RACES OF PEROMYSCUS Abstract The ambient temperatures p r e v a i l i n g in the microhabitats of s i x geographic races of Peromyscus were measured in the summer and winter seasons. A s a t i s f a c t o r y method whereby thermistors were hauled by the mice into t h e i r microhabitats i s described. The r e s u l t s show that the microhabitat provides an environment of moderate, stable temperature where the seasonal extremes of heat and cold can be avoided. Winter microclimate temperatures were never below freezing and were very s i m i l a r among the habitats despite considerable differences i n the gross climates. These r e s u l t s suggest that there i s no s i g n i f i c a n t d i f f e r e n t i a l s e l e c t i v e pressure for temperature adaptation among the s i x races, and support the previously reported findings that metabolic rate i s nonadaptive to climate i n Peromyscus. In the d i s t r i b u t i o n and speciation of Peromyscus, the sel e c t i o n and modification of a suitable microclimate i s of major temperature-adaptive s i g n i f i c a n c e . This p a r t i c u l a r behavioural method of temperature adaptation i s potentiated by small body s i z e and i s discussed i n r e l a t i o n to c l i m a t i c adaptation of small mammals i n general. 71 Introduction In a previous communication (13), i t was shown that basal metabolic rate i s inadaptive to climate in a s e l e c t i o n of geographic races of the deer mouse, Peromyscus sp. In addition, seasonal temperature acclimatization.by means of i n s u l a t i v e or metabolic changes was not found. The metabolic rate differences that did e x i s t could be accounted i n large part by differences i n body s i z e . Since body siz e per se i n these mice was not correlated with c l i m a t i c conditions i n t h e i r respective habitats, only one avenue for temperature adaptation remains. In the scheme of c l i m a t i c adaptation of homeotherms outlined by Scholander et a l (34), that avenue i s reduction of the body-to-a i r temperature gradient. Since body temperature i s e s s e n t i a l l y constant i n these mice (13), the reduction of t h i s gradient must involve behavioural orie n t a t i o n to environmental temperature differences i n the natural habitat. I t i s well known that small mammals such as Peromyscus are able to escape the exigencies of the gross c l i m a t i c environment by the behavioural response of seeking a suitable microclimate. In contrast, larger mammals, by v i r t u e of t h e i r s i z e alone, are unable to u t i l i z e microenvironments to as s i g n i f i c a n t an extent and have r e l i e d , therefore, on processes regulating heat d i s s i p a t i o n (fur, peripheral c i r c u l a t i o n ) i n t h e i r phylogenetic adaptation to hot and cold climates (34). Despite the general knowledge of the use of microclimates by small mammals, l i t t l e quantitative information has been gathered in previous studies of the question of metabolic adaptation to temperature i n small mammals (3, 22, 23, 25). I t would seem to be esse n t i a l that, f o r the interpretation of the r e l a t i o n between a ph y s i o l o g i c a l function such as metabolic rate and the environmental temperature of an animal, measure-ment of the actual temperatures encountered by that animal i s a basic requirement. Except :for two recent cases (2, 36), where microclimatic conditions have been mentioned i n r e l a t i o n to metabolic adaptation, there are many reasonable assumptions, but few data. Bearing witness to t h i s f a c t are the recent statements: "Not enough i s known about the microclimate a c t u a l l y inhabited by either species to allow a guess as to how frequently the conditions of the experiment are encountered by wild populations" (23), and "Unfortunately there are few data on the many desert microclimates" (22). In a description of a model of an energy budget of a wild mouse, McNab (21) was forced to make assumptions about the temperatures i n Peromyscus burrows. In addition, Johnson (14) and Benton and Altmann (1) have expressed the need for more precise evaluation of micro-c l i m a t i c conditions and t h e i r use by small mammals. The data described i n t h i s report may help to r e l i e v e t h i s need. Of primary i n t e r e s t was the te s t i n g of the hypothesis that measurements and comparisons of the temperatures p r e v a i l -ing i n the microhabitats of the s i x geographic races of Peromyscus concerned, may provide important clues to the 73 interpretation of the previous findings on metabolic rate (13). i i Temperature Measuring Technique The d i f f i c u l t y of obtaining temperature readings in the r e s t r i c t e d volume of actively-used Peromyscus burrows may account for the paucity of such data. A method i s described here whereby t h i s problem was met and s a t i s f a c t o r y readings of Burrow temperatures were obtained. The subterranean crevices, burrows and runways that are •potential r e s t i n g places for deer mice are numerous i n a l l habitats. The most v a l i d measure of the microclimate temper-ature i s obtained by simply tying a thermistor or thermocouple to a live-trapped mouse and l e t t i n g i t haul the leads into i t s burrow system as the Schmidt-Nielsens did with kangaroo rats (31). There are two disadvantages to t h i s method. F i r s t l y , by any permanent method of attachment to the mouse, i t w i l l often turn i t s attention to chewing at the lead wires once i t has reached safety i n the burrow. And secondly, i f the mouse does not release i t s e l f from the leads, i t i s l i k e l y to change i t s p o s i t i o n within the burrow system or even come out, e s p e c i a l l y at night. In either case, long-term temperature measurements i n the burrow are impossible. In the present study, a simple device was designed which allowed the investigator to release the thermistor attachment from the mouse when i t had come to a stop i n the burrow. The construction and operation of t h i s 74 "connector" are shown in F i g . 