1 3 J Comp Physiol B (2015) 185:905–915 DOI 10.1007/s00360-015-0928-2 ORIGINAL PAPER Daily and annual cycles in thermoregulatory behaviour and cardio‑respiratory physiology of black and white tegu lizards Colin E. Sanders1 · Glenn J. Tattersall2 · Michelle Reichert1 · Denis V. Andrade3 · Augusto S. Abe3 · William K. Milsom1 Received: 8 April 2015 / Revised: 8 July 2015 / Accepted: 31 July 2015 / Published online: 13 August 2015 © Springer-Verlag Berlin Heidelberg 2015 interplay gave rise to a daily hysteresis in the fH/Tb rela- tionship reflective of the physiological changes associ- ated with warming and cooling as preferred Tb alternated between daytime and nighttime levels. The shape of the hysteresis curve varied with season along with changes in metabolic state and daytime and nighttime body tempera- ture preferences. Keywords Reptiles · Tegu lizards · Torpor · Dormancy · Hibernation · Cardiorespiratory control · Seasonal adjustments Introduction Hibernation is employed by many animals as a strategy to survive periods of limited energy availability in the envi- ronment (Carey et al. 2003). Some of the hallmark traits of endothermic hibernation are a depression of body tem- perature (Tb) and a reduction of basal metabolic rate, accompanied by falls in ventilation and heart rate (Lyman 1982; Willis 1982; Carey et al. 2003; Tøien et al. 2015). While the magnitude and mechanism of metabolism and Tb depression in hibernating endotherms have been well studied (Lyman 1982; Nedergaard et al. 1990; Storey and Storey 1990; Carey et al. 2003), the same aspects of dor- mancy have not been extensively studied in ectothermic hibernators. It is clear that many overwintering ectotherms are capable of similar reductions in metabolism, often inde- pendent of changes in Tb; Mayhew (1965) referred to this as brumation, in order to distinguish ectothermic winter dormancy from endothermic hibernation. Reduction in metabolic rate at the initiation of hiberna- tion involves both lowering of the hypothalamic set point for body temperature regulation and active metabolic Abstract This study was designed to determine the man- ner in which metabolism is suppressed during dormancy in black and white tegu lizards (Tupinambis merianae). To this end, heart rate (fH), respiration rate (fR), and deep body temperature (Tb) were continuously monitored in outdoor enclosures by radio-telemetry for nine months. There was a continuous decline in nighttime breathing and heart rate, at constant Tb, throughout the late summer and fall sugges- tive of an active metabolic suppression that developed pro- gressively at night preceding the entrance into dormancy. During the day, however, the tegus still emerged to bask. In May, when the tegus made a behavioural commitment to dormancy, Tb (day and night) fell to match burrow tem- perature, accompanied by a further reduction in fH and fR. Tegus, under the conditions of this study, did arouse peri- odically during dormancy. There was a complex interplay between changes in fH and Tb associated with the direct effects of temperature and the indirect effects of ther- moregulation, activity, and changes in metabolism. This Communicated by G. Heldmaier. Electronic supplementary material The online version of this article (doi:10.1007/s00360-015-0928-2) contains supplementary material, which is available to authorized users. * William K. Milsom milsom@zoology.ubc.ca 1 Department of Zoology, University of British Columbia, 6270 University Blvd., Vancouver, BC V6T 1Z4, Canada 2 Department of Biological Sciences, Brock University, St. Catharines, ON, Canada 3 Depto de Zoologia, Instituto Nacional de Ciência e Tecnologia em Fisiologia Comparada, Univ Estadual Paulista, Rio Claro, SP 13506-900, Brazil http://crossmark.crossref.org/dialog/?doi=10.1007/s00360-015-0928-2&domain=pdf http://dx.doi.org/10.1007/s00360-015-0928-2 906 J Comp Physiol B (2015) 185:905–915 1 3 suppression (mammals: Heller et al. 1977; Nedergaard et al. 1990; Heldmaier et al. 1992). This is also true of amphibians and reptiles; however, these groups rely on behavioural rather than physiological methods to reduce Tb (Rollinson et al. 2008; Tattersall and Boutilier 1997; Donohoe et al. 1998; Tattersall and Boutilier 1999; Huey and Pianka 1977; Guppy and Withers 1999; Glanville and Seebacher 2006). The manner in which these behavioural and physiological strategies are integrated and employed by ectotherms during entrance into dormancy, however, is not well understood. The black and white tegu (Tupinambis merianae) is a large, diurnal lizard of South America that undergoes winter dormancy in its southern range (Milstead 1961; Abe 1995; Ávila-Pires 1995). During dormancy, tegus retreat into their burrows where they fast and remain inactive from May to August (Abe 1995; Andrade et al. 2004). Preparation for dormancy begins well before environmental conditions become adverse and tegus can depress metabolism to dor- mant levels at any time of the year when inactive in constant cold, darkness and deprived of food (Milsom et al. 2008). By the end of the autumn/beginning of the winter, the final steps leading to dormancy appear to be a behavioural deci- sion to retreat into the burrow, let Tb equilibrate with the surroundings, and abandon behavioural thermoregulation. There also appears to be a progressive reduction in thermal sensitivity (Q10) from summer to winter such that the meta- bolic rate of dormant lizards becomes relatively temperature independent (Abe 1983, 1993, 1995; de Souza et al. 2004). This reduction in Q10 has been proposed to be an advantage to maintaining extremely low metabolic rates even when burrow temperatures fluctuate (Toledo et al. 2008). The previous studies on hibernating tegus have given rise to several questions. First, under natural conditions, can nightly metabolic depression be detected in advance of winter dormancy while the animals are still active during the day? As soon as tegus commit to dormancy and retreat to the burrow they