A Haplotype-resolved reference genome of a long-distance migratory bat, Pipistrellus nathusii (Keyserling & Blasius, 1839).

We present a complete, chromosome scale reference genome for the long-distance migratory bat Pipistrellus nathusii. The genome encompasses both haplotypic sets of autosomes and separation of both sex chromosomes by utilising highly-accurate long-reads and preserving long-range phasing information through the use of 3-dimensional chromatin conformation capture sequencing (Hi-C). This genome, accompanied by a comprehensive protein-coding sequence annotation, provides a valuable genomic resource for future investigations into the genomic bases of long-distance migratory flight in bats as well as uncovering the genetic architecture, population structure and evolutionary history of Pipistrellus nathusii. The reference-quality genome presented here gives a fundamental resource to further our understanding of bat genetics and evolution, adding to the growing number of high quality genetic resources in this field. Here, we demonstrate its use in the phylogenetic reconstruction of the order Chiroptera and in particular, we present the resources to allow detailed investigations into the genetic drivers and adaptations related to long-distance migration.

Effects of tag mass on the physiology and behaviour of common noctule bats.

BACKGROUND: External tags, such as transmitters and loggers, are often used to study bat movements. However, physiological and behavioural effects on bats carrying tags have rarely been investigated, and recommendations on the maximum acceptable tag mass are rather based on rules of thumb than on rigorous scientific assessment. METHODS: We conducted a comprehensive three-step assessment of the potential physiological and behavioural effects of tagging bats, using common noctules Nyctalus noctula as a model. First, we examined seasonal changes in body mass. Second, we predicted and then measured potential changes in flight metabolic rate in a wind tunnel. Third, we conducted a meta-analysis of published data to assess effects of different tag masses on the weight and behaviour of bats. RESULTS: Individual body mass of common noctules varied seasonally by 7.0 ± 2.6 g (range: 0.5-11.5 g). Aerodynamic theory predicted a 26% increase in flight metabolic rate for a common noctule equipped with a 3.8 g tag, equating to 14% of body mass. In a wind tunnel experiment, we could not confirm the predicted increase for tagged bats. Our meta-analysis revealed a weak correlation between tag mass and emergence time and flight duration in wild bats. Interestingly, relative tag mass (3-19% of bat body mass) was not related to body mass loss, but bats lost more body mass the longer tags were attached. Notably, relatively heavy bats lost more mass than conspecifics with a more average body mass index. CONCLUSION: Because heavy tags (> 3 g) were generally used for shorter periods of time than lighter tags (~ 1 g), the long-term effects of heavy tags on bats cannot be assessed at this time. Furthermore, the effects of disturbance and resource distribution in the landscape cannot be separated from those of tagging. We recommend that tags weighing 5-10% of a bat's mass should only be applied for a few days. For longer studies, tags weighing less than 5% of a bat's body mass should be used. To avoid adverse effects on bats, researchers should target individuals with average, rather than peak, body mass indices.

Conversion efficiency of flight power is low, but increases with flight speed in the migratory bat Pipistrellus nathusii.

The efficiency with which flying animals convert metabolic power to mechanical power dictates an individual's flight behaviour and energy requirements. Despite the significance of this parameter, we lack empirical data on conversion efficiency for most species as in vivo measurements are notoriously difficult to obtain. Furthermore, conversion efficiency is often assumed to be constant across flight speeds, even though the components driving flight power are speed-dependent. We show, through direct measurements of metabolic and aerodynamic power, that conversion efficiency in the migratory bat (Pipistrellus nathusii) increases from 7.0 to 10.4% with flight speed. Our findings suggest that peak conversion efficiency in this species occurs near maximum range speed, where the cost of transport is minimized. A meta-analysis of 16 bird and 8 bat species revealed a positive scaling relationship between estimated conversion efficiency and body mass, with no discernible differences between bats and birds. This has profound consequences for modelling flight behaviour as estimates assuming 23% efficiency underestimate metabolic costs for P. nathusii by almost 50% on average (36-62%). Our findings suggest that conversion efficiency may vary around an ecologically relevant optimum speed and provide a crucial baseline for investigating whether this drives variation in conversion efficiency between species.

Flexible Employment of Torpor in Squirrel Gliders (Petaurus norfolcensis): An Adaptation to Unpredictable Climate?

