ABSTRACT

Many hibernating animals thermoregulate during torpor and defend their body temperature (Tb) near 0°C by an increase in metabolic rate. Above a critical temperature (Tcrit), animals usually thermoconform. We investigated the physiological responses above and below Tcrit for a small tree-dwelling bat (Chalinolobus gouldii, ∼14 g) that is often exposed to sub-zero temperatures during winter. Through simultaneous measurement of heart rate (fH) and oxygen consumption (O2), we show that the relationship between oxygen transport and cardiac function is substantially altered in thermoregulating torpid bats between 1 and −2°C, compared with thermoconforming torpid bats at mild ambient temperatures (Ta 5–20°C). Tcrit for this species was at a Ta of 0.7±0.4°C, with a corresponding Tb of 1.8±1.2°C. Below Tcrit, animals began to thermoregulate, as indicated by a considerable but disproportionate increase in both fH and O2. The maximum increase in fH was only 4-fold greater than the average thermoconforming minimum, compared with a 46-fold increase in O2. The differential response of fH and O2 to low Ta was reflected in a 15-fold increase in oxygen delivery per heart beat (cardiac oxygen pulse). During torpor at low Ta, thermoregulating bats maintained a relatively slow fH and compensated for increased metabolic demands by significantly increasing stroke volume and tissue oxygen extraction. Our study provides new information on the relationship between metabolism and fH in an unstudied physiological state that may occur frequently in the wild and can be extremely costly for heterothermic animals.

INTRODUCTION

Extremely low ambient temperatures (Ta) create an energetic hurdle for many animals, particularly small endotherms that must markedly increase heat production to maintain a constant high body temperature (Tb). Torpor, a controlled reduction of metabolic rate (MR), heart rate (fH) and Tb, is therefore critical for many small mammals to save energy during inclement conditions (Ruf and Geiser, 2015). During torpor, the hypothalamic control of temperature regulation is adjusted to a new, lower minimum Tb that is regulated at or just above a critical external temperature (Tcrit). Depending on ambient conditions, animals will thermoconform down to this Tb/Ta (Heller, 1979; Geiser, 2011). However, when Ta falls below Tcrit, animals can either rewarm entirely to a normothermic Tb or remain torpid and increase metabolic heat production to thermoregulate and defend minimum Tb.

Details regarding thermoregulation during torpor at sub-zero temperatures are scant and restricted to medium-sized northern mammals such as ground squirrels (Geiser and Kenagy, 1988; Buck and Barnes, 2000; Richter et al., 2015). However, small hibernators, such as temperate insectivorous bats, often use torpor year round (Eisentraut, 1956; Davis and Reite, 1967; Masing and Lutsar, 2007; Wojciechowski et al., 2007). Thermoregulation during torpor, while still conserving substantial amounts of energy, can be expensive when used over long periods and against a large TbTa differential (Karpovich et al., 2009; Richter et al., 2015). At extremely low Ta, defending Tb above Tcrit is likely to require an alteration of a number of physiological components essential to thermoregulation. The cardiovascular system is vital for maintaining physiological processes during torpor as the heart regulates the supply of blood gases, nutrients and hormones to the body. While there are data on the thermal energetics of torpor at low Ta, detailed information on cardiac function and its relationship to metabolism are entirely lacking.

During torpor at low Tb, the heart must facilitate adequate blood supply under conditions of reduced blood flow and slow ventilation/low oxygen intake (Milsom et al., 2001). In addition, to minimise metabolic costs during torpor, adequate perfusion is only retained in vital tissues and organs as a result of circulatory adjustments (Carey et al., 2003). The hearts of hibernating animals are adapted to function at low Tb and are capable of withstanding temperatures well below the critical point for fibrillation and death in non-hibernating species (Lyman and Blinks, 1959; van Veen et al., 2008). It is widely known that during torpor in thermoconforming hibernators, the heart continues to beat in a co-ordinated rhythm and is resistant to detrimental arrhythmia (Johansson, 1996; van der Heyden and Opthof, 2005). However, when animals are forced to thermoregulate during torpor, the cardiovascular system must adjust to coincide with changes in metabolism.

We investigated the thermal physiology, in particular the interrelationship between fH and MR below and above Tcrit, for torpid Chalinolobus gouldii, a common tree-dwelling bat whose distribution extends across Australia (Churchill, 2008). While temperate zone northern hemisphere bats tend to hibernate in thermally stable caves or mines during winter, many southern hemisphere bats hibernate in tree hollows or under bark. These roosts are thermally labile and often experience sub-zero Ta (Law and Chidel, 2007; Turbill, 2008; Clement and Castleberry, 2013; Stawski and Currie, 2016). Information concerning cardiac function and its relationship to metabolism and thermal energetics in this species, or at such low temperatures in any bat, is entirely lacking. Moreover, explicit investigations of Tcrit and thermogenic capacity in hibernating bats are extremely limited (Reite and Davis, 1966; Stawski and Geiser, 2011).

Given that C. gouldii regularly experience low Ta, we hypothesised that the Tcrit for this species would be close to 0°C. As is the case for other hibernators, we predicted that fH would be substantially reduced during steady-state torpor, with correspondingly low Tb and MR, until Ta reached Tcrit. However, below Tcrit, we hypothesised that bats would increase MR with a proportionate increase in fH, but maintain a steady Tb.

MATERIALS AND METHODS

Six non-reproductive Chalinolobus gouldii (Gray 1841) (3 females and 3 males; capture mass 14.3±2.6 g) were collected from a residential roof near the University of New England (UNE) in Armidale, NSW, Australia, during the austral autumn (April) 2015. Individuals were kept in outdoor aviaries at UNE for 3 months and measurements were conducted from May to July 2015 (austral late autumn/winter). Food (Tenebrio molitor larvae dusted with a supplement of Wombaroo™ Insectivore Mix) and water were provided ad libitum. Prior to release at their capture site, bats were fitted with temperature-sensitive radio transmitters as part of another study (Stawski and Currie, 2016).