generally remain there for the season, but it is not known whether they immediately enter dor- mancy or if the degree of metabolic suppression increases as dormancy progresses. The latter has been reported to occur in Lacerta vivipara (Patterson and Davies 1978), although the metabolic suppression was not sustained throughout the entire season. Finally, it is not clear whether the period of dormancy is always one prolonged bout or whether these animals undergo periodic arousals during which they remain relatively inactive within their burrows, as is seen in other dormant lizards, such as Varanus rosen- bergi (Rismiller and McKelvey 2000). To understand the natural mechanisms involved in win- ter dormancy requires continuous monitoring of behaviour, Tb and, ideally, metabolism throughout the year. Here, we record continuously behaviour and Tb along with heart and breathing rates as physiological surrogates for metabo- lism (Zaar et al. 2004; Butler et al. 2000, 2002; Clark et al. 2004, 2006; Green et al. 2008; Piercy et al. 2015), in a group of black and white tegus, T. merianae, housed out- doors under semi-natural conditions. We hypothesized that metabolic suppression (as indicated by changes in heart rate and breathing frequency) would not be evident until the tegus remained in the burrows for extended periods but that metabolism would then progressively fall and be sus- tained throughout the dormant period. Methods Our study was conducted at the Jacarezario, UNESP Bela Vista Campus, Rio Claro, SP, Brazil. Tegus were captive bred and reared for scientific study and conservation ex situ. The study cohort consisted of 2 males and 2 females. Only 4 animals could be recorded from concurrently as the telemetry base station could only receive and decode 4 signals at the same time. Animals were weighed each month except during dormancy to reduce interference with the dormant state. The tegus weighed 3.2 ± 0.3 kg at the start of the study and 3.5 ± 0.4 kg by the end. All surgeries and experiments were conducted under animal care approval from both the UBC animal care committee and the Universidade Estadual Paulista-Rio Claro (Proto- col #A09-0232). Surgery Animals were anaesthetized using Halothane vapour. An incision was made mid-ventrally from just below the ster- num to just anterior to the post-hepatic septum. A second incision (~3 cm) was then made through the post-hepatic septum and the body of the T29F-7B implantable biopoten- tial/temperature amplifier/encoder (5.7 × 2.8 × 0.92 cm; (60 g or roughly 2 % of body weight)) (Konigsberg Instru- ments, Inc., Pasadena, CA, USA) was inserted through the opening to lie between the fat bodies ventral to the diges- tive tract in the abdominal cavity. To monitor heart rate, 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. To monitor respiration rate, Biopotential leads were sutured into the intercostal mus- cles on the left side, about 1 cm apart vertically, in the area between the fourth and fifth ribs of the lateral body wall. The underlying muscle layers and integument were sutured closed independently, and artificial ventilation with air was continued until the animals regained consciousness. The tegus were treated post-surgery with Baytril (0.1 ml/kg IM, 907J Comp Physiol B (2015) 185:905–915 1 3 every other day) and housed in indoor enclosures for at least a week to ensure full recovery. Study enclosures Outdoor enclosures measuring 2.5 m × 3.5 m enclosed with 1 m high walls were planted with local short blade grass and each enclosure housed a small tree (Eugenia uni- flora). Each enclosure also contained a rectangular burrow (60 cm wide × 80 cm long × 50 cm deep) constructed of brick and cement half buried in the ground with an opening 30 cm × 10 cm. These artificial burrows resemble refuges used in nature; tegus choose to hibernate in concrete, man-made structures (such as under houses) as opposed to under rocks (Winck and Cechin 2008). Over the top of the burrows were pyram- idal lids (60 cm × 80 cm base, 1 m height) constructed of plywood and internally insulated with 2 cm thick Styro- foam insulation. These unorthodox lids reduced solar heat- ing of burrows as well as accommodated infrared cameras to monitor activity in the burrows. Four StowAway TidBit temperature data loggers (Digi-Key Corp., Thief River Falls, MN, USA) programmed to take a reading of the local ambient temperature every 15 min 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 while the south-facing data logger recorded ambient temperatures in the shade. The TidBit data loggers and telemetry implants were calibrated in water baths set at four temperatures (7.2, 23, 29, and 39.7 °C) for at least 30 min and compared against a precision mercury thermometer both before and after the study. Data loggers were placed on a backing of 2 cm thick Styrofoam to insulate them from conductive heat transfer from underlying structures. Antennae to receive telemetric signals from the implants were suspended 1 m above the enclosures to maximize receptivity. Data acquisition Environmental data (rainfall, atmospheric pressure, relative humidity) were collected from the local UNESP meteoro- logical station (22°23′S, 47°32′W, 626.5 m altitude). 