AbstractTorpor is a highly effective response to counter various ecological and physiological bottlenecks in endotherms. In this study, we examined interrelations between thermoregulatory responses and key environmental variables in free-living squirrel gliders (Petaurus norfolcensis) in a habitat with drastic climatic and ecological changes across seasons. To this end, we measured body temperature (Tb) and heart rate (fH) simultaneously throughout the year using implanted data loggers. Squirrel gliders in our study experienced fluctuations in ambient temperature (Ta) between -4.0°C and 44.1°C and expressed torpor at different times during the year. In contrast to our expectations, torpor seemed to be employed flexibly, on demand, and most frequently in spring rather than during the coldest and/or hottest periods. Torpor bouts lasted, on average, about 5 h, and Tb during torpor dropped as low as 17.9°C. The fH during torpor decreased below 50 bpm, which is about one-third of the basal level. The ability to record fH alongside Tb enabled us to also report periods of low fH during thermoconforming hyperthermia at Ta's above 35°C that likely occurred to conserve energy and water. Our findings double the body size of Australian gliders for which data on torpor are available and advance our ecological understanding of the dynamics of torpor expression in wild mammals and of how animals cope with varying conditions. Moreover, they highlight that the flexibility of physiology and thermoregulatory responses are clearly more complex than previously thought.

Pronounced differences in heart rate and metabolism distinguish daily torpor and short-term hibernation in two bat species.

Torpor, and its differential expression, is essential to the survival of many mammals and birds. Physiological characteristics of torpor appear to vary between those species that express strict daily heterothermy and those capable of multiday hibernation, but comparisons are complicated by the temperature-dependence of variables. Previous reviews have compared these different torpor strategies by measuring the depth and duration of torpor in multiple species. However, direct comparison of multiple physiological parameters under similar thermal conditions are lacking. Here, we quantified three physiological variables; body temperature, metabolic rate (MR) and heart rate (HR) of two small heterothermic bats (daily heterotherm Syconycteris australis, and hibernator Nyctophilus gouldi) under comparable thermal conditions and torpor bout durations. When normothermic and resting both MR and HR were similar for the two species. However, during torpor the minimum HR was more than fivefold higher, and minimum MR was 6.5-fold higher for the daily heterotherm than for the hibernator at the same subcutaneous Tb (16 ± 0.5 °C). The data show that the degree of heterothermy defined using Tb is not necessarily a precise proxy for physiological capacity during torpor in these bats and is unlikely to reveal accurate energy budgets. Our study provides evidence supporting a distinction between daily torpor in a daily heterotherm and short term torpor in a hibernator, at least within the Chiroptera with regard to these physiological variables. This exists even when individuals display the same degree of Tb reduction, which has clear implications for the modelling of their energy expenditure.

Accelerometer informed time-energy budgets reveal the importance of temperature to the activity of a wild, arid zone canid.

BACKGROUND: Globally, arid regions are expanding and becoming hotter and drier with climate change. For medium and large bodied endotherms in the arid zone, the necessity to dissipate heat drives a range of adaptations, from behaviour to anatomy and physiology. Understanding how apex predators negotiate these landscapes and how they balance their energy is important as it may have broad impacts on ecosystem function. METHODS: We used tri-axial accelerometry (ACC) and GPS data collected from free-ranging dingoes in central Australia to investigate their activity-specific energetics, and activity patterns through time and space. We classified dingo activity into stationary, walking, and running behaviours, and estimated daily energy expenditure via activity-specific time-energy budgets developed using energy expenditure data derived from the literature. We tested whether dingoes behaviourally thermoregulate by modelling ODBA as a function of ambient temperature during the day and night. We used traditional distance measurements (GPS) as well as fine-scale activity (ODBA) data to assess their daily movement patterns. RESULTS: We retrieved ACC and GPS data from seven dingoes. Their mass-specific daily energy expenditure was significantly lower in summer (288 kJ kg- 1 day- 1) than winter (495 kJ kg- 1 day- 1; p = 0.03). Overall, dingoes were much less active during summer where 91% of their day was spent stationary in contrast to just 46% during winter. There was a sharp decrease in ODBA with increasing ambient temperature during the day (R2 = 0.59), whereas ODBA increased with increasing Ta at night (R2 = 0.39). Distance and ODBA were positively correlated (R = 0.65) and produced similar crepuscular patterns of activity. CONCLUSION: Our results indicate that ambient temperature may drive the behaviour of dingoes. Seasonal differences of daily energy expenditure in free-ranging eutherian mammals have been found in several species, though this was the first time it has been observed in a wild canid. We conclude that the negative relationship between dingo activity (ODBA) and ambient temperature during the day implies that high heat gain from solar radiation may be a factor limiting diurnal dingo activity in an arid environment.

