Nectar-feeding bats fuel their high metabolism directly with exogenous carbohydrates

Authors

  • C. C. VOIGT,

    Corresponding author
    1. Leibniz Institute for Zoo and Wildlife Research, Research Group Evolutionary Ecology, Alfred-Kowalke-Str. 17, 10315 Berlin, Germany; and
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  • J. R. SPEAKMAN

    1. Aberdeen Centre for Energy Regulation and Obesity, School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK
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†Author to whom correspondence should be addressed. E-mail: voigt@izw-berlin.de

Summary

  • 1Mammals usually derive energy from metabolizing fat and glycogen stores combined with exogenous food. Nectarivorous bats mostly consume a diet low in both fat and proteins but rich in simple carbohydrates. Metabolizing exogenous carbohydrates directly to fuel their high mass-specific metabolic rate would save the energetic costs of lipogenesis and gluconeogenesis for nectarivorous bats. Therefore, we expected nectarivorous bats to switch to exogenous carbohydrates rapidly when available and use them predominantly instead of fat or glycogen.
  • 2We first investigated the rate of fractional incorporation of dietary sugars into the pool of metabolized substrates in Glossophaga soricina by measuring the change in 13C enrichment of exhaled CO213Cbreath) when animals were fed glucose, fructose or sucrose that was isotopically distinct from their normal diet. Second, we performed a diet-switch experiment to estimate the turnover rate of fat tissue.
  • 3When fed with sugars, the δ13Cbreath converged quickly on the isotope signature of the ingested sugars, indicating an almost exclusive use of dietary carbohydrates. The time for a 50% carbon isotope exchange in exhaled CO2 equalled 9, 13 and 14 min for fructose, glucose and sucrose, respectively. Nectarivorous bats fuelled 82% of their metabolism with exogenous carbohydrate when fed with fructose, 95% when fed with glucose and 77% when fed with sucrose. Bats depleted 50% of their fat stores each day.
  • 4Although nectarivorous bats consumed most of their body fat each day, this was still barely enough to sustain their diurnal metabolism. The fractional incorporation rates of dietary sugars into the pool of metabolized substrates in G. soricina are the fastest rates ever found in a mammal.

Introduction

Neotropical nectarivorous bats (Glossophaginae; Phyllostomidae) are among the smallest living mammals (< 10 g) and are specialized on the exploitation of flowers (Von Helversen & Winter 2003). Similar to hummingbirds, glossophagine bats consume floral nectar using an energetically costly hovering flight mode (Von Helversen 1986; Voigt & Winter 1999). Glossophagine bats have higher mass-specific metabolic rates than similar-sized terrestrial mammals (Nagy, et al. 1999; Speakman 2000) or similar sized bats with other feeding habits (McNab 1969, 1989; Von Helversen & Reyer 1984; Voigt, Kelm & Visser 2006). Floral nectars of chiropterophilous plants are dilute (Dobat & Peikert-Holle 1985), and consist mostly of sucrose and the monosaccharides glucose and fructose (Baker, Baker & Hodges 1998). These simple sugars are rapidly digested and/or absorbed, hence appropriate for fuelling costly locomotion modes such as hovering flight (e.g. hummingbirds: Suarez et al. 1990; Suarez & Gass 2002).

Glossophagine bats consume large amounts of dilute nectar each night (Von Helversen & Winter 2003): a 10-g nectarivorous bat may ingest up to 150% of its body mass as nectar (Von Helversen & Reyer 1984; Voigt et al. 2005, 2006). In response to their sugary diet, glossophagine bats have large amounts of sucrase in their digestive tract (Hernandez & Martínez del Rio 1992); an enzyme that is important for the hydrolysis of the disaccharide sucrose into its two component monosaccharides. Therefore nectarivorous bats are well equipped to process their sugary diet quickly. Storing fat in small nectarivorous bats may lead to large increases in the energy costs of horizontal and hovering flight (Voigt & Winter 1999; Voigt 2000). Moreover, conversion of carbohydrates into fat is associated with a substantial loss of energy (Wieser 1986). Consequently, although fat allows the bats to store energy, fat tissue has several disadvantages in these animals. Therefore, we expected nectarivorous bats to combust mostly carbohydrates from ingested sugar rather than using endogenous substrates such as glycogen or fat. We hypothesized that glossophagine bats would fuel their energetically expensive life directly with dietary carbohydrates, much in the same way as hummingbirds do. We measured the stable carbon isotope ratio in exhaled CO213Cbreath) of Glossophaga soricina to determine whether δ13Cbreath originates either from exogenous sugars, that is, sucrose, fructose and glucose, or from isotopically distinctive labelled endogenous substrates, that is, fat (Hatch, Pinshow & Speakman 2002a,b). We predicted that the δ13Cbreath of starved nectarivorous bats that had recently fed on sugar water would rapidly converge on the isotopic signature of exogenous sugars, if the bats switched their metabolic substrate use predominantly to the ingested sugars. Alternatively, if nectar-feeding bats use a mixture of endogenous and exogenous substrate, δ13Cbreath should stabilize at a level intermediate between the δ13C of the two isotopically distinct carbon sources. Furthermore, we predicted that G. soricina would incorporate dietary sugars into the pool of metabolized substrates at rates similar to those of hummingbirds (Carleton, Bakken & Martínez del Rio 2006; Welch et al. 2006), and we expected that the fractional incorporation rate of sucrose would be similar to that of the two monosaccharides (glucose and fructose), since sucrase is abundant in the bats’ digestive tracts (Hernandez & Martínez del Rio 1992). Finally, we predicted turnover rates of fat tissues in nectarivorous bats to fall closer to those of hummingbirds (1–2 days in the broad-tailed hummingbird Selasphorus platycercus; Carleton et al. 2006) than to those of terrestrial granivorous rodents (a few weeks in the Mongolian gerbil Meriones unguienlatus: Tieszen et al. 1983) owing to the high metabolic rate and small fat deposits of nectarivorous bats (McNab 1976; Von Helversen & Reyer 1984; Voigt et al. 2006).