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 T29F-7B implantable biopotential/temperature ampli- fier/encoders (Konigsberg Instruments, Inc.) were configured to continuously receive and broadcast the electrocardiogram (ECG), a biopotential recording of chest wall impedance associated with intercostal muscle movements, and body core temperature. Telemeter signals were received and decoded with a TR8-2-2/TD14-10 telemetry signal processor and demodulator (Konigsberg Instruments, Inc.) and the raw decoded voltage signals were collected with a Dataq Instru- ments DI-720 data acquisition system at 250 Hz per channel. These files were later processed with custom-designed Mat- Lab scripts configured to full-wave rectify (only the breath- ing biopotentials), digitally filter (low pass), and detect peaks (using peakdetect.m from Matlab Central) to detect instanta- neous heart (fH), and respiration rates (fR). Automated peak detection was visually verified for accuracy, and to account for any irregularities or interference in electrical signals. Experimental protocol The tegus were housed as pairs (one male and one female) and allowed to roam freely in their enclosures. Active liz- ards were fed to satiation on average every 3 days, their diet consisting of meat mixed with vegetables and fruit with added multivitamin supplement. In the months prior to dormancy, the tegus consumed progressively less food and eventually stopped for the duration of the dormancy period. Water was available at all times. Continuous data recording began on the first of January and continued through to the end of September for all lizards. Recordings ceased at vari- ous times throughout October as the life span of the batter- ies in the telemetry units was reached. Data analysis Average values were calculated for each variable [heart rate (fh), breathing rate (fR), deep body temperature (Tb), burrow temperature (Tburrow), and the temperature in direct sunlight and shade] for each individual for each 15 min time period for the entire study period. These 15 min averages were subsequently averaged over each day, week, and month for each tegu. Daily maximum and daily minimum values were also extracted from each individual for subsequent com- parisons and averaged over each week, and month for each tegu. Seasonal comparisons were made by comparing criti- cal months corresponding to the: active period (February), dormant period (May), and the post-arousal reproductive period (September). Monthly nighttime minimum levels of oxygen consump- tion were calculated from the formula derived by Piercy et al. (2015) from the relationship between heart rate and metabolic rate for this species of tegu lizard under quies- cent conditions. The equation used was: log10 (O2 con- sumption) = −1.47 + 0.67 (log10 (heart rate)). Statistical analysis between averaged values was done by repeated measures one-way ANOVA followed by a Stu- dent–Newman–Keuls post hoc test, unless normalcy tests failed, when a non-parametric repeated measures one-way 908 J Comp Physiol B (2015) 185:905–915 1 3 ANOVA on ranks (Kruskal–Walis test) was used. Within month, data were compared by paired t test. All values are presented as mean ± standard error of the mean (s.e.m). Differences were considered to be statistically significant at the level of P < 0.05. Results Meteorological data In general, the summer time (December to February) in Rio Claro is warm and wet while the winter (June to August) is cool and dry. During this study, the lowest mean daily temperatures occurred from May to August (~15 °C) and corresponded to the periods of highest barometric pressure (716 mmHg) while the highest mean daily temperatures occurred from November to January (~27 °C) and corresponded to the periods of low- est barometric pressure (~705 mmHg). In this particular year, August and September were the driest months (virtually no rainfall) and were the months with the lowest relative humid- ity (~50 %). At this latitude, however, seasonal differences in all these variables, except rainfall, are modest. Seasonal patterns of behaviour January–July Throughout this period, there were days when animals remained in their burrows and did not emerge. Such events were rare from January to March (2–4 events/month of 1–2 days each), and associated with inclement weather. In April, these periods were common (6–8 periods) and lasted ~2–3 days each. During late April/early May, the lizards began block the entrance to the burrows with vegetation and remained inactive in their burrows marking the start of the dormancy period that continued through June and July. Tegus still emerged periodically throughout the dormancy period (3–4 times/month on average for usually 1 day each). August–September Starting in August, the tegus began to emerge from their burrows every day indicating the end of the dormancy period. Animals never remained inactive in their burrows during the daytime in September. Seasonal patterns of physiological change January–March From January to March, daytime temperatures in the enclo- sures (Ta) often rose to 40 °C or higher and nighttime temperatures fell to below 20 °C (Fig. 1a). Burrow tempera- tures (Tburrow) fluctuated little over the day, ranging between 23 and 26 °C (Fig. 2a). Tegus went out to bask each day at roughly the time that Ta rose above Tburrow (as indicated by the red dotted line and the rise in body temperatures for the tegus in February in Fig. 1a). During this period, maximum daytime Tb (~32–35 °C) was remarkably uniform in all ani- mals (Fig. 2b). The tegus entered the burrows in the evening well before Ta began to approach Tburrow (Fig. 1a) and their Tb fell very slowly, equilibrating with burrow temperature by the middle of the night (Fig. 1a). Nighttime Tb minima were also relatively uniform throughout this period (Fig. 2b). In the morning during this period, fH and fR began to rise at a constant Tb an hour or more before the tegus left their burrows to bask, while in the evening fH and fR began to fall in advance of Tb declining (Fig. 1b). Maximum daily fH and fR began to fall significantly (P = 0.031 and <0.05, respec- tively) in March (Fig. 2c, d). Similarly, minimum values of fH and fR during the night fell progressively (P < 0.05) over the three months (Fig. 2c, d) (by 60–75 %) despite the fact that mean Tb was relatively constant. April Although Tburrow in April still fluctuated over the same range as the preceding months, nighttime minimum Tb contin- ued to fall (P = 0.007) (Fig. 2). Maximum daily voluntary temperature also declined (P < 0.001) (Fig. 2), despite the sustained and elevated ambient temperatures in the sun. Dur- ing April, when animals remained in their burrows, daytime maximum Tb remained at the previous night’s minimum Tb. During