Respirometry and electrocardiogram measurements

In the late afternoon, bats were placed in air-tight respirometry chambers in a temperature-controlled cabinet where they remained overnight and into the following day(s). All individuals were exposed to Ta between −2 and 24°C over an experimental period of 8 weeks. Animals were not given access to food or water during respirometry measurements to ensure they were post-absorptive. Bats were weighed to the nearest 0.1 g before and after measurements. The Ta in the chamber was recorded to the nearest 0.1°C using a calibrated thermocouple placed 5 mm within the chamber and read using a digital thermometer.

Respirometry chambers (0.53 l) were made from modified polycarbonate enclosures with clear lids, lined with a small patch of hessian cloth (burlap) from which the bats could roost. Air flow through the chambers was controlled by rotameters and measured with mass flowmeters (Omega FMA-5606, Stamford, CT, USA) that were calibrated prior to the start of experimentation. Flow rate (200 ml min−1) was adjusted to ensure that 99% equilibrium was reached within 12 min. Oxygen concentration was measured using a FC-1B Oxygen Analyser (Sable Systems International Inc., Las Vegas, NV, USA; resolution 0.001%). Measurements were taken from the chamber every minute for 15 min and then switched to outside air for reference readings (3 min) using solenoid valves. Digital outputs of the oxygen analyser, flowmeter and digital thermometer were recorded on a PC using custom-written data-acquisition software (G. Körtner, Centre for Behavioural and Physiological Ecology, Zoology, University of New England, Australia). O2 values were calculated per minute using standardised gas volumes and eqn 3a of Withers (1977), assuming a respiratory quotient of 0.85.

Electrocardiograms (ECG) were recorded from two leads attached to adhesive electrodes on the forearms of each bat following the methods of Currie et al. (2014). Attachment of the electrodes took place once bats were torpid, either following lights on in the morning or during the night following rectal Tb measurements. All bats returned to torpor within 1 h of lead attachment. ECGs were recorded using LabChart v7.3 software and analysed to determine fH by calculating instantaneous fH per second.

Ta above 0°C

For measurements of steady-state torpor of thermoconforming bats at mild Ta, bats were placed in respirometry chambers overnight at Ta between 5 and 15°C. Following ECG lead attachment and a return to steady-state torpor, the Ta of the chamber was increased by 2°C and once the new Ta was reached, this was left unchanged for at least 2 h. The Ta was then progressively increased until animals rewarmed, in response to either changing Ta or the stimulus of lights-off in the evening.

Ta below 0°C

Bats were placed in respirometry chambers overnight at a Ta of between 1 and 5°C. Following lights on and/or the attachment of ECG leads, Ta was progressively decreased in 1°C increments when it was below 3°C. Individuals were exposed to each temperature for a minimum of 2 h before the temperature was further reduced. Previous data (Reite and Davis, 1966; S.E.C., personal observation) suggest that swift reductions in Ta (≥3–5°C) often induce arousal from hibernation. Therefore, we took care to ensure Ta was gradually reduced to obtain an accurate representation of thermoregulatory Tcrit and minimum Ta prior to spontaneous arousal. The minimum temperature reached before animals regularly aroused was −2°C, and therefore no measurements were taken below this temperature. All individuals were exposed to the same range of Ta during experimentation.

Tb measurements

Tb was measured to the nearest 0.1°C using a digital thermometer (Omega, HH-71T) and a calibrated thermocouple probe inserted ∼2 cm into the rectum. Rectal Tb was recorded for animals that had been torpid for at least 2 h, indicated by a low MR and fH. We measured Tb within 1 min of removing the bat from the respirometry chamber and, as the rate of rewarming from torpor is initially slow, rectal Tb was considered to be within 0.5°C of Tb prior to the disturbance. Following rewarming from torpor, in all experiments bats were returned to aviaries where food was available ad libitum.

Analysis and statistics

Bats were considered torpid when O2 fell to less than 75% of the resting MR at the same Ta. During steady-state torpor, fH and O2 were averaged over the same time period, for at least 30 min, corresponding to minimum torpid O2. Bats were deemed to be thermoconforming when Tb was within 2°C of Ta. When Tb was not known, individuals were considered to be thermoconforming when O2 values fell to, or below, those of bats with a Tb within 2°C of Ta, at the same Ta. The Q10 for O2 or fH of thermoconforming torpid bats was calculated using the following equation:
formula
(1)
where R is the O2 or fH at a particular Ta (1 or 2). Animals were considered to be thermoregulating in torpor when minimum O2 was at least double that of minimum average thermoconforming values at the same Ta. Oxygen pulse (OP), the oxygen delivery per heart beat, was calculated for torpid bats by dividing the average O2 (ml min−1) by the corresponding average fH (beats min−1). The percentage contribution of fH to increases in oxygen transport in thermoregulating bats was calculated following Bartholomew and Tucker (1963);
formula
(2)
using fH and OP at Ta below 1°C (Ta,1=0.5°C; Ta,2=−2.1°C).

We calculated average resting fH (fH,rest) and O2 from the period following arousal for at least 5 min when O2 had fallen to ≤75% of maximum O2 at the peak of rewarming. Unfortunately, during arousal at low Ta, shivering associated with rewarming caused artefact on ECG recordings, making calculation of fH extremely difficult. In addition, animals often moved during the final stages of rewarming, which resulted in detachment of ECG electrodes and cessation of fH recording. Therefore, data for fH,rest were only available for three bats across five Ta.

All statistical analyses were performed using R v3.1.3 (http://www.R-project.org/), assuming a significance level of P<0.05. Means are presented ±s.d. for number of animals (n) and number of observations (N). We used analysis of covariance (ANCOVA) to assess whether minimum O2 was significantly different following disturbance associated with ECG lead attachment. Linear mixed effects models (nlme package; https://CRAN.R-project.org/package=nlme) were used to assess the relationship between fH or O2 and Ta in thermoregulating torpid bats and resting bats. To determine whether there were any differences in slope between regressions for resting and thermoregulating O2 with respect to Ta, we performed an ANCOVA using nlme with individual as a random factor. For comparison between the slopes of fH and O2, we log transformed the data prior to ANCOVA using nlme, again with individual as a random factor. Standardised major axis regressions were performed (smatr package; Warton et al., 2012) to assess the correlation between O2 and fH when animals were either thermoconforming or thermoregulating during torpor. We used ANCOVA in smatr to determine whether there was a significant difference in the slopes of fH against O2 between the two torpid states. For all analyses, pseudo-replication was accounted for by using the degrees of freedom from mixed effect linear modelling, which were adjusted for repeated measures. We included sex as an interaction term for all linear models and it was found to have no significant effect; therefore, the data were pooled for further analyses.

