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Quiescent states of sleep, torpor and hibernation in the Sanders, Colin E. 2008

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QUIESCENT STATES OF SLEEP, TORPOR AND HIBERNATION IN THE BRAZILIAN TEGU (Tupinambis merianae Dumeril and Bibron) by Colin E. Sanders BSc.Hon., University of British Columbia 2000  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA Vancouver, Point Grey Campus  April 2008 © Colin E. Sanders, 2008  ABSTRACT  Brazilian tegus (Tupinambis merianae) were instrumented with telemetry encoder implants that measured and broadcast heart rate (HR), breathing rate (fR), and deep body temperature (Tb) and were then allowed to freely roam in outdoor enclosures mimicking their natural environment for a full year (2004) in order to monitor the circadian and circannual patterns in behaviour and cardio-respiratory physiology. The year was divided up into 5 seasons based upon the physiology and behaviour of the tegus: early activity season (Sept.-Nov.), late activity season (Dec.Feb.), entrance into hibernation (March-April), hibernation (May-July), and arousal from hibernation (August). The activity seasons were characterized by warm weather with frequent rainfall which slowly decreased in temperature and precipitation as tegus started entering hibernation so that the end of the dormant season was marked by dry, cold weather. Tegus in the early activity season demonstrated high activity associated with breeding demands, displayed elevated HR and fR, and were able to maintain a large temperature differential (4-7°C) between deep body temperature (Tb) and their respective burrow (Tburrow) during sleep. As the season progressed into late activity season, average Tb remained constant but average HR and fR progressively declined indicating nightly torpor. Periods of inactivity during the active seasons were rare and associated with inclement weather. Tegus entered hibernation through bouts of inactivity that progressively increased in frequency and duration. During this time, Tb  ii  was regulated but declined at different rates in regards to daytime and nighttime values. Heart rate through the entrance into hibernation and hibernation periods frequently demonstrated arrhythmias that increased in duration but decreased with frequency as hibernation progressed. Through hibernation, Tb continued to decline for the first month but HR and fR were constant, demonstrating a temperature independent suppression of metabolism. Through the hibernation season tegus sporadically aroused and emerged from their burrows to warm up and after a short basking period would return to the burrows and swiftly resume hibernation. While hibernating, heart rate was characteristically regular but breathing was sporadic or episodic. Arousals became more frequent towards the end of hibernation so that when they entered arousal from hibernation season most tegus were emerging daily. At this time daily maximum deep body temperature  (Tbmax)  swiftly returned to active  season values but nighttime daily minimum deep body temperature (Tbmirt ) values only showed a gradual increase through August, indicating different body temperature set points (Tbset) for active and sleep states. Changes in heart rate and breathing rate during the year showed greatest correlation with changes in photoperiod, although throughout hibernation HR and fR also showed tight correlation with Tb.  iii  TABLE OF CONTENTS  Topic^ Abstract ^ Table of contents ^ List of Figures ^ List of Abbreviations ^ Acknowledgments ^  Chapter 1: General Introduction ^ 1.1. Terminology ^ 1.1.1.Sleep ^ 1.1.2.Torpor ^ 1.1.3.Hibernation ^ 1.1.4.Dormancy ^ 1.1.5.Brumation ^ 1.2.Sleep, Torpor, and Hibernation in Tegu Lizards ^  Page  ii iv vi  x xi 1  2 2 3 4 5 5 6  Chapter 2: Quiescent states in the Brazilian tegu: Annual cycles in behaviour and cardiorespiratory physiology ^ 10 2.1.Introduction ^ 10 2.2.Methods ^ 14 2.2.1.Surgery ^ 14 2.2.2.Post-Surgical Care ^ 15 2.2.3.Housing/Study Site ^ 16 2.2.4.Data Acquisition ^ 17 2.2.5.Experimental Protocol ^ 18 2.2.6.Data Analysis ^ 19 2.3.Results ^ 20 2.3.1.Meteorological Data ^ 20 2.3.2.Annual Patterns of Behaviour ^ 20 2.3.3.Annual Patterns of Physiological Change ^ 29 2.3.4.Periodic Breathing and Cardiac Asystole; Evidence of Deep Hibernation? ^ 34 2.3.5.Zeitgebers ^ 35 2.4.Discussion ^ 41 2.4.1.Anticipatory Decreases in Metabolic Rate; Programmed Nocturnal Torpor? ^ 42 2.4.2.Daily Torpor/Multi-Day Bouts of Torpor/Hibernation; A Natural Progression? ^ 43 2.4.3.Do Tegus Arouse Periodically? ^ 44 2.4.4.Is Degree of Metabolic Suppression Constant Throughout Hibernation? ^ 46 2.4.5.Zeitgebers ^ 48 2.4.6.Active/Reproductive Season and Body Temperature Regulation ^ 49  iv  Chapter 3: Seasonal changes in daily behavioural and physiological rhythms in the Brazilian tegu ^ 51 3.1.Introduction ^ 51 3.2.Materials and Methods ^ 54 3.2.1.Instrumentation ^ 54 3.2.2.Housing/Study Site ^ 54 3.2.3.Data Acquisition ^ 55 3.2.4.Experimental Protocol ^ 56 3.2.5.Data Analysis ^ 56 3.3.Results ^ 58 3.3.1.Late Active Season ^ 58 3.3.2.Entrance Into Hibernation ^ 61 3.3.3.Hibernation Season ^ 61 3.3.4.Arousal from Hibernation ^ 64 3.3.5.Early Active (Reproductive) Season ^ 64 3.3.6.Heart Rate Hysteresis ^ 69 3.4.Discussion ^ 75 3.4.1.Seasonal Changes in Circadian Cycles of Behaviour and Thermoregulation ^ 75 3.4.2.Seasonal Changes in Heart Rate Hysteresis ^ 77 3.4.3.Conclusions ^ 80  Chapter 4: General Discussion ^ References ^  82 86  LIST OF FIGURES Number^Caption^ Figure 2.1:  Page  Environmental data (rainfall, atmospheric pressure, relative humidity and ambient temperature (dry bulb)) collected from the meteorological station at the Bela Vista campus of UNESP, Rio Claro, SP Brazil. Daily mean atmospheric pressure, relative humidity and temperature were calculated from ^21 three daily readings taken at 09:00, 15:00 and 21:00  Figure 2.2: a-d: The instantaneous heart rate (beats/min), instantaneous breathing rate (breaths/min) and deep body temperature (°C) measured in each tegu during the recording period. Each circle represents the average value for a 15 minute epoch with lines connecting consecutive data points. Tegus 1(a -y) and 2 (bd) shared a common enclosure as did Tegus 3 (c-Y) and 4/5(d-d) 22-25 Figure 2.3:  Top: Percentage of days in each month that tegus emerged from their burrows. Middle: The number of bouts in each month that tegus did not emerge from their burrows. Histograms marked with A are significantly difference from the month of April. Bottom: Duration of non-emergence bouts measured in days for each month ^ 27  Figure 2.4:^Times of sunrise and sunset at Rio Claro, SP Brazil for the year 2004. Time of emergence from the burrow (red symbols) and retreat into the burrow (blue symbols) are also shown for 7 months in the late active season (January), during entrance into hibernation (March), for rare incidents of emergence in hibernation (June, July), during arousal from hibernation (August) and during the early active season (September and December). * denotes statistical significance from respective September values (One-way repeated measures ANOVA; emergence — P<0.001; retreat — P=0.002) ^ 28 Figure 2.5:  Daily minimum (blue), median (black) and maximum (red) values of instantaneous breathing rate (breaths/min), instantaneous heart rate (beats/min), and deep body temperature (°C) of one tegu over the entire recording period. Dotted lines on the bottom graph demonstrate the relatively constant daily Tb max and Tb„„„ witnessed throughout the entire active season.30  Figure 2.6:^Mean monthly values for photoperiod, ambient and burrow temperatures, and core body temperature, heart rate and breathing rate for all tegus over the entire recording period ^ .31 Figure 2.7:  ^  Top: Daily average minimum temperatures of the burrow (green) relative to ambient air (blue). Middle: Daily average minimum temperatures of the tegus (black) relative to the burrows (green). Bottom: The daily average difference at the end of the night between tegu and burrow temperature 33  vi  LIST OF FIGURES Number^Caption^  Page  Figure 2.8:  Representative traces of the electrocardiogram and respiratory signal from one tegu during the photophase (around 13:00-15:00hrs) and during the scotophase (around 02:00 to 04:00hrs) in the late activity season (A) and during entrance into hibernation (B) ^ 36  Figure 2.9:  Representative traces of the electrocardiogram and respiratory signal from one tegu during the scotophase (around 04:00hrs) at different times in the hibernation season. The bottom trace is an expanded portion of the ECG trace from the April recording showing a normal heart beat as well as an example of a type 2 AV block ^ 37  Figure 2.10:  Representative traces of the electrocardiogram and respiratory signal from one tegu during the scotophase (around 04:00hrs) at different times in a single bout of hibernation ^ .38  Figure 2.11:  Representative traces of the electrocardiogram and respiratory signal from one tegu during the photophase (around 13:00-15:00hrs) and during the scotophase (around 02:00 to 04:00hrs) during arousal from hibernation and during the early active season ^ 39 .  Figure 2.12:  Average monthly values of ambient air temperature (°C), photoperiod (min), precipitation (mm), and mean heart rate (beats/min) and mean breathing frequency (breaths/min) for all tegus. Dotted line a indicates where heart rate and breathing rate start increasing after hibernation. Dotted line b indicates where heart rate and breathing rate peak after breeding season. Dotted line c indicates the peak in precipitation and how it correlates with a secondary peak in heart rate and breathing rate ^ 40  vi i  LIST OF FIGURES Page  Number^Caption^ Figure 3.1:  Mean values for ambient temperature, burrow temperature, tegu temperatures, heart rate and breathing rate for all tegus for all days of each month in the late active season (December, January and February). The dotted lines represent the average time when tegus left their burrows in the morning (red) and retreated into their burrows for the evening (blue)....59  Figure 3.2:  The relationships between mean values of Tb and HR for all tegus for all days of each month in the late active season (December, January and February). Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph ^ .60  Figure 3.3:  Mean values for ambient temperature, burrow temperature, tegu temperatures, heart rate and breathing rate for all tegus for all days of each month during entrance into hibernation (March and April). The dotted lines represent the average time when tegus left their burrows in the morning (red) and retreated into their burrows for the evening (blue).....62  Figure 3.4:  The relationships between mean values of Tb and HR for all tegus for all days of each month during entrance into hibernation (March and April). Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph. 63  Figure 3.5:  Mean values for ambient temperature, burrow temperature, tegu temperatures, heart rate and breathing rate for all tegus for all days of each month in the hibernation season (May, June and July). The dotted lines represent the average time when tegus left their burrows in the morning (red) and retreated into their burrows for the evening (blue) during days of .65 emergence ^  Figure 3.6: The relationships between mean values of Tb and HR for all tegus for all days of each month during the hibernation season (May, June and July). Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph. 66  viii  LIST OF FIGURES Number  Caption^  Figure 3.7:  Mean values for ambient temperature, burrow temperature, tegu temperatures, heart rate and breathing rate for all tegus for all days in August when tegus were arousng from hibernation. The dotted lines represent the average time when tegus left their burrows in the morning (red) and retreated into their burrows for the evening (blue) ^ 67  Figure 3.8:  The relationships between mean values of Tb and HR for all tegus for all days in August when tegus were arousing from hibernation. Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph ^ 68  Figure 3.9:  Mean values for ambient temperature, burrow temperature, tegu temperatures, heart rate and breathing rate for all tegus for all days of each month in the early active season (September, October and November). The dotted lines represent the average time when tegus left their burrows in the morning (red) and retreated into their burrows for the evening (blue) ^ 70  Figure 3.10:  The relationships between mean values of Tb and HR for all tegus for all days of each month in the early active season (September, October and November). Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph ^ 71  Figure 3.11:  Mean values for ambient temperature, burrow temperature, and the deep body temperature of two tegus in August, 2003. The dotted line indicates .72 the time at which tegu #3 died ^  Figure 3.12:  The relationships between mean values of Tb and HR for all tegus for all days of all months ^ 73  Figure 3.13:  The relationships between nighttime minimum values of Tb and HR for all tegus for all days of all months colour coded by season. Note the overall hysteresis in the relationship and the difference in heart rates at similar body temperatures for animals in different seasons ^ .74  Page  ix  LIST OF ABBREVIATIONS ECG^Electrocardiogram EEG^Electroencephalogram fR^Breathing Frequency HR^Heart Rate 1^Length of Pipe NREM^Non-Rapid Eye Movement Dynamic Fluid Viscosity PS^Paradoxical Sleep AP^Change in Pressure Q i o^Temperature Quotient r^Radius REM^Rapid Eye Movement SV^Stroke Volume SWS^Slow Wave Sleep T a^Ambient Temperature Tb^Body  Temperature (Deep Body)  Tbmax^Body  Temperature (Deep Body) Maximum  Tbmin^Body  Temperature (Deep Body) Minimum  Tbr^Brain Temperature Tburrow^Burrow  Temperature  Tset^Body Temperature Set Point  ACKNOWLEDGMENTS This has been a project that has involved the input and assistance of a lot of people and without whom the chances of this thesis' completion would likely be microscopically small. A general thanks to the Milsom lab, members past and present, who have either helped out or been there at times when I needed to vent. Most recently, Angelina Fong has been a tremendous help to not only my thesis but also my sanity. My closest friends Bruce `Brick' Trick, John Hare, Charlayne Bozack and Lea 'Mom' Randall have all been a tremendous support, in more ways than I can say. A very special thanks to Erico Nomura who was not only a great friend in Brasil, but also selflessly acted all too frequently as my interpreter and helper. I would like to thank those who gave insights and advice while I was sweating away in Rio Claro: Tobias Wang, Ted Taylor, Tadeu Rantin, A.P. Cruz-Neto, Celio Haddad, Cynthia Prado and Denis Andrade. A special tip of the hat to two special ladies, Alex Reid and Simone Brito. You were both so very instrumental in the success of this project and at the same time were also primary causes for it nearly being a massive failure. I hope some day to return the favour in kind. I appreciate the help, guidance and special consideration of my committee. Particularly, a special thanks to Dr. Colin Brauner, whose insight and attention to detail caught an unforeseen phenomenon in this project that my addled brain had overlooked. How I wish I had listened to your advice, my life would not be in the mess it currently is and this thesis would have been completed in time and with greater skill. I would like to give credit and thanks to Glenn Tattersall. A true friend, colleague, and advisor who wrote the code for processing the data in this study. Without him, I'd likely be bleeding through the eyes and ears as I tried analysing my data for the next 50 years. This project would have not even been possible if it weren't for the contribution of Augusto Shinya Abe. You not only provided me with a place to work and the animals to do research on for two years, but were a great friend and gave me what was without doubt the greatest experience of my life. I sincerely hope we will