1. Ecology
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Cyclic bouts of extreme bradycardia counteract the high metabolism of frugivorous bats

  1. M Teague O'Mara  Is a corresponding author
  2. Martin Wikelski
  3. Christian C Voigt
  4. Andries Ter Maat
  5. Henry S Pollock
  6. Gary Burness
  7. Lanna M Desantis
  8. Dina KN Dechmann
  1. Max Planck Institute for Ornithology, Germany
  2. University of Konstanz, Germany
  3. Smithsonian Tropical Research Institute, Panama
  4. Leibniz Institute for Zoo and Wildlife Research, Germany
  5. University of Illinois at Urbana-Champaign, United States
  6. Trent University, Canada
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Cite as: eLife 2017;6:e26686 doi: 10.7554/eLife.26686

Abstract

Active flight requires the ability to efficiently fuel bursts of costly locomotion while maximizing energy conservation during non-flying times. We took a multi-faceted approach to estimate how fruit-eating bats (Uroderma bilobatum) manage a high-energy lifestyle fueled primarily by fig juice. Miniaturized heart rate telemetry shows that they use a novel, cyclic, bradycardic state that reduces daily energetic expenditure by 10% and counteracts heart rates as high as 900 bpm during flight. Uroderma bilobatum support flight with some of the fastest metabolic incorporation rates and dynamic circulating cortisol in vertebrates. These bats will exchange fat reserves within 24 hr, meaning that they must survive on the food of the day and are at daily risk of starvation. Energetic flexibly in U. bilobatum highlights the fundamental role of ecological pressures on integrative energetic networks and the still poorly understood energetic strategies of animals in the tropics.

https://doi.org/10.7554/eLife.26686.001

eLife digest

To survive, all animals have to balance how much energy they take in and how much they use. They must find enough food to fuel the chemical processes that keep them alive – known as their metabolism – and store leftover fuel to use when food is not available. Bats, for example, have a fast metabolism and powerful flight muscles, which require a lot of energy. Some bat species, such as the tent-making bats, survive on fruit juice, and their food sources are often far apart and difficult to find. These bats are likely to starve if they go without food for more than 24 hours, and therefore need to conserve energy while they are resting.

To deal with potential food shortages, bats and other animals can enter a low-energy resting state called torpor. In this state, animals lower their body temperature and slow down their heart rate and metabolism so that they need less energy to stay alive. However, many animals that live in tropical regions, including tent-making bats, cannot enter a state of torpor, as it is too hot to sufficiently lower their body temperature. Until now, scientists did not fully understand how these bats control how much energy they use.

Now, O’Mara et al. studied tent-making bats in the wild by attaching small heart rate transmitters to four wild bats, and measured their heartbeats over several days. Since each heartbeat delivers oxygen and fuel to the rest of the body, measuring the bats’ heart rate indicates how much energy they are using. The experiments revealed for the first time that tent-making bats periodically lower their heart rates while resting (to around 200 beats per minute). This reduces the amount of energy they use each day by up to 10%, and helps counteract heart rates that can reach 900 beats per minute when the bats are flying.

Overall, these findings show that animals have evolved in various ways to control their use of energy. Future research should use similar technology to continue uncovering how wild animals have adapted to survive in different conditions. This knowledge will help us to understand how life has become so diverse in the tropics and the strategies that animals may use as climates change.

https://doi.org/10.7554/eLife.26686.002

Introduction

Energy intake, incorporation and expenditure are fundamental to animal behavior and evolution (Brown et al., 2004; Weiner, 1992). Animals must balance between generating enough metabolic power to find and acquire food and maintaining sufficient reserves to sustain daily maintenance, and repair and reproduce. This basic requirement of life can drive the foraging strategies of entire clades (Williams et al., 2014) and extensive links among various behavioral and physiological strategies have evolved in response to single ecological pressures including diet and pathogen environments (Cohen et al., 2012). This is largely a consequence of the sequential and linear process of energetic input (feeding), and that energy expenditure is additive across parallel aspects of physiology (Weiner, 1992). Energetic networks then link across physiological systems from mitochondrial oxidation to digestion, to respond to changes in resource availability and maintain physiological integrity. Well-adapted energy metabolisms must then both be able to conserve reserves and deliver enormous energetic power outputs in an efficient and effective manner. However, few animal models currently allow us to follow energy from intake to delivery of energetic currency to fuel metabolism, and finally to the countermeasures taken to slow down energetic expenditure when it is not needed. Furthermore, accumulating evidence shows that data collected in laboratory settings may not reflect the full range of strategies animals employ to deal with this energetic dilemma (Bishop et al., 2015; Bowlin et al., 2005; Calisi and Bentley, 2009; Geiser et al., 2007; Ward et al., 2002). This makes quantitative data from naturally behaving animals in the wild even more important to test the balance and integration of physiological adaptations to energetic limitations.

Flying vertebrates are an excellent example of this balance. While flight is one of the most efficient modes of locomotion per unit distance traveled, it is costlier per unit time than any other mode of locomotion (Norberg, 1990; Schmidt-Nielsen, 1979). To fulfill the exceptional demands of powered flight, both birds and bats have undergone dramatic physiological reorganization that emphasizes the need to supply fuel to large flight muscles (Maina, 2000; Norberg, 1990). Bat flight in particular is an extreme case of vertebrate locomotor energetics. In comparison to those of non-flying mammals of comparable size, hearts and lungs of bats are larger and have higher blood oxygen transport potential, delivering more oxygen per heart beat than non-flying terrestrial mammals (Neuweiler, 2000). Bats use some of the highest mass-specific metabolic rates during flight; 3–5 times greater than any other mammals and maximum increases of 15–16 times minimum resting metabolic rates (Speakman and Thomas, 2003). This may place bats at their energetic ceiling, and integrated physiological networks that allow them to maintain high metabolic rates at or near their limits over extended periods of time may be under equally strong selection to reduce resting energetic expenditure below what is commonly found in mammals.

Bats launch themselves directly into energy-demanding flight at the onset of their activity period and on an empty stomach, fueling flight by limited fat reserves (Voigt et al., 2010). They must then efficiently find and ingest food, and make energy available to their metabolism rapidly, as high metabolic rates and small body size place them at risk of starvation if sufficient food is not found. This risk is enhanced in the many species that specialize on ephemeral food sources. One strategy to cope with this energetic vulnerability is through daily reduction of metabolic rate (torpor) found in small-bodied bat species especially from the temperate zone. By entering a distinct low-energy state characterized by low body temperature, some bats reduce metabolic rates by 99% during torpor when ambient temperatures are lower than their thermoneutral zone (Geiser and Stawski, 2011; Ruf and Geiser, 2015). In tropical and sub-tropical regions where ambient temperatures are high, it may be impossible to lower body temperature beyond these critical minimum temperatures to save energy, therefore reductions in heart rate may reflect reductions in cellular respiration rates and gene expression in multiple pathways and be an effective measure of energetic conservation (Dechmann et al., 2011; Dzal et al., 2015; McNab, 1969; Storey and Storey, 2004). This may be particularly important in those that feed on sugar dense foods as they are at the highest risk of starvation (McNab, 1969; Voigt and Speakman, 2007).

Heart rate has a quadratic relationship with metabolic oxygen consumption (Bishop and Spivey, 2013; Grubb, 1982), and by measuring it directly it is possible to gain insight into energetic expenditure at high temporal resolution. Heart rates in bats may more than double in the transition from rest to flight, reflecting enormous flight power requirements (Thomas, 1975). Controlled experiments in wind tunnels and laboratory conditions have yielded incredible insight into the regulation of metabolism and energy consumption across a wide variety of activities and physiological states. However, heart rates of exercising animals in nature are unpredictable and metabolic rates measured during wind tunnel flight may not indicate the full scope of natural behavior. In a tropical insectivorous bat, heart rate increases from 129 bpm in the roost to 847 bpm during flight, a six-fold increase that is larger than predicted from other captive bats in wind tunnels (Dechmann et al., 2011). Alternatively, heart rates of free-flying animals may be much lower than expected. For example, bar-headed geese traverse the Himalayas with heart rates of 250–475 bpm (Bishop et al., 2015), 20% lower than what is expected from captive measures (Ward et al., 2002), and during migration, heart rates in Swainson’s thrushes are 10% lower than comparable long flights in wind tunnels (Bowlin et al., 2005). This indicates that sustainable metabolic rates possible during exercise may differ greatly from maximal rates or extrapolations in captive studies and we have only been able to get an initial glimpse into the heart rates used by flying bats.

Once they begin to feed, bats fuel their enormous demand for power by directly and rapidly metabolizing ingested food, but this can lead to high risk of starvation via rapid fat turnover (Caviedes-Vidal et al., 2008; Voigt and Speakman, 2007). One mechanism that may help animals to adjust the timing and intensity of shifts in metabolic scopes are glucocorticoids. They are key integrators between the environment and energy balance that ensure rapid response to changes in energetic needs (Cohen et al., 2012). Elevated levels of glucocorticoid hormones in blood plasma suppress glycogen formation and promote gluconeogenesis (Haase et al., 2016), fat oxidation (Brillon et al., 1995), and play a primary role in energy balance (Nieuwenhuizen and Rutters, 2008). Most bats that have been studied show high baseline glucocorticoid concentrations (Reeder et al., 2004; 2006), which indicates that they are in a ready state to rapidly mobilize glucose and glycogen reserves. By manipulating circulating levels of glucocorticoids or those tied to binding globulin, individual use of energy reserves can be modulated (Schneider, 2004).