Echolocation at high intensity imposes metabolic costs on flying bats.

Vocalizations are of pivotal importance for many animals, yet sound propagation in air is severely limited. To expand their vocalization range, animals can produce high-intensity sounds, which can come at high energetic costs. High-intensity echolocation is thought to have evolved in bats because the costs of calling are reported to be negligible during flight. By comparing the metabolic rates of flying bats calling at varying intensities, we show that this is true only for low call intensities. Our results demonstrate that above 130 dB sound pressure level (SPL, at a reference distance of 10 cm), the costs of sound production become exorbitantly expensive for small bats, placing a limitation on the intensity at which they can call.

Responding to the weather: energy budgeting by a small mammal in the wild.

Energy conservation is paramount for small mammals because of their small size, large surface area to volume ratio, and the resultant high heat loss to the environment. To survive on limited food resources and to fuel their expensive metabolism during activity, many small mammals employ daily torpor to reduce energy expenditure during the rest phase. We hypothesized that a small terrestrial semelparous marsupial, the brown antechinus Antechinus stuartii, would maximize activity when foraging conditions were favorable to gain fat reserves before their intense breeding period, but would increase torpor use when conditions were poor to conserve these fat reserves. Female antechinus were trapped and implanted with small temperature-sensitive radio transmitters to record body temperature and to quantify torpor expression and activity patterns in the wild. Most antechinus used torpor at least once per day over the entire study period. Total daily torpor use increased and mean daily body temperature decreased significantly with a reduction in minimum ambient temperature. Interestingly, antechinus employed less torpor on days with more rain and decreasing barometric pressure. In contrast to torpor expression, activity was directly related to ambient temperature and inversely related to barometric pressure. Our results reveal that antechinus use a flexible combination of physiology and behavior that can be adjusted to manage their energy budget according to weather variables.

Fatty acid profiles of the European migratory common noctule bat (Nyctalus noctula).

In animals, fatty acids (FA) are essential as structural components in membranes and for energy storage in adipocytes. Here, we studied the relative proportions of FA in a mammal with extreme changes in metabolic rates. Common noctule bats (Nyctalus noctula) switch from energetically demanding long-distance migration at high metabolic rates to regular torpor with extremely low metabolic rates. We found that composition of FA categories differed between adipose tissue types (white adipose tissue (WAT) vs brown adipose tissue (BAT)) and muscle tissue types (skeletal vs heart), but not between sexes. We found oleic acid to be the most abundant FA in all studied tissues. Concentrations of polyunsaturated FA (PUFA) were not always higher in muscular tissue compared with adipocyte tissue, even though high concentrations of PUFA are considered beneficial for low body temperatures in torpor. In all tissues, we observed a high content in monounsaturated fatty acids (MUFA), possibly to compensate for a low PUFA content in the diet. Ratios of ω6/ω3 were lower in the heart than in skeletal muscles of common noctules. Three FA (palmitic, oleic, and linoleic acid) accounted for about 70% of the FA in adipose tissue, which is similar to proportions observed in migrating birds, yet migrating birds generally have a higher PUFA content in muscle and adipose tissues than bats. Bats seem to contrast with other mammals in having a high MUFA content in all tissues. We conclude that FA profiles of bats differ largely from those of most cursorial mammals and instead are-with the exception of MUFA-similar to those of migrating birds.

Valuable carcasses: postmortem preservation of fatty acid composition in heart tissue.