be able to collaborate again in the future. Most of all, I owe my greatest thanks to my supervisor, and friend, Dr. William Milsom. You have been shaping and influencing my mind (in a positive manner) for a quarter of my life, you have always supported me, and have been there all too often to help out when my life has fallen apart (I shudder to think how frequent that has been). While many have marvelled at the enormity of my project and credited me with ambition, I've usually only felt like the monkey at the controls — this project is without question a greater reflection of your work than mine. I will always be indebted to you and thank you for putting me to this task and giving me this opportunity. Perhaps most importantly for this project, a special mention should go to Kenny, Cartman, Stan, Kyle, Timmy, Bebe, Wendy, Ms. Crabtree and Marsh, the tegus who gave everything they could for a scientific cause. It was a wonderful time working with you. Lastly, a nod to the Brasilian liquid gold, cachaca and it's wonderful concoction the caiparinha, my liquid saviour. This project was funded by NSERC Canada and FAPESP Brasil. xi  1: GENERAL INTRODUCTION  As ectotherms derive their body heat almost exclusively from external sources, and as their metabolism is strongly influenced by body temperature, the external environment has a profound influence on every aspect of an ectotherm's life. This makes it necessary for ectotherms to synchronize their biological rhythms with cycles in their environment, both daily and seasonally (Cloudsley-Thompson, 1999). Synchronization is enhanced by an ability to predict major environmental changes that serve to reduce the possible deleterious or even lethal consequences of environmental change. Ultimately, two astronomical phenomena either directly or indirectly provide external environmental cues or zeitgebers that are responsible for the cyclic nature of the biorhythms observed in most organisms. The earth's revolution about its polar axis creates daily cycles wherein heat and photonic energy are divested to the ecosystem as any particular point on the surface of the planet revolves towards and away from the sun. This produces the circadian phenomena of day and night. Similarly, the tilt of Earth's polar axis and the entire planet's revolution around the sun interact to produce seasonal fluctuations in the overall energy received by particular hemispheres, producing the circannual phenomena represented by the seasons. This thesis focuses on circadian and circannual rhythms in heart rate, and breathing rate and their implications for temperature regulation and changes in metabolism in tegu lizards. These physiological changes are associated with changes in behaviour that give rise to a variety of quiescent states; sleep, torpor, hibernation and dormancy. The definitions of these quiescent states have not been uniformly applied in the literature and hence their use within this thesis requires some explanation. 1  1.1 Terminology 1.1.1 Sleep^The state of sleep as described in mammals is characterized by recurrent spontaneous bouts of inactivity with concomitant elevated thresholds of sensory response and greatly reduced cognitive function or unconsciousness (McGinty & Beahm, 1984). Sleep is differentiated from other states of unconsciousness, such as coma, by being readily reversible. Electroencephalographic (EEG) studies show mammals and birds possess two distinct phases of sleep, slow wave sleep (SWS) and paradoxical sleep (PS). Slow wave sleep, also known as non-rapid-eye-movement (NREM) sleep, is associated with synchronized cortical EEG activity of high amplitude and low frequency with occasional high frequency spikes, while paradoxical sleep (PS), also known as rapid-eyemovement (REM) or activated sleep, is associated with desynchronized EEG activity of low amplitude very similar to that seen in the awake state (Steriade, et al., 1993). In endotherms, sleep is associated with a progressive decline in brain temperature (TO and metabolism as sleep duration increases (for review, see Heller & Glotzbach, 1977). Sleep also has behavioural correlates such as specific sleep sites or postures and a distinct latency of behavioural response to stimulation (for review, see McGinty & Beahm, 1984). Many of the criteria that have been used to identify sleep states in mammals and birds do not describe the sleeping condition seen in less encephalized animals such as reptiles. Attempts to quantify reptilian cortical EEG activity using mammalian characteristics have been relatively unsuccessful (e.g. Huntley, et al., 1977; Meglasson & Huggins, 1979; Huntley & Cohen, 1980). While spike EEG activity homologous with that recorded from mammalian limbic systems does arise from the forebrain of reptiles during sleep (Diaz, et al., 1973; Hartse, et al., 1979), and is absent in both taxa during  2  wakefulness, sleep states in reptiles with EEG profiles comparable to those seen in mammals are usually no longer than a few seconds occurring in bouts of "behavioural sleep" lasting several hours (Huntley & Cohen, 1980; K. Rogers & W.K. Milsom, unpublished data). Sleep in reptiles therefore is usually quantified based on behavioural criteria, which include posture, closed eyelids, increased latency of responses to stimulation, and reduced but regular ventilation and cardiac frequencies (Flanigan, 1973, 1974; Flanigan, et al., 1973, 1974; Huntley, et al., 1977; Huntley & Cohen, 1980). 1.1.2 Torpor Torpor was first used to describe the phenomenon during which many endotherms allow their body temperatures to drop below levels considered normal for sleep, but not as low as the levels (within a few degrees of ambient) typically observed in hibernation (Morrison & Ryser, 1959; Tucker, 1962; MacMillen, 1964). As such, torpor usually involves a drop in Tb greater than 5°C and as much as 15°-20°C below normal, a reduction in resting metabolic rate by more than 25%, and usually occurs on a daily basis during the normal quiescent period of sleep. It may, however, extend over several days (Heller, et al., 1978; Hudson & Scott, 1979; KOrtner & Geiser, 2000). The term torpor has never been defined clearly as it pertains to ectotherms, and this has lead to its free use to describe nearly any quiescent state. There are researchers that posit the idea that torpor is simply a function of reduced body temperature (e.g. Hock, 1958) and others that use torpor and hibernation interchangeably to denote the winter quiescent period (e.g. Binyon & Twigg, 1965; Ultsch, 1989). Most researchers have adopted the term in the mammalian sense to describe a profound daily reduction in metabolism due to declining ambient temperature during the inactive phase (e.g. Potter & Glass, 1931; Holzapfel, 1937; Willis, et al., 1956). As such, it involves a daily reduction in  3  metabolism to a level below that predicted by changes in body temperature alone, is readily reversible and involves a greater latency of responsiveness to stimulation than that associated with sleep. 1.1.3 Hibernation^In general usage, to hibernate is to spend the winter in an inactive, sleep-like state and occurs in practically every phylum. For endothermic vertebrates it generally involves a seasonal reduction in body temperature to levels within a few degrees of ambient temperature (T a ). Hibernation occurs in bouts that last for periods of days, weeks, or even months that are interspersed by short periods of arousal during which the animals spontaneously rewarm (K6rtner & Geiser, 2000). The duration of these bouts varies from species to species as well as with ambient temperature and position in the winter season (early, mid, late) within species (Twente & Twente, 1965; Twente, et al., 1977; Kenagy, 1981; French 1982; Wilz & Heldmaier, 2000). Hibernation in ectothermic vertebrates, unfortunately, is not well defined. However, there do appear to be some common physiological phenomena correlated with hibernation observed in ectotherms and in particular, in reptiles (Bennett & Dawson, 1976.) Preparation for entrance into hibernation begins well before the winter season with many reptiles spontaneously initiating fasting in the fall (Cagle, 1950; Hernandez & Coulson, 1952; Musacchia & Sievers, 1956; Hutton & Goodnight, 1957; Coulson & Hernandez, 1964; Mayhew, 1965; Chabreck & Joanen, 1969; Fitch & von Achen, 1977; Abe, 1983; de Andrade & Abe, 1999). It also involves what has been termed by some as "inverse acclimatization" wherein metabolic rate is suppressed in relation to cooler temperatures (Mayhew, 1965; Jacobson & Whitford, 1970; Gatten, 1978; Patterson & Davies, 1978; Johansen & Lykkeboe, 1979; Abe, 1983). Many reptiles increase their metabolic rate to  4  compensate for cold temperatures (Roberts, 1968; Tinkle and Hadley, 1973; Dutton and Fitzpatrick, 1975; Ruby, 1977) and some develop an insensitivity of metabolic rate to changes in body temperature (Gelineo, 1967). This acclimation usually occurs in reptiles inhabiting regions with little or moderate seasonal variation and allows them to continue to function normally at lower temperatures. Inverse acclimatization, or hibernation, on the other hand occurs in reptiles that inhabit regions with more pronounced seasonality and involves active metabolic suppression. 1.1.4 Dormancy^There appears to be some confusion in the literature surrounding the terms dormancy and hibernation, especially when used with respect to reptiles. Many authors interchange the terms and give them equal meaning; others use dormancy to denote a specific type of behaviour within hibernation (e.g. Mayhew, 1965; Gregory, 1982). Hibernation in common usage refers to winter inactivity, while dormancy has no similar temporal qualification. For the purpose of this thesis, dormancy is used as a general reference to any quiescent state involving reduced metabolic rate, including sleep, torpor, aestivation (a quiescent state associated with excessive heat) and hibernation. 1.1.5 Brumation^Mayhew (1965) proposed the term brumation to define inactivity and physiological changes identified with winter dormancy in ectotherms that occur independent of body temperature, to differentiate it from simple cold-induced inactivity, and hibernation seen in mammals. While some authors have adopted the term (e.g. Gaffney and Fitzpatrick, 1973; Huey & Pianka, 1977; Hutchison, 1979; Rismiller & Heldmaier, 1982), it has not achieved wide acceptance, and most authors overlook the distinction of the state being temperature independent.  5  1.2^Sleep, Torpor and Hibernation in Tegu Lizards Tegu lizards (genus Tupinambis) are large diurnal reptiles with a broad distribution throughout South America east of the Andes (Avila-Pires, 1995). The genus is comprised of two major clades (Colli et al., 1998; Fitzgerald et al., 1999). The teguixin Glade is comprised of four species (Tupinambis teguixin, T palustris, T quadrilineatus and T longilineus) whose distribution is more northerly and mostly restricted to the Amazon basin. The merianae Glade, comprised of three species (T. merianae, T. duseni and T rufescens) is distributed primarily south of the Amazon river extending down into Argentina and Uruguay. Like all diurnal ectotherms, tegus are active during the day and predominantly regulate their body temperature behaviourally by heliothermy, using the sun as the primary external source of heat (Bartholomew and Tucker, 1963; Tattersall, et al., 2004). Periods of activity are highly correlated to ambient air temperature rather than other environmental variables such as substrate temperature, relative humidity or wind velocity (Milstead, 1961). Their preferred daytime body temperature (Tb) is usually greater than 33°C (Vitt & de Carvalho, 1995; Vitt & Zani, 1996), and they rarely emerge from their shelters before ambient temperatures rise to 23°C in the morning (Milstead, 1961). As nightfall advances, tegus retreat into shelters where they become quiescent and sleep. If the weather turns inclement and cool, they will remain in their burrows throughout the day. During the winter months (the cold, dry season from May to September) the Brazilian tegu (Tupinambis merianae) in the southern reaches of its range undergoes a winter dormancy which involves a seasonal, temperature-independent, reduction in  6  metabolic rate (i.e. an active metabolic suppression) (Abe, 1995). The tegus retreat permanently into their burrows where they remain inactive and fast for 3-4 months (Abe, 1995; Andrade et al., 2004). As with most hibernators, preparation for hibernation begins well before environmental conditions become adverse. Milsom, et al., (2008) have shown that in juvenile T. merianae, the depressed metabolism observed during the dormant winter season can be produced at any time of the year if the animals are faced with prolonged periods of inactivity by administration of constant cold, darkness and forced aphagia. Abe (1983; 1993; 1995) has shown that in nature, by autumn tegus begin to fast, their metabolic rates are already depressed, and the lizards are ready to enter dormancy where a further minor temperature-dependent lowering of metabolism will occur. By the end of the autumn/beginning of the winter, the final step in the suite of events leading to dormancy appears to be the behavioral decision to retreat into the burrow, let body temperature equilibrate with the surroundings, and abandon the daily increases in body temperature associated with active thermoregulation. There also appears to be a progressive reduction in thermal sensitivity (Q 10) from summer to winter such that the metabolic rate of dormant lizards becomes relatively temperature independent (Abe, 1983; 1993; 1995; de Souza, et al., 2004). This has been proposed to have the selective advantage of maintaining extremely low metabolic rates even when burrow temperatures rise slightly. While these data appear to paint a clear picture, the vast majority of studies investigating the circannual rhythms of reptiles in general and tegus in particular present snap shots of data from specific seasons which may not tell the complete story. Both based on studies in hibernating endotherms, and anecdotal observations in tegus, several  7  questions arise. While animals appear to depress metabolism in the fall in anticipation of the hibernation season, this observation arises from studies in which animals were confined in the dark for several days. During this season tegus are still active in the day and still warm to usual temperatures. This raises the question of the extent to which metabolism falls under natural conditions and whether the metabolic depression only occurs at night. Is this a form of daily torpor? Also, during this period the lizards may remain in their burrows for several days. During these times, do they enter multi-day bouts of torpor? Are the progressive increases in the daily periods of inactivity progressive increases in depth and length of daily torpor, or are they periods of sleep through which the animals enter hibernation (Larkin and Heller, 1996)? Once tegus commit to the burrow for the season, do they immediately enter hibernation or does the degree of metabolic suppression increase as the hibernation bout proceeds? The latter has been reported to occur in Lacerta vivipara during the initial phase of hibernation (Patterson & Davies, 1978). Is the period of hibernation one prolonged bout or do these animals undergo periodic arousals during which they remain relatively inactive within their burrows? It is commonly thought that once reptiles become inactive, they have entered the hibernating state and remain in this state until spring when ambient temperatures are high enough again to allow activity. A recent study, however, has shown that hibernating Varanus rosenbergi spontaneously arouse fairly frequently during their hibernating season (Rismiller & McKelvey, 2000). Finally, if tegus can undergo periods of daily torpor, do they only do this during the period leading up to hibernation or do they do this at other times of the year as a function of energy balance? To answer these questions requires continuous monitoring of behaviour, body temperature and, ideally, metabolism throughout the year. In the present study this was  8  attempted by continuously recording behaviour, body temperature, heart rate and breathing rate as physiological surrogates for metabolism in a group of tegu lizards housed outdoors under pseudo-natural conditions.  