1. A mouse that was live-trapped outside a burrow system was l i g h t l y anaesthetized with ether for s u f f i c i e n t time to attach a small wire r i n g to the loose dorsal skin by means of a needle and thread. When the mouse was completely revived (2-3 min.), the connector was attached to the r i n g and the mouse was then ready to be released into the burrow (Fig. 2). When released, the mouse would drag the thermistor and l i g h t , f l e x i b l e leads behind. Such a load imposed l i t t l e curtailment during escape, and the mouse would usually take 3 to 4 feet of lead into the burrow before i t stopped. After waiting b r i e f l y to see that the mouse would proceed no farther, any slack i n the lead wires was taken up and a gentle tug at the fin e nylon l i n e would release the connector and thermistor from the mouse. With the thermistor in p o s i t i o n , the temperature readings were commenced using a telethermometer. Several connectors and thermistors were constructed with leads about 20 f t . long. With these, concurrent recordings i n several burrows were obtainable. Mice retrapped with rings s t i l l attached to t h e i r backs showed no i l l - e f f e c t s or l o c a l inflammation at the s i t e of attachment. The connector-thermistor device described has proven very s a t i s f a c t o r y for the purposes of t h i s study, and should be adaptable for use on a v a r i e t y of small mammals. During the summer of 1963 and the following winter, microclimate temperature recordings i n r e l a t i o n to outside 75 F i g . 1. A drawing of the connector-thermistor device in i t s two operating positions. The hinged bar at the posterior ejid of the connector i s made to f i t t i g h t l y enough that a gentle tug on the nylon l i n e i s s u f f i c i e n t to release the mouse. 1 C M . I 1 TELETHERMOMETER connected —- released 76 F i g . 2. This photograph shows a deer mouse with the connector and thermistor attached. The connector device i s e a s i l y made by a jewellery mechanic who has the small hinges necessary. The thermistor i s a Fenwall GB32P8. 77 fluctuations were carried out i n the s i x habitats concerned. "Outside" temperatures were recorded one foot above the ground in the shade. Temperatures were measured hourly for periods of at l e a s t 24 hours. Habitat Descriptions The locations from which the s i x races of Peromyscus were sampled have been presented i n a previous report (12). Somewhat more d e t a i l as to habitat and climate i s now required. A. Alpine. (Peromyscus maniculatus nebrascensis, trapped about 30 miles south, by road, of Red Lodge, Montana). This alpine habitat i n the Bear tooth Plateau of Northern Wyoming l i e s generally between 10,000 and 11,000 f t . elevation, and i s over 1,000 f t . above t r e e - l i n e . The mean annual temperature i s around -3° to -1°C with f a l l , winter and spring p r e c i p i t a t i o n occurring as snow. Pr e v a i l i n g winds keep certain exposed areas l i g h t l y covered by snow i n the winter, but snow p e r s i s t s i n the accumulation areas throughout summer. Peromyscus i n t h i s habitat u t i l i z e the burrows dug by pocket gophers (Thomomys sp.) and are common i n the areas where masses of large rocks provide shelter. B. Puget Sound Lowlands. (P_. m. austerus, trapped near Vancouver, B. C ) . This coastal habitat has a moderate climate. The winters are mild, summers cool, and r a i n f a l l i s 78 heavy. The mean annual temperature i s 9° - 10°C. The climax forest i s Douglas F i r (Pseudotsuga menziesii) with shrub ground cover of s a l a l (Gaultheria shallon). C. High-altitude Desert. (P. m. sonoriensis, trapped near S i l v e r Peak, Nevada). In thi s t y p i c a l area of Nevada desert (4,800 f t . elevation and approximately 100 miles north of Death Va l l e y ) , the summers are very hot and winters cold. Annual p r e c i p i t a t i o n i s less than 5 inches. The f l a t , tree-less v a l l e y bottoms are broken by islands of b a s a l t i c outcrops which o v e r l i e o l d lake sediments. Numerous crevices i n the ba s a l t and sediments, and easy-digging i n the talus rubble provide many suitable microhabitats for deer mice. D. Osoyoos Arid Zone. (P. m. artemisiae, trapped near Oliver, B. C ) . This small zone extends about 30 miles along the Okanagan V a l l e y i n southern B r i t i s h Columbia and i s bordered by the Dry Forest Association. The summers are hot and winters mild. The general vegetation in the v a l l e y i s t y p i c a l of a moist desert with antelope-bush (Purshia  tridentata) and b i g sage (Artemisia tridentata) dominant. E. Subalpine Forest. (J?. m. oreas, trapped i n Manning Park, B. C ) . Engelmann spruce (Picea engelmanni) and alpine f i r (Abies lasiocarpa) form the dominant tree species in t h i s zone which occurs above 4,000 f t . elevation in the southern part of the province. The summers are short and winters moderately cold with snowfall generally heavy. The mean annual temperature i s 1-3°C. F. Queen Charlotte Islands. (P_. sitkensis prevostensis, trapped on Hotspring Island). This large group of coastal islands which l i e s between 52° and 54°N l a t i t u d e i s heavily-forested with Sitka spruce (Picea sitkensis) and western hemlock (Tsuga heterophylla). The smaller islands such as Hotspring have a very moderate climate with temperatures much below 0°C or above 20°C occurring infrequently. The d i v e r s i t y of gross c l i m a t i c conditions within and between these s i x