In order to effectively conserve species, we must understand the structure and function of integral mechanisms at all levels of organismal organisation, from intracellular biochemistry to whole animal ecophysiology. The accuracy of biochemical analyses depend on the quality and integrity of the samples analysed. It is believed that tissue samples collected immediately postmortem provide the most reliable depiction of the living animal. Yet, euthanasia of threatened or protected species for the collection of tissue presents a number of ethical complications. Polyunsaturated fatty acids (PUFA) are essential to the cardiovascular system of all animals and the structure of PUFA can be degraded by peroxidation, potentially modifying the fatty acid composition of the tissue over postmortem time. Here, we assessed the composition of PUFA in cardiac tissue of bats (Carollia perspicillata) over the course of 12-h postmortem. We show that PUFA are resistant to naturally occurring postmortem degradation in heart tissue, with no difference in the overall composition of fatty acids across all time classes (0, 3, 6 or 12-h postmortem). Our results suggest that carcasses that would otherwise be discarded may actually be viable for the assessment of fatty acid composition in a number of tissues. We hope to spur further investigations into the viability of carcasses for other biochemical analyses as they may be an untapped resource available to biologists.

No effect of season on the electrocardiogram of long-eared bats (Nyctophilus gouldi) during torpor.

Heterothermic animals regularly undergo profound alterations of cardiac function associated with torpor. These animals have specialised tissues capable of withstanding fluctuations in body temperature > 30 °C without adverse effects. In particular, the hearts of heterotherms are able to resist fibrillation and discontinuity of the cardiac conduction system common in homeotherms during hypothermia. To investigate the patterns of cardiac conduction in small insectivorous bats which enter torpor year round, I simultaneously measured ECG and subcutaneous temperature (Tsub) of 21 Nyctophilus gouldi (11 g) during torpor at a range of ambient temperatures (Ta 1-28 °C). During torpor cardiac conduction slowed in a temperature dependent manner, primarily via prolongation along the atrioventricular pathway (PR interval). A close coupling of depolarisation and repolarisation was retained in torpid bats, with no isoelectric ST segment visible until animals reached Tsub <6 °C. There was little change in ventricular repolarisation (JT interval) with decreasing Tsub, or between rest and torpor at mild Ta. Bats retained a more rapid rate of ventricular conduction and repolarisation during torpor relative to other hibernators. Throughout all recordings across seasons (> 2500 h), there was no difference in ECG morphology or heart rate during torpor, and no manifestations of significant conduction blocks or ventricular tachyarrhythmias were observed. My results demonstrate the capacity of bat hearts to withstand extreme fluctuations in rate and temperature throughout the year without detrimental arrhythmogenesis. I suggest that this conduction reserve may be related to flight and the daily extremes in metabolism experienced by these animals, and warrants further investigation of cardiac electrophysiology in other flying hibernators.

Can bats sense smoke during deep torpor?

While torpor is a beneficial energy-saving strategy, it may incur costs if an animal is unable to respond appropriately to external stimuli, which is particularly true when it is necessary to escape from threats such as fire. We aimed to determine whether torpid bats, which are potentially threatened because they must fly to escape, can sense smoke and whether respiration rate (RR), heart rate (HR) and reaction time of torpid bats prior to and following smoke introduction is temperature-dependent. To test this we quantified RR and HR of captive Australian tree-roosting bats, Nyctophilus gouldi (n=5, ~10g), in steady-state torpor in response to short-term exposure to smoke from Eucalyptus spp. leaves between ambient temperatures (Ta) of 11 and 23°C. Bats at lower Ta took significantly longer (28-fold) to respond to smoke, indicated by a cessation of episodic breathing and a rapid increase in RR. Bats at lower Ta returned to torpor more swiftly following smoke exposure than bats at higher Ta. Interestingly, bats at Ta<15°C never returned to thermoconforming steady-state torpor prior to the end of the experimental day, whereas all bats at Ta≥15°C did, as indicated by apnoeic HR. This shows that although bats at lower Ta took longer to respond, they appear to maintain vigilance and prevent deep torpor after the first smoke exposure, likely to enable fast escape. Our study reveals that bats can respond to smoke stimuli while in deep torpor. These results are particularly vital within the framework of fire management conducted at Ta<15°C, as most management burns are undertaken during winter when bats will likely respond more slowly to fire cues such as smoke, delaying the time to escape from the fire.

Cold-hearted bats: uncoupling of heart rate and metabolism during torpor at sub-zero temperatures.