Bats are then faced with an energetic dilemma where they must rapidly power flight, but quickly switch to conserving energetic stores gained during foraging. To better understand the interplay of energy expenditure and conservation, we describe the daily energetic life of Peters’ tent-making bat (Uroderma bilobatum, family Phyllostomidae) in Gamboa, Panamá. These bats are central-place foragers that leave a stable roost location to feed primarily on juice extracted from ripe figs (Ficus spp). We hypothesized that they would not use torpor during their regular daily life and that their energy intake and turnover rates would be high. Daily energy intake and expenditure should then be closely matched, resulting in a specialized life-style at the energetic edge. Testing our hypotheses was made possible by newly miniaturized heart rate transmitters to describe both the activity patterns of the species and their instantaneous energetic expenditure throughout the day, including the first flying heart rates of free-ranging individuals. We also tested how these bats fuel their metabolism through measurement of metabolic incorporation rates and fat turnover from stable isotope ratios in their breath in short-term captivity (Voigt and Speakman, 2007). In combination with an estimate of energy mobilization potential via elevated circulating cortisol, this allows us a more complete view into how these small-bodied, high-metabolic frugivores meet daily energetic demands.

Results

Activity patterns

We tracked the heart rates (Figure 1—figure supplement 1) of four bats for 13.6 ± 4.9 hr (mean ± SD) each day for two to four days (13 days total). This included 4.03 ± 0.05 hr of activity outside of the roost at night and the approximately 12 hr that bats spend in their roost during the day for 350 hr of total recording time. All bats left their roosts between 18:00 – 18:30 and flew three to seven minutes to their initial foraging sites. Bats executed multiple short flights of 1–2 min each (mean ± SD: 1 ± 1.5 min) that were consistent with flying to a fruiting tree, selecting a fruit, and carrying it to a separate feeding perch. During our tracking, flight accounted for 13 ± 6% (30.6 ± 15.6 min) of the time outside of the roost. We were able to locate several food trees, all of which were Ficus insipida, but all bats also fed for short periods across the Panama Canal at sites inaccessible during tracking. Bats returned to their day roosts between 22:30 – 06:00. When bats returned early in the night, they left for an additional one to two hours later in the morning. The minimum time that we tracked a bat foraging, including short bouts away from the roosts was two hours and the maximum total time outside of the roost was 10 hr. All bats returned to their home roost each night where they remained for the rest of the day.

Field metabolic rates and cyclic bradycardia

Uroderma bilobatum used a large range in heart rates (fH) across the day, ranging from 173 to 1066 bpm (Figure 1—figure supplement 2). Analysis of activity-specific fH shows that bats expend 4.9 ± 0.8 kJ h−1 during flight (mean ± SD; fH: 766 ± 56 bpm, Figure 1). Amplitude fluctuations of the fH radio signals show that minimum fH of flying bats was 750 bpm. Maximum recorded flying fH was 1066 bpm. Uroderma bilobatum then needed to generate a minimum of 0.98 W to fly (3.5 kJ h−1), but flight typically had higher costs of 1.36 ± 0.23 W with a maximum recorded output of 2.3 W. This is a mass-specific metabolic power of 75.89 ± 11.9 W kg−1 and a maximum mass specific power of 145.6 W kg−1. Nightly non-flight activity when bats were stationary required 2.2 ± 1.1 kJ h−1 (fH: 492 ± 128 bpm, Figure 1).

Figure 1 with 4 supplements see all
Heart rate and energetic expenditure of U. bilobatum recorded across 350 hr of observation.

(A) 30 min examples of continuous heart rates of Uroderma bilobatum during daily activities and (B) the distribution of energetic costs estimated for these activities from heart rate.

https://doi.org/10.7554/eLife.26686.003

Surprisingly, U. bilobatum periodically lower fH to 200–250 bpm from a mean fH of 374 ± 112 bpm throughout their daily resting periods where they remain relatively inactive in their roosts, and during which they consume 1.2 ± 0.8 kJ h−1 (0.33 — 0.23 W or 18.84 ± 13.59 W kg−1, Figure 2). During these periods bats are typically sitting quietly, although bats can be alert during these times and engage in bouts of agonism, grooming, and may fly from the roost due to disturbances around the roosting sites. Bats suppress fH by 30% 2–3 times per hour (mean: 1.54 ± 1.18 sd times per hour) for 5–7 min throughout the day (Figure 2). This cyclic bradycardia is a yet undescribed strategy that was only detectable through complete sampling of daily heart rate recordings. These lowered heart rates were followed by a return to the more stable rates between 300–400 bpm, or often to a brief arousal state with elevated heart rates above resting rates. All bats employed these reduced heart rates but one individual only used them on two of the four days it was observed.

Example heart rate recordings of one individual (bat 1) from 2014-12-07.

(A) Twenty-four hours of observation include periods of missing data when the bat was out of tracking range (grey boxes). Black and white bars above indicate night and day. Inset B shows more detail from the same time period (13–16 hr) to highlight the daily, cyclic bradycardia executed by these bats that save up to 10% of their daily energetic expenditure.

https://doi.org/10.7554/eLife.26686.008

This lowered heart rate resulted in a median resting metabolic rate (RMR) of 0.54 ± 0.01 kJ h−1 compared to RMR 0.75 ± 0.04 kJ h−1 at higher mean fH. Using the mean energetic expenditure by each bat on each night it was tracked (Supplementary file 1) we can estimate typical field metabolic rate (FMR) of 45.79 kJ if a bat spends 2 hr in flight and executes daily cyclic bradycardia (Figure 3). Two hours may be an over-estimate of time flying in the resource dense region where we tracked bats, but likely reflects areas with more dispersed fruit trees. Based on median values for each individual mean metabolic scope was 5.39 ± 1.80. This short, cyclic bradycardia then saved U. bilobatum 0.3–0.5 kJ h−1 or 3.5–6 kJ total over the 12 hr resting phase which is 10% (7.6–13.1%) of their total FMR.

Mean field metabolic rate ±95% CI estimated by the number of hours spent in flight with (solid line) and without (dashed line) daily cyclic bradycardia.

A conservative estimate of two hours flight and a mean FMR of 45.79 kJ day−1 is based on our radio tracking observations of free-flying bats in their natural environment. This is within the estimates from the Speakman (2005) scaling relationship (grey box) for the range of body masses (16–19 g) measured in this population.

https://doi.org/10.7554/eLife.26686.009

Metabolic incorporation rates of resting bats

We used a diet switching experiment that transitioned bats from a natural diet, dominated by figs with low δ13C values, to an experimental diet with high δ13C values (agave sugar) to model the speed at which ingested sugar enters metabolism by measuring the changes in the δ13C composition of exhaled CO2. After a baseline sample, bats (n = 8) were fed a solution of agave nectar. Their exhaled breath was rapidly enriched in 13C and reached an asymptotic value of −16.5 ± 2.0 ‰ 50 min after initial feeding (Figure 4A, Supplementary file 2) which is lower than the δ13C value of the diet and indicates that fat or glycogen stores continued to be metabolized (δ13Cdiet = −12.0 ± 0.1 ‰, t = −12.6, df = 32, p<0.001). Overall δ13C breath enrichment followed a mean single pool incorporation model of δ13C breath(t)=−16.575–12.841e-0.081t, with 50% of metabolism fueled by ingested food after only 8 min (t50 = 8.1 ± 15.6 min). The large standard deviation in t50 is due to one distinctive bat (Individual A) that showed a nearly linear enrichment curve with no asymptote (Supplementary file 2). If this bat is excluded, t50 drops to 7.6 min and an incorporation curve of δ13C breath(t)=−16.497–13.138e-0.091t. Bats fed with ripe Ficus indica (n = 6) did not show any change in δ13Cbreath over the course of the following 90 min (Figure 4A, F1, 24 = 2.614, p=0.113).

δ13C measured from exhaled CO2 post feeding on agave nectar (black circles) and Ficus insipida (blue squares).

(A) Uroderma fueled metabolism from ingested food immediately upon feeding on agave nectar (black) and fueled 50% (t50) of their metabolism within 8 min. There was no change in δ13C when bats were fed figs that comprise their natural diet. (B) When fed agave nectar over 72 hr bats reached a t50 for fat replacement after 13 hr and approached asymptotic values at 48 hr.

https://doi.org/10.7554/eLife.26686.010

Over the course of the next three days, bats kept in captivity and fed on agave nectar showed increasingly 13C enriched baseline δ13C breath values at the beginning of the night (Figure 4B) and after not eating for the entire day, which is typical of feeding patterns of these bats. We estimated a mean single-pool exponential model of δ13Cbreath(t)=−16.412–12.901e-0.801t, with a t50 = 13.2 ± 4.6 hr, and by the third night bats approached an asymptotic starting value of −17.06 ± 1.27 ‰ which is not different from the asymptotic value of the initial feeding experiment (t13 = 0.91, p=0.38). This indicates that fifty percent of an individual’s fat reserves are then exchanged after 13–17 hr, and a carbon atom has a residency period of 1–2 days, with a full exchange of fat after 3 days.

Glucocorticoids and energy mobilization

Bats captured at their roosts (15 F, 6 M) showed low baseline values of circulating cortisol concentrations (ng ml−1) that did not differ by sex (F: 64.81 ± 158.81 ng ml−1, M: 57.66 ± 137.07 ng ml−1, F1,19 = 0.009, p=0.92). When restrained in a cloth bag for one hour they showed a strongly sex biased response: restraint-induced values were 10–15 times baseline conditions (Figure 5), and were two-fold greater in females than in males (F: 989.50 ± 450.78 ng ml−1, M: 428.34 ± 94.45 ng ml−1, F1,19 = 0.8.89, p=0.008).