All procedures were approved by the University of New England Animal Ethics Committee and New South Wales National Parks and Wildlife Service. Data are available from the Dryad digital repository (Currie et al., 2017; doi:10.5061/dryad.jr74d).

RESULTS

Rest

At rest, following arousal from torpor, O2 and fH of normothermic bats increased with decreasing Ta in a qualitatively similar linear pattern (Fig. 1A,B). Resting O2 ranged from 1.44 to 10.04 ml g−1 h−1 as Ta fell from 24.1 to −0.4°C (Fig. 1A; n=6, N=81). Over a similar Ta range (19.4–1.7°C), the values we were able to obtain for fH,rest increased from 412 to 698 beats min−1 (Fig. 1B; n=3, N=6); however, the relationship between fH and Ta was not statistically significant when animal was included as a random factor (nlme; r2=0.96, P=0.07).

Fig. 1.

Oxygen consumptionand heart ratefor Chalinolobus gouldii at rest and during torpor. Oxygen consumption (O2, A) and heart rate (fH, B) are shown as a function of ambient temperature (Ta) for normothermic individuals at rest [white circles, solid line; O2=9.69−0.27(Ta), r2=0.91, P<0.001; O2 n=6 animals, N=17 observations, fHn=3, N=6] and during torpor when thermoconforming (black circles, n=6; O2 N=62, fHN=54) or thermoregulating (grey circles, n=6; O2 N=18, fHN=15).

Fig. 1.

Oxygen consumptionand heart ratefor Chalinolobus gouldii at rest and during torpor. Oxygen consumption (O2, A) and heart rate (fH, B) are shown as a function of ambient temperature (Ta) for normothermic individuals at rest [white circles, solid line; O2=9.69−0.27(Ta), r2=0.91, P<0.001; O2 n=6 animals, N=17 observations, fHn=3, N=6] and during torpor when thermoconforming (black circles, n=6; O2 N=62, fHN=54) or thermoregulating (grey circles, n=6; O2 N=18, fHN=15).

Thermoconforming torpid bats

All bats entered torpor at all Ta and reached low O2 and fH values indicative of steady-state torpor within 3 h of disturbance associated with ECG lead attachment. There was no impact of this disturbance evident, as minimum O2 following ECG lead attachment was not significantly different from the minimum O2 beforehand (P=0.85). Below Ta of 20°C, most individuals remained torpid until the lights went off, except for a few times when individuals hibernated for up to 48 h or rewarmed in response to low Ta. Importantly, all bats were capable of spontaneously rewarming from the lowest Ta they were exposed to in our study.

When bats were in steady-state torpor and thermoconforming, O2 (n=6, N=62) and fH (n=6, N=54) tracked Ta in a curvilinear manner. Tb fell to within 1.0±0.9°C of Ta in thermoconforming individuals (n=6, N=20). The minimum average O2 of thermoconforming bats was 0.02±0.01 ml g−1 h−1 (n=6, N=9) recorded at a Ta of 2.1±0.3°C, which was <1% of resting values at a similar Ta (1.4±0.5°C, O2=10.23±1.58 ml g−1 h−1, n=2, N=2). fH fell to an absolute minimum of 3 beats min−1 at Ta=1.0°C while the average minimum torpor fH (fH,torpor) was 8±2 beats min−1 (n=6, N=7) and only 1.1% of fH,rest recorded at 1.4°C (698 beats min−1, n=1, N=1). Even when bats were torpid at mild Ta (19.6°C), fH and O2 were <11% of the corresponding resting rates (fH,torpor=10.3%; O2=3.7%). When O2 was plotted against fH in thermoconforming individuals, there was a significant positive linear correlation (r2=0.88, P<0.001; Fig. 2). On occasion, animals maintained a low O2 and fH when Ta fell below 1°C and one animal thermoconformed down to a Ta of −1°C with a Tb of 0.6°C and a corresponding O2 equal to 0.03 ml g−1 h−1 and fH of 7 beats min−1.

Fig. 2.

The relationship between fH andO2 in torpid C. gouldii. Data are given for thermoconforming individuals [O2=0.004(fH)−0.016, r2=0.88, P<0.001; n=6, N=54] or thermoregulating individuals [white circles, dashed line; O2=0.039(fH)−0.272, r2=0.95, P<0.001; n=6, N=15].

Fig. 2.

The relationship between fH andO2 in torpid C. gouldii. Data are given for thermoconforming individuals [O2=0.004(fH)−0.016, r2=0.88, P<0.001; n=6, N=54] or thermoregulating individuals [white circles, dashed line; O2=0.039(fH)−0.272, r2=0.95, P<0.001; n=6, N=15].

The transport of oxygen per heart beat (OP) was qualitatively similar to fH and O2 in bats during steady-state torpor; however, it declined only slightly with decreasing Ta down to 2°C (n=6, N=53; Fig. 3). The Q10 for O2 was 3.8 in thermoconforming bats, determined between a Ta of 19.6 and 2.1°C. When compared with the basal MR previously determined for this species (1.44 ml g−1 h−1; Hosken and Withers, 1997), average Q10 was also 3.8. However, the corresponding Q10 for fH during torpor in thermoconforming bats was only 2.6.

Fig. 3.

Oxygen pulse (OP) as a function of Ta in torpid C. gouldii. Data are given for thermoconforming (n=6, N=54) or thermoregulating (n=6, N=15) individuals. The relationship with Ta changes from curvilinear (thermoconforming) to linear when animals begin thermoregulating [solid line; OP=0.0045−0.0021(Ta), r2=0.87, P<0.001].

Fig. 3.