April, fH and fR no longer began to rise in the morn- ing before Tb, but rose only when the tegus left their burrows to bask. Both maximum daytime and minimum nighttime rates were lower in April than in March (for fH min P < 0.05, and for max P0.031; for fR both min and max P < 0.05) and the magnitude of the daily changes in Tb, fH, and fR was reduced. May–July During the dormancy period, the tegus largely remained in their burrows that were at their lowest temperatures for the year. At this time, Tb equalled Tburrow. When a tegu did emerge, it was late in the day and while Tb at such times did rise, it was only to moderate levels (18–22 °C) and for brief amounts of time. Once the animals entered dormancy, mean fH and fR remained low and relatively constant throughout the day and night (Fig. 2). August–September In August, animals began frequently to emerge from their burrows and by September they were emerging every day. 909J Comp Physiol B (2015) 185:905–915 1 3 Again, emergence occurred only once Ta exceeded Tburrow (as indicated by the rise in body temperatures for the tegus in September in Fig. 1). Periods of basking were longer and maximum daytime Tb increased to 33–37 °C (Fig. 2). Ani- mals entered the burrows after Ta began to fall, but while Ta was still well above Tburrow (as indicated by the blue dotted line for September in Fig. 1a). Mean Tburrow was beginning to increase at night and during the day (P < 0.001) (Fig. 2). During this period, Tb never fell to the level of Tburrow dur- ing the night (Figs. 1, 2). In the morning during this period, fH and fR again began to rise at a constant Tb before the tegus left their burrows to bask and began to fall in advance of Tb in the evening (Fig. 1). Maximum daily fH and fR began to increase progressively (P = 0.011 and <0.05, respectively) through August and September, as did nighttime fH, fR and Tb (P < 0.05, P < 0.05 and P = 0.002, respectively) (Fig. 2). Heart rate hysteresis From January through March, the rate of increase (with respect to Tb) in fH during warming exceeded the rate of decrease during cooling and thus there was a large hys- teresis in the correlation between fH and core Tb (Fig. 3). During the dormancy period, there was less hysteresis in the relationship between fH and Tb (Fig. 3). Beginning in August, but most evident in September, are dramatic increases in fH before Tb rises in the morning, and falls in heart rate before Tb falls in the evening (Figs. 1, 3). Fig. 1 Mean (±SEM) values for a ambient temperature, burrow tem- perature, tegu temperatures, and b heart rate for all tegus for each 15 min period on all days during February (the active season), May (the dormancy season) and September (the post-arousal, reproductive season). 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 emergence. c The relationships between mean values of Tb and heart rate for all tegus for all days of each of these months. Times when tegus, on average, emerged to bask (red dot) or retreated to their burrows to rest for the night (blue dot) are indicated in the upper left corner of each graph 910 J Comp Physiol B (2015) 185:905–915 1 3 Nightime metabolic rate In Fig. 4, the monthly nighttime minimum values of Tb, Tburrow, fH and fR have been re-plotted along with rates of oxygen consumption calculated from the formula derived by Piercy et al. (2015) from the relationship between heart rate and metabolic rate for this species of tegu lizard under quiescent conditions. From this figure, it is clear that fH, fR and calculated levels of O2 consumption fell progressively at night from January through April (P < 0.001, 0.05 and 0.001, respectively) despite the fact that Tb was constant. From April into May, however, there was a further progres- sive drop in fH, fR and estimated O2 consumption (only the latter was significant P = 0.037) at night but this was asso- ciated with a further, non-significant drop in Tb. In June and July, all variables remained relatively constant while in August and September, all variables increased (P = 0.003 for O2 consumption, <0.001 for fH, <0.05 for fR, <0.05 for Tb and <0.001 for Tburrow). Discussion The pattern of metabolic rate reduction leading into dormancy One of the goals of this study was to describe the pattern by which metabolism falls during the autumn under natural conditions. Does it occur progressively during the day and night, does it occur only at night, or does it occur only dur- ing multi-day periods of inactivity in the burrow? Previous studies have shown that tegu lizards depress metabolism in advance of dormancy (Abe 1983, 1993, 1995). In all these studies, measurements were made on animals confined in Fig. 2 Mean (±SEM; error bars may be smaller than symbols) monthly values for maximum and minimum a ambient temperatures and burrow temperatures, b tegu body temperatures, c heart rate, and d breathing rate for all tegus over the entire recording period. Maxi- mum values are indicated by open symbols while minimum values are indicated by filled symbols. In a the grey shading links the maximum and minimum ambient temperatures while the black shading links the maximum and minimum burrow temperatures. Note how well the burrows are buffered from ambient temperature swings. Asterisk indicates values that are significantly lower than January values. + indicates values that are significantly elevated compared to January values. All minimum values are lower than maximum values except for those indicated with a # 911J Comp Physiol B (2015) 185:905–915 1 3 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; Toledo et al. 2008). By contrast, in nature during this period tegus are still active and warm themselves to active temperatures during the day while being exposed to progressive changes in photoperiod and ambient temperature (Köhler and Langerwerf 2000). We found that from January to March, tegus regulated their maximum daily Tb from 33 to 37 °C, except on days with inclement weather when Ta did not permit behav- ioural