Methods

equilibration of study animals to two isotopically distinct diets

We captured 56 individuals (27 males, 29 females) of G. soricina, Pallas (Glossophaginae, Phyllostomidae) from a captive breeding colony maintained in greenhouse facilities at the University of Erlangen-Nuremberg (Germany). Animals were assigned arbitrarily to two treatments, that is, they were either fed a diet based on C4/CAM plant products (groups A and B) or a diet based on C3 plant products (groups C, D and E) for 20 days prior to the actual measurements. Group A consisted of 8 bats, group B of 16 bats, group C of 16 bats, and groups D and E of 8 bats each. The sizes of flight rooms were either 2 × 2 × 3 m3 or 5 × 3 × 4 m3. The C4/CAM plant based sugary diet of groups A and B was based on 18% mass/mass Agave syrup water to which we added supplemental food (Nido Superkid, Nestlé, South Africa) and the C3 plant based sugary diet of groups C, D and E was based on 18% (m/m) honey water to which we added Aletemil (Alete, Nestlé, Germany). The C4/CAM plant based diet consisted of c. 62% fructose, 16% glucose and 22% sucrose. The C3 plant based diet consisted of c. 32% fructose, 28% glucose and 40% sucrose. These values were calculated according to the ratio at which the base diet (either Agave syrup or honey) was mixed with the supplemental food (Nido Superkid and Aletemil) and according to the manufacturers’ specifications. The dietary stable carbon isotope ratio of groups A and B was –11·6 and that of groups C, D and E –26·0 (Table 1). Animals from groups A and B are called C4 bats and animals from groups C, D and E are called C3 bats. Ambient temperature in the flight rooms was 27 °C, relative humidity 70% and the photoperiod was 12:12 h. Individuals of each group were marked by labelling the toes with white marker fluid (Tipp-Ex, Nodef, Germany) prior to the experiment. After the study all individuals were returned to the breeding colony. The average body mass of the bats was 9·9 ± 1·1 g (n = 56).

Table 1.  Experimental treatment of bat groups, average volume of sugar water ingested (mL) per individual and experiment (± 1 standard deviation) and stable carbon isotope signature of sugars (‰)
δ13C of maintenance diet (‰)Sugar switched to during experimentGroupSugar water ingested (mL)δ13Csugar (‰)
−11·6C3 fructoseB1·61 ± 0·42−26·77
C3 glucoseA1·56 ± 0·32−26·52
C3 sucroseB1·55 ± 0·15−25·65
−26·0C4 fructoseC1·55 ± 0·32−10·65
C4 glucoseD1·61 ± 0·22−10·56
C4 sucroseC1·49 ± 0·25−12·23

fractional incorporation of dietary sugars into the pool of metabolized substrate

The fractional incorporation of dietary sugars into the pool of metabolized substrates was assessed using a diet-switching protocol on bats in groups A–D. During each experimental day we tested only a single type of sugar. We performed two experiments each with a specific sugar with bats of group A and B. The two experimental days were separated by 1 day. Details of the switching protocol for each sugar and carbon source are depicted in Table 1. Each experimental run lasted 60 min and consisted of six breath collection events. For breath sampling, bats were transferred singly into silk bags (1 × 5 × 7 cm3) that were put each into a larger plastic bag (10 × 10 cm2; volume 200 mL; ZiplockTM). Ambient air was washed of CO2 using NaOH and flushed through the silk bag via a plastic tube (diameter 3 mm) at a flow through rate of 700 mL min−1. The outlet of the plastic bag consisted of a small slit of 4 cm (width 0·2 cm). Then we sealed the plastic bag for 1·5 min to let CO2 accumulate in it. Glossophaga soricina have a resting metabolic rate of c. 1·14 mL O2 min−1 (Cruz-Neto & Abe 1997). Therefore, we expected CO2 to accumulate to c. 0·5% during this time span. For breath collection we used evacuated vacutainers (LabcoTM, Buckinghamshire, UK). Approximately 10 mL of air were sucked from close to the position of the bat in the silk bag via a second plastic tube (diameter 1 mm, length 4 cm) when a needle hermetically fused to the tube's end outside the bag penetrated the Teflon membrane of a vacutainer. After each breath collection the plastic bag was unsealed again and CO2 free air was flushed through the bag. Breath collection was repeated after 5, 10, 20, 40 and 60 min following the first feeding event, since we expected an exchange of stable carbon isotopes in exhaled CO2 during this time period. Bats were fed repeatedly after 20 and 40 min following the first feeding event to ensure that the bats’ breath was equilibrated isotopically to the new diet. The average amount of ingested sugar water per individual is listed in Table 1. On each experimental day, bats were fed with a single type of sugar water, either mixed with C3 labelled fructose, glucose (both Sigma Aldrich, Munich, Germany) and sucrose (beet sucrose; Kristallzucker, Südzucker, Germany) or C4 labelled fructose, glucose and sucrose (commercially available sugars based on corn). Sugar was mixed with water to a concentration of c. 30% (mass/mass; Atego refractometer, Atego Ltd, Tokyo, Japan). To measure baseline stable carbon isotope ratios of exhaled CO2 in unfed animals, we collected breath samples of five unfed individuals from groups A and C at the same time intervals as the other experiments, and tested whether stable carbon isotope signatures of exhaled CO2 changed during the course of the experiment using a repeated measures anova. All bats were released into the flight rooms after experimental runs and afterwards to the breeding colony in the greenhouses.