Baseline and restrained plasma cortisol values from female (n = 15) and male (n = 6) U. bilobatum.

There were no differences between sexes in baseline values, but females had higher circulating plasma cortisol values after one hour of restraint.

https://doi.org/10.7554/eLife.26686.011

Discussion

We tracked the heart rates free-ranging bats throughout the 24 hr period, including foraging, to estimate total energetic costs. As hypothesized, we found that heart rate derived energy expenditure of U. bilobatum during flight is high and this is achieved through rapid incorporation of ingested food into their metabolism. We found flying heart rates that were 4–5 times higher than resting rates during the day and twice the heart rates of bats roosting at night. These bats replace nearly half of their fat reserves within a single day, resulting in short potential starvation times. Uroderma bilobatum counter this high energetic expenditure by spending relatively little time in flight and they have exceptionally low circulating cortisol values at rest during the day. These low basal values promote conservation of glucose reserves, but can be elevated up to 15 times, at least in response to stress, and could be used to generate the high metabolic power needed for flight. Most surprising, we found that by cyclically lowering heart rates during the day, they save 10% of their energy budget. This cyclic bradycardia is a novel strategy that minimizes energetic expenditure at relatively high ambient temperatures and allows U. bilobatum to maintain a FMR expected for their size. Only by completely sampling these high-resolution data from naturally behaving bats were we able to detect these lowered heart rates and quantify their effect on bat energetic strategies.

Using the energetic expenditure derived from median heart rates of resting bats in their natural roosts during the day (0.54 kJ h−1 or 0.27 W) we can estimate a RMR of 13.0 kJ day−1, which closely approximates previous measures of BMR (12.8 kJ day−1 [McNab, 1969]). After commuting to the foraging patch, figs are collected during short flights of 1–2 mins and most of the remaining time is spent more or less at rest in their night feeding roosts resulting in only 30 mins per night in actual flight. Although this may differ among sites or during periods of less favorable food availability, this perch-resting with short fruit collection flights is an important part of their energy saving strategy. The subsequently low heart rates and activity patterns estimate an estimated FMR of ca. 46 kJ day−1 (Bishop and Spivey, 2013), which is within the general predictions for FMR based on body mass from a broad taxonomic sampling of studies using doubly-labeled water (Speakman, 2005). While energetic expenditure met estimates for 16–19 g bats, this was only possible due to U. bilobatum restriction of total active flying time to less than about two hours per night (Figure 3) and the cyclic suppression of heart rates while resting during the day.

The cyclic bradycardia during daytime rest in our study is unprecedented. It is possible that these cycles are linked to REM and pre-REM sleep, but when humans and cats sleep their heart rate slows immediately prior to the elevation of heart rates during REM cycles (Taylor et al., 1985; Verrier et al., 1998). In both taxa the change in heart rate lasts only seconds and amounts to a total change of 3–5% from the resting heart rate as compared to the minutes-long 30% reduction in U. bilboatum. A reversed pattern in heart rate is found in hibernating ground squirrels that show irregular heart rates that speed up for 30–50 s before slowing again to a steady rate (Milsom et al., 1993; Milsom et al., 1999). The regular occurrence of this cyclic bradycardia suggests that it is a standard and regular aspect of the way that U. bilobatum rests and is likely a further extension of the energy conservation of sleep (Benington and Heller, 1995; Kilduff et al., 1993; Schmidt, 2014; Walker et al., 1979). Bradycardia is common aspect of the dive response where diving mammals slow their heart rates to conserve oxygen when submerged for long periods (Noren et al., 2012). Mammals in torpor are also bradycardic (Currie et al., 2014; Dechmann et al., 2011; Elvert and Heldmaier, 2005; Heldmaier et al., 2004), but the cyclic and varying nature of the heart rate depressions we find in U. bilobatum are not characteristic of any of these physiological states. Animals enter torpor and hibernation through controlled reductions of heart rate via increased inter-beat interval and skipped beats (Elvert and Heldmaier, 2005; Milsom et al., 1999). It may be that the slowed heart rates in U. bilobatum reflect the initial descent into short and shallow torpor events with a decrease in heart rate preceding the shift to torpor. Further investigation into the nervous control of bradycardic states in U. bilobatum would clarify both how these reductions are executed and any similarity to a sleep-torpor transition (Milsom et al., 1999).

Thus far, bats and hummingbirds in torpor and at rest have showed low and constant heart rates without any indication of the cycling we observe (Currie et al., 2014; Dechmann et al., 2011; Schaub and Prinzinger, 1999). Species that are capable of daily torpor generally lower their body temperatures to maximize energetic savings, particularly when exposed to cold temperatures (Ruf and Geiser, 2015), and this commonly occurs in tropical and sub-tropical mammals at temperatures below 24°C (Canale et al., 2012; Geiser and Stawski, 2011). However, a similar torpor response is not possible for the tropical U. bilobatum. Instead, they actively defend their body temperatures when exposed to cold, and more than triple metabolic rates to maintain a body temperature of 36°C at ambient temperatures of 10°C vs 30°C (McNab, 1969). In our respirometry calibrations, U. bilobatum maintained a constant body temperature around 37°C across the measured range of heart rates of 300–800 bpm. In fact, they may alter heart rate dynamics independently of body temperature (Tøien et al., 2011). The low heart rates we observed are similar to the minimum resting heart rates of small bats (i.e., 200–400 bpm) in thermonetural conditions (Currie et al., 2015; Kulzer, 1967; Leitner, 1966; Leitner and Nelson, 1967). The thermoneutral zone for U. bilobatum is reported to be 29–35°C (McNab, 1969; Rodríguez-Herrera et al., 2016), which is still slightly higher than the ambient temperature of our field site (mean: 25.87 ± 1.21°C, range: 23.38–28.24°C. It is unclear if the frequency and intensity of the cycling we observed are in response to ecological and energetic interactions, such as lowered foraging success, that decouples resting metabolic rates from overall FMR (Nilsson, 2002; Welcker et al., 2015). Decoupling resting metabolic rates from total energetic expenditure is hypothesized to be found in animals that live near their energetic ceilings (Welcker et al., 2015) with high metabolic rates, like U. bilobatum. We found the number of cycles per hour varied both within and among individuals, but we do not yet have enough information on the relationship between total nightly energy expenditure, energy intake, and the lowering of heart rates. However, it is unclear why these bats move between two apparently stable low energy states at rest. Further investigation into the potential relationship between this heart rate change and more commonly perceived torpor states would help understand energetic adaptations in tropical environments.

There are relatively few data on the heart rates of free-flying animals (Green, 2011) all of which are larger than U. bilobatum. Furthermore, accurately estimating energy consumption during flight under controlled conditions has often been unpractical or impossible due to the conditions needed in wind tunnels and mask respirometry. Our bats' heart rates and metabolic power are surprisingly low and variable when compared to flight tunnel studies. The heart rate derived estimate for cost of flight in U. bilobatum (1.36 ± 0.23 W, range 0.98–2.3 W) was slightly lower than mass loss estimates for bats of similar sizes and wing shapes (1.96–2.45 W: (von Busse et al., 2013; Winter and von Helversen, 1998). However, U. bilobatum show a mass specific power of 76 W kg−1 with a maximum output of 145 W kg−1, which is within the power requirements of bats that are up to 44 times larger (Carpenter, 1986; Thomas, 1975). While our estimates of energy consumption were not directly calibrated with flying bats, they provide the best potential estimates available based on broad patterns of the relationships among heart rate, stroke volume, and oxygen consumption during exercise (Bishop and Spivey, 2013), and must be interpreted with some caution. The large changes in heart rate among activity states remain lower than would be expected based on body size and reinforce the emerging pattern of lower energy consumption by free-flying animals versus those in controlled laboratory conditions (Bishop et al., 2015; Bowlin et al., 2005; Ward et al., 2002). Metabolic power of bat flight may be difficult to predict as a function of body size, but more likely the context in which the animals fly plays a strong role in determining the energy used. Laboratory experiments have been our best window into animals' physiological possibilities, but it is increasingly important to study energetics in relevant ecological settings to understand how these physiological mechanisms evolve.

The ability to rapidly fuel metabolism through ingested food seems to be a common adaptation among hummingbirds and bats with diets of simple carbohydrates and the fastest incorporation rates measured for vertebrates (Welch et al., 2016). Nectarivorous hummingbirds (3–5 g) and bats (10 g) fuel 50% of their metabolism within 3–9 min of feeding (Suarez et al., 2011; Voigt and Speakman, 2007; Welch et al., 2008). At three times their body size, U. bilobatum shows similar fractional incorporation rates. In contrast, other fruit-eating bats use incorporation rates of 10–12 min regardless of body size (Amitai et al., 2010). These comparative data indicate strong pressure on all flying frugivores, regardless of size, to mobilize ingested food to power flight and this is mediated through paracellular absorption (Caviedes-Vidal et al., 2008; Price et al., 2014). While initiating flight on stored energy, U. bilobatum and other sugar-focused bats rely heavily on ingested carbohydrates to supplement rapidly depleted glycogen at the onset of flight, further taxing the sugar oxidation cascade to push energy to muscle as quickly as possible (Suarez et al., 2011; Welch et al., 2016). Frugivorous bats deplete the large glycogen stores in their liver within 24 hr (Pinheiro et al., 2006). Our fat turnover experiments also showed that half of fat and sugar storage is mobilized within a single day. Specialization on foods rich in simple but rapidly incorporated carbohydrates seems to come with high risks that necessitate additional physiological and behavioral strategies to ensure energetic stability.