9  2. QUIESCENT STATES IN THE BRAZILIAN TEGU: ANNUAL CYCLES IN BEHAVIOUR AND CARDIO-RESPIRATORY PHYSIOLOGY  2.1 INTRODUCTION Tegu lizards (genus Tupinambis) are large diurnal reptiles with a broad distribution throughout South America east of the Andes (Avila-Pires, 1995). The genus is comprised of two major clades (Colli, et al., 1998; Fitzgerald, et al., 1999). The teguixin Glade is comprised of four species (Tupinambis teguixin, T palustris, T. quadrilineatus and T. longilineus) whose distribution is more northerly and mostly restricted to the Amazon basin. The merianae Glade, comprised of three species (T merianae, T duseni and T. rufescens) is distributed primarily south of the Amazon river extending down into Argentina and Uruguay. Like all diurnal ectotherms, tegus are active during the day and predominantly regulate their body temperature behaviourally by heliothermy, using the sun as the primary external source of heat (Bartholomew and Tucker, 1963; Tattersall, et al., 2004). Periods of activity are highly correlated to ambient air temperature rather than other environmental variables such as substrate temperature, relative humidity or wind velocity (Milstead, 1961). Their preferred daytime body temperature (Tb) is usually greater than 33°C (Vitt & de Carvalho, 1995; Vitt & Zani, 1996) and they rarely emerge from their shelters before ambient temperatures rise to 23°C in the morning (Milstead, 1961). As nightfall advances, tegus retreat into shelters where they become quiescent and sleep. If the weather turns inclement and cool, they will remain in their burrows throughout the day.  10  During the winter months (the cold, dry season from May to September) the Brazilian tegu (Tupinambis merianae) in the southern reaches of its range undergoes a winter dormancy which involves a seasonal reduction in metabolic rate (i.e. an active metabolic suppression) that is independent of temperature(Abe, 1995). It is believed that tegus retreat into their burrows for the entirety of the season where they remain inactive and fast for 3-4 months (Abe, 1995; Andrade, et al., 2004). As with most hibernators, preparation for hibernation begins well before environmental conditions become adverse. Milsom, et al., (2008) have shown that in juvenile T. merianae, the depressed metabolism observed during the dormant winter season can be produced at any time of the year if the animals are faced with prolonged periods of inactivity by administration of constant cold, darkness and forced aphagia. Abe (1983; 1993; 1995) has shown that in nature, by autumn tegus stop eating, their metabolic rates are already depressed, and the lizards are ready to enter dormancy where a further minor temperature-dependent lowering of metabolism will occur. By the end of the autumn/beginning of the winter, the final step in the suite of events leading to dormancy appears to be the behavioral decision to retreat into the burrow, let body temperature equilibrate with the surroundings, and abandon the daily increases in body temperature associated with active thermoregulation. There also appears to be a progressive reduction in thermal sensitivity (Q10) from summer to winter, such that the metabolic rate of dormant lizards becomes relatively temperature independent (Abe, 1983; 1993; 1995; de Souza, et  al., 2004). This has been proposed to have the selective advantage of maintaining extremely low metabolic rates even when burrow temperatures rise slightly.  11  While these data appear to paint a clear picture, the vast majority of studies investigating the circannual rhythms of reptiles in general and tegus in particular present snap shots of data from specific seasons which may not tell the complete story. Both based on studies in hibernating endotherms, and anecdotal observations in tegus, however, several questions arise. While animals appear to depress metabolism in the fall in anticipation of the hibernation season, this observation arises from studies in which animals were confined in the dark for several days. During this season tegus are still active in the day and still warm to usual temperatures. This raises the question of the extent to which metabolism falls under natural conditions and whether the metabolic depression only occurs at night. Is this a form of daily torpor? Also, during this period the lizards may remain in their burrows for several days. During these times, do they enter multi-day bouts of torpor? Are the progressive increases in the daily periods of inactivity progressive increases in depth and length of daily torpor, or are they periods of sleep through which the animals enter hibernation? Once tegus commit to the burrow for the season, do they immediately enter hibernation or does the degree of metabolic suppression increases as the hibernation bout proceeds? The latter has been reported to occur in Lacerta vivipara (Patterson & Davies, 1978), although the metabolic suppression was not sustained throughout the entire season. This in turn raises the question: is the period of hibernation one prolonged bout or do these animals undergo periodic arousals during which they remain relatively inactive within their burrows? It is commonly thought that once reptiles become inactive, they have entered the hibernating state and remain in this state until spring when ambient temperatures are high enough again to allow activity. A recent study, however, has shown that hibernating Varanus rosenbergi spontaneously arouse fairly  12  frequently during their hibernating season (Rismiller & McKelvey, 2000). Finally, if tegus can undergo periods of daily torpor, do they only do this during the period leading up to hibernation or do they do this at other times of the year as a function of energy balance? To answer these questions requires continuous monitoring of behaviour, body temperature and, ideally, metabolism throughout the year. In the present study this was attempted by continuously recording behaviour, body temperature and heart rate and breathing rate as physiological surrogates for metabolism in a group of tegu lizards housed outdoors under pseudo-natural conditions.  13  2.2 MATERIALS AND METHODS Tegus for this study were acquired from Augusto S. Abe at the Jacarezario, UNESP Bela Vista Campus in Rio Claro, Sdo Paulo State, Brazil. The tegus are from a colony where all the individuals are captive bred and reared for scientific study. Hatchlings were raised indoors in large plastic containers and then transferred to outdoor pens following their first hibernation season when they were nearly a year old. Juveniles were reared in groups with communal burrows until they reach an adult size of —2 kg, after which they were separated into outdoor breeding pens comprised of groups consisting of one male with up to three females. They were reared and maintained on a diet consisting of whole chicks, mice, rats, whole ground chicken, bananas, papaya, acerola, pitanga and palm fruits. For the present study, adult tegus weighing at least 2 kg were selected to accommodate the telemeter implants. Animals of this size were needed not so much to meet the conservative field telemetry guidelines of 5% of the animals body mass (implant mass = 0.0454 kg) but to be able to accommodate the size of the implant without much displacement of the internal organs (body of the implant measured 5.7 X 2.8 X 0.92 cm). The study cohort consisted of 3 males and 4 females. Only 4 animals could be recorded from concurrently as the telemetry base station was only capable of receiving and decoding 4 signals simultaneously. Hence 2 females and 1 male were held in reserve as contingency. One of these was subsequently used (see below). 2.2.1 Surgery  All 7 tegus were initially anaesthetized by being placed in a sealed container for about thirty minutes along with a handful of cotton wadding soaked in Halothane. Once the animals were compliant, they were intubated, placed on a tidal respirator (20-30cc per  14  breath at 5-10 ventilations per minute). Halothane was then administered (5% in air) until a surgical plane of anesthesia was achieved as determined by lack of response to toe pinching. Before surgery, their integument was sterilized with 100% ethanol and then washed with Betadine solution. During the surgical procedure, the level of Halothane was reduced to —1% in air. An incision was made midventrally from just below the sternum, caudally about 10cm to just anterior to the post-hepatic septum. An incision (-3cm) was then made through the post-hepatic septum and the body of the T29F-7B implantable biopotential/temperature amplifier/encoder (Konigsberg Instruments, Inc.) was inserted through the opening to lay between the fat bodies and ventral to the digestive tract in the abdominal cavity. The tissue of the post-hepatic septum was then sutured tightly closed around the leads of the implant using 000 silk. The ECG leads were affixed to the medial pleuroperitoneal membrane along the body wall with PeriAcryl glue and mersiline mesh so that the negative (-) contact lead rested near the apex of the heart and the positive (+) contact lead was near the conus arteriosus. Biopotential leads were sutured into the intercostal muscles on the left side, about 1 cm apart vertically, in the area between the fourth and fifth ribs of the lateral body wall. The antenna and on/off switch were left to float relatively free in the thoracic cavity. The underlying muscle layers and integument were sutured closed independently, and artificial ventilation with air was continued until animals regained consciousness. Polysporin was applied to gauze secured over the surgical wound. 2.2.2 Post-surgical Care The tegus were treated post-surgery with Baytril (0.1m1/kg IM, every other day) and Anestesico L (lidocaine containing epinephrine, topically, twice daily). Gauze  15  bandaging was changed daily (or as needed) with polysporin applied for preventative measure. Animals were housed in indoor enclosures (1.5 m X 1.5 m) with clean wood shavings for bedding for at least a week to ensure full recovery before being placed into the outdoor study arenas. Food was offered every other day during this period. 2.2.3 Housing/Study Site Outdoor enclosures measuring 2.5 m X 3.5 m enclosed with 1 m high walls were constructed in the Jacarezario compound of the Bela Vista campus of UNESP, Rio Claro, Sfto Paulo, Brazil (22°23'S, 47°32'W, 626.5m altitude). The enclosures were designed to mimic a natural savannah habitat and were planted with local short blade grass and each enclosure housed a tree. Each enclosure was also provided with a rectangular burrow (60 cm wide X 80 cm long X 50 cm deep) constructed of brick and cement buried in the ground. Over top of the burrows were pyramidal lids (60 cm X 80 cm base, 1 m height) constructed of plywood painted white and internally insulated with 2cm thick Styrofoam insulation. These lids accommodated infrared cameras to monitor activity in the burrows. Four StowAway TidBit temperature data loggers programmed to take a reading every 15 minutes were placed around the enclosures, one in each burrow, one affixed on the north facing wall of one enclosure and one on the opposite south facing side of the wall, about 75 cm above the ground. The data logger on the north face recorded temperatures in direct sunlight (reflecting the basking potential of the tegus). The data logger on the south face recorded ambient temperatures in the shade in all seasons except mid-summer (late December-early January) when, due to the low latitude of the study location, the mid-day sun was directly overhead and the south facing data logger was also in direct sunlight. However, this also indicated that at this time there was no shade available within the  16  enclosures aside from the burrows. Data loggers were placed on a backing of 2 cm thick Styrofoam to insulate them from conductive heat transfer from underlying structures. Antennae were suspended 1 m above the enclosures via bamboo poles to maximize receptivity. The TidBit data loggers and telemetry implants were calibrated in water baths set at four temperature points (7.2°C, 23°C, 29°C, 39.7°C) and were compared against a precision mercury thermometer. Devices were left in each individual temperature water bath for 30 minutes to allow equilibration before being moved to the next bath. 2.2.4 Data Acquisition Environmental data (rainfall, atmospheric pressure, relative humidity) was collected from the local UNESP meteorological station. Daily mean atmospheric pressure and relative humidity were calculated by UNESP personnel from three daily readings taken at 09:00, 15:00 and 21:00. The tegus' physiological variables were measured via T29F-7B implantable biopotential/temperature amplifier/encoders (Konigsberg Instruments, Inc.) that were configured to receive and broadcast 3 real-time signals: electrocardiogram (ECG), a biopotential of movement/impedence (configured to indicate breathing frequency at rest when focused on the movement of the lateral thoracic wall), and body core temperature. Telemeter signals were received and decoded with a TR8-2-2/TD14-10 telemetry signal processor and demodulator (Konigsburg Instruments, Inc.) and the raw decoded signals were collected with a Dataq Instruments DI-720 data acquisition system. The information was recorded using WinDaq software set to record at 250 Hz per channel (3 channels per tegu, 12 channels in total) in one hour files. The WinDaq program was  17  configured to automatically close each one hour recording upon completion and automatically and instantly open a new file and continue recording sequentially ad  infinitum. The files were stored initially on hard drive and then backed-up onto CD-R disks. These files were later processed with MatLab software configured to do peak detections and calculate average instantaneous frequency (based on point-to-point timing) measurements of heart rate (HR), respiratory rate (fR) and average deep body temperature (Tb) for every 15 minutes. 2.2.5 Experimental Protocol As male tegus are prone to fighting with each other, the tegus were housed as pairs (one male and one female) and allowed to roam freely in their enclosures. They were fed to satiation on average every three days, subject to food availability and weather. Their diet consisted of whole mice/rats/chicks, chicken heads and necks, whole ground chicken or ground beef mixed with vegetables and fruit (arugula, collard greens, kale, dandelion, acerola, papaya, mango, jabuticaba, apple and/or pitanga, equal parts by volume of meat and plant matter) with added multivitamin supplement. During entrance into hibernation, the tegus consumed progressively less and so feeding frequency was reduced and then stopped for the duration of the hibernation season. Water was present at all times of the year. Animals were weighed approximately each month except during hibernation to reduce interference with the hibernating state. Recordings were made continuously from January to the first week of December in 2004. During this period one of the tegus escaped and one died. Both events occurred in the reproductive season. One tegu (tegu 2) died unexpectedly of undetermined causes on September 22nd and was not replaced. Tegu 4  18  escaped (looking for nesting sites) on October 10th and was replaced a couple of days later with a reserve tegu of the same sex. As a consequence there are data for 4 tegus from the start of the year until September 22nd and for 3 tegus from that point on. After October 10th the data set is for two of the original tegus and one reserve tegu that had been living under identical conditions.  