Oxygen pulse (OP) as a function of Ta in torpid C. gouldii. Data are given for thermoconforming (n=6, N=54) or thermoregulating (n=6, N=15) individuals. The relationship with Ta changes from curvilinear (thermoconforming) to linear when animals begin thermoregulating [solid line; OP=0.0045−0.0021(Ta), r2=0.87, P<0.001].

Thermoregulating torpid bats

Below a Tcrit of 0.7±0.4°C (n=6, N=7), both fH and O2 substantially increased and bats began thermoregulating (Fig. 1A,B). Average Tb at Tcrit was 1.8±1.2°C (n=5, N=5), and as Ta fell to −2°C, bats defended Tb at 2.3±1.6°C (n=2, N=2). The majority of animals rewarmed spontaneously when Ta fell below −1.3°C; however, one animal remained torpid down to a Ta of −2.1°C (O2=0.61 ml g−1 h−1; fH=22 beats min−1). Thermoregulating torpid bats exposed to a Ta below zero exhibited up to a 46-fold increase in O2 when compared with the minimum O2 during torpor at a Ta of 2°C (n=6, N=15). In contrast, fH in torpid thermoregulating bats only increased on average 2-fold (range 1- to 4-fold) over the same Ta range (n=6, N=15). The maximum fH recorded in thermoregulating torpid bats was 31 beats min−1 at Ta −1.1°C with a corresponding O2 of 0.93 ml g−1 h−1.

There was a significant linear correlation between fH and O2 in thermoregulating bats (r2=0.95, P<0.001) and this was significantly steeper than the relationship for thermoconforming bats at a Ta greater than 1°C (r2=0.88, P<0.001; ANCOVA, P<0.001; Fig. 2). Both O2 and fH increased linearly with decreasing Ta in thermoregulating individuals (fH, r2=0.71, P<0.01; MR, r2=0.61, P<0.01; Figs 4 and 5). However, following log transformation, O2 showed a significantly steeper response to declining Ta than fH (ANCOVA; P<0.05; Fig. 6). This disproportionate increase in fH and O2 was also evident via a significant linear increase in OP as Ta fell below ∼1°C (r2=0.89, P<0.001; Fig. 3).

Fig. 4.

O2 as a function ofTa in thermoregulating C. gouldii. Data are for bats that are normothermic at rest [O2=9.69−0.27(Ta), r2=0.91, P<0.001; n=6, N=17] or thermoregulating during torpor [inset; O2=0.31−0.21(Ta), r2=0.62, P<0.01; n=6, N=18]. There was no significant difference between the slopes of O2 and Ta in resting versus thermoregulating torpid individuals (ANCOVA; P=0.63).

Fig. 4.

O2 as a function ofTa in thermoregulating C. gouldii. Data are for bats that are normothermic at rest [O2=9.69−0.27(Ta), r2=0.91, P<0.001; n=6, N=17] or thermoregulating during torpor [inset; O2=0.31−0.21(Ta), r2=0.62, P<0.01; n=6, N=18]. There was no significant difference between the slopes of O2 and Ta in resting versus thermoregulating torpid individuals (ANCOVA; P=0.63).

Fig. 5.

fH as a function of Ta in thermoregulating C. gouldii. Data are for bats that are normothermic at rest (n=3, N=6) or thermoregulating during torpor [inset; fH=14.27−5.72(Ta), r2=0.71, P<0.01; n=6, N=15].

Fig. 5.

fH as a function of Ta in thermoregulating C. gouldii. Data are for bats that are normothermic at rest (n=3, N=6) or thermoregulating during torpor [inset; fH=14.27−5.72(Ta), r2=0.71, P<0.01; n=6, N=15].

Fig. 6.

fH andO2 as a function of Ta in thermoregulating C. gouldii during torpor. log10-transformed fH [log10fH=1.11−0.18(Ta), r2=0.82, P<0.001; n=6, N=15] and O2 [log10O2=−0.71−0.37(Ta), r2=0.71, P<0.001; n=6, N=18] increased disproportionately with decreasing Ta. O2 increased at a greater rate than fH below 1°C.

Fig. 6.

fH andO2 as a function of Ta in thermoregulating C. gouldii during torpor. log10-transformed fH [log10fH=1.11−0.18(Ta), r2=0.82, P<0.001; n=6, N=15] and O2 [log10O2=−0.71−0.37(Ta), r2=0.71, P<0.001; n=6, N=18] increased disproportionately with decreasing Ta. O2 increased at a greater rate than fH below 1°C.

There was an almost 15-fold increase in average OP from 2°C to −2°C (5.5×10−4 ml O2 beat−1 to 80×10−4 ml O2 beat−1, respectively) and the contribution of fH to changes in oxygen transport was minimal, at only 31% (calculated using Eqn 2). Interestingly, the slope of the relationship between O2 and Ta, an indicator of thermal conductance, did not differ between thermoregulating torpid and resting bats (ANCOVA; P=0.63; Fig. 4).

DISCUSSION

While thermoregulating in torpor below Tcrit, animals must not only produce enough heat to maintain the gradient between Tb and Ta but also ensure adequate cardiovascular function to support thermogenic needs. Our study is the first to provide simultaneously recorded data of metabolism and cardiac function in normothermic and torpid bats exposed to Ta at or below 0°C. Confirming our hypothesis, we found that torpid C. gouldii are capable of coping with low Tb while maintaining coordinated cardiac function. While our sample size is limited, we report decidedly lower minimum fH values than anticipated for animals this size, which compare to torpid fH values found in much larger hibernators (Lyman, 1951; Augee and Ealey, 1968; Swoap et al., 2017). We also established a new Tcrit for thermoregulation of around 1°C, substantially lower than previous estimates for this species (Kulzer et al., 1970; Hosken and Withers, 1997). Below Tcrit, animals began to defend Tb by increasing metabolic heat production and fH, and all animals in our study were able to rewarm from the lowest Ta (−2°C).