thermoregulation to this extent. Minimum nighttime Tb and Tburrow from January to April also remained con- stant. By contrast, nighttime values of fH and fR declined progressively over this period and daytime maximum lev- els also began to decline in March. The declines in night- time fH and fR at constant Tb suggest that metabolic rate was being suppressed actively and progressively over this period of time. Based on the calculations in Fig. 4, night- time metabolic rate appears to have been suppressed by approximately 45 % from January to April. This is simi- lar to the progressive decline in metabolism seen in L. vivipara, although for this species the decline occurred dur- ing the dormancy only and not preceding it (Patterson and Davies 1978). While we do not have data that reveal the mechanism underlying this reduction, altered right-to-left intra-cardiac shunting, reducing O2 delivery to the tissues, has previously been implicated in metabolic suppression (Hicks and Wang 2004) and is a definite possibility. Daytime fH fell in March, suggesting that daytime meta- bolic rate may also have begun to fall prior to entrance into dormancy. However, the animals were active to varying degrees during the day and not in a steady state; therefore, heart rate could not be used to estimate metabolic rate. The decline in maximum Tb that occurred in April–may was indicative of an endogenous seasonal rhythm of body temperature and metabolism. Although it was possible for the tegus to achieve higher maximum Tb through behav- ioural thermoregulation, that they did not suggest an endog- enous seasonal rhythm, similar to that seen in the sleepy lizard, T. rugosa (Firth and Belan 1998; Ellis et al. 2008). From April into May, there was a further significant night- time drop in fH and fR indicating a further suppression in metabolism, but this was associated with a significant fall in Tb. This amounted to a 30 % decrease in O2 consump- tion (a Q10 of 2.1) bringing the metabolic rate to levels that were 45 % of those calculated in January. As indicated by the low but consistent levels of Tb, fH and fR (day and night) from May through July, metabolism was relatively uniform during dormancy. The values of metabolic rate estimated for the tegus in dormancy (0.18–0.21 ml O2/min/kg) are similar to those measured in previous studies on dormant tegus (0.15–0.30 ml O2/min/kg; Abe 1995; Andrade and Abe 1999; Milsom et al. 2008; de Souza et al. 2004; Toledo et al. 2008), indicating that our methods for estimating met- abolic rate were consistent with previous studies. In August and September, all variables increased to lev- els significantly greater than those recorded from tegus at similar body temperatures in May and April, suggesting that they were due not only to the increases in Tb, but also due to removal of the active metabolic suppression. The increasing incidence of arousals associated with slowly increasing nighttime heart rate and breathing in August is also suggestive that the degree of metabolic suppres- sion was decreasing as the period of arousal progressed, which has been documented in other lizard species as they approach arousal (Patterson and Davies 1978). Fig. 3 The relationships between mean values of body temperature and heart rate for all tegus for all days of all months (error bars are omitted for clarity) 912 J Comp Physiol B (2015) 185:905–915 1 3 Arousal during dormancy While species of reptiles that undergo dormancy are not likely to emerge from their burrows in mid-winter when environmental conditions are extreme, they may still arouse from dormancy and remain within the burrow. To date, however, there is no documentation that this occurs. Species of reptiles that go dormant in subtropical regions should be less constrained to remain in their burrows during periods of arousal and it has been shown that Varanus rosenbergi spontaneously arouse frequently during dormancy (Ris- miller and McKelvey 2000). In the present study, T. meri- anae 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 period of dormancy and in the phase of the dormant period (early versus late) dur- ing which these events occurred. There was no synchrony to the occurrence of arousals in tegus inhabiting the same burrow suggesting that they were not tightly correlated to local factors such as temperature change, noise or distur- bance. Arousals appeared to occur randomly, with no dis- tinct pattern in any animal, suggesting that they were not the consequence of an underlying biological rhythm. This does not preclude the existence of an internal clock control- ling arousal from dormancy or the onset of reproduction. Many species that are arrhythmic in winter can be rhythmic at other seasons (see Revel et al. 2007; Ellis et al. 2008). It is possible that 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 and were thus spacious and left the animals relatively exposed. Natural burrows tend to be more constrictive and possibly deeper in the substrate where daily fluctuations in temperature would be absent. Animals rarely, if ever, leave them during the dormancy season. A more constant temperature and tactile stimulation Fig. 4 a Mean (±SEM; error bars may be smaller than symbols) monthly values for heart rate (fH), breathing rate(fR) and minimum burrow and tegu body temperatures for all tegus over the entire recording period. b Resting levels of estimated oxygen consumption over the same period (derived from the equation of Piercy et al. 2015). Note the fall in heart rate, breathing rate and rate of oxygen consumption at constant burrow and tegu temperatures from January to April. Asterisk indicates values that are significantly lower than Janu- ary values. + indicates values that are significantly elevated compared to January values. Vertical dotted lines indicate the entrance into and the emergence from dormancy 913J Comp Physiol B (2015) 185:905–915 1 3 may promote dormancy and eliminate periods of arousal. At present, the underlying cause of the