habitats should provide a good sample for interpretation of the importance of the microclimate temperature. Results Subsequent to the measurement of the microclimate temperature, the thermistor lead wires were traced to where the thermistor and connector l a y i n the burrow. None was found i n conspicuous nests, which suggests that the mice either had no nests at the time or did not run far enough when released to reach t h e i r usual r e s t i n g place. I t i s , therefore, more appropriate to specify that the burrow temperatures recorded are those of the "potential micro-climate" . The data presented are for p a r t i c u l a r days in the summer (July-August) and winter (January-February). An endeavour was made to select days representative of these seasons with respect to weather. A t y p i c a l summer recording taken in the subalpine habitat of £. m. oreas i s shown in F i g . 3. The d a i l y temperat-ure fluctuations in three d i f f e r e n t burrows are compared with outside fluctuations. Although the outside temperatures fluctuate 15.2°C (4.6° — 19.8°C), the burrow temperatures at t h i s season are grouped around 10°C and fluctuate very l i t t l e (0.3° - 0.6°C). These t y p i c a l underground burrows of deer mice provide, therefore, a moderate, stable temperature environment. The burrow temperatures are a c t u a l l y the equilibrium value between the burrow a i r and the surrounding s o i l . Accordingly, the numerous data on s o i l temperature p r o f i l e s (6, 30, 37) are valuable indications of p o t e n t i a l microclimates for small, burrow-dwelling mammals. The effectiveness of the burrow systems in providing shelter from the extremes of summer or winter temperatures i s demonstrated i n F i g . 4. In t h i s figure, the r e l a t i o n of burrow to outside temperature i s compared i n summer and winter and among a l l six habitats. The most s t r i k i n g feature i s that i n winter the burrow temperatures do not f a l l below 0°C and are not midway between the d a i l y outside extremes as i s general i n summer. I t i s apparent that in winter the heat reservoir of the lower s o i l l e v e l s i s being continually dissipated towards the surface as described by Geiger (6), and in so doing keeps a l l but the top few inches of s o i l above freezing. 81 F i g . 3. A t y p i c a l summer recording of outside and burrow temperatures over a 24-hour period (subalpine habitat) . 82 F i g . 4. Comparison of outside ( s o l i d lines) and burrow (broken lines) temperatures i n summer and winter and i n a l l six habitats. The shaded areas are temperatures warmer than the burrows i n summer and colder than the burrows i n winter. P m. nebrascensis, P m. ouster us, P m. sonoriensis, P m. ortemisioe, P m. ore as, R sitkensis, alpine mesic desert arid subalpine mesic HOUR OF THE DAY 83 The shaded areas between the outside and burrow temperature curves in Fig„ 4 point out the "amount" of, temperature that i s warmer than the burrow i n summer and colder than the burrow i n winter. These areas or "amounts" of temperature are expressed as the product of temperature (°C) and time (hours). They may be most simply determined by the well-known geometric procedure of counting squares on g r i d paper. The value so obtained allows a quantitative description of the seasonal temperature regimes. The r a t i o of the amount of temperature that i s warmer than the burrow i n summer to the t o t a l amount of temperature d i f f e r e n t from the burrow averages 0.58 for a l l habitats. This same r a t i o considering the amount of temperature colder than the burrow in winter i s 0.98. I t i s obvious i n these terms that the burrow i s much more important as a refuge from temperature extremes i n winter than i n summer. The most s i g n i f i c a n t summer burrow temperatures with respect to su r v i v a l value are those recorded i n the Nevada desert. On the day presented, the outside temperature rose to 44°C (111°F). Despite t h i s high external temperature, a mouse re s t i n g i n a burrow at 26°C would have no thermo-regulatory problem (13). Sim i l a r l y , Carpenter (2) showed that subsurface s o i l temperatures i n summer permitted two species of kangaroo rats (Dipodomys) to spend a l l of t h e i r i n active periods at thermoneutral temperatures. 84 As was pointed out e a r l i e r , the burrow temperatures tend to be very stable, although there i s a greater burrow temperature fluctuation i n those habitats lacking forest cover (Wyoming alpine and Nevada desert). In winter, when snow cover adds i t s i n s u l a t i v e influence, burrow temperatures are exceptionally stable. The l e a s t stable burrow temperatures were recorded i n the Nevada desert, a habitat which lacks both forest cover and, winter snow. Discussion I t has been shown that the microhabitats u t i l i z e d by Peromyscus provide a moderated, r e l a t i v e l y stable temperature environment. Important to the assessment of the sur v i v a l value of such an environment i n the adaptation to cold i s the understanding that i t i s when an animal i s r e s t i n g that environmental temperature w i l l exert i t s major influence. For example, a deer mouse which may be exposed to short periods of subfreezing temperatures in i t s nocturnal a c t i v i t i e s i s not subject to thermoregulatory stress due to the heat increment associated with such a c t i v i t y . This heat increment may p a r t i a l l y substitute f o r the energy required for thermo-regulation, although Hart (9) has contrary evidence. I t i s when the mouse i s rest i n g that the most moderate temperatures must p r e v a i l i n the int e r e s t of energy conservation. This i s esp e c i a l l y important during winter conditions when food supplies 85 may be li m i t e d . I t was seen (Fig. 4) that moderate temperatures are avai l a b l e to these mice for t h e i r r e s t i n g periods. In conjunction with these temperatures, the adjustment of outside a c t i v i t y periods in small mammals in order to expend the l e a s t energy for thermoregulation per se i s an added expedient for which there i s much supporting evidence (7, 10, 28). The importance of microclimates to small mammals inhabit-ing deserts has been pointed out i n several investigations (2, 11, 31, 40). Much more has been said about winter microclimates, e s p e c i a l l y the influence of snow cover and the si g n i f i c a n c e of the subnivean a i r layer to small mammals (4, 14, 16, 19, 27, 28, 29, 39). In the present study, the int e r e s t i n the microclimates used by the s i x races of deer mice i s p r i m a r i l y from a comparative viewpoint. The es s e n t i a l question i s : -are the temperatures i n these microhabitats s u f f i c i e n t l y d i f f e r e n t such that temperature may have played a s e l e c t i v e r o l e i n the d i s t r i b u t i o n and speciation of these mice? To f a c i l i t a t e t h i s comparison, the data of F i g . 4 have been condensed i n the manner shown i n F i g . 5. The highest outside summer temperature and lowest outside winter temperature f o r the six habitats are compared with the average burrow temperatures at the same times. The range o of outside temperatures (58 C) i s much reduced i n the burrows (26°C), but the most i n t e r e s t i n g observation i s that, in winter, burrow temperatures are very si m i l a r (0° - 6°C) 86 F i g . 5. A comparison of the seasonal outside-temperature extremes i n each habitat with the average burrow temperatures at the same times. The broken l i n e s indicate how the burrow temperatures are moderated i n comparison to the seasonal extremes. This diagram emphasizes the p o t e n t i a l a v o i d a b i l i t y of temperatures capable of inducing thermo-regulatory stress i n deer mice. 45r 4 0 35 3 0 summer maxima 2 5 2 0 15 10 O o o o o o o o summer ZERO winter - 5 -io winter minima -15 OUTSIDE TEMPERATURES BURROW TEMPERATURES 87 i n a l l habitats. From t h i s i t does not appear l i k e l y that there i s a s i g n i f i c a n t d i f f e r e n t i a l s e l e c t i v e pressure for temperature adaptation i n these s i x races. Presumably, t h i s accounts for the fac t that metabolic rate was not found to be temperature-adaptive in these races (13). In addition to the s i m i l a r i t y of the microclimate temperatures measured, the actual temperature at which these mice r e s t i n winter may be even warmer than the present recordings when the evidence concerning the in s u l a t i v e value of nest-building and huddling i s considered. For example, in the present study, a deer mouse was kept i n a cold-room at 1° - 3°C and allowed to b u i l d a nest with dried leaves and moss. Later, the temperature recorded within the nest was 13.5°C. Other studies with Peromyscus (15, 24, 35) and Microtus (38) have shown that temperatures of occupied nests are from 7° to 24°C higher than the immediate surroundings. Huddling of two or more individuals i n the nests has also been shown to be common during winter conditions (5, 24). There i s also evidence that nest-building a c t i v i t y i n Peromyscus i s greater i n winter than i n summer (5, 35). From another approach, Pearson (26) demonstrated a reduced energy expenditure by harvest mice at low temperatures that i s a r e s u l t of nest-building and huddling. From the foregoing, i t i s a reasonable hypothesis that the combined influences of microclimate and seasonally-regulated nest-building and huddling provide a resting deer 88 mouse with an environmental temperature above 10°C and below t h e i r lower c r i t i c a l temperature (about 26°C) i n any habitat or season. Although these temperatures w i l l require energy production greater than the basal l e v e l i n Peromyscus (13), they cannot be considered of a stressing magnitude. Supporting p h y s i o l o g i c a l evidence that Peromyscus l i v e at moderated micro-climate temperatures i n t h e i r natural habitats i s provided by Sealander (36). In these mice, the use of a subterranean microclimate i s of considerable temperature-adaptive s i g n i f i c a n c e and i s a concomitant of small body s i z e . For t h i s reason, one might suspect that even within the genus Peromyscus there may be a sel e c t i o n for small body s i z e associated with t h e i r widespread d i s t r i b u t i o n . Interesting evidence to t h i s e f f e c t i s provided by Grinnel and Orr (8). They noted the d i s t r i b u t i o n s of four species of Peromyscus i n C a l i f o r n i a and point out that there i s an inverse c o r r e l a t i o n of si z e with extent and continuity of range- the larger the species, the more r e s t r i c t e d i t i s i n general geographic d i s t r i b u t i o n . Notably, in the present study, the largest species (P. sitkensis) i s an islan d form with r e s t r i c t e d d i s t r i b u t i o n . Also, the smallest race of the maniculatus species (j?. m. nebrascensis) inhabits the alpine environment where the only e f f e c t i v e shelters are small tunnels and narrow crevices i n rocky s o i l s . This l a s t example i s testimony to the apparent overriding influence of se l e c t i o n for small body siz e i n the temperature adaptation of these 89 small rodents as opposed to -the concept given by Bergmann's Rule. Similar findings regarding a d i f f e r e n t i a l a b i l i t y of species of Peromyscus to use t e r r e s t r i a l holes and crevices were discussed by McCabe and Blanchard (20). In t h i s same context, i t has been emphasized (4, 28) that there i s a maximum siz e for small