2.2.6 Data Analysis Average values were calculated for each variable [instantaneous heart rate (HR), instantaneous breathing rate (fR), deep body temperature (Tb), burrow temperature (Tb urrow ), and the temperature in direct sunlight and shade] for each individual for each 15 minute time epoch for the entire year. These data were subsequently averaged over each epoch, day, week, and month to calculate the means for the entire group of tegus over each time domain. Statistical analysis was performed between averaged values by repeated measures one-way ANOVA followed by a Student-Newman-Kuels post-hoc test, unless normalcy tests failed, when a non-parametric repeated measures one-way ANOVA on ranks was used (SigmaStat 3.5). All values are presented as mean ± S.E.M. Differences were considered to be statistically significant at the level of P < 0.05.  19  2.3  RESULTS  2.3.1 Meteorological Data As a rule, the summer time (December to February) in Rio Claro is warm and wet while the winter (June to August) is cool and dry. Figure 2.1 shows the changes that occurred in relative humidity, precipitation, barometric pressure and ambient temperature (dry bulb in the shade) at the weather station at UNESP in Rio Claro (less than 1 km from where the tegus were housed) over the year of this study. Of note is that the lowest mean daily temperatures occurred from May to August and corresponded to the periods of highest barometric pressure. In this particular year, while rainfall began to decrease in April, August and September were the driest months and were the months with the lowest relative humidity. Overall, at this latitude, seasonal differences in all of these variables, except rainfall, were modest. 2.3.2 Annual Patterns of Behaviour Figure 2.2 (a-d) illustrates the mean heart rate, breathing rate and core body temperature data obtained for each of the tegus used in this study every 15 minutes over the entire study period. In many of the figures to follow, mean values are shown that were derived from the data illustrated here.  Fall - Winter decreases in activity: One of the most significant observations to be made from Figure 2.2 is the individual variation present in periods of reduced activity as reflected in sustained low daily body temperature, heart rate and breathing rate. These reflect days spent in the burrow during which animals did not emerge. These could occur at any time of the year (except September when all animals were active every day) (Fig. 2.3) but were  20  ▪ 100  100 80  80  2 =Its" 60  - 60  CI) 't,5 .> (,)^40  - 40  E  a  o  oc^20  20  0  0 955 z (I)^950 cn 2 22 0_ (15 945 -2  t c D E 2^940 as  i I  s)  1  DI Y  f  I  935 30 25 -  0  1 05 Jan^Mar May^Jul^Sep Nov Date  Figure 2.1 Environmental data (precipitation, barometric pressure, relative humidity and ambient temperature (dry bulb)) collected from the meteorological station on the Bela Vista campus of UNESP, Rio Claro, SP Brazil. Reported daily mean barometric pressure, relative humidity and ambient temperature were calculated from the mean of three daily readings taken at 09:00, 15:00 and 21:00.  21  ^ ^  ^_  CT  O H^  _  _  o  _ —  -  •^ ▪•  - )  U  ,--. ._c ct .4 E r-. , c(.4,71  C^ M^ I lif)  a) H  "0 = LID  li =^M a)  0 (.4 > ^-0 fa, cu .....‘  0_^  -.= a ) cu — a) c = ;_. U)^-'-71 1.) "Ft  Ob „,....  L..„ ,. ct u.  1.. ,^,.4 ,a, ‘,.._, ,, „.., u 4, c, •  .  U +_-2 7E,  0 ^c„.) s—, -tX)^ct cl..) cf) cn -10  r..n c 5. ,..0  c a) a) C I-. i•-■ _^a) sa■ '-'7s = a) ,..c al s. v)  —m  4—^  !  . ^—,  .  a ct c)  [..:,A4*:-.....^ — ozS ^--- ..c c-.1 ^ 2  E  ,— ri.....1 -7-1 = = ct  cr, "CD  el-) b.0 cr)  C.) r. ct an <1.) .- ^  2  — ct  o H  ct = = ccs^,_, . ^e4)  ^L ^—  :  .:...--:1.ftti.-...i..  1.) U ...c a)^• `” N c a 6 = 0„ c t)i) 9-' C ct^Z-S1 C:- czt bl.) %I=  b.0  U cl-) = c) E_, ..c a) c) cA ct c .-:^ci) 0  .,--,  cr5^'5 ,73 c "CS 111111^—D^7:3^ (1.) cn  C) CD CD 0 0 0 C) CO (r) 71" C 0 CO CO C\J CD in C) 1.1) CD 110 CD 1.1) C) 0‘.1 C) OD CO^C\.1^ 7f CO^CO C\.1 N 7— T 1—  (wwisieaq)^(wwisinealq)^(00) aieu 6willeale ammiadwai area peaH snoaueweisw snoauewelsw Apog dean  22  0)  a)  O  '  111.11•••••-•  ^..•••  •-• •  III^I^I^I^I^I^I^I^I^I^I^1^I^I  (ulLuisqlealq)^(on) area 6u!ineais amiwadwai snoeueluelsui^Apos deal]  0 0 0 0 0 0 0 0 CO CO N 0 CO CO tco 1.0 0 10 0 o O to 0 CV 0 CO CO 71 - N NT TT 1- 1-- ^d t CO CY) N N T 1-  (u!Lu/sieeq) .13^ajeld peaH snoeuejueisui  23  CO D 0)  a) H  1-- .7--  -  -  -  -  -  N 0  (u!wist.nealq) eiebi 6umeai8 snoeuewelsui  0 0 0 0 0 0 O(0 '71" NC CO CO 71 1- 1 1 1 N 0 CO CD 71 N  (u!wisleaq) c.)^Ojebi JeOH snoeuelueisui  -  ,  C Ct --)  2  > - o Z  1- -f-  o LU c) N N  Lc)  IIIII1 .  10 c) up c) 7t co co .1  (00)  annwedwai Apo dean 24  LO  0)  N  F-  a)  F-  -  11111 -  •  -  :ffi:t2tlittf;:-''..  ^•  00000000 CO '71" CV 0 CO CO CV 0 LO 0 LO 0 1.0 0 LO 0 T T T 71 C\1 0 CO CD 71 C 71 •7t CO CO C\I CV 1- 11- 1-- T--  ^ ^ (u!w/sieaq) (wwisinewq) (30) aieti 6u!inewg amiwadwai ^ ^snoeuelueisui snoeueweisui Apo daaa  o  25  rare from October to December, and associated with periods of inclement weather from January to March. In April and May they increased in frequency, were no longer associated strictly with the weather patterns and gave rise to multi-day periods of inactivity and time spent in the burrow. During May the lizards began to plug up the entrance to the burrows with vegetation indicating a behavioural commitment to remain inactive in their burrows, marking the start of the hibernation period that continued through June and July. Note, however, that while there was tremendous individual variation, all tegus still emerged periodically throughout the hibernation season. In conjunction with the trends seen in days when animals did not emerge from their burrows were trends in the timing of emergence from the burrows on days when they were active as well as in the times they retreated to their burrows in the afternoon. During the late fall (March, Fig. 2.4) animals emerged from their burrows progressively later in the morning, stayed out for shorter periods of time, and re-entered the burrows earlier. Tegus that emerged from the burrows during the hibernation season emerged even later and remained out for very short periods of time (June, July in Fig. 2.4).  Spring - Summer increases in activity: Starting in late July, the tegus began to emerge from their burrows every day indicating the end of the hibernation season. They showed a linear increase in their minimum daily body temperature reflecting the changes in ambient temperature (Figs. 2.1 and 2.2). August was the driest month of the year and all tegus emerged from their burrows most days. Days of inactivity where animals remained in their burrows were rare throughout the reproductive season (September to December) (Fig. 2.3). During this period, tegus emerged from their burrows early in the morning, stayed out longer, and retreated to the burrows later in the day (Fig. 2.4).  26  120 0 100 0)  -  T  a) 80  T  E W 60 U)  ca 40 20 -  0  0 S 2  8  i  0  m= o6 8 E c 4,1 -  z:N) >, 4 a) c w  E  a. 2 C 0 O L.Z LL  A  A  T  A  0 25  0 CO^20 ,w„,„ • al C "0 15 CD ■-•••  E c5 G) CD  -  .  0  z  10  5 0  r-1^  sras,_V;s0 :60,,,,,scn  1-17  e 0?)-'\ ye  I^I^I^III  ■).\, 4 .^ ec kcic0.„(\ ec see043' X) .  Figure 2.3: Top: Percentage of days in each month that tegus (n=4) emerged from their burrows. Middle: The number of bouts in each month that tegus did not emerge from their burrows. Histograms marked with A are significantly different from the month of April. Bottom: Duration of non-emergence bouts measured in days for each month.  27  Sunset 17:00:00 -  ^Retreat * 13:00:00 -  ^  *  ^- -^I  09:00:00 - ir  Emergence -  ---------'^Sunrise -------------------___^ 05:00:00 ^ ,\\),•k•e\O e*c^ek^e.c^,k ^ ^^ (0 ON\ \\ z' \;,C^ NO° 0 No^cc O. \, 0 ^643 q -^c\-^ c c\ Ge \ 0 \e ^‘>-\' C ) N Jg'•• e \..v) ye f  .  Month Figure 2.4: Times of sunrise and sunset at Rio Claro, SP Brazil for the year 2004. Time of emergence from the burrow (red symbols) and retreat into the burrow (blue symbols) are also shown for 7 months in the late active season (January), during entrance into hibernation (March), for rare incidents of emergence in hibernation (June, July), during arousal from hibernation (August) and during the early active season (September and December). * denotes significant difference from respective September values (One-way repeated measures ANOVA; emergence — P<0.001; retreat — P=0.002).  28  2.3.3. Annual Patterns of Physiological Change Figure 2.5 illustrates the maximum (day time) and minimum (night time) breathing rate, heart rate and body temperature for one representative tegu (tegu 4) throughout the recording period. Several things are of note. The first is the uniformity of maximum day time voluntary (basking) body temperature (-37°C) throughout the active season (January to April and September to December) on the days when this animal emerged from the burrow. All animals showed similar patterns (Fig. 2.2a-d). During entrance into hibernation (April to May), the maximum daily voluntary temperature of this tegu remained relatively constant although in other tegus it declined progressively (Fig. 2.2a-c). Also on days when tegus emerged during hibernation, the maximum daily voluntary temperature was generally lower than that seen in the active season (Figs. 2.2 and 2.5). Maximum day time ambient temperatures were such that tegus should easily achieve body temperatures of 35-37°C throughout the year if they chose. Night time minimum body temperature (usually the body temperature at the end of the night) was also relatively uniform throughout the active season. During entrance into hibernation, when animals remained in their burrows, day time maximum body temperature fell to match daily minimum body temperature (Fig. 2.5). The mean burrow temperature closely followed the mean daily ambient temperature (Fig. 2.6) and was relatively stable at —22-25 °C during the active season from January to March and September through December. In roughly mid-April mean burrow temperatures began to decline progressively reaching values of —16-17 °C during the hibernation period in June and July (Fig. 2.6). However, the burrows were sufficiently buffered against  29  140 "8^120 1  100 N  80 ct.  60 40  ca  20 0 45 40 35 o. (^30 8' 25 -  co^20 Q.  a)  15  March^May^July^Sept^Nov  Figure 2.5: Daily minimum (blue), median (black) and maximum (red) values of instantaneous breathing rate (breaths/min), instantaneous heart rate (beats/min), and deep body temperature (°C) of tegu 4 over the entire recording period. Dotted lines on the bottom graph demonstrate the relatively constant daily T bmax and T bmin witnessed throughout the entire active season.  30  S\\)':;.C\N(61 6\\`Z•SC" ^  .c kW\ Jae 0 \O Noe  cc\N ‘).,\^e^  c  Clc .\o43 eoe De 8  850 -  2  - 30 O. CO  E 800 -  cu CC - 20 4.„  0 "C 750 a) Q. 0 *6 700 -c  -  a)  6  715  2  5^(1) ca CC 4 0) C  0  2 600^I^I^I^I^,^„  I  co  1 2  c,\\^ „s6k ,06kcc\v 0^ e^4\. 3\.^,oe (6S ‘)..9 kV- ) c\ ecc. ^ \).^c ,,\N ^‘ e ^c^ kl`S\> PS\ •o^ e  e'C^1/4-) ..vz)N1^eo O  Figure 2.6: Mean monthly values for photoperiod, ambient and burrow temperatures, and core body temperature, heart rate and breathing rate for all tegus over the entire recording period.  31  ambient temperature extremes that the minimum daily burrow temperatures were always above and more stable than the minimum daily ambient temperatures (Fig. 2.6). Given the relatively short periods of time the tegus spent out basking and active (maximum of seven hours in the reproductive season) the mean body temperature for each animal averaged over each 24 hour period was more similar to the minimum than the maximum temperature (Fig. 2.5).The difference between average body temperature and burrow temperature (Fig. 2.6) reflected two things. The first was the amount of time the animals spent basking during the day (Fig. 2.4) and the second was a combination of heat retention and thermogenesis as reflected by the ability of the lizards to maintain a body temperature (Tb) to burrow temperature (Tburrow) (Tb-Tburrow) differential (Fig. 2.6). This differential was largest (4-7 °C) in the active season and disappeared in the hibernation season. It was surprisingly large in April through June during entrance and early hibernation but this probably reflects thermal inertia as the tegus cooled progressively while the ambient and burrow temperatures fluctuated widely. Day time maximum heart rate was also relatively constant (-100-120 bpm) throughout the active season (Fig. 2.5) but night time heart rate and daytime and night time breathing rate were not. These trends are explored for the mean data of the group of lizards in Figure 2.7. Note that while the mean deep body temperature was relatively constant through the active period (September to December and January to March) (Fig. 2.7), mean heart rate and breathing rate peaked in October/November during the reproductive season and then began to decline through December as well as from January through until May (Fig. 2.7). As a consequence, from November until March, heart rate and breathing rate were progressively falling (by 60 to 75%) despite the fact mean body temperature was  32  30 25 0  burrow  -  Ii  20  (2 15 cu E 10 -  Ea)  ambient  50 30  25  0  w  V 20 a)  a_ E  a 15 )  10 10  tegu-burrow difference  \1  1 11  Jan^May^Sep^Jan  Figure 2.7: Top: Daily average minimum temperatures of the burrow (green) relative to ambient air (blue). Middle: Daily average minimum temperatures of the tegus (black) relative to the burrows (green). Bottom: The daily average difference at the end of the night between tegu and burrow temperature. 33  relatively constant. As noted above, while these are mean daily values, they largely reflect the values for quiescent tegus in the burrows. Once the animals entered hibernation, mean heart rate and breathing rate remained low and relatively constant (May to July). From August to October, along with mean body temperature, mean heart rate and breathing rate rose rapidly as the animals entered the reproductive season (Fig. 2.7). 2.3.4. Periodic Breathing and Cardiac Asystole; Evidence of Deep Hibernation? While heart rate, breathing rate, and presumably metabolic rate, fell progressively at night from October/November through into hibernation in May, the manner in which heart rate and breathing rate fell was not regular. Starting during entrance into hibernation, tegus began to exhibit arrhythmias consisting of skipped heart beats as well as a breathing pattern with increasing periods of apnea (Fig. 2.8). These were not just confined to nighttime but also occurred during the day. Once animals were in hibernation, skipped beats were infrequent but present in all tegus and occasionally gave rise to periods of several minutes with no detectable heart beat (Figs, 2.9, 2.10). In the most extreme cases animals went with no detectable heart beat for three to four minutes. Interestingly, during early entrance, the skipped beats could be seen on closer observation to consist of atrial depolarization, with a noticeable P wave in the ECG, but no QRST complex (Fig. 2.9), suggestive of the presence of a secondary AV block. As the season progressed, while skipped beats remained common, evidence of secondary AV block disappeared (Fig. 2.9). Breathing also became very irregular and frequently episodic in hibernation. During arousal from hibernation, heart rate and breathing once again were elevated during daytime active periods but continued to fall to very low levels at night (Figs. 2.2, 2.5, 2.11). As the animals entered  34  into the reproductive, or early active season, body temperature, heart rate and breathing rate were once again sustained at higher levels during the night (Figs. 2.2, 2.5, 2.11). 2.3.5. Zeitgebers  Comparing the data in figures 2.1 and 2.7 it can be seen that although hibernation in tegu lizards is generally associated with the cool dry winter, these animals begin to show signs of metabolic depression at night (lowered heart rate and breathing rate) as soon as the reproductive season ends and they are beginning to emerge from hibernation during the driest period of the year. It can be seen from figure 2.12 that the increases in heart rate and breathing on arousal (dotted line a) show a better correlation with photoperiod than with changes in ambient temperature or levels of precipitation. The peak in heart rate and breathing does not correlate well with any of the environmental variables (dotted line b), although there is a secondary fall in heart rate and breathing shortly after ambient temperature and photoperiod begin to decline at the peak of the wet season (dotted line c, Fig. 2.12).  