When animals were exposed to Ta below 1°C, there was a disproportionate response of the cardiovascular and metabolic systems as O2 increased at a greater rate than fH to support elevated heat production. The bats we studied increased oxygen consumption by 46-fold, compared with a maximum increase of only 4-fold in fH. During this phase, Tb remained low and therefore the heart was cold. Although hibernators' hearts are capable of withstanding very low Tb, muscle contractility and rate are still limited by temperature (Michael and Menaker, 1963; Smith and Katzung, 1966; South and Jacobs, 1973). In addition, it is possible that excessive cardiac stimulation and fH acceleration at low Tb could induce detrimental arrhythmias. Therefore, changes in circulation and the relationship between fH and O2 must be altered to maintain adequate supply under increasingly demanding conditions as Ta falls. In particular, the increased needs of the tissues must be met by an increase in oxygen delivery per heart beat or via increased oxygen extraction rates. Our results are the first to demonstrate a substantial upward shift in oxygen pulse during this phase of almost 15-fold, with fH only contributing to 31% of the elevated oxygen supply to tissues. Accordingly, we are also the first to illustrate a significant difference in relationship between fH and O2 for thermoregulating torpid individuals compared with thermoconforming individuals with a greater O2 at almost every fH recorded for thermoregulating bats.

The novel finding of our data is the differential response of the cardiovascular and metabolic systems to thermogenic requirements at low Ta, which would be unknown had we not recorded these variables simultaneously. Following the Fick equation [O2 (ml min−1)=fH×SV×(CaO2CvO2)], the significant change in oxygen delivery per beat that we recorded for thermoregulating C. gouldii must be the result of substantial increases in stroke volume (SV) and/or oxygen uptake by the tissues (arteriovenous difference in oxygen content; CaO2CvO2). Because of the difficulties associated with the measurement of SV and blood characteristics, data for these variables in hibernating animals remain scarce with virtually none available for bats in any physiological state. Under the assumption that SV increases to its maximum capacity (∼0.03 ml min−1, calculated using eqn 7 of Bishop, 1997; using the heart mass reported for C. gouldii from Bullen et al., 2009), we calculate that arteriovenous difference must increase to a maximum of 26.23 ml O2 dl−1 blood at −2°C. This value is approaching maximal tissue oxygen extraction capacity, and is similar to oxygen extraction rates thought to occur during extreme exercise and flight (Bishop, 1997). To achieve such high levels of oxygen extraction, haemoglobin (Hb) content of the blood must be at least 21 g dl−1 blood and 100% saturated in thermoregulating torpid bats. Previous reports of Hb content of bat blood vary, with only one report for hibernating individuals (Wołk and Bogdanowicz, 1987); however, values ≥20 g dl−1 blood have been reported in resting bats and would suggest our results are not outside the expectations for oxygen carrying capacity in these animals (Jürgens et al., 1981; Arévalo et al., 1987; Bishop, 1997). In the light of our findings for oxygen pulse, and in line with these calculations, our results suggest that both SV and tissue oxygen extraction approach maximal capacity when bats are thermoregulating during torpor at sub-zero Ta.

There remains some debate as to the control mechanisms behind the cardiac rhythms we see during torpor (Lyman and O'Brien, 1963; Milsom et al., 1999; Zosky and Larcombe, 2003; Braulke and Heldmaier, 2010). Although the autonomic nervous system plays an essential role in reducing fH at the onset of torpor, at low Tb nervous input is reduced, partially related to the dampening effects of temperature on nerve function but also probably as a result of withdrawal of control (Milsom et al., 2001). While we did not directly measure neurotransmitters or experimentally test for nerve response at the heart, our results suggest that once thermoconforming bats are in steady-state torpor and the heart is cold, temperature becomes the driving factor behind the patterns that we observe. This is supported by the Q10 values of fH in thermoconforming bats and also in part by the limited response of fH to decreasing Ta in thermoregulating torpid animals. While nervous control is not entirely withdrawn, we interpret our results to suggest that during thermoregulation in torpid bats at low Tb, excessive cardiac stimulation is probably inhibited to protect against arrhythmia.

Additionally, the change in relationship between fH and O2 could also be the result of an alteration in blood supply across the body. During torpor, circulation to the periphery is restricted with perfusion of only vital organs such as the heart, lungs and brain retained (Lyman and O'Brien, 1963). This restriction of blood flow results in dramatic changes in blood pressure, enabling animals to maintain sufficient supply to tissues at low Tb when blood viscosity is increased (Lyman et al., 1982). It is possible that when thermoregulating during torpor, animals increase blood supply to essential thermogenic organs, such as brown adipose tissue or muscle, to defend Tb against increasing differentials with Ta. This may enable individuals not only to remain torpid for longer but also to rewarm swiftly should the external temperature drop below a level they are capable of withstanding. It would be interesting to investigate how blood flow during this phase is altered, and whether the increased peripheral resistance and restriction of circulation is partially withdrawn. Our results show that in thermoregulating torpid bats, thermal conductance is similar to that in resting bats, as reported for other heterotherms (Henshaw, 1968; Geiser, 2004), and this may be the result of such changes in blood flow. We have previously suggested that this may be the case in bats during passive rewarming (Currie et al., 2015), and it may be that changes in blood flow influence the relationship between metabolism and heart rate when animals are thermoregulating during torpor.

When thermoregulation is activated during torpor, although it still conserves substantial energy compared with normothermic thermoregulation at low Ta (99% less at −1°C), it is more energetically demanding than thermoconforming during torpor (∼26-fold greater at −1°C than 2°C). When arctic ground squirrels (Urocitellus parryii) and golden-mantled ground squirrels (Callospermophilus lateralis and Callospermophilus saturatus) were forced to thermoregulate during hibernation at decreasing Ta (down to −30°C), the frequency of spontaneous arousals increased and the amount of body mass lost during the hibernation season almost doubled compared with free-living animals (Geiser and Kenagy, 1988; Richter et al., 2015). Smaller hibernators are unlikely to be able to cope with such large TbTa differentials during torpor because of their larger surface area to volume ratios and comparatively higher costs of thermoregulation. We show that small bats rewarmed before Ta fell below −3°C, a TbTa differential of only a few degrees. It is likely that more frequent arousals and a rapid depletion of fat stores occurs in small hibernating mammals exposed to very low temperatures for long periods.