mammals above which the important sub-nivean air-space becomes uninhabitable. I t i s concluded that in non-hibernating small mammals such as Peromyscus the behavioural resppnses to environmental temperature s t i m u l i (use of microclimate, nest-building, huddling) are of such e f f i c i e n c y , that i n s u l a t i v e (fur, peripheral c i r c u l a t i o n changes) and phy s i o l o g i c a l (metabolic rate) adaptations to cl i m a t i c temperature play an i n s i g n i f i c -ant r o l e i n d i s t r i b u t i o n and speciation. This conclusion adds v a l i d i t y to the statement by Johnson (14) who, i n reference to a r c t i c and subarctic small mammals, suggests that- " I t may well be that these l i g h t l y clad forms survive only because they l i v e i n a microclimate so warm that very thick in s u l a t i o n i s unnecessary". Apparently, the temperature regulation of small mammals has much i n common with that of man. Scholander (32) points out that "In the eskimo, the main adaptation l i e s not i n physiology, but i n an age-long experience and technical s k i l l i n ducking the cold". Selection of a suitable microclimate i s also a potent factor i n the cl i m a t i c adaptation of t e r r e s t r i a l poikilotherms (33) . 90 In future work on the microclimate of small mammals, the use of miniature, temperature-sensitive radio transmitters -"endoradiosondes" (18) - that are small enough to attach externally to wild individuals, would be a valuable refinement. 91 References 1. Benton, A. H. and Altmann, H. J. 1964. A study of fleas found on Peromyscus i n New York. J. Mammal. 45, 31-36. 2. Carpenter, R. E. 1963. A comparison of thermoregulation and water metabolism in the kangaroo rats, Dipodomys a g i l i s and Dipodomys merriami. Dissert. Abst. _24, 899-900. 3. Cook, S. F. and Hannon, J. P. 1954. Metabolic differences between three strains of Peromyscus maniculatus. J. Mammal. _35, 553-560. 4. Coulianos, C. C. and Johnels, A. G. 1963. Note on the subnivean environment of small mammals. Arkiv. Zool. 15, 363-370. 5. F i t c h , H. S. 1958. Home ranges, t e r r i t o r i e s , and seasonal movements of vertebrates of the Natural History Reservation. Univ. Kansas Publ., Mus. Natural History 11, 63-326. 6. Geiger, R. 1950. The climate near the ground. Harvard Un i v e r s i t y Press, Cambridge, Mass. 7. Gentry, J . B. and Odum, E. P. 1957. The e f f e c t of weather on the winter a c t i v i t y of o l d - f i e l d rodents. J . Mammal. 38, 7 2-77. 8. G r i n n e l l , J . and Orr, R. T. 1934. Systematic review of the c a l i f o r n i c u s group of the rodent genus Peromyscus. J . Mammal. 15, 210-220. 9. Hart, J . S. 1950. Interrelations of d a i l y metabolic cycle, a c t i v i t y , and environmental temperature of mice. Can. J . Res., D. _28, 293-307. 10. H a t f i e l d , D. M. 1940. A c t i v i t y and food consumption in Microtus and Peromyscus. J. Mammal. 21, 29-36. 11. Hayward, J . S. 1961. The a b i l i t y of the wild rabbit to survive conditions of water r e s t r i c t i o n . C.S.I.R.O. W i l d l i f e Res. 6, 160-175. 12. 196_. The gross body composition of s ix geographic races of Peromyscus. Submitted for pub l i c a t i o n . (Included i n t h i s t h e s i s ) . 92 13. 196_„ Metabolic rate and i t s temperature-adaptive si g n i f i c a n c e in six geographic races of Peromyscus. Submitted for pu b l i c a t i o n . (Included in t h i s t h e s i s ) . 14. Johnson, H. M. 1951. Preliminary ecological studies of microclimates inhabited by the smaller a r c t i c and subarctic mammals. Proc. Second Alaska S c i . Conf. Alaska Div. A.A.A.S. £,125-131. 15. Johnson, M. S. 1926. A c t i v i t y and d i s t r i b u t i o n of certa i n wild mice i n r e l a t i o n to b i o t i c communities. J . Mammal. 1_, 245-277. 16. Kayser, C. H. 1961. The physiology of natural hibernation. Pergamon Press, New York. 17. Linduska, J. P. 1947. Winter den studies of the cotton-t a i l i n southern Michigan. Ecol. 28, 448-454. 18. MacKay, R. S. 1961. Radio telemetering from within the body. Science 134, 1196-1202. 19. Mayer, W. V. 1960. H i s t o l o g i c a l changes during the hibernating cycle i n the a r c t i c ground s q u i r r e l . B u l l . Mus. Comp. Zool. 124, 131-154. 20. McCabe, T. T. and Blanchard, B. D. 1950. Three species of Peromyscus. Rood Associates, Santa Barbara, C a l i f o r n i a . 21. McNab, B. R. 1963. A model of the energy budget of a wild mouse. Ecol. 44, 521-532. 22. and Morrison, P. 1963. Body temperature and metabolism in subspecies of Peromyscus from a r i d and mesic environments. Ecol. Monog. _33, 63-82. 23. Murie, M. 1961. Metabolic c h a r a c t e r i s t i c s of mountain, desert and coastal populations of Peromyscus. E c o l . 42, 723-740. 24. Nicholson, A. J. 1941. The homes and s o c i a l habits of the wood-mouse (Peromyscus leucopus  noveborascensis) i n southern Michigan. Amer. Midland N a t u r a l i s t 25, 196-223. 25. Pearson, A. M. 1962. A c t i v i t y patterns, energy metabolism, and growth rate of the voles Clethrionomys rufocanus and C. glareolus i n Finland. Ann. Zool. Soc. "Vanamo" 24, 1-58. 93 26. Pearson, O. P. 1960. The oxygen consumption and bioenergetics of harvest mice. Physiol. Zool. 3*3, 152-160. 27. P r u i t t , W. 0. 1959. Microclimate and l o c a l d i s t r i b u t i o n of small mammals on the George Reserve, Michigan. Misc. Publ. Mus. Zool., Univ. Michigan, No. 109. 28. and Lucier, C. V. 1958. Winter a c t i v i t y of red s q u i r r e l s i n i n t e r i o r Alaska. J. Mammal. 39, 443_444, — 29. Quimby, D. G. 1944. A comparison of overwintering populations of small mammals i n a northern coniferous forest for two consecutive years. J . Mammal. 25, 86-87. 30. Rickard, W. H. and Murdock, J . R. 1963. S o i l moisture and temperature survey of a desert vegetation mosaic. Ecol. 44, 821-824. 