Uroderma bilobatum further control energetic incorporation and conservation by maintaining exceptionally low baseline cortisol levels that then are elevated to some of the highest recorded naturally induced values for mammals (Sapolsky et al., 2000). Basal glucocorticoid values of other bat species are especially high for mammals of their size (100–800 ng ml−1; (Reeder et al., 2004; 2006) and show large potential maximal output when challenged with ACTH (Lewanzik et al., 2012). However, the difference between baseline and restraint-induced circulating cortisol especially in female U. bilobatum is more similar to the extremes found in lemmings (Lemmus trimucronatus) that seasonally elevate their baseline corticosterone values by 10–80 times to concentrations of over 4000 ng ml−1 (Romero et al., 2008) or in flying squirrels (Glaucomys sp) that elevate cortisol values 38–40% above already high baseline values (Desantis et al., 2016). The low baselines we found may be a consequence of capturing resting or sleeping animals in their day roosts at least 4 hr after sunrise when circulating glucocorticoids were at their lowest (Sapolsky et al., 2000). However, this cannot explain peak values 1.5x greater than those observed in other mammals. We suggest that rapid increases in circulating cortisol levels during the acute stress response act in concert to mobilize energy stores, but more importantly, by suppressing glucocorticoid secretion during rest these bats are able to further minimize energetic expenditure and lower their metabolic rates (Haase et al., 2016; Nieuwenhuizen and Rutters, 2008; Palme et al., 2005) and minimize additional fat oxidation (Brillon et al., 1995).

Unpredictable fruit availability can have dramatic effects on survival and some bats, including U. bilobatum take advantage of their roosts to leverage social information and identify newly available food items (O'Mara et al., 2014a; Ramakers et al., 2016). Furthermore, the potential for rapid declines in food availability has likely shaped conservative physiological strategies in these bats to minimize energy expenditure while allowing for rapid resource mobilization needed for powered flight. These dynamic energetic strategies likely contribute to the success and diversity of the over 1300 bat species throughout the world (Simmons, 2005).

Materials and methods

All methods were approved by the Autoridad Nacional del Ambiente, Panama (SE/A-88–13; SE/AP-12–14; SE/A-73–14) and by the Institutional Animal Care and Use Committee of the Smithsonian Tropical Research Institute (2012-060-2015; 2014-0701-2017). All data presented are available at the Dryad Digital Repository (doi: 10.5061/dryad.n821p)

Capture and transmitter attachment

We captured 4 adult Uroderma bilobatum (2f/2m, 18.1 ± 1.5 g body mass) from their day roosts in Gamboa, Panamá in December 2014. Bats were fitted with a heart rate transmitter (ca 0.8 g; SP2000 HR Sparrow Systems, Fisher, IL USA) that emitted a continuous long-wave signal modulated by cardiac muscle potentials (Bowlin et al., 2005). This added 4.5 ± 0.04% of body mass and is within the range of the additional loading (5%) that should have minimal impact on behavior and physiology of bats and birds (Aldridge and Brigham, 1988; Barron et al., 2010; Elliott, 2016; O'Mara et al., 2014b), particularly broad-winged understory foragers like U. bilobatum. We trimmed the dorsal fur below the shoulder blades. A topical analgesic was then applied (Xylocaine gel, Astra Zeneca, Wedel Germany) and after disinfecting the electrodes and back of the bats with 70% EtOH, the transmitter’s two copper plated gold electrodes were inserted ca. 3 mm through a puncture made with a 23 G sterile needle. The transmitters were mounted on thin, flexible cloth and glued over the electrode insertion points using a silicone-based skin adhesive (Sauer Hautkleber, Manfred Sauer, Germany). The electrodes are flexible and do not appear to disturb the animals, and we expect superficial healing of the small punctures within one hour. While behavioral responses may not directly reflect physiological stress (Ditmer et al., 2015), our radio tracking data show typical behavior for this species, and both the large variation and temporal consistent heart rate data we collect do not indicate that bats are either under excessive stress or that habituation was needed to accommodate the added load of the transmitter (O'Mara et al., 2014b). After calibration of heart rate versus oxygen consumption (below) animals were tracked for 2–6 days (mean: 3.75 d). We recaptured three of the four bats and removed their transmitters. Bats lost 0.0–0.5 g (0.17 ± 0.29 g) which is within the daily mass fluctuations (1–2 g) observed in this species (O’Mara, unpublished data).

Calibration of heart rate versus oxygen consumption

We measured rates of oxygen consumption (V˙O2) carbon dioxide production (V˙CO2), heart rate (fH), and body temperature (Tb) of these four bats with an open-flow, push-through respirometry system. External air (>75% relative humidity, ~26°C) was dried with Drierite (WH Hammond Driertie Co, Ltd, Xenia, OH, USA) and pumped through a mass flow controller (FB8, Sable Systems International, Las Vegas, NV, USA) into a 1 L respirometry chamber fitted with a thermocouple within a 20 L insulated cooler that was dark and temperature controlled (PELT5, Sable Systems). Flow rate was 600 ml min−1, chamber temperature was maintained at 28–29°C, and relative humidity and vapor production were measured with a RH-300 (Sable Systems), and an additional empty chamber served as a reference to the animal chamber. After drying the air leaving the chamber with Drierite we measured CO2 concentration, and after scrubbing the air of CO2 with Ascarite (Thomas Scientific, Swedesboro NJ, USA) we determined O2 concentrations (FOXBOX, Sable Systems). Chamber temperature, CO2, O2, and relative humidity were recorded directly with Expedata via the UI-2 data acquisition interface (Sable Systems). V˙O2 and V˙CO2 were then calculated across five minute intervals (Lighton, 2008). Bat Tb was monitored with a temperature sensitive PIT-tag (BioThermo13, Biomark Inc, Boise ID, USA) injected dorsally under the skin and recorded every minute (Stockmaier et al., 2015). Bats remained normothermic throughout the experiment with Tb = 36.9 ± 1.6°C (Figure 1—figure supplement 3). Heartbeat of bats in the respirometry chamber was recorded as a sound file (see below), and fH was averaged over the one minute preceding each Tb measurement. This gave five Tb and fH measures for each measurement of V˙O2 and V˙CO2. After three hours bats were released at their roosts. Respirometry measures were taken between 19:00 – 04:00 hr. Heart rate provided a better fit in a single factor generalized linear mixed effect model (bat identity as a random effect) of energy consumption than body temperature (R2adjusted = 0.758 vs R2adjusted = 0.145, respectively), and the inclusion of Tb in a two-factor model did not improve the model’s predictive ability. In the best-fit model, energy expenditure was related to fH as (kJ h−1)=0.004 * fh - 0.3228 (Figure 1—figure supplement 4).

Heart rate telemetry and estimated field energy expenditure

We recorded fH of the four free-ranging bats during 2–6 days and nights using telemetry receivers (AR8000, AOR Ltd) connected to 3-element Yagi antennae (Sparrow Systems). This was then recorded via mini-dv output to a wave file (44.1–48 kHz) on a digital recorder (Tascam DR-05). Receivers were placed under roosts to record fH during the full inactive cycle during daylight hours. One to two people then followed the bats at emergence (ca 18:00) for 4–8 hr during the night’s activity and continuously recorded estimated activity (flight, inactivity, grooming) via fluctuations in the amplitude of the transmitted signal. Transmitter signal could be detected within 70–100 meters in the forest and up to a kilometer over open space (the Panama Canal). This gave us 18–20 hr of heart rate recordings per individual per day for a total of 350 hr. Daytime mean ambient temperature was 25.87 ± 1.21°C (mean daytime minimum to mean maximum: 23.38–28.24), and mean nightly ambient temperature was 23.74 ± 0.50°C (mean nightly minimum to mean maximum: 22.74–24.78°C). Ambient temperature was recorded by the Autoridad del Canal de Panamá for Gamboa and provided by the Smithsonian Tropical Research Institute’s Physical Monitoring Program.

Heart rate from radio transmitters was scored previously by visually measuring the interval needed to encompass 5–10 heart beats at sampling intervals of 0.5–10 min apart (Barske et al., 2014; Bowlin et al., 2005; Dechmann et al., 2011; Sapir et al., 2010; Steiger et al., 2009). We fully sampled the recorded data using an automated approach in R 3.2 (Core Team, 2016) to identify and count all heartbeats (Figure 1—figure supplement 1). We used a finite impulse response filter in seewave with a window length of 1500–2000 samples to select the carrier frequency of the transmitter. We counted individual heartbeats by applying a timer function in seewave that ran over non-overlapping windows of 500 samples. This created a resolution of 88–98 sampling windows per second. We then applied a kernel density filter in KernSmooth to further eliminate noise that was outside of the 90% quantile. This approach is conservative in that it may have eliminated some heart rate outliers, but the autocorrelated nature of heart rate allowed us to filter out errors likely induced by static or other interference in the recordings. Automated samples were inspected periodically to validate the filtering method, particularly in periods with high variation.