However, it has been suggested that the comparative costs of arousals are reduced when animals are exposed to decreasing Ta, as the costs of maintaining Tb during torpor increase (Karpovich et al., 2009; Richter et al., 2015). However, unlike rodents that hibernate in thermally stable hibernacula, C. gouldii roost in thermally labile environments, such as tree roosts and buildings (Lumsden et al., 2002). The individuals we studied were originally removed from the roof of a building and were radio-tracked to tree roosts for at least 17 days following their release (Stawski and Currie, 2016). In these circumstances, bats may be able to passively rewarm, thus mitigating much of the cost of arousal from low Ta/Tb. Indeed, we found evidence of passive rewarming in these same individuals when radio-tracked following their release, with 83% of all recorded arousals during winter involving passive rewarming (Stawski and Currie, 2016). Although poorly insulated roosts mean that animals may experience temperatures below their torpor Tcrit, the ability to passively rewarm may outweigh some costs of thermoregulation during torpor.

Red bats (Lasiurus borealis) are also a tree-dwelling species and continue to remain in thermally labile roosts even though the Ta may often fall below freezing (Mormann and Robbins, 2007). When exposed to Ta below 0°C during torpor, L. borealis showed a similar range of fH to that of C. gouldii in this study, increasing from an average of 12 beats min−1 at 5°C to 25–40 beats min−1 at −2°C (Reite and Davis, 1966). Like C. gouldii, L. borealis also take advantage of mild winter days and passively rewarm (Dunbar and Tomasi, 2006), but have been found to remain torpid unless external temperatures reach at least 15°C (Davis and Lidicker, 1956; Davis, 1970). The physiological similarities between L. borealis and C. gouldii with regard to their response to low Ta during torpor probably reflect similar roosting habits over winter. In contrast, little brown bats (Myotis lucifugus), which overwinter in stable environments, responded to decreasing Ta by increasing fH to ∼100 beats min−1 at −2°C (Reite and Davis, 1966). However, these data may be more indicative of animals initiating or attempting the arousal process, as Reite and Davis (1966) note that after 4 h, animals kept at −5°C became hypothermic and died. This suggests that bats which hibernate in unstable microclimates are more likely to remain torpid as Ta falls below zero, while bats that overwinter in thermally stable places are more likely to arouse as they can change roost location to select warmer areas within the cave to avoid excessively low temperatures. Our results show that Tcrit is probably a response of selection related to the long-term environmental temperatures of their habitat and can be close to zero, even though sub-zero temperatures may only be experienced for a few days throughout the year.

Conclusion

Our data provide fundamental information regarding the costs of thermoregulation while in torpor, which has implications for understanding energy budgets of heterothermic animals that regularly experience low Ta in the wild. Disturbances such as sound (Speakman et al., 1991), smoke (Stawski et al., 2015) and disease (Verant et al., 2014) have all been shown to increase Tb and therefore metabolic rate during torpor (Geiser, 2004), as well as inducing arousal. Increased arousal frequency during winter has been the cause of devastating losses of bats infected with white nose syndrome (Willis, 2017), and this could possibly be the result of increased metabolic rate during torpor. As animals are increasingly susceptible to disturbances associated with anthropogenic activity and climate change, it is crucial that we understand the costs associated with thermoregulation during torpor as our data show that it is distinctly different from torpor when bats are thermoconforming. Our results suggest that while fH is limited by low Tb and the heart continues to beat relatively slowly, other aspects of the cardiovascular system must be increased to near-maximal levels to supply sufficient oxygen during this demanding phase. An increased incidence of thermoregulation during torpor in the wild could have dramatic implications for survival in many heterothermic species, and this is an area of great interest that is virtually unstudied in nature.

Acknowledgements

We thank Heidi Kolkert for her help with capturing bats, Anna Doty for her assistance with animal care and Brian Shaw for access to his property. We also appreciate the insights of Bill Milsom and Philip Currie on the study and their constructive comments on the manuscript.

Footnotes

Author contributions

Conceptualization: S.E.C., C.S.; Methodology: S.E.C., C.S., F.G.; Validation: S.E.C.; Formal analysis: S.E.C., C.S.; Investigation: S.E.C.; Resources: C.S., F.G.; Data curation: S.E.C.; Writing - original draft: S.E.C.; Writing - review & editing: S.E.C., C.S., F.G.; Visualization: S.E.C.; Supervision: F.G.; Project administration: S.E.C., F.G.; Funding acquisition: S.E.C., F.G.

Funding

This research was supported by an Alexander von Humboldt-Stiftung Postdoctoral Research Fellowship awarded to S.E.C., an Australian Research Council Discovery Early Career Researcher Award to C.S., and a grant from the Australian Research Council awarded to F.G.

Data availability

Data are available from the Dryad digital repository: http://datadryad.org/resource/doi:10.5061/dryad.jr74d.