arousals seen in this study is not clear. Periodic arousals are a hallmark of most mammalian hibernation (Willis 1982) and here too it is not clear what the underlying cause is (Barnes et al. 1993; Wang 1993; Carey et al. 2003). One hypothesis is that transcription and translation of genetic material are inhibited by low temper- atures and that animals must arouse periodically to under- take essential maintenance activities (Van Breukelen and Martin 2002; Carey et al. 2003). The occurrence of peri- odic arousals is normally rhythmic in mammals (Twente and Twente 1967), but at present there is no consensus on what triggers these arousals. The incidence and role of peri- odic arousals in both mammalian and ectothermic hiberna- tion are, therefore, areas that require further study. Heart rate hysteresis and implications for body temperature regulation Heart rate hysteresis has been well described in reptiles, and its role in temperature regulation has received much attention. To maximize the period where body temperature exceeds ambient temperature, many reptiles increase cuta- neous blood flow in the morning to maximize heat gain. A concomitant rise in heart rate due to the baroreflex leads to an appropriate increase in cardiac output that main- tains blood pressure constant (Bartholomew and Tucker 1963; Galli et al. 2004; Crossley et al. 2015). A decrease in cutaneous blood flow and heart rate in the evening con- serves heat by reducing the rate of heat loss (Morgareidge and White 1969; Langille and Crisp 1980; Galli et al. 2004; Clark et al. 2006). This gives rise to hysteresis in the rela- tionship between fH and Tb in which the rate of change in fH reflects the effects of temperature on fH and metabolic rate (Q10 effects), the effects of activity and feeding (Zaar et al. 2004), and the effects of thermoregulatory processes associated with reaching/retaining preferred Tb (Seebacher 2000; Seebacher and Franklin 2001, 2005). From January to March, and again in August and Septem- ber, on days when the tegus emerged from their burrows to bask, fH and fR began to rise in the morning, at constant Tb, even before the tegus left their burrows. The most extreme case was in September when fH more than doubled, reach- ing almost maximum daytime levels over a two hour period before the tegus emerged from their burrows. This corre- lated with the period of greatest reproductive mating activ- ity, highest daytime Tb and heart rate, and longest periods spent active. This suite of changes is not uncommon in rep- tiles during mating season and has been attributed to “mat- ing unrest,” which can be accompanied by an elevation in preferred Tb (Huey and Bennett 1987; Rismiller and Held- maier 1982, 1991; Luiselli and Akani 2002; Seebacher and Franklin 2005). Once mating occurs, preferred Tb in preg- nant females may increase (Hoplodactylus maculatus, Wer- ner and Whitaker 1978; Thamnophus sirtalis, Stewart 1965; Gerrhonotus coeruleus, Stewart 1984) or decrease (Lacerta vivipara, Patterson and Davies 1978; Scleroperuscyanog- enys, Garrick 1974; Scleroporus jarrovi, Beuchat 1986). This rapid initial increase in fH was most likely due to changes in activity state (sleep to alert) and activity in the burrow. Throughout the fall, this pre-emergence increase in fH slowly decreased and by April, fH and fR no longer began to rise in the morning before Tb, instead only rising when the tegus left their burrows to bask. In all seasons, once tegus left the burrow and began to warm, fH and fR increased further, with the rates of these changes varying across the seasons (Fig. 3). Not surprisingly, the higher the fH at the time of emergence from the burrow, the lower the rate of rise until the maximum daily fH and Tb were reached. The rate of rise at this time must reflect the effects of temperature on fH and metabolic rate (Q10 effects), the effects of activity, and the effects of thermoregulatory processes associated with reaching preferred Tb. In the evenings of the non-dormant periods, fH and fR began to fall in advance of Tb with the greatest changes occurring in September. These rapid changes most likely reflect increases in total peripheral vascular resistance asso- ciated with vasoconstriction of peripheral beds for heat retention as described above (Seebacher 2000; Clark et al. 2004, Galli et al. 2004; Seebacher and Franklin 2005). This rapid fall in fH was absent during dormancy when tegu Tb fell rapidly to approximate Tburrow within hours of entering the burrows, suggesting that peripheral vascular resistance was not increased and, therefore, heat retention was not actively occurring as it was during the non-dormant peri- ods. The abandonment of heat retention strategies during dormancy may be a strategy to maintain a reduced meta- bolic rate, as much as it may be related to the reduction in preferred body temperature. There were seasonal differences in the complex inter- play between changes in fH due to the direct effects of tem- perature and the indirect effects of thermoregulation, activ- ity, and changes in metabolism. The base hysteresis (i.e. that in each monthly loop) is reflective of the physiological changes associated with daily warming and cooling as pre- ferred Tb alternated between day time and nighttime levels. Changes in the shape and position of the hysteresis curves reflect the fact that these daytime and nighttime preferences change with the seasons. Conclusion While this study was largely observational, the continu- ous recording of body temperature along with heart rate 914 J Comp Physiol B (2015) 185:905–915 1 3 and breathing rate in black and white tegu lizards provides insight into the physiological correlates of changes in behav- iour patterns. In particular, the data suggest that there was a continuous decline in nighttime metabolic rate, at constant Tb throughout the late summer and fall during the lead up to the dormancy period. This is indicative of an active metabolic suppression that develops progressively, but