35  A C5 >- C) 0  Late Activity Season  C  16,  .  0) cc  0C 2 (i)  w cc  Entrance Into Hibernation  B  >a 00010110000000^  4  '400milioot 00-1bvs14001—  C)  z  0. U) a)  cc  1 min Figure 2.8: Representative traces of the electrocardiogram and respiratory signal from one tegu during the photophase (around 13:00-15:00hrs) and during the scotophase (around 02:00 to 04:00hrs) in the late activity season (A) and during entrance into hibernation (B).  36  April 24, 2004 -04:00 C.)  t  C 0  June 18, 2004 -04:00 Cs  ^ 1^  C O  H  co a. N CC  July 13, 2004 -04:00 0 u.1  O  a.  iteit,1/40.0**10.41404010N4hoofeerh*Olyk  a) CC  C.)  5 sec  Figure 2.9: Representative traces of the electrocardiogram and respiratory signal from one tegu during the scotophase (around 04:00hrs) at different times in the hibernation season. The bottom trace is an expanded portion of the ECG trace from the April recording showing a normal heart beat as well as an example of a type 2 AV block.  37  June 30, 2004 -04:00 w 0  CO  a) Cc  July 8, 2004 -04:00 0 0  w  44  0  "WI -  a_U)  ,  a)  July 13, 2004 -04:00 0 0  w  0  -  a a)  cc  Figure 2.10: Representative traces of the electrocardiogram and respiratory signal from one tegu during the scotophase (around 04:00hrs) at different times in a single bout of hibernation.  38  Arousal from Hibernation  A  Ilil^ill! II I^1,1^iil^II 1,,  \tsio4o 1**■ F-  z Early Activity Season  B 0  w  a 0. a) cc  ht++\\-11  H+*)  ,,  1""'  0  C  Z "a a)co cc  1 min Figure 2.11: Representative traces of the electrocardiogram and respiratory signal from one tegu during the photophase (around 13:00-15:00hrs) and during the scotophase (around 02:00 to 04:00hrs) during arousal from hibernation (A) and during the early active season (B)  39  Temperature Photoperiod (min)  a^b^c  Precipitation (mm)  28 -  850  400  26 -  800  300  22 -  750  200  20 -  700  100  24 -  181614 -  650 600  40 35 30 25 20 15 10 5 0  Heart Rate  Breathing Frequency  OC) Figure 2.12: Average monthly values of ambient air temperature (°C), photoperiod (min), precipitation (mm), and mean heart rate (beats/min) and mean breathing frequency (breaths/min) for all tegus. Dotted line a indicates where heart rate and breathing rate start increasing after hibernation. Dotted line b indicates where heart rate and breathing rate peak after breeding season. Dotted line c indicates the peak in precipitation and how it correlates with a secondary peak in heart rate and breathing rate.  40  2.4 DISCUSSION The yearly cycle of a tegu can be divided into five main seasons based upon the physiological-behaviour states exhibited by the lizards: early activity, late activity, entrance (into hibernation), hibernation, and arousal (from hibernation). Mid-September through to November is a period of high activity associated with courtship and breeding (here termed the early activity season). December-February (here termed the late activity season) is a post-reproductive period of reduced activity. Throughout the active season (early and late), the lizards are foraging and replenish energy stores. These lizards enter hibernation with significant fat reserves. It has been shown that other teiids draw upon these stores during dormancy (Bostic, 1966; Brackin, 1979) but do not significantly deplete these fat reserves as their energy requirements during hibernation are dramatically reduced (see Derickson, 1974; Bartlett, 1976; Jameson and Allison, 1976 for examples in other lizards). Most of these reserves, however, are consumed after arousal during the reproductive season (Lopes and Abe, 1999; Herrera and Robinson, 2000). Active foraging throughout the active season replenishes these stores in preparation for the coming year. During March to mid April the lizards slowly enter into hibernation. This season is marked by a drastic decrease in activity and complete aphagia (Lopes and Abe, 1999). Hibernation occurs from mid April through to the end of July during which time the tegus spend almost all their time inactive in their burrows. In August to mid-September they then arouse. During this season the tegus progressively emerge from the burrows with increasing frequency and show a linear increase in their minimum daily body temperature.  41  2.4.1. Anticipatory Decreases in Metabolic Rate; Programmed Nocturnal Torpor? Previous studies have shown that tegus depress metabolism in the fall in anticipation of the hibernation season. These observations, however, arise from studies in which animals were confined in the dark for several days, usually at constant temperature (Abe, 1983; 1993; 1995; de Souza, et al., 2004; Andrade and Abe, 1999; Milsom, et al., 2008). By contrast, in nature during this season tegus are still active in the day, still warm to usual active temperatures and are still exposed to progressive changes in photoperiod and ambient temperature (Kohler and Langerwerf, 2000). This raises the question of the extent to which metabolism falls during this season under natural conditions. In the present study we found that throughout the active season, from September to February, tegus regulated their maximum daily Tb around a fairly consistent set point; Tbmax ranged from 33 to 37°C (interestingly similar to mammalian T b ). The only exception was on days with inclement weather when ambient temperatures did not permit behavioural thermoregulation to this extent. Daytime maximum levels of heart rate and breathing showed similar trends. Likewise, during this season unless weather did not allow it, tegus emerged to bask in the mornings and retreated into their burrows to sleep for the night at relatively consistent times. By contrast, night time values of HR and fR started declining as early as December and decreased progressively as time passed. Night time Tb through December to March, on the other hand, was fairly constant (Tb min hovered around 25°C). If HR is correlated to metabolic rate under steady state conditions, as suggested by several studies (Butler, et al., 2000, 2002; Clark, et al., 2004), falls in nighttime HR at constant Tb would suggest that there was a nightly torpor that increased in magnitude over this period of time.  42  As the year progressed, during the late active season and during entrance into hibernation (March and April), tegus started to emerge from their burrows later in the morning and retreat into their burrows earlier in the afternoon. In most tegus, daytime maximum Tb also began to drop slightly. Daytime maximum levels of heart rate and breathing again showed similar trends. These data suggest that a behavioural metabolic suppression was also occurring during the day late in the active season and during the entrance phase, but the extent of this was small.  2.4.2. Daily Torpor / Multi-Day Bouts of Torpor / Hibernation; A Natural Progression? At the onset of the active season, day time quiescence was rare and associated with inclement weather. If rainfall only occurred for half a day, tegus still usually emerged once the rain stopped. When full days were spent in the burrow due to unfavourable conditions, Tb, fR, HR, and presumably metabolic rate remained low. Tb, however, remained elevated relative to burrow temperature suggesting that this was simple quiescence. There were no signs of nightly torpor until November. As indicated above, from November through April, tegus entered a nightly torpor. This progressed as the hibernation season approached and may also have been expressed, to some small extent, during the day. Throughout the late active season and the period leading up to hibernation the tegus began to spend more time in the burrow, often for more than one day, even when weather was favourable, with Tb falling to equal the temperature of the burrow. This became particularly pronounced in April when, although rainfall was infrequent and light, the tegus spent roughly half the month in their burrows. The hallmark of final entrance into hibernation was when the burrow entrances were routinely blocked with detritus and litter, indicating a behavioural commitment to hibernate.  43  These data suggest there is a continuum from sleep to nightly torpor to multi-day bouts of torpor to hibernation. This is consistent with a recent report that tegu lizards can reduce their metabolism to the low rates seen in hibernation at all times of the year when given sufficient time in the cold and dark (Milsom, et al., 2008). It is also consistent with the views of some (Wilz and Heldmaier, 2000; Heldmaier and Elvert, 2004) but not all (Geiser and Ruf, 1995; Geiser, 2004) studies of mammalian hibernation. 2.4.3. Do Tegus Arouse Periodically? One of the hallmarks of mammalian hibernation is the phenomenon of periodic arousal (Willis, 1982). While there is still much debate over the causes and benefits of this behaviour (Barnes, et al., 1993; Wang, 1993; Carey, et al., 2003), it is common to all mammalian hibernators except the bears. The current hypothesis is that transcription and translation of genetic material are inhibited by low temperatures and that animals must arouse periodically to undertake essential maintenance activities (Van Breukelen and Martin, 2002; Carey, et al., 2003). The occurrence of periodic arousals is normally very rhythmic, however, with the lengths of the hibernation bouts increasing in the early season and then decreasing again in the spring (Twente and Twente, 1967). At present there are no indications of what triggers these arousals. Throughout most of the hibernation season mammals do not leave their burrows during these bouts of periodic arousal but remain underground (Willis, 1982). While species of reptiles that hibernate in temperate zones are not likely to emerge from their hibernacula in mid-winter when environmental conditions are extreme, they too may still arouse from hibernation and remain within the hibernacula. To date, however, there is no documentation that this occurs. Species of reptiles that hibernate in subtropical  44  regions should be less constrained to remain in their burrows during periods of arousal, and in a recent study it has been shown that hibernating Varanus rosenbergi spontaneously arouse and warmed to active temperatures fairly frequently during their hibernating season (Rismiller & McKelvey, 2000). In the present study, Tupinambis merianae also exhibited periodic bouts of arousal accompanied by short bouts of emergence. Amongst the four individuals in this study there was a wide range of variability in the occurrence of this behaviour, both in the number of times an individual aroused over the hibernation season and in the phase of the hibernation season (early versus late) during which these events occurred (Fig. 2.2). Arousals appeared to occur randomly, with no distinct pattern in any animal, suggesting that they were not the consequence of an under-riding consistent biological rhythm. There was also no synchrony to their occurrence in tegus inhabiting the same burrow, suggesting that they were not tightly correlated to local factors such as temperature change, noise or disturbance. At present it is not clear what the underlying cause of the arousals was. There is also some evidence to indicate that these tegus may have aroused on occasion without leaving the burrow. One such indication is the occurrence of peaks in HR„, a), during long periods with no emergence (Figs. 2.2 and 2.5). Again, if HR under steady state conditions reflect metabolic rate, then these daily increases in HR may well reflect short intermittent periods of arousal without emergence. Consistent with this is the observation that when other artificial burrows at the facility were opened during the hibernation season, some animals would be found to be fully alert while others would be comatose and unresponsive.  45  It is also not clear to what extent these arousals were the consequence of the experimental design. The artificial burrows were designed for ease of access and to allow infra-red recording of activity within the burrow. They were spacious and left the animals relatively exposed, albeit under quantities of litter and detritus. Natural burrows tend to be very constrictive and possibly deeper in the substrate where daily fluctuations in temperature would be absent. A constant temperature and tactile stimulation may promote deeper hibernation and eliminate periods of arousal and further studies along these lines are needed to address this question. 2.4.4. Is the Degree of Metabolic Suppression Constant Throughout Hibernation? In an early study of L. vivipara, Patterson & Davies (1978) showed that in the early stages of hibernation metabolism progressively declined even when temperatures remained constant. They also reported that metabolism measured towards the end of the hibernation season, at the same temperatures, was similar to that measured at the very beginning of the hibernation season, indicating that the degree of metabolic suppression was decreasing as the period of arousal approached. Similar cycles in hibernation depth are well documented in mammals (Twente and Twente, 1967). In the case of T. merianae in the present study, the progressive increase in metabolic suppression appeared to occur during nightly and multi-day bouts of torpor before the animals entered hibernation, and, as indicated by the low but consistent levels of HR and fR from May through July, when the tegus were in a hibernating state in the burrow, their metabolism was relatively uniform throughout this period. The increasing incidence of arousals associated with slowly increasing night time heart rate and breathing in August is also suggestive that the degree of metabolic suppression was decreasing as the period of arousal progressed.  