References

Arévalo
,
F.
,
Pérez-Suárez
,
G.
and
López-Luna
,
P.
(
1987
).
Hematological data and hemoglobin components in bats (Vespertilionidae)
.
Comp. Biochem. Physiol.
88A
,
447
-
450
.
Augee
,
M. L.
and
Ealey
,
E. H. M.
(
1968
).
Torpor in the Echidna, Tachyglossus aculeatus
.
J. Mammal.
49
,
446
-
454
.
Bartholomew
,
G. A.
and
Tucker
,
V. A.
(
1963
).
Control of changes in body temperature, metabolism, and circulation by the Agamid lizard, Amphibolurus barbatus
.
Physiol. Zool.
36
,
199
-
218
.
Bishop
,
C. M.
(
1997
).
Heart mass and the maximum cardiac output of birds and mammals: implications for estimating the maximum aerobic power input of flying animals
.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
352
,
447
-
456
.
Braulke
,
L. J.
and
Heldmaier
,
G.
(
2010
).
Torpor and ultradian rhythms require an intact signalling of the sympathetic nervous system
.
Cryobiology
60
,
198
-
203
.
Buck
,
C. L.
and
Barnes
,
B. M.
(
2000
).
Effects of ambient temperature on metabolic rate, respiratory quotient and torpor in an arctic hibernator
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
279
,
R255
-
R262
.
Bullen
,
R. D.
,
McKenzie
,
N. L.
,
Bullen
,
K. E.
and
Williams
,
M. R.
(
2009
).
Bat heart mass: correlation with foraging niche and roost preference
.
Aust. J. Zool.
57
,
399
-
408
.
Carey
,
H. V.
,
Andrews
,
M. T.
and
Martin
,
S. L.
(
2003
).
Mammalian hibernation: cellular and molecular responses to depressed metabolism and low temperature
.
Physiol. Rev.
83
,
1153
-
1181
.
Churchill
,
S.
(
2008
).
Australian Bats
, 2nd edn.
New South Wales
:
Allen and Unwin
.
Clement
,
M. J.
and
Castleberry
,
S. B.
(
2013
).
Tree structure and cavity microclimate: implications for bats and birds
.
Int. J. Biometeorol.
57
,
437
-
450
.
Currie
,
S. E.
,
Körtner
,
G.
and
Geiser
,
F.
(
2014
).
Heart rate as a predictor of metabolic rate in heterothermic bats
.
J. Exp. Biol.
217
,
1519
-
1524
.
Currie
,
S. E.
,
Noy
,
K.
and
Geiser
,
F.
(
2015
).
Passive rewarming from torpor in hibernating bats: minimizing metabolic costs and cardiac demands
.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
308
,
R34
-
R41
.
Currie
,
S. E.
,
Stawski
,
C.
and
Geiser
,
F.
(
2017
).
Data from: Cold-hearted bats: uncoupling of heart rate and metabolism during torpor at subzero temperatures
.
Dryad Digital Repository
.
Davis
,
W. H.
(
1970
).
Hibernation: ecology and physiological ecology
. In
Biology of Bats
(ed.
W.
Wimsatt
), pp.
265
-
294
.
New York
:
Academic Press
.
Davis
,
W. H.
and
Lidicker
,
W. Z.
(
1956
).
Winter range of the red bat, Lasiurus borealis
.
J. Mammal.
37
,
280
-
281
.
Davis
,
W. H.
and
Reite
,
O. B.
(
1967
).
Responses of bats from temperate regions to changes in ambient temperature
.
Biol. Bull.
132
,
320
-
328
.
Dunbar
,
M. B.
and
Tomasi
,
T. E.
(
2006
).
Arousal patterns, metabolic rate, and an energy budget of eastern red bats (Lasiurus borealis) in winter
.
J. Mammal.
87
,
1096
-
1102
.
Eisentraut
,
M.
(
1956
).
Der Winterschlaf mit seinen ökologischen und physiologischen Begleiterscheinungen
.
Jena
:
G. Fisher
.
Geiser
,
F.
(
2004
).
Metabolic rate and body temperature reduction during hibernation and daily torpor
.
Annu. Rev. Physiol.
66
,
239
-
274
.
Geiser
,
F.
(
2011
).
Hibernation: Endotherms
.
Encyclopedia of Life Sciences
.
Hoboken, NJ
:
John Wiley & Sons, Ltd
.
Geiser
,
F.
and
Kenagy
,
G. J.
(
1988
).
Torpor duration in relation to temperature and metabolism in hibernating ground squirrels
.
Physiol. Zool.
61
,
442
-
449
.
Heller
,
H. C.
(
1979
).
Hibernation: neural aspects
.
Annu. Rev. Physiol.
41
,
305
-
321
.
Henshaw
,
R. E.
(
1968
).
Thermoregulation during hibernation: application of Newton's law of cooling
.
J. Theor. Biol.
20
,
79
-
90
.
Hosken
,
D. J.
and
Withers
,
P. C.
(
1997
).
Temperature regulation and metabolism of an Australian bat, Chalinolobus gouldii (Chiroptera: Vespertilionidae) when euthermic and torpid
.
J. Comp. Physiol. B Biochem. Syst. Environ. Physiol.
167
,
71
-
80
.
Johansson
,
B. W.
(
1996
).
The hibernator heart - nature's model of resistance to ventricular fibrillation
.
Cardiovasc. Res.
31
,
826
-
832
.
Jürgens
,
K. D.
,
Bartels
,
H.
and
Bartels
,
R.
(
1981
).
Blood oxygen transport and organ weights of small bats and small non-flying mammals
.
Respir. Physiol.
45
,
243
-
260
.
Karpovich
,
S. A.
,
Tøien
,
Ø.
,
Buck
,
C. L.
and
Barnes
,
B. M.
(
2009
).
Energetics of arousal episodes in hibernating arctic ground squirrels
.
J. Comp. Physiol. B Biochem. Syst. Environ. Physiol.
179
,
691
-
700
.
Kulzer
,
E.
,
Nelson
,
J. E.
,
McKean
,
J. L.
and
Möhres
,
F. P.
(
1970
).
Untersuchungen über die Temperaturregulation australischer Fledermäuse (Microchiroptera)
.
Z. Vergl. Physiol.
69
,
426
-
451
.
Law
,
B. S.
and
Chidel
,
M.
(
2007
).
Bats under a hot tin roof: comparing the microclimate of eastern cave bat (Vespadelus troughtoni) roosts in a shed and cave overhangs
.
Aust. J. Zool.
55
,
49
-
55
.
Lumsden
,
L. F.
,
Bennett
,
A. F.
and
Silins
,
J. E.
(
2002
).
Selection of roost sites by the lesser long-eared bat (Nyctophilus geoffroyi) and Gould's wattled bat (Chalinolobus gouldii) in south-eastern Australia