31. Schmidt-Nielsen, B. and Schmidt-Nielsen, K. 1950. Evaporative water loss i n desert rodents i n t h e i r natural habitat. Ecol. _31, 75-85. 32. Scholander, P. F. 1955. Evolution of cl i m a t i c adaptation in homeotherms. Evolution 15-26. 33. , Flogg, W., Walters, V., and Irving, L. 1953. Climatic adaptation i n a r c t i c and t r o p i c a l poikilotherms. Physiol. Zool. 26, 67-92. 34. , Hock, R., Walters, V. and Irving, L. 1950c. Adaptation to cold i n arctic.and t r o p i c a l mammals and bir d s i n r e l a t i o n to body temperature, insulation, and basal metabolic rate. B i o l . B u l l . 99, 259-271. 35. Sealander, J . A. 1952. The relat i o n s h i p of nest protection and huddling to survival of Peromyscus at low temperature. Ecol. J33, 63-71. 36. 1962. Seasonal changes i n blood values of deer mice and other small mammals. Ecol. 43, 107-119. 37. Shanks, R. E. 1956. A l t i t u d i n a l and microclimatic relationships of s o i l temperature under natural vegetation. Ecol. 3 2 , 1-7. 94 38. Stark, H. E. 1963. Nesting habits of the C a l i f o r n i a vole, Microtus c a l i f o r n i c u s , and microclimatic factors a f f e c t i n g i t s nests. Ecol. 44, 663-669. 39. Swan, L. W. 1952. Some environmental conditions influencing l i f e at high a l t i t u d e s . Ecol. 33, 109-111. — 40. Vorhies, C. T. 1945. Water requirements of desert animals i n the Southwest. Univ. Arizona Tech. B u l l . No. 107, 487^525. 95 APPENDIX A SIMPLE ELECTROLYTIC RESPIROMETER FOR SMALL ANIMALS* by J. S. Hayward, H. C. Nordan,^ and A. J. Wood Abstract A simple e l e c t r o l y t i c respirometer i s described. Its design embodies several features advantageous for the accurate measurement of oxygen consumption. Representative re s u l t s using two species of mice are presented. Introduction There i s a continuous requirement for an e f f i c i e n t respirometer for the study of animal metabolism. For t h i s reason, numerous devices have been described, most of which measure changes i n volume or pressure of a closed system. * published i n the Canadian Journal of Zoology 41, 63-68. 1963. ^ D i v i s i o n of Animal Science, U n i v e r s i t y of B r i t i s h Columbia. * Department of Zoology, U n i v e r s i t y of B r i t i s h Columbia* 96 Brody (2) and Swift and French (11) have reviewed the various techniques. More recently, Morrison (7, 8), Smith (9), and Bailey est a_L. (1) have described volumetric respirometers. There exists a need for an apparatus which adequately combines the features of s i m p l i c i t y of construction, ease and r e l i a b i l i t y of measurement, long-term operation at constant gas composition, and a d a p t a b i l i t y to animals of various siz e s . The object of t h i s paper i s to present a description of the design and operation of an apparatus constructed to s a t i s f y the above requirements. Examples of the r e s u l t s obtained with i t s use for the determination of oxygen consumption of two species of mice are also given. P r i n c i p l e The apparatus i s of the closed c i r c u i t , volumetric type. The p r i n c i p l e of i t s operation i s that the oxygen consumed by an animal i n the sealed chamber i s almost continuously replaced by oxygen generated e l e c t r o l y t i c a l l y . E l e c t r o l y t i c respirometers have been used extensively i n s o i l microbiology studies (6, 10) where constancy of gas composition i s e s s e n t i a l . In such cases oxygen production has been measured by c o l l e c t i o n of the hydrogen evolved at the negative electrode. Such a method i s not too suitable for long-term measurement on small mammals and has the disadvantage that a large volume of gas must be maintained at 97 constant temperature. An elaborate e l e c t r o l y t i c respirometer for small animals has been described by Werthessen (12) which relates oxygen production to current flow and embodies a recording ammeter. The special apparatus required and i t s r e l a t i v e l y complicated construction seem to have c u r t a i l e d i t s general usage. Description of the Respirometer A diagrammatic representation of the present apparatus i s shovln i n F i g . 1. Its major components are l i s t e d below. 1. An e l e c t r o l y s i s apparatus (A) which, i n t h i s case, i s a product of Southern Instruments Ltd.* (model A1653). The e l e c t r o l y t e solution i s 30% NaOH. 2. A small, d e l i c a t e l y balanced spirometer (B) which maintains constant pressure i n the system and has a " t i d a l " volume of 1.6 cc. The spirometer i s made from the thin brass casing of an automobile d i s t r i b u t o r condenser and i s p a r t i c u l a r l y suitable for the purpose. The spirometer rests i n a well (C) made from lh-in. inside diameter p l a s t i c tubing mounted on a p l a s t i c base. 3. A low-torque microswitch (110 vo l t , 5 amp) (D) which i s actuated by the balance arm of the spirometer. The microswitch turns the oxygen generator (A) and the e l e c t r i c clock (E) on or o f f together. The simple operational c i r c u i t * Southern Instruments Ltd., Frimley Road, Camberley, Surrey, England. 98 F i g . 1. A diagrammatic representation of the respirometer. The spirometer i s shown i n two positions. 99 i s shown i n the inset of F i g . 1 . 4. The animal chamber (F) i s a pyrex glass vessel previously described (1), which has a volume of 750 cc. It i s f i t t e d with a l i d with a ground-glass seal and has two o u t l e t tubes. 5. A rectangular animal cage (G) of h-in. mesh hardware cloth which i s designed for a mouse. Its dimensions are 7 cm high, 5 cm wide, and 8 cm long and i t i s mounted on a metal droppings pan. 6. A magnetic s t i r r e r (K) which slowly turns the magnetic bar (H) thereby mixing the KOH solution in the bottom of the animal chamber. This insures a maximum rate of carbon dioxide absorption. 