46  There are also indications that hibernation was not a totally uniform state during the hibernation season itself. While heart rate, breathing rate, and presumably metabolic rate, fell progressively at night from October/November through into hibernation in May, part of the slowing of heart rate was due to arrhythmias consisting of skipped heart beats, and part of the slowing of breathing was due to an increasing incidence of long apneas. Once animals were in hibernation, skipped beats were less frequent but common in all tegus and occasionally gave rise to periods of several minutes with no detectable heart beat. As noted above, amongst the four individuals in this study there was a wide range of variability in both the number of times an individual aroused over the hibernation season and in the phase of the hibernation season (early versus late) during which these events occurred. For the most part, however, the longest periods of hibernation appeared to occur early in the hibernation season. Interestingly, early in the hibernation season the skipped beats often included incidents of atrial depolarization, with a noticeable P wave in the ECG, but no QRST complex, suggestive of a secondary AV block. As the season progressed, while skipped beats remained common but less frequent, evidence of secondary AV block disappeared. Many mammals also exhibit falls in heart rate before there is any noticeable fall in body temperature, and the progressive decrease in heart rate is the result of skipped beats and regular asystoles as well as a slowed heart beat (Lyman, 1982). These events give way to a uniform heart beat in deep hibernation at low temperatures where both parasympathetic and sympathetic tone appear absent (Milsom, et al., 1999). These changes in the regularity of heart beat in hibernators occur over several days of each hibernation bout, at constant Tb and metabolic rate, and have given rise to the concept of "deep hibernation" and the view of hibernation as a non-static state in which physiological  47  changes continue to take place in response to internal and external stimuli despite the fact standard measurements of Tb, heart rate and oxygen consumption have reached a minimum level (Lyman, 1982). In the case of the tegus in the present study, as hibernation progressed the asystoles became less frequent and the heart beat became regular between asystolic events. Breathing was very hard to quantify in hibernation as patterns were inconsistent both within and between individuals. Tegus appeared to frequently change from continuous to episodic or sporadic breathing, but the quality of the respiratory signal in hibernation was poor, making it difficult to draw any firm conclusions. While we were not able to analyze breathing traces closely in the present study, episodic breathing has been documented in tegu lizards hibernating at 17°C, but not at 25°C (Andrade and Abe, 1999). Episodic breathing is also very common in some species of hibernating mammals (Milsom, 1991). Taken together, these data and observations are suggestive that the hibernation state in tegus is also not a static state despite the relatively low but consistent levels of HR and fR from May through July. 2.4.5. Zeitgebers  To initiate physiological changes in preparation for seasonal environmental challenges, organisms rely on both internal rhythms and external environmental cues (Licht, 1972; Cloudsley-Thompson, 1999). Although hibernation in tegu lizards is generally associated with the cool dry winter, in the present study rainfall did not begin to decrease until April, ambient temperature until May and rainfall and humidity until August. Clearly if metabolic rate is correlated to HR and Tb (which it must be), it fell well in advance of any major changes in the environment; as soon as the reproductive season  48  ended. With mammalian hibernators, it would appear that the physiological changes associated with hibernation and emergence have evolved to occur in anticipation of environmental change rather than as a consequence of environmental change (Lyman, 1982). It can be seen from figure 12 that the peak in heart rate and breathing did not correlate well with any of the environmental variables (Fig. 2.12, dotted line b). There was a secondary fall in heart rate and breathing shortly after ambient temperature and photoperiod began to decline at the peak of the wet season, however, (Fig. 2.12, dotted line c) and the increases in heart rate and breathing on arousal (Fig. 2.12, dotted line a) show a better correlation with photoperiod than with changes in ambient temperature or levels of precipitation. Consistent with this, L.vivipara show a distinct preference for higher Tb during days of prolonged photoperiod and reduced Tb during days of shorter photoperiod, irrespective of actual environmental season (Rismiller and Heldmaier, 1982). 2.4.6. Active / Reproductive Season and Body Temperature Regulation Shortly after emerging from hibernation, the biological priority of the tegus was to reproduce. Activity from September through to November was elevated as tegus not only sought food to replenish stores depleted by hibernation, but also engaged in courtship, combat, mating and subsequently, egg laying. Not surprisingly, concomitant with this elevated activity was an elevated metabolism as evidenced by high day time and night time HR and fR. This suite of changes is not uncommon in reptiles (Rismiller & Heldmaier, 1991) and can be accompanied by an elevation in preferred Tb and is attributed to "mating unrest" (Weber, 1957; Rismiller and Heldmaier, 1982). In the tegus in the present study we also saw an increase in preferred day time Tb in August and most notably, an increase in nighttime Tb that exceeded the increse in nighttime burrow temperature. This gave rise to a  49  nighttime difference between Tb and Tburrow that was maintained throughout this period. At its peak, the Tb- Tburrow difference was as much as 6-8°C. The underlying cause(s) of this  Tb- Tburrow  differential is unclear. It can not have  been due to activity as it occurs during the nighttime quiescent period. Certainly, basic thermal inertia due to body size should contribute to a modest extent. This could be accentuated by changes in vascular tone and peripheral blood flow acting to decrease thermal conductance (see next Chapter). Thermal conductance is inversely proportional to body mass in reptiles, and the Scholander-Irving model predicts that with a lower thermal conductance, the difference between deep body temperature in an ectotherm and ambient temperature will dissipate more slowly (McNab & Auffenberg, 1976). This could also be enhanced by behavioural means, including the use of bedding/nesting material in the burrows as well as social huddling. Finally, it is also possible that this also reflects increased thermogenesis during this season. Whatever the combination of factors involved, the data show that a large  Tb Tburrow differential -  was maintained over several days if the  weather turned inclement, and the animals remained in their burrows with no indication that it could not be sustained indefinitely during this season.  50  3. SEASONAL CHANGES IN DAILY BEHAVIOURAL AND PHYSIOLOGICAL RHTYHMS IN THE BRAZILIAN TEGU  3.1 INTRODUCTION Chapter 2 described the seasonal changes that occur in quiescent behaviour in tegu lizards throughout the year and the accompanying changes that occur in mean heart rate, breathing rate and body temperature. Of note, we observed that mean HR progressively fell at night throughout the post-reproductive period (November - April) until animals entered hibernation in the winter (May-July). This occurred despite the fact these animals maintained a relatively constant Tb at night over much of this period. By contrast, day time HR continued to rise to similar values from November through February, in association with similar day time body temperatures. The primary regulated variable in the cardiovascular system is blood pressure. Each tissue regulates blood flow locally to satisfy ever changing needs. This is done by local changes in vaso-motor tone that in turn lead to changes in peripheral resistance. As a consequence of these changes, if blood pressure is to be maintained constant, then cardiac output (through changes in stroke volume or heart rate) must reflexly change such that total cardiac output matches the flow demands of all the peripheral beds. Most changes in local vaso-motor tone are associated with local changes in metabolism, with associated demands for delivery of substrates and removal of wastes, and most are accommodated by changes in heart rate (rather than stroke volume). As a consequence, it is possible under many circumstances to equate changes in heart rate with changes in total metabolic rate (Butler, et al., 2000, 2002; Clark, et al., 2004).  51  Changes in vaso-motor tone in some tissues, however, can take place for other reasons. There has been renewed interest of late in those changes associated with thermoregulation in reptiles, and their consequences for the regulation of heart rate (see Seebacher, 2000; Seebacher and Franklin, 2005, for review). Many reptiles have been reported to regulate peripheral blood flow, and hence heart rate, such that they can control their rates of heating and cooling, allowing them to extend the period of the day during which they remain within their preferred temperature range (Seebacher and Franklin, 2005). In squamate reptiles, this control is in small part cholinergic and f3-adrenergic (Seebacher and Franklin, 2001), but most of the heart rate response appears to be a function of prostaglandins (Seebacher and Franklin, 2003). The interesting conundrum that arises here is that changes in body temperature will give rise to changes in heart rate due to both direct thermal (Q 10 ) effects as well as to indirect effects on metabolic rate and substrate demands by the tissues. Changes in heart rate, on the other hand, during basking in the morning and cooling in the evening, can give rise to changes in body temperature due to changes in blood flow distribution for thermoregulation. Thus, changes in both heart rate and body temperature can be cause or effect. Adding to this complexity, changes in metabolism at constant temperature will also give rise to changes in heart rate. As a result, the seasonal trends observed in day/night differences in HR described in Chapter 2 are not straight forward to interpret. While it is tempting to hypothesize that peak day and night heart rates reflect steady state metabolism while transient changes during the morning and evening reflect thermoregulatory behaviour, this remains to be shown. In the present chapter we wished to analyze circadian changes in HR and breathing across the various seasons to provide further insight into  52  seasonal changes in metabolism (night time sleep and torpor and seasonal hibernation) and thermoregulation (day time emergence and basking) and to begin to address this issue.  53  3.2 MATERIALS AND METHODS The data presented here were collected as part of the study described in Chapter 2. As a consequence, the methods are identical. In brief, adult tegus captive bred at the Jacarezario, UNESP Bela Vista Campus in Rio Claro, Sao Paulo State, Brazil and weighing at least 2 kg were used in the present study. 3.2.1 Instrumentation In all tegus a T29F-7B implantable biopotential/temperature amplifier/encoder (Konigsberg Instruments, Inc.) was implanted in the pleuroperitoneal cavity just posterior to the hepatic septum as described previously (Chapter 2). ECG leads from the encoder were affixed to the medial pleuroperitoneal membrane along the body wall with PeriAcryl glue and mersiline mesh so that the negative (-) contact lead rested near the apex of the heart and the positive (+) contact lead was near the conus arteriosus. Biopotential leads to monitor breathing movements were sutured into the intercostal muscles on the left side, about 1 cm apart vertically, in the area between the fourth and fifth ribs of the lateral body wall. The antenna and on/off switch were left to float relatively free in the thoracic cavity. 3.2.2 Housing/Study Site Animals were housed in outdoor enclosures measuring 2.5 m X 3.5 m enclosed with 1 m high walls. The enclosures were designed to mimic a natural savannah habitat and were planted with local short blade grass and each enclosure housed a tree and each was provided with a rectangular burrow (60 cm wide X 80 cm long X 50 cm deep) constructed of brick and cement buried in the ground. Four StowAway TidBit temperature data loggers programmed to take a reading every 15 minutes were placed around the enclosures, one in each burrow, one affixed on the north facing wall of one enclosure and one on the opposite  54  south facing side of the wall, about 75 cm above the ground. The data logger on the north face recorded temperatures in direct sunlight (reflecting the basking potential of the tegus). The data logger on the south face recorded ambient temperatures in the shad in all seasons except summer (late December-early January) when, due to the low latitude of the study location, the mid-day sun was directly overhead and the south facing data logger was also in direct sunlight. This indicated that at this time there was almost no shade available within the enclosures aside from the burrows. Data loggers were placed on a backing of 2 cm thick Styrofoam to insulate them from conductive heat transfer from underlying structures. Antennae were suspended 1 m above the enclosures via bamboo poles to maximize receptivity.  3.2.3 Data Acquisition Environmental data (rainfall, atmospheric pressure, relative humidity) was collected from the local UNESP meteorological station. The tegus' physiological variables were measured via the T29F-7B implantable biopotential/temperature amplifier/encoders (Konigsberg Instruments, Inc.). Telemeter signals were received and decoded with a TR82-2/TD14-10 telemetry signal processor and demodulator (Konigsburg Instruments, Inc.) and the raw decoded signals were collected with a Dataq Instruments DI-720 data acquisition system using WinDaq software set to record at 250 Hz per channel (3 channels per tegu, 12 channels in total) in one hour files. These files were later processed with MatLab software configured to calculate average instantaneous frequency (based on pointto-point timing) measurements of heart rate (HR), respiratory rate (fR) and average deep body temperature (Tb) for every 15 minutes.  55  3.2.4 Experimental Protocol The tegus were housed as pairs (one male and one female) and allowed to roam freely in their enclosures. They were fed on average every three days, subject to food availability and weather. During entrance into hibernation, the tegus consumed progressively less and so feeding frequency was reduced and then stopped for the duration of the hibernation season. Water was present at all times of the year. Animals were weighed approximately each month except during hibernation to reduce interference with the hibernating state. Recordings were made continuously from January to the first week of December in 2004. During this period one of the tegus escaped and one died. Both events occurred in the reproductive season. One tegu (tegu 2) died unexpectedly of undetermined causes on September 22nd and was not replaced. Tegu 4 escaped (looking for nesting sites) on October 10th and was replaced a couple of days later with a reserve tegu of the same sex. As a consequence there are data for 4 tegus from the start of the year until September 22nd and for 3 tegus from that point on. After October 10th the data set is for two of the original tegus and one reserve tegu that had been living under identical conditions. 3.2.5 Data Analysis Average values were calculated for each variable [instantaneous heart rate (HR), instantaneous breathing rate (fR), deep body temperature (Tb), burrow temperature (Tbui-row), and the temperature in direct sunlight and shade] for each individual for each 15 minute time epoch for the entire year. These data were subsequently averaged over each epoch, day, week, and month to calculate the means for the entire group of tegus over each time domain. Statistical analysis between averaged values was by repeated measures one-  56  way ANOVA followed by a Student-Newman Keuls post-hoc test, unless normalcy tests failed when a non-parametric repeated measures one-way ANOVA on ranks was used. All values are presented as mean ± S.E.M. Differences were considered to be statistically significant at the level of P < 0.05.  57  3.3 RESULTS 3.3.1. Late Active Season Figure 3.1 depicts the mean data for all tegus for all days of each month in the late active season (December through February) averaged over 15 minute epochs. This was the start of our recording period. During this period, day time ambient temperatures in direct sunlight (T a) rose to 40°C or higher and nighttime temperatures fell to just below 20°C. Burrow temperatures fluctuated little over the day, ranging between 23 and 26°C. Tegus went out to bask each day at roughly the time that ambient temperature rose above that of the burrow and warmed to deep body temperatures (Tb) between 33 and 37°C. They entered the burrows in the evening well before T a began to approach Tburrow) and their body temperatures fell very slowly, usually only just reaching burrow temperatures by the end of the night. The heart rate at the end of the night fell progressively over these three months (Figs 3.1 and 3.2). In the morning, heart rate and breathing began to rise, at constant  Tb,  before the tegus left their burrows to bask (Figs. 3.1 and 3.2). These increases could precede the increase in  Tb by  an hour or more. As the tegus warmed, heart rate and  breathing increased further at rates well within the range expected for the changes in body temperature. In the evening, heart rate and breathing began to fall in advance of body temperature. As body temperature began to fall, heart rate and breathing continued to fall relatively rapidly at first, and then progressively more slowly throughout the night. The rate of increase during warming exceeded the rate of decrease during cooling and thus there was a significant hysteresis in the correlation between heart rate and deep body  Tb.  58  CI.  64  .  •• I •)  ;7",  •  . FD.  ?1)  fl N  rD  B_  H  ▪  E  CD  2  00  0 -  •  V.  O. 0 0 0  O.  '0 0  0O.  0 °o -  '00  0^O. 11,)^'00  0  CD^ 0  .0O.  -  0 0 v '0 0 ." '0 0 0 cP. '0 0  0 0 0O  0  . 00 '0  °O. ' 0 0 0 O. .  3^0 cp. O  0  ,c..1^0 O.  O.  / 0• co.^.  0 o 0  co. .0  CD  0  SID  •  CD  •  0  r )  t  • p  CD  CM  < CD  CD Cm  CD^ .1)  °0  O 0 0 0  rp^  ^t  O.  T.D 0  O.  ■-t^'00 CD^ C'D  ▪  0  '00. CD^". 0  Cr^  -  fTQ^  O  0)^co  O  Instantaneous Heart Rate (beats/min) O  co  Instantaneous Breathing Rate (breaths/min)  O. 0 0 O 0 ^ 00  °.  O°  -^  CD^■-t^'0 CD^0 0  •  CD -  o -t  cr -t  r7) oil fin  Cr p •  a . F4 Po -t < 0  CD  O  a-  r"'D  •  0. co  N  •  C. 14 CD  C2.  •  c-D 1=1., p CD  -  O  $1 7-  cr.  CD  o<  cp  _^  5  CD  CD CD c./) C) •  • (-4  Fp' E  • ••  'O o  0 0  0 0 -  . '0 0  O.  O G  O O  O.  .0  00  00.  o,  To  7  O  o.  o  c.D  0 (9 (P. '0  oa -0  00 0 0 0  O.  '0 O. *C2 0 0  '0 00  °o  0 0  —A.  O  O  O  O  Temperature ( ° C)  0  0  C1)  CD) 7  •  cn  •  01 0  59  3.3.2. Entrance into Hibernation The trends described above continued through March and into April (Figs. 3.3 and 3.4). By April, however, the burrow temperature began to fall, the tegus spent less time basking, and their maximum (day time) and minimum (nighttime) Tb began to fall. Of particular note, their body temperatures fell rapidly to approximate burrow temperatures within hours of entering the burrows. The heart rate at the end of the night continued to fall progressively during this period (Figs 3.3 and 3.4). By April, heart rate and breathing no longer began to rise in the morning before Tb. They only rose when the tegus left their burrows to bask (Figs. 3.3 and 3.4). The rate of increase during warming still exceeded the rate of decrease during cooling, and thus there was still a significant hysteresis in the correlation between heart rate and deep body temperature. In the late afternoon and evening, heart rate and breathing fell in parallel with body temperature, although the rate of decrease was more rapid at first and progressively slower through the night reflecting the decrease in the thermal gradient between the animal and the burrow. Note that in April, the magnitude of the daily changes in Tb and heart rate were tremendously reduced.  3.3.3. Hibernation Season During the hibernation season, the days still got warm but for the most part, the tegus did not. They remained in their burrows and burrow temperatures were at their lowest for the year. In July, while the animals are in the burrow their body temperatures equaled burrow temperature. This was not always the case in May and June, but this reflected large fluctuations in Tburrow on some days that were not tracked by Tb. Night time heart rate was uniformly low throughout this period. In May there was no hysteresis in the  61  •  0 •  ••  50  50  40  0 40  30  TO 30  20  ,D 20 E 1--  10  10  • • O  April  Tegus Burrows Direct Sunligh  •• ••  a.  OO 013^o^• 0 ..0° 0°^ O.^0.0 160' 63 . O •^ 0°' .0.Nr?.:-: •.°^ Q;P ° p 0 ^ O P '. 9 :2 O. N 0o 0^0'680 a)  ^8  cc  """".  .6c)^c9 .QP•^sP• o(3 *^o'e.^.01 80  4  0 6(.,. 00  '^  C10, •^(t)0'  a) - 8 cc a)  cc^— cc a) cc  60 ▪ 60 ^1E^F a CIS "E CU C 6^a) E^•E  we  -  o •  C  r)  (  40 -  co  0)  ^(1)  011 1 1:94141 lifillitati ve40  2 s)  -6 1 9 'E ta cr‘ 4 o _a c  40 -  ^„,„  4 o c 2  ^cp  cCo *E.^20 co ^*C -.A' rn 2 in c C ^0  20 -  ^(  ^.63 .63 .63 .6) .63^00 63. .63 • 0Q' 0Q' • .CP • .CP • (P •^b`•^0(6.^'■?'.^NC°.^q5)^CP.  Time of Day  ^  -2 ^  ......^,^.......^,  C  .6) .6) 00 .63 00 .63 .63 .6)^•^63 ' .6) * .63 ' .63 ' .63 ' .63 ' 63 '^61'; •^66 *^1q"^NC° .^2,,Q' •^CP •  Time of Day  Figure 3.3: Mean values for ambient temperature, burrow temperature, tegu temperatures, heart rate and breathing rate for all tegus for all days of each month during entrance into hibernation (March and April). The dotted lines represent the average time when tegus left their burrows in the morning (red) and retreated into their burrows for the evening (blue).  62  as CC •  20  a) c  co  E 0^  O co  15  20  25  30  35  40  CD CD .0 80 ^  cow a as^ 60 co a  15^20^25^30^35  ^  40  Deep Body Temperature ( ° C)  Figure 3.4: The relationships between mean values of Tb and HR for all tegus for all days of each month during entrance into hibernation (March and April). Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph.  63  relationship between heart rate and Tb (Fig.3.6). The animals also did not emerge during this month. In June and July there were some days when different tegus emerged giving rise to increases in Tb and heart rate. At these times a hysteresis reappeared in the relationship between HR and Tb. When a tegu did emerge, it was late in the day (Fig. 3.5), and while Tb at such times did rise to near normal levels (Chapter 2), it was not for long and so mean Tb did not rise much. 3.3.4. Arousal from Hibernation In August, animals began to emerge from hibernation on a more regular basis. On days when this occurred, it was once T a exceeded Tb urrow (Fig. 3.7). Periods of basking were longer and maximum daytime Tb increased to 33-37°C. Animals entered the burrows after T a began to fall, but while T a was still well above Tburrow. Mean Tburrow was beginning to increase and larger circadian oscillations appear (Fig. 3.7). Heart rate and breathing once again began to increase in anticipation of the tegus leaving the burrow. The slope of the relationship between HR and Tb decreased progressively through the night, i.e. HR dropped rapidly for small drops in Tb initially and then dropped little for large drops in Tb later on (Fig. 3.8). 3.3.5. Early Active (Reproductive) Season This was the season (Chapter 2) when the lizards arose early and stayed out longest. In September the greatest anticipatory increase in HR at constant Tb occurred (Figs. 3.9, 3.10). The greatest day time HR of the year occurred in October (Fig. 3.9). Despite the long days basking, animals still re-entered the burrows while T a exceeded Tburrow. They seemed to enter the burrow when T a equaled Tb; i.e when they could no longer sustain their  64  ^  ^ ^ ^  ▪  Instantaneous Heart Rate (beats/min)  O  0  0  cs) 0  Temperature ( ° C)  0o OD  *0  ^  0 00  0 C, 57  •c2o a 0 -0  cfl  ,,---. == - t^. . )^ 00  ^.--%^-0 CIO CD^ o  '0 0 .  CD Cr Cr^'0 ^aL,  .......,^CD^,..7.'.  ^=  P  ^CD  .00  '00  '0 ,-,^ c.) 0 •  ^rp  0  rn  61  P^=^  0  O.  5 I,^  2^0.  =  0 ■-•■•^ 0 0 .--t^(.. r. CD^0. P -t P P^ °O 0 Fr) ci, O 0^o 0 = .-t^0. - . .--.. 0)^'00 =^ '0  CD  ^CL  a 0 0  O  O  O  O  O  O  01 O  cr^0 0 ^F: ') ,4 ,-. .,^= -  5  O  o^0(3).  .- -t t..  .-7' t•^  0  -0a  CS = ■=ta -I 2- 0 O (-D (-;^ ^■-.1 P 5^s''D. 0 -I^ 3 ^0 = CD.'75^ 0 0 CD 0 6'. c.....,^,--t cr^A)^ '0 O h = 0 a 0 ^C `--< .1^0)^ ,..) ■--t^„CD^.< 0. 0  ^0  CD H FD'  Crq^oo rD CD  o  O.  °O.  '0  0 0  ^a  a CD o... ;v. ,^-o C =^o 0 -  ,... -^--)^a -0 ^CIsC) CD CD,^a 0 0  0 0 00 . 0 0 0  ^■-t^  P^v. '0 = a ^0 0-t^-0  ^.  ^ 01 CO 0 O O  -  ct,^cr^rp^(...., ,.._..,^cP. o cr^-0 C. CD ”^a o  ^--t  P^'o Cr^ a Cfc) CD^00 =^..,6'. 0 AD rg. 0 t,.< CD.)^ s.3 '0 cr^ ^CD p^ 0. 0 O A, =^'0 ^o-h < C.-^0 0 ^CD CD Cr ^0 0 0-e^,-t 0 P Cl,^ -0 0 CD OAD .-t 0^' o (Pa^-',9 CD .-■.... -.. b = = 0,q n CD CI -t P 0- D CD ..-4-) O 0 g ,-s (1,0 P -O cr^■:-_-,(1), CT CV) ,-h 0 v)  O  ^CD  6'  oo  '0 0  O 0  oo  0)^C)  Instantaneous Breathing Rate (breaths/min)  65  80  May  60 40 20 0 15  a) ca•  80  as  60  w z  40 -  • •  CZ  a) c  O 15 a) c ---  20  25  30  35  40  30  35  40  June  11:30 14:30  20  0 80  20  25  60 40 20 0  15^20^25^30^35^40  Deep Body Temperature ( ° C) Figure 3.6: The relationships between mean values of Th and HR for all tegus for all days of each month during the hibernation season (May, June and July). Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph. 66  • O O  50 0  6.  4  Tegus Burrows •• Direct Suntight  40 30  a. E cD 20  fI  I-  af'falr(1*(atROVOI(00  10  -(9 .6)^.6)1 .6) .(9 .6) .6) .(9. ^.6). ,BCD. ,  -  (9*^or'' ^66 '^4-*^ 80  •  .  ^6).  a) E1  4  8 Cc a) C  :E  1 1 ,1i  . 1: 1 1 1: 1 ll II II!  Ft) 40 CS a)  -  .0  6 7‘3 co c) 4 z a) ca  (1)  i?.?.?iNNI(Kw.wq(041 .  0  .6)^.6)^.6)^.6)^.6)^.6)^.(9. (9 .6) qz,.6 .6 .6 ^.6)• `6.^\9" .^\Cci^?,C) • .  .  ).  ).  )•  Time of Day  Figure 3.7: Mean values for ambient temperature, burrow temperature, tegu temperatures, heart rate and breathing rate for all tegus for all days in August when tegus were arousng from hibernation. The dotted lines represent the average time when tegus left their burrows in the morning (red) and retreated into their burrows for the evening (blue).  67  1114r 11  15  20  25  ‘^  30^35  40  Deep Body Temperature ( ° C)  Figure 3.8: The relationships between mean values of Tr and HR for all tegus for all days in August when tegus were arousing from hibernation. Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph.  68  preferred Tb. They maintained a significant  Tb-Tburmw  differential throughout the night  (Fig. 3.9). If the weather was inclement (rare) and animals stayed in the burrow, this differential could be sustained the entire time (Fig. 3.10). This was not just due to changes in thermal conductance as seen by comparing the values for an animal that died at this time with its burrow mate (Fig. 3.11). 3.3.6. Heart Rate Hysteresis Fig. 3.12 shows all the hysteresis curves for the relationship between mean heart rate and Tb over the year in one figure for comparison, while Fig. 3.13 compares the log minimum heart rate versus minimum Tb (i.e. end of the night). There is also a circannual hysteresis (Fig. 3.12), which is evident in the plot of log minimum daily heart rate versus minimum daily Tb (Fig. 13). In Fig. 3.13, values from all five seasons overlap at 26-27°C and it can be seen that at constant temperature, HR was lowest during the entrance period through into hibernation, a little higher on arousal (a mixed state), highest in the early active season, dropping again in the late active season. Also note that the Q  io  for the  relationship between minimum HR and minimum Tb was extremely high (11.5) as the animals entered hibernation, indicative of an active metabolic suppression, but was within normal limits for most physiological processes while the animals were in hibernation and during the arousal phase (Qios of 2.4 to 4.0) (Fig. 3.13).  69  :11 = -"' cm  cr S-'  CD 0 < -t  2  (--7‘ P  v) CD  crq = o (:,  F.D.' ‘-< (/' tw .)  CD C. =  '0^  ,  O.^  00. '^0 0 co.  cr 0 ^a CD  -t^0 '0 0 "" CD SD,D v. 0 ..t-..^ '-t^,......,^ 0O. t-, ^,-t^ 0  --  ?  --, al- qc ^o ,-,^o o. 0 0 Z °O. -, -, .o no o. 0 0 (1, B oo. < = CS cr ,- a^  `.‹  ,-.) t^0.  0 t-t0 P.) < '--'" CD p.,  ,-,- clq  CD = =  2 ('2 CD c") -I.) 0  ,-t- c6  ^SID^t1D CJO '—‘  ^--t^•  ^CD  0  cc) ,-...-'C.^'0O. '0 Cr 0 ^0 .1^0 0. ^'CS CD^'o ^-t p^a ^CD t-r^ 0 i vi ^0  ^E  =^CD^o 0 CD H ,,c-r-'^d). °O. ="' CD ='-' CD C^ 0^,"DC.^ ,^'yam c--). . CD^ C) .1^ O 0 ^B“ ,-' '-'^ ,.). 0 ^(JO (1) 2,^ 61 Cl. 7_,^ 0 ^( Id^ O. Cr -^ V  ^Cr  ^2  5  °a0  a -  00 0  , ID -.30 0 tF-,^-00.  ^O  0 O.  ^ :00 • :_.*---: ^ O.  Instantaneous Breathing Rate (breaths/min)  OD^CO  '00  0 O C7  ° .0 *0  0.  o Q 7 0  0  °Q  °o  O  2. '0 0  0  ' 00 Qo o 7.  '0 '0^ 0. 0^ C7 ^.= ^00. 0 0 0.^  00. D(I) 0 -t 0 2-6' 0 -...  Fi^ j ,.,^'c'  ,-t CD ...^-c. ,-,0 CD Fij :t^-1 -t  -0 '0  6 0 /  ^-^o0 - 0o.^ ^..=. 00^----^o o 0 0. o^  0 cp 0 0^  f^ a,^ID 0 ,___,^0. ^77 V]^0 ^. 0 ^a^ .71 °nc7  ,--. cf) -t 0 N^.-I  = 0, 0 ,-t C1C7^ AD p  0  <= rD -0.^.-cs cf,  - •  CD^= P t-,2 r) CD  -  o. -t o ^.0 ,..., = ,-< no^....-^0 o.  0 -0  *0 0 0 0  .0 ^ -^ 0^ o 0 oQ^=-= 0 a_.— ___^ Y.  o o^ co o o^_p..^0)^ N ^o^o^c)^CD '0O. °O.^ Q  Instantaneous Heart Rate (beats/min)  0^ =^CD 0 t-tx -- ry) 0 0^ -1 0 S 0^*0 O. "^ .-t .' '0 0 0 ^SID^y ' s -) . — co = 0 = .%oo^  ^ ^ ^  0  O  O  O  Co O  O  O  01  CT  0  01 O  O  ^ O  Co  N.)^CO^-1=. CD^0^CD^CD^0  Temperature ( ° C)  70  80 60 -  • •  08:30 15:30  September  40 20 0^ 15 a) ca cc  20^25^30^35^40  80  60 c^ 0  • •  08:00 15:30  VS 40 co a) 0  ca ▪  E  20 -  cu  .  C  0^ 15  20^25^30^35^40  80 ^ 60 -  • •  08:00 15:00  November  40 20 0 15^20^25^30^35^40  Deep Body Temperature ( ° C)  Figure 3.10: The relationships between mean values of Th and HR for all tegus for all days of each month in the early active season (September, October and November). Epochs corresponding to times when tegus, on average, emerged to bask (red) or retreated to their burrows to rest for the night (blue) are indicated with the corresponding times listed in the upper left corner of each graph. 71  60 -  50 ezitIP  co  0  40  Burrow Ambient Tegu 4 Tegu 3  a) 30  E 20  a)  10  Mon 11  Tue 12  Wed 13  Figure 3.11: Mean values for ambient temperature, burrow temperature, and the deep body temperature of two tegus in August, 2003. The dotted line indicates the time at which tegu #3 died.  72  ▪  •  80 -  August September AD- October o^ November -  C  0 u) 80 cis  4-0 a) 60 ca CC 3  a)  a) a  40  20  ccs  a 0 +as th. 80 a 4  j  -  March April -4 — May June o^July --4*—  60  ,  40 20 0-  15^20^25^30  35  Deep Body Temperature ( ° C)  Figure 3.12: The relationships between mean values of T h and HR for all tegus for all days of all months.  73  ^,  sA  0 ^  CD  ■••••J  =  Crq  CDI  C  S31)  13 (D  3  '"1  CD  cn  ct  rf  ,-,..,  -t •-• 0  N.) 01  ND  O  01  0  ^  2 pzJ^0  =-'  CD^  P ,-, •^= Cl.  ,  Frj 0 —t^'—', CD^1...i  .  = 0. rc, 4z,^c,,  p 0^...."  <-4-^<  =  crcr^I-, •  CD  -  gp  CD  (  '73 cr -7 Cr  co n" o rt)^0  t7.4-  •-•■• 0^<4.)  ^  _L  -  -  L1117111T1 1  • —^  z-= ;;E:  111.1111111  ELM+ lq.1 1!  !!  .. !.131. 11116 • if !!"  1 gram  "VC:It:14  14111.1111111ii MINN  IN I  rz;  111 1 111111 1 11,^  11111  opal r-'1:22!gel tr,-1161.0.11.14  --  7.!  0^  Instantaneous Heart Rate (beats/min) 0  o  74  3.4 DISCUSSION 3.4.1. Seasonal Changes in Circadian Cycles of Behaviour and Thermoregulation Morning warming From the data in the present study we see that from August through November, as animals began to emerge from hibernation and entered their active season, they generally went out to bask each day shortly after the ambient temperature rose above that of the burrow and warmed to relatively consistent preferred day time deep body temperatures (Tb); between 33 and 37°C. The periods of basking and activity at this time were relatively long (7.5 hours). From December through March, animals still went out to bask shortly after the T a rose above Tburrow but the periods of activity got progressively shorter (just over 4 hours by March) and the maximum day time preferred temperature began to decrease. From April to July, during late entrance and into the hibernation season, if tegus emerged from their burrows, it was quite some time after T a exceeded Tbuffow and the animals did not warm to the same extent (20-25°C). Clearly there were seasonal changes in thermoregulatory behaviour, and either tegus had a lowered preferred day time maximum Tb  at this time, or physical thermodynamics precluded them from attaining a higher Tb. Similar observations have been made in other reptiles. North American chuckwallas  (Sauromalus obesus) collected from the wild at various times of the year and put into artificial thermogradients showed a trend in seasonal preferred  Tb  in which winter animals  generally preferred cooler temperatures and spent more time in their dens while spring and summer lizards generally preferred warmer temperatures and exhibited greater basking activity (Case, 1976). A more complex seasonal thermoregulatory scenario was demonstrated in Lacerta viridis, wherein both preferred Tbmax in photophase and preferred  75  Tbmin in scotophase changes with the seasons (Rismiller and Heldmaier, 1982). These changes in preferred  Tb were  influenced by photoperiod (Regal, 1967; Rismiller and  Heldmaier, 1982). These observations support the suggestion that T merianae have separate daytime (active) and nighttime (sleep) body temperature set points (Tset) that decline with photoperiod (see Chapter 2) during hibernation. Evening cooling As just described, in the early active season, tegus stayed out basking or shuttling much of the day. They re-entered the burrows only once T a fell to equal their preferred day time Tb; i.e. once they could no longer maintain their preferred Tb through passive means. Once they entered their burrows in the late afternoon, they maintained a significant TbTburrow differential  throughout the night. If the weather was inclement and animals remained  in the burrow throughout the day, this differential was sustained. This was not the case for one tegu in a previous study (2003) that unfortunately, but fortuitously died just after entering its burrow one evening, clearly demonstrating that maintenance of the Tb-Tbun-ow differential was not simply due to thermal inertia. By the late active season the tegus entered the burrows in the evening usually well before ambient temperatures fell below their preferred Tb, and their body temperatures fell very slowly, usually only just reaching burrow temperatures, or remaining slightly above burrow temperature, by the end of the night. It should be noted, however, that burrow temperatures at this time were the highest of the year. The net result was that throughout the active season (both early and late) Tb at the end of the night was about 25°C. By contrast, during March and April as the animals entered into hibernation, on days when they emerged their body temperatures fell rapidly to approximate burrow temperatures (that were now falling) within hours of entering the  76  burrows. During the hibernation season, Tb, for the most part, was equal to Tbun-ow. There were days in May and June when this was not the case, but this reflected occasional large fluctuations in Tb„„„ due to inclement weather that were not tracked by Tb. The latter in turn probably reflects a decreased thermal conductance (due to vasoconstriction of peripheral tissues associated with a reduced metabolism) along with insulation from burrow litter. 