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 (V̇O2 ), 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 V̇O2 The maximum increase in fH was only 4-fold greater than the average thermoconforming minimum, compared with a 46-fold increase in V̇O2 The differential response of fH and V̇O2  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.

Post-fire recovery of torpor and activity patterns of a small mammal.

To cope with the post-fire challenges of decreased availability of food and shelter, brown antechinus (Antechinus stuartii), a small marsupial mammal, increase the use of energy-conserving torpor and reduce activity. However, it is not known how long it takes for animals to resume pre-fire torpor and activity patterns during the recovery of burnt habitat. Therefore, we tested the hypothesis that antechinus will adjust torpor use and activity after a fire depending on vegetation recovery. We simultaneously quantified torpor and activity patterns for female antechinus from three adjacent areas: (i) the area of a management burn 1 year post-fire, (ii) an area that was burned 2 years prior, and (iii) a control area. In comparison to shortly after the management burn, antechinus in all three groups displayed less frequent and less pronounced torpor while being more active. We provide the first evidence that only 1 year post-fire antechinus resume pre-fire torpor and activity patterns, probably in response to the return of herbaceous ground cover and foraging opportunities.

Black or white? Physiological implications of roost colour and choice in a microbat.

Although roost choice in bats has been studied previously, little is known about how opposing roost colours affect the expression of torpor quantitatively. We quantified roost selection and thermoregulation in a captive Australian insectivorous bat, Nyctophilus gouldi (n=12) in winter when roosting in black and white coloured boxes using temperature-telemetry. We quantified how roost choice influences torpor expression when food was provided ad libitum or restricted in bats housed together in an outdoor aviary exposed to natural fluctuations of ambient temperature. Black box temperatures averaged 5.1°C (maximum 7.5°C) warmer than white boxes at their maximum daytime temperature. Bats fed ad libitum chose black boxes on most nights (92.9%) and on 100% of nights when food-restricted. All bats used torpor on all study days. However, bats fed ad libitum and roosting in black boxes used shorter torpor and spent more time normothermic/active at night than food-restricted bats and bats roosting in white boxes. Bats roosting in black boxes also rewarmed passively more often and to a higher skin temperature than those in white boxes. Our study suggests that N. gouldi fed ad libitum select warmer roosts in order to passively rewarm to a higher skin temperature and thus save energy required for active midday rewarming as well as to maintain a normothermic body temperature for longer periods at night. This study shows that colour should be considered when deploying bat boxes; black boxes are preferable for those bats that use passive rewarming, even in winter when food availability is reduced.

Measuring subcutaneous temperature and differential rates of rewarming from hibernation and daily torpor in two species of bats.

Prolonged and remote measurement of body temperature (Tb) in undisturbed small hibernators was not possible in the past because of technological limitations. Although passive integrated transponders (PITs) have been used previously to measure subcutaneous temperature (Tsub) during daily torpor in a small marsupial, no study has attempted to use these devices at Tbs below 10°C. Therefore, we investigated whether subcutaneous interscapular PITs can be used as a viable tool for measuring Tb in a small hibernating bat (Nyctophilus gouldi; Ng) and compared it with measurements of Tb during daily torpor in a heterothermic bat (Syconycteris australis; Sa). The precision of transponders was investigated as a function of ambient temperature (Ta) and remote Tsub readings enabled us to quantify Tsub-Tb differentials during steady-state torpor and arousal. Transponders functioned well outside the manufacturer's recommended range, down to ~5°C. At rest, Tsub and rectal Tb (Trec) were strongly correlated for both bat species (Ng r(2)=0.88; Sa r(2)=0.95) and this was also true for N. gouldi in steady-state torpor (r(2)=0.93). During induced rewarming Tsub increased faster than Trec in both species. Our results demonstrate that transponders can be used to provide accurate remote measurement of Tb in two species of bats during different physiological states, both during steady-state conditions and throughout dynamic phases such as rewarming from torpor. We show that, at least during rewarming, regional heterothermy common to larger hibernators and other hibernating bats is also present in bats capable of daily torpor.

Passive rewarming from torpor in hibernating bats: minimizing metabolic costs and cardiac demands.