only at night in the early stages. Although lizards dedicate shorter periods of daytime to basking during the late summer and fall, they still reach the same Tb values seen in spring and early summer. In May, when the tegus made a behavioural commitment to dormancy, there was a decrease in Tb associated with a decrease in Tburrow and accompanied by a further reduction in heart rate, breathing rate and metabolic rate. Dormancy was a fairly uniform state from which the tegus, under the condi- tions of this study, did arouse periodically. The sum of the data suggests that tegu lizards can actively suppress metabo- lism in a complex and temperature independent fashion for which the underlying mechanism remains to be explored. Acknowledgements This research was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to ASA, from the Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Fundação para o Desenvolvimento da Unesp (FUNDUNESP) to DVA, and the Natural Sciences and Engi- neering Research Council of Canada to WKM and GJT. References Abe AS (1983) Observations on dormancy in tegu lizards—Tupinam- bis teguixim (Reptilia, Teiidae). Naturalia 8:235–239 Abe AS (1993) Effect of the environment on ventilation in reptiles. In: Bicudo JEPW (ed) The vertebrate gas transport cascade: adap- tations to environment and mode of life. CRC Press Inc., Boca Raton, pp 87–93 Abe AS (1995) Estivation in South American amphibians and reptiles. Braz J Med Biol Res 28:1241–1247 Andrade DV, Abe AS (1999) Gas exchange and ventilation during dormancy in the tegu lizard Tupinambis merianae. J Exp Biol 202:3677–3685 Andrade DV, Brito SP, Toledo LF, Abe AS (2004) Seasonal changes in blood oxygen transport and acid-base status in the tegu lizard, Tupinambis merianae. Respir Physiol Neurobiol 140:197–208 Ávila-Pires TCS (1995) Lizards of Brazilian Amazonia (Reptilia: Squamata). Zoologische Verhandelingen 1995:3–706 Barnes BM, Omtzigt C, Daan S (1993) Hibernators periodically arouse in order to sleep. In: Carey C, Florant GL, Wunder BA, Horwitz B (eds) Life in the cold: ecological, physiological, and molecular mechanisms. Westview Press, Boulder, pp 555–558 Bartholomew GA, Tucker VA (1963) Control of changes in body temperature, metabolism, and circulation by the agamid lizard, Amphibolurus barbatus. Physiol Zool 36:199–218 Beuchat CA (1986) Reproductive influences on the thermoregulatory behavior of a live-bearing lizard. Copeia 1986:971–979 Butler PJ, Woakes AJ, Bevan RM, Stephenson R (2000) Heart rate and rate of oxygen consumption during flight of the barnacle goose, Branta leucopsis. Comp Biochem Physiol A 126:379–385 Butler PJ, Frappell PB, Wang T, Wikelski M (2002) The relationship between heart rate and rate of oxygen consumption in Galapagos marine iguanas (Amblyrhynchus cristatus) at two different tem- peratures. J Exp Biol 205:1917–1924 Carey HV, Andrews MT, Martin SL (2003) Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature. Physiol Rev 83:1153–1181 Clark TD, Butler PJ, Frappell PB (2004) Digestive state influences the heart rate hysteresis and rates of heat exchange in the varanid liz- ard Varanus rosenbergi. J Exp Biol 208:2269–2276 Clark TD, Butler PJ, Frappell PB (2006) Factors influencing the pre- diction of metabolic rate in a reptile. Funct Ecol 20:105–113 Crossley DA, Wearing OH, Platzack B, Hartzler LK, Hicks JW (2015) Acute and chronic temperature effects on cardiovascular regula- tion in the red-eared slider (Trachemys scripta). J Comp Physiol B 185:401–411 de Souza SCR, de Carvalho JE, Abe AS, Bicudo JEPW, Bianconcini MSC (2004) Seasonal metabolic depression, substrate utilisation and changes in scaling patterns during the first year cycle of tegu lizards (Tupinambis merianae). J Exp Biol 207:307–318 Donohoe PH, West TG, Boutilier RG (1998) Respiratory, metabolic, and acid-base correlates of aerobicmetabolic rate reduction in overwintering frogs. Am J Physiol 274:R704–R710 Ellis DJ, Firth BT, Belan I (2008) Interseasonal variation in the circa- dian rhythms of locomotor activity and temperature selection in sleepy lizards, Tiliqua rugosa. J Comp Physiol [A] 194:701–712 Firth BT, Belan I (1998) Daily and seasonal rhythms in selected body temperatures in the Australian lizard Tiliqua rugosa (Scincidae): field and laboratory observations. Physiol Zool 71:303–311 Galli G, Taylor EW, Wang T (2004) The cardiovascular responses of the freshwater turtle Trachemys scripta to warming and cooling. J Exp Biol 207:1471–1478 Garrick LD (1974) Reproductive influences on behavioral thermoreg- ulation in the lizard, Sceloporus cyanogenys. Physiol Behaviour 12:85–91 Glanville EJ, Seebacher F (2006) Compensation for environmental change by complementary shifts of thermal sensitivity and ther- moregulatory behaviour in an ectotherm. J Exp Biol 209:4869–4877 Green JA, Frappell PB, Clark TD, Butler PJ (2008) Predicting rate of oxygen consumption from heart rate while little penguins work rest and play. Comp Biochem Physiol 150A:222–230 Guppy M, Withers P (1999) Metabolic depression in animals: physiologi- cal perspectives and biochemical generalizations. Biol Rev 74:1–40 Heldmaier G, Ruf T (1992) Body temperature and metabolic rate during natural hypothermia in endotherms. J Comp Physiol B 162(8):696–706 Heller HC, Colliver GW, Beard J (1977) Thermoregulation during entrance into hibernation. Pflug Arch Eur J Phy 369:55–59 Hicks JW, Wang T (2004) Hypometabolism in reptiles: behavioural and physiological mechanisms that reduce aerobic demands. Resp Physiol Neurobiol 141:261–271 Huey RB, Bennett AF (1987) Phylogenetic studies of coadaptation: preferred temperatures versus optimal performance temperatures of lizards. Evolution 41:1098–1115 Huey RB, Pianka ER (1977) Seasonal variation in thermoregulatory behavior and body temperature of diurnal Kalahari lizards. Ecol- ogy 58:1066–1075 Köhler G, Langerwerf B (2000) Tejus: Lebensweise, Plege, Zucht. Herpeton, Offenbach Langille BL, Crisp B (1980) Temperature dependence of blood vis- cosity in frogs and