We then estimated total energy consumption in two ways. First, we used the five minute V˙CO2 production from the respirometry chamber estimate total energy consumption using a conversion of 1 mL CO2 ≅ 26 J and matched this to an average of the preceding one minute Tb and fH measurements. While we attempted to get a range of fH within the respirometry chamber, we could not attain the high heart rates typical of U. bilobatum during flight or the very low fH we observed during day rest. Furthermore, high fH of animals due to factors other than exercise, such as our respirometry chamber, may under-estimate energy consumption caused by changes in stroke volume and oxygen extraction efficiency during exercise (Bishop and Spivey, 2013). However, we can use the relationship between heart mass (Mh, a proxy for stroke volume), and body mass (Mb) to model oxygen consumption as function of fH as V˙O2 = 0.0402 Mb0.328±0.05Mh0.913±0.045fh2.065±0.03 (Bishop and Spivey, 2013). This estimate is based on the exercise response of 24 species of endotherms across 5 orders of magnitude of body size. This model is able to accurately estimate energy consumption during the primary mode of locomotion (Ward et al., 2002), which has been the major shortfall of experimental calibration of heart rate against V˙O2 in respirometry conditions where locomotion is restricted. We estimated individual heart mass as 1% of body mass at capture (Canals et al., 2005). Because the bulk of U. bilobatum diet is carbohydrate we then converted V˙O2 estimates to energy by assuming that 1 ml O2 ≅ 21.11 J.

Metabolic incorporation rates

We used a feeding experiment to measure the change in δ13C values in exhaled CO2 and estimate the time needed for ingested food to enter metabolic processes and exit as waste CO2. Uroderma bilobatum feed on figs with a low enrichment of 13C, typical for a C3 plant. By feeding an enriched 13C source from a CAM plant (agave nectar) we could measure how quickly sugar entered metabolism (McCue and Welch, 2016; Voigt and Speakman, 2007). Bats were captured from their day roosts and housed individually in mesh-lined cages. At time zero, bats were removed from their cage and immobilized by gently wrapping them in cotton gauze, excluding their heads and feet. They were then placed into a 6 × 6 × 4 cm plastic container with an 18 G needle hermetically attached. After sealing the container, ambient air was washed of CO2 using NaOH and flushed through the plastic container at a flow rate of 700 mL min−1. The flushing gas exited the container through the attached needle. The pump was turned off 2 min prior to collection to allow breath to accumulate in the plastic box. To collect accumulated CO2 we pierced the teflon membrane of an exetainer (LabCo Exetainer Buckinghamshire, UK) with the needle tip attached to the plastic container. This vacuumed approximately 4.5 ml of headspace into the vacutainer.

After the initial sample collection (time 0), bats were removed from the container and fed either freshly-collected Ficus insipida, or approximately 1.5 ml of a solution of 20% (w/w) agave nectar (Organic Blue Agave Nectar, Wholesome Sweeteners, Sugar Land Texas, USA), 2% (w/w) Nutri-Cal (Vétoquinol Prolab Inc, Princeville, Québec, Canada) and water using a transfer pipette. Breath samples were collected at 0, 10, 20, 30, 40, 60, and 90 min after the initial feeding. Bats fed figs (n = 6) were placed back in their home cage and allowed to feed ad libitum after sample collection at 40 mins and 60 mins. Bats fed agave nectar (n = 8) were fed an additional 0.5 ml of the agave nectar solution after sample collection at 30 and 60 min to ensure that the bats’ breath was equilibrated isotopically with the new diet. Additional samples at 50 and 70 mins post initial feeding were collected during the agave feeding experiments. Bats fed on figs were returned to their capture site after the last sample collection.

To measure fat turnover following (Voigt and Speakman, 2007), bats fed agave nectar were given an additional 1 ml of agave solution after the final sample collection and returned to their home cages. They were maintained on the agave nectar solution supplemented with Nutrical (tomlyn, Fort Worth USA; δ13C Agave + Nutri-Cal: 12.023 ± 0.11 ‰) as their only source of food for the following three nights. Bats were offered agave nectar and water ad libitum and were also fed by hand every 3 hr to ensure that they were feeding consistently. Food was removed during the day and bats were fasted for at least 10 hr prior to sample collection. At the beginning of each night were removed from their holding cages to collect a single breath sample as in the previous experiment to measure their baseline δ13C. Following breath collection, bats were fed with the agave nectar solution and returned to their holding cages. Body mass and body condition were monitored throughout the experiment to ensure animal optimum health, and one animal was released after night 2 because of weight loss.

Breath samples were then shipped to the stable isotope laboratory of the Leibniz Institute for Zoo and Wildlife Research where δ13CO2 was analyzed in a blind protocol using a GasBench (Thermo Scientific, Bremen Germany) connected to a stable isotope ratio mass spectrometer (Delta V Advantage Thermo Scientific, Bremen Germany). Samples were analyzed together with a laboratory standard gas that was previously calibrated with the international 13C reference materials NBS 19 and L-SVEC. Ratios of 13C and 12C were expressed relative to the international standard (Vienna-PeeDee Belemnite) using the δ notation in parts per mill (‰): δ13CV-PDB = Rsample/Rstandard-1) x 103 where Rsample/Rstandard is the ratio of heavy and light carbon isotopes (13C/12C) in the sample and the standard. Precision was always better than ±0.06‰ (1 SD). To measure the isotopic composition of the agave nectar solution a sample was dried in a drying oven until constant mass and 3 subsamples were then separated, weighed, and loaded in a tin capsule. Samples were analysed together with laboratory standards of known stable carbon isotope ratios using an elemental analyser (Flash elemental analyser, Thermo Scientific, Bremen, Germany) connected in continuous mode via a Conflo III to a stable isotope mass spectrometer (Delta V Advantage; Thermo Scientific, Bremen, Germany). Samples were combusted under chemically pure helium gas in the analyser and resulting gases were then routed to the IRMS for the analysis of stable carbon isotope ratios. The analytical precision was always better than 0.13 per mille (one standard deviation).

We estimated the fractional rate of isotopic incorporation (k) using a one-pool model for each individual bat as δ13Cbreath (t) = δ13Cbreath(∞) + [δ13Cbreath(0) – δ13Cbreath (∞)] e-kt; where δ13Cbreath (t) is the isotope composition, δ13Cbreath (∞) is the asymptotic equilibrium isotope composition, and k is the fractional rate of isotope incorporation. The time at which 50% of carbon isotopes are exchanged in the animal’s breath is calculated as t50=-ln(0.5)/k. The reciprocal of the fractional incorporation rate (k−1) estimates the average residence time of a carbon atom in fat reserves. We used a one-compartment model as this typically reflects isotopic incorporation into breath better than models with more complicated dynamics (Martínez Del Rio and Anderson-Sprecher, 2008). Non-linear least-squares models based on one-pool dynamics were fit to individual bats.

Energetic mobilization

We sampled circulating cortisol values from bats (15 F, 6 M) captured from their natural day roosts under the roofs of houses in Gamboa, Panama using a hoop net between 10–12 hr in November 2013 when females were not palpably pregnant or with dependent young. Baseline cortisol samples were collected by puncturing the antebrachial or cephalic vessels with a sterile 23 G needle and collecting ca. 70 uL of blood in heparin-coated hematocrit tubes. Blood samples were collected within 3 min of capture and placed on ice. Bats were placed in a soft cloth bag for 60 min and then a second blood sample of equal volume was collected. After the second blood collection, bats were fed 30% sugar water and released at the capture site. Blood samples were spun in a centrifuge for seven minutes at 7000 g, the plasma removed and snap frozen in liquid nitrogen before storage at −30°C. Samples were then mailed on dry ice to Trent University where total plasma cortisol was measured in duplicate using a commercially available radioimmunoassay (MP Biomedicals ImmuChem Coated Tube Cortisol 125I RIA Kit; MP Biomedicals, LLC, Diagnostic Division, Orangeburg, NY, USA). This kit was validated for parallelism with plasma from U. bilobatum. Tests for differences between slopes on log-transformed data showed that the serially diluted plasma curve was parallel to the assay standard curve (F1,9 = 0.21, p=0.66). The intra-assay coefficient of variation (CV) was 2.4% and all samples were run in a single assay. Seven generalized linear mixed effects models in lme4 were used to evaluate circulating cortisol concentrations (ng ml−1) for effects of sex and time point sampled (baseline or restraint) using animal identity as a random effect.

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Decision letter

  1. David Lentink
    Reviewing Editor; Stanford University, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Cyclic bouts of extreme bradycardia counteract the explosive metabolism of frugivorous bats" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors and the evaluation has been overseen by the Reviewing Editor and Ian Baldwin as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Fritz Geiser (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. Whereas we realize the number of essential revisions is rather extensive in this particular case, we believe that rewriting this manuscript such that it is more rigorous is entirely feasible based on the excellent style of the submitted manuscript. In general, we are mostly concerned with overstatements and a lack of essential information needed to place the findings in context; we do not question your key field observations.

Please comply to your best ability to the comments and provide point-by-point responses indicating at the start of every response if you comply, explain, or respectfully disagree.

Summary:

The manuscript by O'Mara et al., reports the discovery of a new cyclic bradycardic state in tropical fruit bats that saves energy by reducing the heart rate to extremely low values for a series of short moments during day rest. This is a unique and exciting find that advances our understanding of the mammalian cardiovascular system and metabolism. The technical accomplishment of measuring heart rate in freely flying individuals over several days in their natural habitat is impressive. Overall the use of heart rate as a measure of energy expenditure in small free-ranging mammals is commendable, the study was well constructed and the manuscript well written. The findings will be of interest to physiologists, ecologists, comparative biomechanists and organismal biologists in general.

Essential revisions:

Overall, we believe that the manuscript needs some careful reorganization to ensure scientific rigor: That the discussion and conclusions are placed within the context of the studies limitations.