Endothermic arousal from torpor is an energetically costly process and imposes enormous demands on the cardiovascular system, particularly during early stage arousal from low body temperature (Tb). To minimize these costs many bats and other heterothermic endotherms rewarm passively from torpor using solar radiation or fluctuating ambient temperature (Ta). Because the heart plays a critical role in the arousal process in terms of blood distribution and as a source of heat production, it is desirable to understand how the function of this organ responds to passive rewarming and how this relates to changes in metabolism and Tb. We investigated heart rate (HR) in hibernating long-eared bats (Nyctophilus gouldi) and its relationship to oxygen consumption (V̇o2) and subcutaneous temperature (Tsub) during exposure to increasing Ta compared with endogenous arousals at constant low Ta. During passive rewarming, HR and V̇o2 remained low over a large Tsub range and increased concurrently with increasing Ta (Q10 2.4 and 2.5, respectively). Absolute values were higher than during steady-state torpor but below those measured during torpor entry. During active arousals, mean HR and V̇o2 were substantially higher than during passive rewarming at corresponding Tsub. In addition, partial passive rewarming reduced the cost of arousal from torpor by 53% compared with entirely active arousal. Our data show that passive rewarming considerably reduces arousal costs and arousal time; we suggest this may also contribute to minimizing exposure to oxidative stresses as well as demands on the cardiovascular system.

Torpor and hypothermia: reversed hysteresis of metabolic rate and body temperature.

Regulated torpor and unregulated hypothermia are both characterized by substantially reduced body temperature (Tb) and metabolic rate (MR), but they differ physiologically. Although the remarkable, medically interesting adaptations accompanying torpor (e.g., tolerance for cold and ischemia, absence of reperfusion injury, and disuse atrophy) often do not apply to hypothermia in homeothermic species such as humans, the terms "torpor" and "hypothermia" are often used interchangeably in the literature. To determine how these states differ functionally and to provide a reliable diagnostic tool for differentiating between these two physiologically distinct states, we examined the interrelations between Tb and MR in a mammal (Sminthopsis macroura) undergoing a bout of torpor with those of the hypothermic response of a similar-sized juvenile rat (Rattus norvegicus). Our data show that under similar thermal conditions, 1) cooling rates differ substantially (approximately fivefold) between the two states; 2) minimum MR is approximately sevenfold higher during hypothermia than during torpor despite a similar Tb; 3) rapid, endogenously fuelled rewarming occurs in torpor but not hypothermia; and 4) the hysteresis between Tb and MR during warming and cooling proceeds in opposite directions in torpor and hypothermia. We thus demonstrate clear diagnostic physiological differences between these two states that can be used experimentally to confirm whether torpor or hypothermia has occurred. Furthermore, the data can clarify the results of studies investigating the ability of physiological or pharmacological agents to induce torpor. Consequently, we recommend using the terms "torpor" and "hypothermia" in ways that are consistent with the underlying regulatory differences between these two physiological states.

Heart rate as a predictor of metabolic rate in heterothermic bats.

While heart rate (fH) has been used as an indicator of energy expenditure, quantitative data showing the relationship between these variables are only available for normothermic animals. To determine whether fH also predicts oxygen consumption ( ) during torpor, we simultaneously measured , fH and subcutaneous body temperature (Tsub) of a hibernator, Gould's long-eared bats (Nyctophilus gouldi, 9 g, N=18), at ambient temperatures (Ta) between 0 and 25°C. At rest, fH of normothermic resting bats was negatively correlated with Ta, with maximum fH of 803 beats min(-1) (Ta=5°C). During torpor, the relationship between fH and Ta was curvilinear, and at low Tsub (~6°C), fH fell to a minimum average of 8 beats min(-1). The minimum average values for both and fH in torpor reported here were among the lowest recorded for bats. The relationship between fH and was significant for both resting (r(2)=0.64, P<0.001) and torpid bats (r(2)=0.84, P<0.001), with no overlap between the two states. These variables were also significantly correlated (r(2)=0.44, P<0.001) for entire torpor bouts. Moreover, estimates of from fH did not differ significantly from measured values during the different physiological states. Our study is the first to investigate the accuracy of fH as a predictor of during torpor and indicates the reliability of this method as a potential measure of energy expenditure in the field. Nevertheless, fH should only be used to predict within the range of activities for which robust correlations have been established.