3.4.2. Seasonal Changes in Heart Rate Hysteresis Morning Rise in Heart Rate From August to March, on days when the tegus emerged from their burrows to bask, heart rate and breathing began to rise in the morning, at constant Tb, before the tegus left their burrows. The most extreme case was in September when heart rate more than doubled, reaching almost maximum daytime levels over a two hour period before the tegus emerged. This correlated with the period of greatest reproductive mating activity, highest day time Tb and heart rate, and longest periods spent active. The rapid initial increase in HR could have been due to 1) removal of a nighttime metabolic suppression, 2) activity in the burrow, 3) vasodilation in anticipation of going out to bask or 4)other strategies to "prime the pump" in preparation for warming. If the rapid increase in HR at constant Tb had been due to removal of metabolic suppression associated with nightly torpor, it should have increased from November to April as the degree of nightly torpor increased. Not only was this not the case, the opposite occurred. As the degree of nightly torpor increased, animals were still warming to the same day time maximum Tb but the anticipatory increase was absent and the HR hysteresis was diminished. The rapid increase in HR while still in the burrow was also not likely due to activity in the burrow since activity disrupted the  77  radiotelemeter signals and was easy to detect. It is also difficult to attribute the anticipatory increase in HR to skin and peripheral vasodilation in anticipation of going out to bask. An increase in heart rate, associated with elevated cardiac output and distribution of blood flow to peripheral tissues would increase heat transfer between the tegu and its environment. Since the tegus were still warmer than their burrows at the end of the night at this time of the year, they should have lost heat and cooled to burrow temperature. Thus at this time it is not clear exactly what the underlying cause of the "anticipatory" increase in HR was, but it could have resulted from some combination of the factors listed above, or a change in any other component of Poiseuille's law (HR x SV = 2t(AP)r 4/8n1). As blood (a liquidsolid suspension) is the convective medium, the increase in HR before basking may serve to reduce blood viscosity and increase turbulence, which would facilitate heat convection (Ozbelge and Somer, 1994; Ku, et al., 2000). The importance of blood viscosity in reptilian thermoregulation is often overlooked (Langille and Crisp, 1980; Firth and Turner, 1982). However, changes in blood flow that cannot be attributed to any change in blood vessel diameter (Drane and Webb, 1980) suggest that viscosity is also an important component in thermoregulation. Throughout the active season, this anticipatory increase in heart rate slowly decreased, and by April, heart rate and breathing no longer began to rise in the morning before Tb. They only rose when the tegus left their burrows to bask. Day time Changes in Heart Rate and Maximum Daily Heart Rate In all seasons, once tegus left the burrow and began to warm, heart rate and breathing increased further. The rates of these changes varied across the seasons (Fig 3.13) Not surprisingly, the higher the heart rate once the tegus emerged from the burrow, the lower the rate of rise until the maximum daily heart rate and Tb were reached. The rate of  78  rise at this time must reflect both the effects of temperature on heart rate and metabolic rate (Qio effects), the effects of activity, as well as the effects of thermoregulatory processes associated with reaching preferred Tb. As noted previously, the maximum daily HR was surprisingly consistent from August through March associated with a consistent maximum daily Tb. Evening Falls in Heart Rate In the evening, during the active season, heart rate and breathing began to fall in advance of body temperature, with the greatest changes occurring in the early active season. These rapid changes occurred at the same time that Tb was sustained at levels well above Tburrow through the night and most likely reflect increases in total peripheral vascular resistance associated with vasoconstriction of peripheral beds for heat retention. Not surprisingly, this rapid initial fall was absent from May through August when tegu body temperatures fell rapidly to approximate burrow temperatures within hours of entering the burrows and nightly torpor was progressively greater. Nighttime Changes in Heart Rate and Minimum Daily Heart Rate In the active season, after the initial rapid fall in HR, the slope of the relationship between HR and Tb decreased progressively through the night. This most likely reflects a relatively normal Q10 effect and the decreasing thermal gradient for heat dissipation. From March through July, however, if animals emerged from their burrows for the day, the rate of fall in HR with decreasing Tb in the evening was much greater, suggestive of a change from heat conservation to heat dissipation.  79  3.3.3. Conclusion Perhaps one of the more profound observations to come out of this analysis was that, despite being ectotherms requiring heat gain via passive processes, at no point in the year did it appear that Tb regulation was abandoned and left completely at the mercy of ambient temperature fluctuations. What we saw was 1) a high day time preferred Tb and a drive to maximize time spent at this Tb in the early active (reproductive) season. This was the most metabolically demanding season. The burrows at this time, due to their own intrinsic thermal inertia, were cool. There was also an attempt to retain heat through the night and signs of possible thermogenesis (a maintained Tb-Tbuirow differential that could be maintained over several days of inactivity). 2) Then, in the late active season more time was spent in the burrow and signs of thermogenesis disappeared. This seemed largely due to the warmer burrows, but the data suggest that the tegus also had both a preferred daytime and a preferred nighttime Tb that were maintained throughout this period. Ironically, despite the relatively warm end-night Tb, there were progressive night time falls in HR and fR from November through April. Thus metabolic suppression appears to have been occurring despite retention of relatively constant day and nighttime Tb set points. From March on, even daytime preferred Tb was reduced, and the amount of time spent at it was greatly reduced. At night there was now an equilibrium with Tb falling in association with an increase in nightly torpor. Thus both day time and nighttime Tb set points seemed to be falling but at very different rates. The net result of this complex interplay between changes in heart rate associated with the direct effects of temperature and the indirect effects of thermoregulation, activity, and changes in metabolism was a circannual hysteresis (Fig. 3.13). The base daily  80  hysteresis is reflective of the physiological changes associated with warming and cooling as preferred body temperatures alternate between day time and nighttime levels, while the secondary hysteresis reflects the fact that these day time and nighttime preferences change with the seasons. In the latter case, the Qio for the relationship between minimum HR and minimum Tb was extremely high (11.5) as the animals entered hibernation, indicative of an active metabolic suppression, while Qio values were within normal limits for most physiological processes when the animals were in hibernation and during arousal and the active season (Q i os of 2.4 to 4.0) (Fig. 3.13). While heart rate hysteresis has been well described in reptiles, and its role in temperature regulation has received much attention (Seebacher, 2000; Seebacher and Franklin, 2001, 2003, 2005), we know of no other studies that have examined this phenomenon as a function of season.  81  4. GENERAL DISCUSSION  In the field of quiescent states, there is a contemporary debate over whether sleep, torpor and hibernation represent discrete physiological states (Geiser, 1988, 2003; Geiser and Ruf, 1995; KOrtner and Geiser, 2000) or if they are degrees of magnitude on a continuum of quiescence (Heller, et a/.,1978; Heller and Glotzbach, 1977; Larkin and Heller, 1996). While mammals and birds have been the subjects of this debate, this project provides new insight from a different taxa. In early activity season, tegus infrequently show periods of non-emergence that are associated with inclement weather. During these events, Tb remains elevated while HR and fR progressively decrease slowly as the bout continues,  similar to sleep. As the season progresses into the late activity period, average Tb remains consistent while average HR and fR progressively drop, indicating nightly torpor is taking place. During late activity progressing to entrance into hibernation, non-emergence bouts become more frequent, have greater duration and are no longer associated with inclement weather. Average Tb begins to decline and average HR and fR decline to a greater degree until hibernation is entered wherein HR, fR and Tb are all greatly reduced. Through hibernation, the tegus exhibit arousals wherein active Tbs reach levels comparative to active season values, but then quickly return back to dormancy within hours of retreating into the burrow. Milsom, et al. (2008) have demonstrated that metabolic rates measured during hibernation can be achieved at any time of the year by constraining tegus to constant dark and cool temperatures while withholding food over several days. Integrated together, these findings support the view that the quiescent states of sleep, torpor and hibernation are degrees of magnitude on a continuum.  82  Although the continuum hypothesis is supported here, some would question the validity of applying the term hibernation to reptiles (see Gregory, 1982). The parallels between winter dormancy in mammals and the tegus in this study are numerous. The tegus started preparing for hibernation via metabolic suppression (as seen with nightly torpor) as early as January, well before ambient conditions became unfavourable. Behaviourally, tegus demonstrated voluntary aphagia as early as mid-February and spent considerable time gathering litter and detritus for their hibernacula. In hibernation, we saw spontaneous arousals that were not tightly correlated with zeitgebers, just as seen with mammals (KOrtner and Geiser, 2000). Similar to mammals, the frequency of these arousals greatly declined in the early phase of hibernation and then slowly increased in frequency again as the season progressed (Twente and Twente, 1967). Physiologically, the state of hibernation is a regulated state in regards to the three variables we recorded: HR, fR and Tb. Heart rate during the early phase of hibernation was characterized by arrhythmia and frequent `skipped beats' that later in the season became regular. Breathing was continuous in the active seasons but in hibernation became sporadic and episodic. About the only endothermic hibernation trait not observed in the tegus was non-shivering thermogenesis via brown fat. Based on this, perhaps a re-evaluation of the phylogenetics of hibernation needs to be considered. Reptiles represent the closest ancestral root to both of these groups and therefore may present an ancestral form of hibernation. What cues are responsible for initiating hibernation in reptiles remains unclear. Data from this study are anecdotal and need further investigation. However, HR and fR are most closely correlated with photoperiod, both during entrance into hibernation and arousal from hibernation. Through the entrance into hibernation, hibernation and arousal from  83  hibernation seasons, however, HR and fR also show very tight correlation to Tb. Whereas Tb is  not classically considered a zeitgeber, ultimately Tb does derive from the external  environment in ectotherms. Interaction between photoperiod and Tb are likely responsible for the total hibernation suite. Whereas temperature regulation has long been demonstrated in reptiles, some show very precise regulation and others only moderate ability to thermoregulate (see Huey, 1982). A previous study demonstrated different preferred Tb for day and night that varied over the year in Lacerta viridis (Rismiller and Heldmaier, 1982), but this is the first study we have found that provides evidence for separate regulation of Tbset in active and sleeping reptiles: ie. not only two different Tb set , one in day and the other night, but each affected differently by changes in the season. Through most of the active season, daytime Tbset appears quite consistent. However, night time  Tbset  starts declining in the entrance into  hibernation season well before daytime Tbset shows a similar reduction. Likewise, during arousal from hibernation, tegus swiftly resumed daytime Tbset of regular activity seasons, but night time Tbset showed gradual increase through the whole arousal season until night Tb was comparable to regular active season, but now substantially elevated above Tbun ow.  The profound differential between Tb and Tbunow observed in the tegus during the early active season was undoubtedly a result of cardiopulmonary adjustments to decrease thermal conductance (see Bartholomew, 1982) but may also represent active thermogenesis in a reptile. Previously in reptiles, thermogenesis has been shown in relation to postprandial elevations in metabolic rate (Tattersall, et al., 2004) and from spasmodic contraction of axial musculature for the purpose of incubating eggs in snakes (Hutchison, et al., 1966; Van Mierop and Barnard, 1976, 1978). We propose thermogenesis may be a  84  component of the Tb  -  Tburrow differential  seen in the early activity season as when a tegu  coincidentally but suddenly died upon retreating into its burrow during this time in a previous 2003 study. Thermal inertia was not enough to maintain the differential for even one night. 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