Please discuss how estimates of energy expenditure and energy savings are extrapolations from a small sample of HR and MR under limited conditions and a single activity state. Further, the initial calibrations of HR against MR seem limited in that they were only conducted over a short time frame (3hrs) and at a single high ambient temperature very close to the thermoneutral zone. This gives a likelihood that animals with high HR (around 800bpm may have been exhibiting stress associated with the respirometry procedure, which can impact the derived regression equation as stress response alters blood pressure and cardiac output and is not indicative of true resting conditions.

While we agree that these short bouts of bradycardia are unlikely to be representative of torpor. We note that even small reductions in Tb can be reflected in reductions in HR and energy savings. Especially, because at rest many tropical and subtropical bats can reduce their body temperature by up to 6 degrees.

We appreciate the authors may make inferences of the costs of flight based on their resting calibration, however, these extrapolations may be inaccurate and this should be discussed appropriately. Alternatively, the paragraph in the discussion about flight costs (in the Discussion section) could simply be removed altogether without detracting from the findings improving the overall rigor of the manuscript.

To assist the reader please clarify how the regression calculations were conducted. How long were the HR values averaged with VO2? What time of day were the animals placed in the respirometry chamber? And were issues of autocorrelation and repeated measures addressed in the calculation of the regression equation?

Please further clarify the method by which HR was analyzed so the reader does not need to find and read the Cochran and Wikelski, 2005 reference but can simply fully rely on the Materials and methods section of the present manuscript to appreciate both its strengths and limitations.

Please clarify how many male and female bats were sampled for circulating cortisol, reading the manuscript we were confused by the different statements.

Title: An explosive metabolism would interfere with survival of the organisms. Another adjective may be more appropriate.

Bat stress related comments:

Based on the Introduction we wondered how well the bats function with the backpack after the surgical procedure. This also holds for the discussion in the Introduction and Discussion section "truly evolve" (too strong wording). Both the previous wind tunnel and invasive field experiments performed here are stressful to the animals unless habituation and positive reinforcement training have been implemented with all stressors removed. Only non-invasive non-stress experiments may possibly give this insight.

Reading the Materials and methods the 23ga needle is rather large, the metabolism of the bats is high, how quickly are they expected to heal up compared to the duration of the experiment? Please discuss this in the Materials and methods.

The Materials and methods states the backpack weighs 0.8 gram, which seems a high percentage of body weight. Heavy backpacks are a continues stressor of an animal with a high metabolic rate close to starvation. Please give the percentage backpack weight versus body weight and discuss it in the context of bat and bird experiments in which the effect of backpacks on stress, metabolics and general energetics have been studied. Please integrate this in the discussion if the backpack weighs more than 5% (limit for bird studies without recapture) bodyweight, otherwise integration in the Materials and methods section is sufficient.

Figure 2A; there appears to be a data gap beyond 22:00 hours, please explain and discuss. Panel B also appears to overlap with the data of panel A causing another gap, or there is another gap. Please plot the entire data of panel A without gaps from 0-24hrs if possible, otherwise indicate gaps using a gray area / bar with the explanation of why gaps exist.

Materials and Methods: What is the body mass and sample size of the bats? Sample sizes also need to be integrated into the Results section. Also include the average mass and std when discussing backpack mass in the Materials and methods and discuss the size of the backpack with respect to the size measure of the bat.

Table 1: include the mass and a measure of size of the individuals.

Abstract: Clearly much more than the cardiovascular system is involved here, for example digestion among other functions will be crucial in all this.

Introduction: Statement on homeostasis. There are other approaches than simply maintaining homeostasis in animal physiology.

Introduction:. …15-16 times 'of' minimum metabolic rates. Is this in comparison to BMR? If so, state. BTW some small bats at rest and without flight can do 300-400 times of the minimum metabolic rate during torpor.

Introduction: It is more complex than just lowering heart rate and body temperature and bats can reduce metabolic rate during torpor by up to 99% even when compared to BMR and more when compared to RMR at low ambient temperatures. Rephrase.

Introduction: There are published data on reduction of metabolism by 50% within the TNZ. Moreover, tropical bats are not always in thermo-neutrality, see the above reference. The sentence also does not make sense as written because they should be able to lower body temperature outside of thermo-neutral conditions. And reductions in heart rates will not only reduce metabolism. Rephrase.

Introduction: quadratic?

Introduction: Here it appears that all of the energy comes from food, further down stores of glycogen are needed. See also your results. Rephrase.

Introduction: By manipulating circulating levels of what?

Introduction: Define 'central-place forager'

Introduction: The reference in the Indroduction lists a number of phyllostomids using torpor (some described by McNab from lab studies as homeothermic) and also other nectarivorous/frugivorous bat.

Introduction: How would not using torpor result in a high metabolic scope -- should it not be the opposite?

Introduction: Further up it is argued that lab work is no good, now lab work is used. An explanation as to why this needs to be done in the laboratory would help.

Results: Large range in heart rates. To put this into perspective, six–fold is pretty good, but humans can do about four–fold and hibernating bats about eighty–fold.

Results: Were these bats captured or in captivity? If so, where and how?

Discussion: Entire range? Did they not fly over the canal?

Discussion: Replacement of half the fat reserves. The results on this need to be made clearer.

Discussion: 10% is not a lot and perhaps insignificant with regard to FMR.

Discussion: The short foraging times are interesting and are known from other small mammals in the field.

Discussion: Predictions of FMR from body mass?

Discussion: See comments above regarding McNab and reference in the Introduction. Differences between field and laboratory data are not only observed for flight, but also torpor expression and there is even a review on that. Rephrase this section.

Discussion: High sustained metabolic rates. Further up in the Discussion section it is stated their FMR is low.

Discussion: How will cyclic bradycardia maintain a semi-vigilant (and why semi?) state?

Discussion: Fuel metabolism from what?

Discussion: Is 50% of fat a majority?

Materials and methods: The statement is insufficient. In bears it was observed that behavioral responses to stressors can be minimal even if physiological responses are significant:http://www.cell.com/current-biology/abstract/S0960-9822(15)00827-1 Please discuss these issues briefly and fairly.

Materials and methods: Please include detailed information on the following: were animals retrieved during the experiment or after the experiment? Was the heart rate transmitter removed during or after the experiment, was the animal sacrificed or released after the experiment? If it was released in which state was the animal released?

Materials and methods: How was body temperature tracked? Provide details.

Materials and methods: What were the ambient temperatures during field work? If you can, provide daily minima, maxima and averages.

Materials and methods: Address assumptions and define abbreviations.

Materials and methods: Were the Figures and agave nectar analyzed for isotopes?

Materials and methods: This needs more explanation.

Materials and methods: What was in the vacutainers?

Materials and methods: This was queried above, were these in the wild? If so, please describe how bats were found, captured etc.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Cyclic bouts of extreme bradycardia counteract the high metabolism of frugivorous bats" for further consideration at eLife. Your revised article has been favorably evaluated by Ian Baldwin (Senior editor), a Reviewing editor, and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) Throughout the results and discussion energy expenditure in free ranging bats should be clarified as estimated, calculated or "HR derived" or equivalent. (E.g. Discussion section and other lines.)

2) The bradycardia experienced by these animals at rest seem to fall within the range expected for basal heart rate and values found for other species of bats of a similar size or smaller (see Kulzer, 1967 and Currie et al., 2015), therefore related statements should be clarified accordingly.

https://doi.org/10.7554/eLife.26686.017

Author response

Essential revisions:

Overall, we believe that the manuscript needs some careful reorganization to ensure scientific rigor: That the discussion and conclusions are placed within the context of the studies limitations.

Please discuss how estimates of energy expenditure and energy savings are extrapolations from a small sample of HR and MR under limited conditions and a single activity state. Further, the initial calibrations of HR against MR seem limited in that they were only conducted over a short time frame (3hrs) and at a single high ambient temperature very close to the thermoneutral zone. This gives a likelihood that animals with high HR (around 800bpm may have been exhibiting stress associated with the respirometry procedure, which can impact the derived regression equation as stress response alters blood pressure and cardiac output and is not indicative of true resting conditions.

This is an excellent point, and one we have attempted to circumvent using the allometric relationships based on heart and body mass developed by Bishop & Spivey (2013). Under stress conditions, it is unclear what the relationship between heart rate and oxygen consumption truly is. First, as a semantic distinction it is unclear why the reviewers assume that only high heart rates are indicative of stress. In our experience with frugivorous phyllostomids, such as Uroderma bilobatum, the most dangerous and stressful situations are when these animals enter low energy states, reduce Tb and fH. While rare, in these stress-induced states, animals are unlikely to recover. Furthermore, high stress can induce torpor conditions in many, including tropical, bat species (e.g., Molossidae). The physiological adjustments animals make to increase blood pressure, mobilize glucose reserves, and hyperventilate, to name only a few, all could yield contradictory energetic results. Furthermore, increased heart rate in these conditions may not have the full cardiac expansion and contraction of exercising heart rates.

One point of confusion that we would like to highlight is how we extrapolated field metabolic rates from the heart rate data. There has been quite a bit of discussion in the literature about the applicability and limitations of respirometry-based calibration of fH since most animals in these situations are not capable of exercising. Therefore, the calibrations of fH that we, and many others have used, fail to include exercise values of animals engaging in their typical modes of locomotion. The allometric equations developed by Bishop and Spivey, (2013) are a breakthrough in overcoming this limitation. Instead of the resting-state calibrations we conducted, we instead apply Bishop and Spivey's relationships using body mass and heart mass of our bats. This was noted in the original submission in subsection “Heart rate telemetry and estimated field energy expenditure”, but it was not made clear that this is the fundamental relationship that defined the energetic expenditure of our field study. This has been applied to studies on bar-headed geese (Bishop et al., 2015, Science doi: 10.1126/science.1258732; Hawkes et al., 2014, PloS one doi: 10.1371/journal.pone.0094015), great frigate birds (Wiemerskirch et al., 2016; Science doi: 10.1126/science.aaf4374), and golden-collared manakins (Barske et al., 2014, Proc R Soc B doi: 10.1098/rspb.2013.2482) We have clarified this in the Materials and methods, the Introduction, and in the Results.