7. A thermistor (J) i s inserted into the animal chamber and permits an accurate account of temperature (L). A general view of the apparatus i s shown in F i g . 2. Operation of the Respirometer P r i o r to a metabolism run, the animal i s enclosed i n the cage and placed i n the chamber without the l i d for a period of 24 hours. Except for determinations of r e s t i n g or basal metabolism, food and water are provided. One hour before the run i s to begin, the chamber i s sealed with the l i d and placed i n the water bath for temperature regulation. At the same time, the operational c i r c u i t , except the clock, 100 F i g . 2 . A general view of the respirometer. o 101 i s turned on. The following events then occur: as the animal respires, the carbon dioxide produced i s absorbed by the KOH solution and the oxygen consumed res u l t s i n a decrease i n the pressure of the system. This allows the small spirometer to descend slowly to the point at which the microswitch i s actuated to turn on the e l e c t r o l y s i s apparatus. Oxygen i s immediately li b e r a t e d at the p o s i t i v e electrode and enters into the closed system. The increasing pressure raises the spirometer to the point at which the microswitch turns o f f the e l e c t r o l y s i s apparatus, one cycle of the apparatus then being completed. This continues u n t i l the temperature has s t a b i l i z e d and the animal i s re s t i n g . The measurement of oxygen consumption can begin when the system i s at i t s maximum volume, that i s , when the e l e c t r o l y s i s apparatus has ju s t turned o f f . At t h i s point, time i s recorded on a stopwatch and the e l e c t r i c clock i s turned on. Thereafter, any operation of the e l e c t r o l y s i s apparatus as i t replaces the oxygen consumed i s timed by the e l e c t r i c clock i n a p a r a l l e l c i r c u i t . This e s s e n t i a l l y automatic operation permits runs of long duration with a minimum of attention. The run can be terminated at any time recordings are obtained and from these data the rate of oxygen consumption can be calculated. 102 C a l i b r a t i o n and Calculation The primary r e q u i s i t e for the c a l i b r a t i o n of t h i s apparatus i s that the e l e c t r o l y t i c current i s constant. This i s dependent upon two factors: 1. The constancy of the input voltage. This has been s a t i s f a c t o r i l y achieved by the use of a voltage s t a b i l i z e r between the mains and the operational c i r c u i t . 2. Changes i n the conductivity of the e l e c t r o l y t e solution. As e l e c t r o l y s i s proceeds, there i s a gradual increase i n the concentration of the e l e c t r o l y t e solution which i s approximately 1 to 2% for a 24-hour metabolism determination with a mouse* This increase i s n e g l i g i b l e since the conductivity of strong e l e c t r o l y t e s such as NaOH changes very l i t t l e even with large changes i n concentration (4). If the e l e c t r o l y t i c current strength (I) i s accurately determined, the oxygen consumption can be calculated from the r e l a t i o n s h i p : I x t x e  m = 96,500 where m = grams of oxygen l i b e r a t e d by e l e c t r o l y s i s (convertible to volume at S.T.P.), t i s the t o t a l e l e c t r o l y s i s time i n seconds as recorded on the e l e c t r i c clock, e i s the equivalent weight of oxygen, and 96,500 i s the value of 1 faraday of current expressed i n coulomb units. 103 An al t e r n a t i v e method of c a l i b r a t i n g the apparatus for the r e l a t i o n s h i p between duration of e l e c t r o l y s i s and oxygen consumed i s to connect the closed system to a burette from which accurately measured aliquots of water can be slowly drained. This r e s u l t s i n a decrease i n pressure of the closed a i r system and simulates oxygen consumption by an animal. A l i n e a r r e l a t i o n s h i p between volume increase and time of e l e c t r o l y s i s i s obtained. Since t h i s c a l i b r a t i o n i s carried out at room temperature, the gas volume must be corrected to S.T.P. This method i s an easy c a l i b r a t i o n check and can be car r i e d out before each experiment. When the respirometer has been calib r a t e d by either of the two methods outlined above, a simple recording of time can be d i r e c t l y converted to a reading of oxygen consumption. Results and Discussion The u t i l i t y of t h i s apparatus has been tested by several hundred determinations of oxygen consumption with mice over the temperature range 0° to 35°C and for periods from 5 minutes to 24 hours. Figure 3 shows the influence of ambient temperature upon the r e s t i n g rate of oxygen consumption of a fasted, mature Swiss albino mouse. Thermal n e u t r a l i t y i s i n the region of 27° to 28°C and at t h i s point the rate of oxygen consumption i s about 1.9 cc/gram body weight per hour. Assuming a postabsorptive respiratory 104 F i g . 3. An example of the re s u l t s obtained with the respirometer showing the rela t i o n s h i p between environmental temperature and the res t i n g rate of oxygen consumption of a 25-gm Swiss albino mouse. CC. OXYGEN PER GRAM BODY WEIGHT PER HOUR 105 quotient of 0.75, the d a i l y heat production would be 5.39 Calor i e s . On the basis of the equation; basal metabolic rate = 0 7 70.5W ' (2), the d a i l y heat production of a 25.0 g mature animal would be 5.09 Calories. The example given here i s very close to the t h e o r e t i c a l value and i s sim i l a r to res u l t s reported elsewhere for the white mouse (1, 3, 5). The respirometer i s e s p e c i a l l y suitable for long-term measurement of oxygen consumption. This maybe i l l u s t r a t e d by Pig. 4, where fluctuations i n the d a i l y a c t i v i t y cycle of a wild mouse (Peromyscus maniculatus) are r e f l e c t e d i n the oxygen consumption averaged for 1-hour i n t e r v a l s . The advantages of t h i s apparatus can be b r i e f l y summarized as follows: 1. A standard e l e c t r o l y s i s instrument i s the only major item required. 