.
J. Zool.
257
,
207
-
218
.
Lyman
,
C. P.
(
1951
).
Effect of increased CO2 on respiration and heart rate of hibernating hamsters and ground squirrels
.
Am. J. Physiol.
167
,
638
-
643
.
Lyman
,
C. P.
and
Blinks
,
D. C.
(
1959
).
The effect of temperature on the isolated hearts of closely related hibernators and non-hibernators
.
J. Cell. Comp. Physiol.
54
,
53
-
63
.
Lyman
,
C. P.
and
O'Brien
,
R. C.
(
1963
).
Autonomic control of circulation during the hibernating cycle in ground squirrels
.
J. Physiol.
168
,
477
-
499
.
Lyman
,
C. P.
,
Willis
,
J. S.
,
Malan
,
A.
and
Wang
,
L. C. H.
(ed.) (
1982
).
Hibernation and Torpor in Mammals and Birds
.
New York
:
Academic Press
.
Masing
,
M.
and
Lutsar
,
L.
(
2007
).
Hibernation temperatures in seven species of sedentary bats (Chiroptera) in northeastern Europe
.
Acta Zool. Lituanica
17
,
47
-
55
.
Michael
,
C. R.
and
Menaker
,
M.
(
1963
).
The effect of temperature on the isolated heart of the bat Myotis lucifugus
.
J. Cell. Comp. Physiol.
62
,
355
-
358
.
Milsom
,
W. K.
,
Zimmer
,
M. B.
and
Harris
,
M. B.
(
1999
).
Regulation of cardiac rhythm in hibernating mammals
.
Comp. Biochem. Physiol. A Physiol.
124
,
383
-
391
.
Milsom
,
W. K.
,
Zimmer
,
M. B.
and
Harris
,
M. B.
(
2001
).
Vagal control of cardiorespiratory function in hibernation
.
Exp. Physiol.
86
,
791
-
796
.
Mormann
,
B. M.
and
Robbins
,
L. W.
(
2007
).
Winter roosting ecology of eastern red bats in southwest Missouri
.
J. Wildlife Manag.
71
,
213
-
217
.
Reite
,
O. B.
and
Davis
,
W. H.
(
1966
).
Thermoregulation in bats exposed to low ambient temperatures
.
Proc. Soc. Exp. Biol. Med.
121
,
1212
-
1215
.
Richter
,
M. M.
,
Williams
,
C. T.
,
Lee
,
T. N.
,
Tøien
,
Ø.
,
Florant
,
G. L.
,
Barnes
,
B. M.
and
Buck
,
C. L.
(
2015
).
Thermogenic capacity at subzero temperatures: how low can a hibernator go?
Physiol. Biochem. Zool.
88
,
81
-
89
.
Ruf
,
T.
and
Geiser
,
F.
(
2015
).
Daily torpor and hibernation in birds and mammals
.
Biol. Rev.
90
,
891
-
926
.
Smith
,
D. E.
and
Katzung
,
B.
(
1966
).
Mechanical performance of myocardium from hibernating and non-hibernating mammals
.
Am. Heart J.
71
,
515
-
521
.
South
,
F. E.
and
Jacobs
,
H. K.
(
1973
).
Contraction kinetics of ventricular muscle from hibernating and nonhibernating mammals
.
Am. J. Physiol.
225
,
444
-
449
.
Speakman
,
J. R.
,
Webb
,
P. I.
and
Racey
,
P. A.
(
1991
).
Effects of disturbance on the energy expenditure of hibernating bats
.
J. Appl. Ecol.
28
,
1087
-
1104
.
Stawski
,
C.
and
Currie
,
S. E.
(
2016
).
Effect of roost choice on winter torpor patterns of a free-ranging insectivorous bat
.
Aust. J. Zool.
64
,
132
-
137
.
Stawski
,
C.
and
Geiser
,
F.
(
2011
).
Do season and distribution affect thermal energetics of a hibernating bat endemic to the tropics and subtropics?
Am. J. Physiol. Regul. Integr. Comp. Physiol.
301
,
R542
-
R547
.
Stawski
,
C.
,
Matthews
,
J. K.
,
Körtner
,
G.
and
Geiser
,
F.
(
2015
).
Physiological and behavioural responses of a small heterothermic mammal to fire stimuli
.
Physiol. Behav.
151
,
617
-
622
.
Swoap
,
S. J.
,
Körtner
,
G.
and
Geiser
,
F.
(
2017
).
Heart rate dynamics in a marsupial hibernator
.
J. Exp. Biol.
220
,
2939
-
2946
.
Turbill
,
C.
(
2008
).
Winter activity of Australian tree-roosting bats: influence of temperature and climatic patterns
.
J. Zool.
276
,
285
-
290
.
van der Heyden
,
M. A. G.
and
Opthof
,
T.
(
2005
).
The hidden secrets of the hibernator's heart may protect against arrhythmias
.
Heart Rhythm
2
,
976
-
978
.
van Veen
,
T. A. B.
,
van der Heyden
,
M. A. G.
and
van Rijen
,
H. V. M.
(
2008
).
The secrets of hibernators’ cardiac conduction reserve
.
Heart Rhythm
5
,
1597
-
1598
.
Verant
,
M. L.
,
Meteyer
,
C. U.
,
Speakman
,
J. R.
,
Cryan
,
P. M.
,
Lorch
,
J. M.
and
Blehert
,
D. S.
(
2014
).
White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host
.
BMC Physiol.
14
:
10
.
Warton
,
D. I.
,
Duursma
,
R. A.
,
Falster
,
D. S.
and
Taskinen
,
S.
(
2012
).
smatr 3- an R package for estimation and inference about allometric lines
.
Methods Ecol. Evol.
3
,
257
-
259
.
Willis
,
C. K. R.
(
2017
).
Trade-offs influencing the physiological ecology of hibernation in temperate-zone bats
.
Integr. Comp. Biol.
,
icx087
.
Withers
,
P. C.
(
1977
).
Measurement of VO2, VCO2, and evaporative water loss with a flow-through mask
.
J. App. Physiol.
42
,
120
-
123
.
Wojciechowski
,
M. S.
,
Jefimow
,
M.
and
Tęgowska
,
E.
(
2007
).
Environmental conditions, rather than season, determine torpor use and temperature selection in large mouse-eared bats (Myotis myotis)
.
Comp. Biochem. Physiol. A Physiol.
147
,
828
-
840
.
Wołk
,
E.
and
Bogdanowicz
,
W.
(
1987
).
Hematology of the hibernating bat: Myotis daubentoni
.
Comp. Biochem. Physiol.
88A
,
637
-
639
.
Zosky
,
G. R.
and
Larcombe
,
A. N.
(
2003
).
The parasympathetic nervous system and its influence on heart rate in torpid western pygmy possums, Cercatetus concinnus (Marsupialia: Burramyidae)
.
Zoology
106
,
143
-
150
.

Competing interests

The authors declare no competing or financial interests.