Resting calibrations of animals in respirometry have been widely applied using the heart rate methods, and likely over-estimate energy consumption at the low ends of the calibration values, and more importantly dramatically under-estimate energy consumption at high heart rates. Bishop and Spivey, (2013) highlights the work by Dechmann and colleagues (2011) and the implausibly low field metabolic rates due to underestimates of flying energy consumptions via exercise restricted heart rate / metabolic rate calibrations. Outside of Bishop and Spivey’s particularly thorough work, the quadratic relationship driving this is nicely shown by Ward et al., (2002) and the differences in metabolic rates of geese under their primary and non-primary modes of exercise locomotion. There are few data from flying vertebrates that have captured this so completely.

While we agree that these short bouts of bradycardia are unlikely to be representative of torpor. We note that even small reductions in Tb can be reflected in reductions in HR and energy savings. Especially, because at rest many tropical and subtropical bats can reduce their body temperature by up to 6 degrees.

The use of torpor and heterothermy by tropical and subtropical bats when ambient temperature drops below 24°C is an important detail and we have noted this in the Discussion section. In other work, we have even found that a free-tailed bat species in Panama is capable of reducing energetic expenditure to values lower than the minimal torpor metabolic rates of many sub-tropical and temperate zone mammals, but at body temperatures greater than 32°C, largely by lowering heart rate independently of lowered body temperatures (O’Mara et al., in review, Proc R Soc B). We also note in this paragraph that at least in previous work with captive animals by McNab, U. bilobatum actively defends body temperatures and will not undergo heterothermy. It would have been ideal if we were able to also simultaneously measure body temperature of free-ranging bats, but their roosts would not accommodate the PIT tag antenna.

We appreciate the authors may make inferences of the costs of flight based on their resting calibration, however, these extrapolations may be inaccurate and this should be discussed appropriately. Alternatively, the paragraph in the discussion about flight costs (in the Discussion) could simply be removed altogether without detracting from the findings improving the overall rigor of the manuscript.

Flight is obviously the most energetically demanding aspect of a bat’s daily life, and with so few data from free-flying animals we feel that discussing this is worthwhile. We have added a qualifying statement of the difficulty of accurately measuring in-flight metabolic rates in subsection “Heart rate telemetry and estimated field energy expenditure”.

To assist the reader please clarify how the regression calculations were conducted. How long were the HR values averaged with VO2? What time of day were the animals placed in the respirometry chamber? And were issues of autocorrelation and repeated measures addressed in the calculation of the regression equation?

We have clarified this in the Materials and methods to show that heart rates were averaged over the one minute preceding the VO2 measure, as well as the time of day when the experiments were conducted (subsection “Metabolic incorporation rates.”). To account for potential confounds of repeated measures we included individual identity as a random effect. We did not attempt to correct for autocorrelation in the heart rate data, but treated the five-minute samples as independent units.

Please further clarify the method by which HR was analyzed so the reader does not need to find and read the Cochran and Wikelski, 2005 reference but can simply fully rely on the Materials and methods section of the present manuscript to appreciate both its strengths and limitations.

We are unclear to what this comment is referring since the 2005 Cochran & Wikelski chapter focuses on the spatial tracking of thrushes, not physiological sampling. We have added the sampling windows and intervals for how heart rate was previously visually counted in subsection “Metabolic incorporation rates.” so that it is clear how our current method differs.

Please clarify how many male and female bats were sampled for circulating cortisol, reading the manuscript we were confused by the different statements.

This is noted in subsection “Glucocorticoids & energy mobilization” and in Figure 4.

Title: An explosive metabolism would interfere with survival of the organisms. Another adjective may be more appropriate.

We have adjusted the title accordingly to read ‘high metabolism’.

Bat stress related comments:

Based on the Introduction we wondered how well the bats function with the backpack after the surgical procedure. This also holds for the discussion in the Introduction and Discussion sections "truly evolve" (too strong wording). Both the previous wind tunnel and invasive field experiments performed here are stressful to the animals unless habituation and positive reinforcement training have been implemented with all stressors removed. Only non-invasive non-stress experiments may possibly give this insight.

The impact of tag placement on an animal’s behavior is always of concern, and it is unlikely that any tag attachment, no matter how small or streamlined will have no impact on an animal. The best we can do is to minimize this impact on the limited number of animals that we study. The 0.8 g tag we used represents 4-5% of body mass (4.5 ± 0.04%). We have included this summary of body mass and tag mass as a percent of body mass in the Discussion section. We have also revised the wording for the statements. For all studies conducted in wind tunnels extensive habituation and positive reinforcement are needed to get animals to fly in these environments. Animals must be trained to use these devices. We therefore need to acknowledge that the results of these wind tunnel experiments represent normal unstressed states for animals under those conditions. As we note throughout the manuscript, however, the more we learn from free-ranging animals, even those wearing any biologging device, the more we understand that there is great behavioral, mechanical, and physiological flexibility that we have underestimated in these study species.

Reading the Materials and methods the 23ga needle is rather large, the metabolism of the bats is high, how quickly are they expected to heal up compared to the duration of the experiment? Please discuss this in the Materials and methods.

Healing time is difficult to estimate, but we also don't believe that the 23ga needle is large (⌀ = 0.6414 mm). The needle punctures are difficult to find for lead insertion and we expect superficial healing within an hour. In comparison, human insulin needles are typically 26–31 ga (⌀ = 0.4636–0.2604 mm), and most intramuscular and subcutaneous injections for human infants are done with 23ga needles. Furthermore, cannulae used to inject PIT tags that are commonly used on small mice and bats including Uroderma in our study populations are 12ga ((⌀ = 2.769) and no negative impact has been observed. We have added additional statement regarding potential healing time in subsection “Calibration of heart rate versus oxygen consumption.”

The Materials and methods states the backpack weighs 0.8 gram, which seems a high percentage of body weight. Heavy backpacks are a continues stressor of an animal with a high metabolic rate close to starvation. Please give the percentage backpack weight versus body weight and discuss it in the context of bat and bird experiments in which the effect of backpacks on stress, metabolics and general energetics have been studied. Please integrate this in the discussion if the backpack weighs more than 5% (limit for bird studies without recapture) bodyweight, otherwise integration in the Materials and methods section is sufficient.

This additional mass represents 4.5 ± 0.04% of body mass. This, and the general limits of additional loading on flying animals, has been added in the Material and methods.

Figure 2A; there appears to be a data gap beyond 22:00 hours, please explain and discuss. Panel B also appears to overlap with the data of panel A causing another gap, or there is another gap. Please plot the entire data of panel A without gaps from 0-24hrs if possible, otherwise indicate gaps using a gray area / bar with the explanation of why gaps exist.

There are indeed gaps in the data. We have amended Figure 2 to note these gaps and explain in the figure that this is when bats were out of range of radio telemetry (i.e., they flew faster through the forest than we could run) or when they left the roost when recording had been automated.

Materials and Methods: What is the body mass and sample size of the bats? Sample sizes also need to be integrated into the Results section. Also include the average mass and std when discussing backpack mass in the Materials and methods and discuss the size of the backpack with respect to the size measure of the bat.

Noted in subsection “Heart rate telemetry and estimated field energy expenditure”. The sample sizes, both in the number of individuals tracked (or sampled) and the duration of tracking, were integrated in each section in subsections “Activity patterns”; “Field metabolic rates and cyclic bradycardia“, “Metabolic incorporation rates of resting bats“; and Figure 4.

Table 1: include the mass and a measure of size of the individuals.

Added.

Abstract: Clearly much more than the cardiovascular system is involved here, for example digestion among other functions will be crucial in all this.

This has been deleted to simplify.

Introduction: Statement on homeostasis. There are other approaches than simply maintaining homeostasis in animal physiology.

Revised to ‘maintain physiological integrity’.

Introduction:. …15-16 times 'of' minimum metabolic rates. Is this in comparison to BMR? If so, state. BTW some small bats at rest and without flight can do 300-400 times of the minimum metabolic rate during torpor.

Clarified to resting metabolic rate.

Introduction: It is more complex than just lowering heart rate and body temperature and bats can reduce metabolic rate during torpor by up to 99% even when compared to BMR and more when compared to RMR at low ambient temperatures. Rephrase.

Rephrased

Introduction: There are published data on reduction of metabolism by 50% within the TNZ. Moreover, tropical bats are not always in thermo-neutrality, see the above reference. The sentence also does not make sense as written because they should be able to lower body temperature outside of thermo-neutral conditions. And reductions in heart rates will not only reduce metabolism. Rephrase.

Rephrased.

Introduction: quadratic?

Corrected.

Introduction: Here it appears that all of the energy comes from food, further down stores of glycogen are needed. See also your results. Rephrase.

Rephrased.

Introduction: By manipulating circulating levels of what?

Clarified.

Introduction: Define 'central-place forager'

Clarified.

Introduction: The reference in the Indroduction lists a number of phyllostomids using torpor (some described by McNab from lab studies as homeothermic) and also other nectarivorous/frugivorous bat.