turtles: effect on heat exchange with environ- ment. Am J Physiol 239:R248–R253 Luiselli L, Akani GC (2002) Is thermoregulation really unimportant for tropical reptiles? Comparative study of four sympatric snake species from Africa. Acta Oecol 23:59–68 Lyman CP (1982) Entering Hibernation. In: Lyman CP, Willis JS, Malan A, Wang LCH (eds) Hibernation and torpor in mammals and birds. Academic Press, New York, pp 37–53 915J Comp Physiol B (2015) 185:905–915 1 3 Mayhew WW (1965) Hibernation in the Horned Lizard, Phrynosoma m’calli. Comp Biochem Physiol 16:103–119 Milsom WK, Andrade DV, Brito SP, Toledo LF, Wang T, Abe AS (2008) Seasonal changes in daily metabolic patterns of tegu liz- ards (Tupinambis merianae) placed in the cold (17°C) and dark. Physiol Biochem Zool 81:165–175 Milstead WW (1961) Notes on the Teiid lizards in southern Brazil. Copeia 1961:493–495 Morgareidge K, White FN (1969) Cutaneous vascular changes dur- ing heating and cooling in the Galápagos marine iguana. Nature 223:587–591 Nedergaard J, Cannon B, Jaenicke R (1990) Mammalian hibernation. Phil Trans R Soc Lond B 326:669–686 Patterson JW, Davies PMC (1978) Energy expenditure and metabolic adaptation during winter dormancy in the lizard Lacerta vivipara Jacquin. J Therm Biol 3:183–186 Piercy J, Rogers K, Andrade D, Abe AS, Tattersall G, Milsom WK (2015). The relationship between body temperature, heart rate, breathing rate, and the rate of oxygen consumption in the tegu lizard (Tupinambis merianae) at various levels of activity. J Comp Physiol B (Submitted) Revel FG, Herwig A, Garidou ML, Dardente H, Menet JS, Masson- Pevet M, Simonneux V, Saboureau M, Pevet P (2007) The circa- dian clock stops ticking during deep hibernation in the European hamster. Proc Natl Acad Sci 104:13816–13820 Rismiller PD, Heldmaier G (1982) The effect of photoperiod on tem- perature selection in the European green lizard, Lactera viridis. Occologia 53:222–226 Rismiller PD, Heldmaier G (1991) Seasonal changes in daily meta- bolic patterns of Lacerta viridis. J Comp Physiol B 161:482–488 Rismiller PD, McKelvey MW (2000) Spontaneous arousal in reptiles? Body temperature ecology of Rosenberg’s Goanna, Varanus rosenbergi. In: Heldmaier G, Klingenspor M (eds) Life in the Cold. Springer-Verlag, New York, pp 57–64 Rollinson N, Tattersall GJ, Brooks RJ (2008) Overwintering habitats of a northern population of Painted Turtles (Chrysemys picta): winter temperature selection and dissolved oxygen concentra- tions. J Herpetol 42:312–321 Seebacher F (2000) Heat transfer in a microvascular network: the effect of heart rate on heating and cooling in reptiles (Pogona barbata and Varanus varius). J Theor Biol 203:97–109 Seebacher F, Franklin CE (2001) Control of heart rate during thermoreg- ulation in the heliothermic lizard Pogona barbata: importance of cholinergic and adrenergic mechanisms. J Exp Biol 204:4361–4366 Seebacher F, Franklin CE (2005) Physiological mechanisms of thermoregulation in reptiles: a review. J Comp Physiol B 175:533–541 Stewart GR (1965) Thermal ecology of the garter snake Thamnophis sirtalis concinnus (Hallowell) and Thamnophis orfinoides (Baird and Girard). Herpetologica 21:81–102 Stewart JR (1984) Thermal biology of the live bearing lizard Ger- rhonotus coeruleus. Herpetologica 40:349–355 Storey KB, Storey JM (1990) Metabolic rate depression and biochem- ical adaptation in anaerobiosis, hibernation and estivation. Q Rev Biol 65:145–174 Tattersall GJ, Boutilier RG (1997) Balancing hypoxia and hypother- mia in cold-submerged frogs. J Exp Biol 200:1031–1038 Tattersall GJ, Boutilier RG (1999) Behavioural oxy-regulation by cold-submerged frogs in heterogeneous oxygen environments. Can J Zool 77:843–850 Tøien Ø, Blake J, Barnes BM (2015) Thermoregulation and ener- getics in hibernating black bears: metabolic rate and the mys- tery of multi-day body temperature cycles. J Comp Physiol B 185:447–461 Toledo LF, Brito SP, Milsom WK, Abe AS, Andrade DV (2008) Effects of season, temperature and body mass on the standard metabolic rate of tegu lizards (Tupinambis merianae). Physiol Biochem Zool 81:158–164 Twente JW, Twente JA (1967) Seasonal variation in the hibernating behaviour of Citellus lateralis. In: Fisher KC, Dawe AR, Lyman CP, Schöbaum E, South FE Jr (eds) Mammalian Hibernation III. Elsevier Inc., New York, pp 47–63 Van Breukelen F, Martin SL (2002) Reversible depression of tran- scription during hibernation. J Comp Physiol B 172:355–361 Wang LCH (1993) Neurochemical regulation of arousal from hiber- nation. In: Carey C, Florant GL, Wunder BA Horwitz B (eds) Life in the cold: ecological, physiological, and molecular mecha- nisms. Westview Press, Boulder, pp 559–561 Werner YL, Whitaker AH (1978) Observations and comments on the body temperatures of some New Zealand reptiles. N Z J Zool 5:375–393 Willis JS (1982) The mystery of the periodic arousal. In: Lyman CP, Willis JS, Malan A, Wang LCH (eds) Hibernation and torpor in mammals and birds. Academic Press, New York, pp 92–103 Winck GR, Cechin SZ (2008) Hibernation and emergence pattern of Tupinambis merianae (Squamata: Teiidae) in the Taim Ecologi- cal Station, southern Brazil. J Nat Hist 42:239–247 Zaar M, Larsen E, Wang T (2004) Hysteresis of heart rate and heat exchange of fasting and postprandial savannah monitor lizards (Varanus exanthematicus). Comp Biochem Phys A 137:675–682 Daily and annual cycles in thermoregulatory behaviour and cardio-respiratory physiology of black and white tegu lizards Abstract Introduction Methods Surgery Study enclosures Data acquisition Experimental protocol Data analysis Results Meteorological data Seasonal patterns of behaviour January–July August–September Seasonal patterns of physiological change January–March April May–July August–September Heart rate hysteresis Nightime metabolic rate Discussion The pattern of metabolic rate reduction leading into dormancy Arousal during dormancy Heart rate hysteresis and implications for body temperature regulation Conclusion Acknowledgements References