2. I t i s suitable f o r measurements of long duration and i s e s s e n t i a l l y automatic. 3. Normal atmospheric gas composition i s maintained in the chamber. 4. Only a small volume of a i r i s required in the closed system which minimizes the influence of temperature and pressure v a r i a t i o n upon the readings of oxygen consumption. 5. No correction factors need to be applied to the measurement of oxygen consumption such as occur i n many 106 F i g . 4. A record of the 24-hour oxygen consumption of a deer mouse (Peromyscus maniculatus) at 25°C. Points are the average of 1-hour i n t e r v a l s . 107 kinds of apparatus where oxygen consumption i s measured by the emptying of a spirometer of known volume. In such cases there i s no measurement of oxygen consumed during the short period that the spirometer i s r e f i l l i n g with oxygen. 6. The e a s i l y and accurately measurable quantity, time, i s the index to oxygen consumption. The respirometer i s adaptable to the measurement of carbon dioxide production and respi r a t o r y quotient. The carbon dioxide absorbed by the KOH solution maybe followed by t i t r i m e t r i c or conductivity methods. There i s a p r a c t i c a l upper l i m i t to the rate of oxygen production by the p a r t i c u l a r e l e c t r o l y s i s instrument described which l i m i t s the s i z e of the animal i t w i l l handle. The maximum rate of oxygen production i s 1.7 l i t e r s per hour. For mammals larger than a rabbit either an e l e c t r o l y s i s instrument of greater current capacity or an al t e r n a t i v e technique could be used. 108 References 1. Bailey,-C. B., K i t t s , W. D., and Wood, A. J. 1957. A simple respirometer for small animals. Can. J. Animal S c i . 37, 68-72. 2. Brody, S. 1945. Bioenergetics and growth. Reinhold Publ. Corp., New York. 3. Davis, J . E. and Van Dyke, H. B. 1933. The oxygen consumption of f a s t i n g white mice. J . B i o l . Chem. 100, 455-462. 4. Glasstone, S. 1946. The elements of physical chemistry. Van Nostrand Co., New York. 5. Hart, J . S. 1950. Interrelations of d a i l y metabolic cycle, a c t i v i t y and environmental temperature of mice. Can. J. Res. D. 28, 293-307. 6. McGarity, J. W., Gilmour, C. M., and Bollen, W. B. 1958. Use of an e l e c t r o l y t i c respirometer to study d e n i t r i f i c a t i o n in s o i l . Can. J. Microbiol. 4, 303-316. 7. Morrison, P. R. 1947. An automatic apparatus for the determination of oxygen consumption. J. B i o l . Chem. 169, 667-679. 8. 1951. An automatic manometric respirometer Rev. S c i . Instr. 22, 264-267. 9. Smith, E. 1955. A new apparatus for long term measurement of oxygen consumption i n small mammals. Proc. Soc. B i o l . Med. 89, 499-501. 10. Swaby, R. J . and Passey, B. I. 1953. A simple macro-respirometer for studies i n s o i l microbiology. Australian J. Agr i . Res. 4, 334-339. 11. Swift, R. W. and French, C. E. 1954. Energy metabolism and n u t r i t i o n . Scarecrow Press, Washington, D. C. 12. Werthessen, N. T. 1937. An apparatus f o r the measurement of the metabolic rate of small animals. J . B i o l . Chem. 119, 233-239. 109 EPILOGUE "With the i n f a l l i b l e judgment of hindsight, i t i s e a s i l y seen that .. . . ." J. M. Sherman* I t may be appropriate at t h i s juncture to indulge i n some "hindsight". Several improvements and extensions to t h i s study can be suggested on the basis of the experience obtained. I t would be in t e r e s t i n g to follow the seasonal changes in body composition of a single race much more closely, e s p e c i a l l y with regard to f a t content. As t h i s study has suggested, an extensive investigation of f a t l e v e l s i n desert-adapted rodents may help c l a r i f y the reason for the apparent lower basal metabolic rates of these animals. Although the respirometer used i n t h i s study has been very s a t i s f a c t o r y , i t i s d i f f i c u l t to use at temperatures below freezing. An open c i r c u i t respirometer using conventional gas analysis recorders would have permitted measurements of the lower summit metabolism. The r a c i a l differences i n slope of the basal metabolic rate:body weight regressions require more concentrated study with many individuals of fewer races. For such a study, i t would be valuable to have laboratory-reared individuals of known age and n u t r i t i o n a l h i s t o r y . The influence of duration of * In attempts to reveal sex i n bacteria; with some l i g h t on gerraentive v a r i a b i l i t y i n the Coli-aerogenes group. J . Bact. 33, 315-321. (1937). 110 f a s t i n g on the basal metabolic rate of individuals of d i f f e r e n t sizes may help to explain these slope differences. Continuous recordings of the temperatures i n the immediate environment of a mouse during normal a c t i v i t i e s i n i t s natural habitat would be expected to y i e l d f a s c i n a t i n g r e s u l t s with respect to temperature regulation. With the recent development of miniature radio transmitters which monitor p h y s i o l o g i c a l parameters, such continuous recordings may be possible. The author f e e l s that the main value of t h i s contribution has been the inclusion, i n one study, of three important types of data necessary for the interpretation of comparative basal metabolic rates. 

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