This is true; however; McNab has previously shown that this species does not use torpor.

Introduction: How would not using torpor result in a high metabolic scope -- should it not be the opposite?

The breadth of metabolic scope is not a focus for our MS, so we have removed it from this statement.

Introduction: Further up it is argued that lab work is no good, now lab work is used. An explanation as to why this needs to be done in the laboratory would help.

We did not intend to say that lab work is no good – only that what has been seen is that there are substantial differences between the responses of the same species under laboratory conditions and when they are placed into more complicated ecological circumstances. In addition to the work we highlight, this has also been shown by Professor Geiser’s group, and others, regarding torpor and homeothermy expression (e.g, Geiser et al. 2007 doi:10.1007/s00360-007-0147-6; Geiser et al. 2000 doi:10.1007/978-3-662-04162-8_10; and Audet & Thomas 1997 doi: 10.1007/s003600050058). We have added a sentence in the Introduction to hopefully minimize these concerns and reaction about the importance of controlled experiments.

Results: Large range in heart rates. To put this into perspective, six–fold is pretty good, but humans can do about four–fold and hibernating bats about eighty–fold.

Thank you for the additional context.

Results: Were these bats captured or in captivity? If so, where and how?

Clarified.

Discussion: Entire range? Did they not fly over the canal?

This sentence highlights the range of activities, not necessarily spatial range. We have revised accordingly. We did not track the bats across the canal, but were able to maintain radio contact with them due to the large open space and that the bats apparently did not venture far into the forest on the south side of the canal.

Discussion: Replacement of half the fat reserves. The results on this need to be made clearer.

We believe that the results for this are clear in subsection “Glucocorticoids & energy mobilization” and Figure 4B, but we would appreciate further guidance as to what should be clarified.

Discussion: 10% is not a lot and perhaps insignificant with regard to FMR.

We respectfully disagree, particularly in light of the rapid incorporation rates and fat turnover that we also document.

Discussion: The short foraging times are interesting and are known from other small mammals in the field.

We agree, and this seems to be the norm for other frugivorous bats as well.

Discussion: predictions of FMR from body mass?

Clarified.

Discussion: see comments above regarding McNab and reference in the Introduction. Differences between field and laboratory data are not only observed for flight, but also torpor expression and there is even a review on that. Rephrase this section.

We are unclear about the specifics of this comment as the reference in the Discussion section focuses on past work by McNab which shows that U. bilobatum defends Tb across a wide range of temperatures and does not enter classic torpor. We have added reference in the previous sentence that is more focused on lowering Tb in torpor to also include tropical and sub-tropical species. We would like to note as well that torpor appears to be more likely to be document in laboratory conditions than in free-ranging animals.

Discussion: High sustained metabolic rates. Further up in the Discussion section it is stated their FMR is low.

It is high for a mammal of their size, but what is expected for a frugivorous bat. We have rephrased this.

Discussion: How will cyclic bradycardia maintain a semi-vigilant (and why semi?) state?

We have removed this statement.

Discussion: Fuel metabolism from what?

Clarified.

Discussion: Is 50% of fat a majority?

Rephrased.

Materials and methods: The statement is insufficient. In bears it was observed that behavioral responses to stressors can be minimal even if physiological responses are significant:http://www.cell.com/current-biology/abstract/S0960-9822(15)00827-1 Please discuss these issues briefly and fairly.

This is an excellent point and the citation is spot on, particularly since it includes heart rate data. We have added further discussion in the Materials and methods section.

Materials and methods: Please include detailed information on the following: were animals retrieved during the experiment or after the experiment? Was the heart rate transmitter removed during or after the experiment, was the animal sacrificed or released after the experiment? If it was released in which state was the animal released?

3 of the 4 bats were recaptured and the transmitter was removed. Bats lost 0.0–0.5g (0.17 ± 0.29 g) which is within the daily mass fluctuations of 1–2g observed in this species (487 bats 92 of which have been captured more than once; O’Mara, unpublished data).

Materials and methods: How was body temperature tracked? Provide details.

This was described in subsection “Calibration of heart rate versus oxygen consumption” of the original submission. There may be confusion with the word "tracked" in that sentence. We did not measure Tb in free-ranging animals, only in respirometry. This has been clarified.

Materials and methods: What were the ambient temperatures during field work? If you can, provide daily minima, maxima and averages.

Added in subsection “Energetic Mobilization”.

Materials and methods: address assumptions and define abbreviations.

Added.

Materials and methods: Were the Figures and agave nectar analyzed for isotopes?

We measured the value of the agave nectar solution but not the Figures.

Materials and methods: This needs more explanation.

We have expanded these methods and hopefully they are now clear.

Materials and methods: What was in the vacutainers?

Rephrased.

Materials and methods: This was queried above, were these in the wild? If so, please describe how bats were found, captured etc.

We have clarified these methods: These were all wild bats in their natural roost.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

1) Throughout the results and discussion energy expenditure in free ranging bats should be clarified as estimated, calculated or "HR derived" or equivalent. (E.g. Discussion section and other lines.).

We have corrected this accordingly by adding ‘heart rate derived’ where appropriate (e.g., Discussion section).

2) The bradycardia experienced by these animals at rest seem to fall within the range expected for basal heart rate and values found for other species of bats of a similar size or smaller (see Kulzer 1967 and Currie et al., 2015), therefore related statements should be clarified accordingly.

This is true and is it interesting how consistent basal heart rates are in bats. We have added detail related to this in the Discussion section that gives the range basal heart rate of small bat species and also notes the thermoneutral zone for U. bilobatum relative to the ambient temperatures of our field site for readers to consider.

https://doi.org/10.7554/eLife.26686.018

Article and author information

Author details

  1. M Teague O'Mara

    1. Department of Migration and Immuno-ecology, Max Planck Institute for Ornithology, Radolfzell, Germany
    2. Department of Biology, University of Konstanz, Konstanz, Germany
    3. Smithsonian Tropical Research Institute, Panama City, Panama
    4. Zukunftskolleg, University of Konstanz, Konstanz, Germany
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    teague.omara@gmail.com
    Competing interests
    No competing interests declared
    ORCID icon 0000-0002-6951-1648
  2. Martin Wikelski

    1. Department of Migration and Immuno-ecology, Max Planck Institute for Ornithology, Radolfzell, Germany
    2. Department of Biology, University of Konstanz, Konstanz, Germany
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
  3. Christian C Voigt

    Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany
    Contribution
    Conceptualization, Resources, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  4. Andries Ter Maat

    Department of Behavioural Neurobiology, Max Planck Institute for Ornithology, Starnberg, Germany
    Contribution
    Resources, Software, Formal analysis, Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  5. Henry S Pollock

    Program in Ecology, Evolution and Conservation Biology, University of Illinois at Urbana-Champaign, Urbana, United States
    Contribution
    Resources, Formal analysis, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  6. Gary Burness

    Department of Biology, Trent University, Peterborough, Canada
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon 0000-0002-1695-7179
  7. Lanna M Desantis

    Environmental and Life Sciences Graduate Program, Trent University, Peterborough, Canada
    Contribution
    Formal analysis, Investigation, Methodology, Writing—original draft
    Competing interests
    No competing interests declared
  8. Dina KN Dechmann

    1. Department of Migration and Immuno-ecology, Max Planck Institute for Ornithology, Radolfzell, Germany
    2. Department of Biology, University of Konstanz, Konstanz, Germany
    3. Smithsonian Tropical Research Institute, Panama City, Panama
    Contribution
    Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon 0000-0003-0043-8267

Funding

National Geographic Society (GEFNE124-14)

  • M Teague O'Mara
  • Martin Wikelski
  • Dina KN Dechmann

Max Planck Institute for Ornithology

  • M Teague O'Mara
  • Martin Wikelski
  • Dina KN Dechmann

University of Konstanz

  • M Teague O'Mara
  • Martin Wikelski
  • Dina KN Dechmann

Marie Sklodowska-Curie Actions

  • M Teague O'Mara

Natural Sciences and Engineering Research Council of Canada (RGPIN-04158-2014)

  • Gary Burness
  • Lanna M Desantis

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We would like to thank Rachel Page, the Gamboa Bat Lab, the Smithsonian Tropical Research Institute, and the Autoridad del Canal de Panamá for facilitating this work. Tom Faughnan, Bart Kranstauber, Inge Müller, Nele Herdina, Sebastian Stockmaier, and Sebastian Rikker helped with data collection. We would also like to thank the homeowners in Gamboa for allowing us access to their homes, particularly Hubert Herz, Daisy Dent, . Steve Paton (STRI) provided the daily ambient temperature data for Gamboa. We are grateful to Karin Grassow who analyzed the breath samples for stable isotope ratios, and to Sharon Swartz who provided valuable insight on a previous draft. This work was funded, in part, by the National Geographic Society (GEFNE124-14), The Max Planck Society, the Marie Skłodowska-Curie Actions, and the University of Konstanz.

Ethics

Animal experimentation: All methods were approved by the Autoridad Nacional del Ambiente, Panama (SE/A-88-13; SE/AP-12-14; SE/A-73-14) and by the Institutional Animal Care and Use Committee of the Smithsonian Tropical Research Institute (2012-060-2015; 2014-0701-2017).

Reviewing Editor

  1. David Lentink, Reviewing Editor, Stanford University, United States

Publication history

  1. Received: March 9, 2017
  2. Accepted: August 13, 2017
  3. Version of Record published: September 19, 2017 (version 1)

Copyright

© 2017, O'Mara et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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