turnover of fat tissue

We changed the diet of group E for 4 days from C3 plant products to C4/CAM plant products (same diet as previously used for equilibration of groups A and B). On the first (day 0) and last day (day 3) of the diet switch experiment all bats were weighed (electronic balance, Mettler PM-100, Greifensee, Switzerland, accuracy 0·01 g). Between 17:00 and 19:00 h preceding day 0 of the experiment, and at the same time of day on the following 3 days, bats were caught with hand nets and put singly into silk bags. Breath collection followed the procedure described above except we collected only a single breath sample and released the animals immediately afterwards into their flight cage.

isotope analysis

Stable carbon isotope ratios of sugar samples were measured following the description in Voigt & Kelm (2006) at the Department of Geology and Mineralogy of the University of Erlangen-Nürnberg. Breath samples were measured in an ISOCHROM-µG isotope ratio mass spectrometer (Micromass, UK; Perkins & Speakman 2001). The breath samples were automatically flushed from the vacutainers in a stream of chemically pure helium, after which a gas chromatograph separated the CO2 gas from the other gases before admitting it into the mass spectrometer in a continuous flow. Breath samples together with internal standards that had been previously characterized relative to an international 13C standard (IAEA-CO-1) were analyzed in duplicates. All 13C/12C were expressed relative to the international standard using the δ notation in parts per mil (‰) and the following eqn 1:

image( eqn 1)

with 13C/12C representing the isotope ratio in either the breath sample or the standard. Precision was better than ±0·01 (1σ). All samples were analyzed using a blind experimental protocol. Carbon dioxide concentration was too low in some samples of one individual of group B that was fed C3 fructose and therefore we excluded this individual from further analysis.

calculation of equation parameters

We expected that changes in isotopic composition follow a single-pool exponential model (e.g. Tieszen et al. 1983; Voigt et al. 2003). Hence, we calculated equations of the following type for each individual and experiment according to Carleton & Martínez del Rio (2005):

image( eqn 2)

In eqn 2, δ13Cbreath(t) is the stable carbon isotope ratio of exhaled CO2 at time t, δ13Cbreath(∞) the asymptotic stable carbon isotope ratio of exhaled CO2 when animals are equilibrated to the stable carbon isotope signature of their diet, δ13Cbreath(0) the stable isotope ratio of exhaled CO2 at time 0 of the experiment, and k the fractional rate of isotope incorporation into exhaled CO2. Estimation of k was performed on an iterative basis using SigmaPlot (spss, Version 8·0). For each experiment, we averaged regression coefficients over individual values. To test for differences in mean δ13Cbreath(∞) and the stable carbon isotope ratio of dietary sugars we performed one sample Student's t-tests.

We calculated the time at which 50% of carbon isotopes are exchanged in the animals’ breath (t50) according to the following equation: t50 = ln(0·5)/k, with ln representing the natural logarithm and 0·5 the exchange of 50% isotopes. All values are given as means ± 1 standard deviation and all statistical tests were performed two-tailed if not stated otherwise.

In the fat tissue turnover experiment, we estimated the asymptote value of δ13Cbreath(∞) of eqn 2 in the following way, since the asymptotic increase of δ13Cbreath did not reach a plateau. We averaged the initial values of unfed bats equilibrated to the C3 plant based diet (groups A and B; n = 24) and the C4/CAM plant based diet (groups C and D; n = 24) and calculated the difference between the average δ13Cbreath of bats of group A and B, and the animals of the fat turnover experiment (group E). To determine the expected asymptotic value of δ13Cbreath(∞), we subtracted this difference from the mean δ13Cbreath of the animals of groups C and D. The expected δ13Cbreath(∞) was used for estimating the other regression coefficient in the single-pool exponential model.

Results

equilibration of study animals to two isotopically distinct diets

Glossophaga soricina that were maintained on a constant diet based on C4/CAM plant products (C4 bats) had a mean δ13Cbreath of −11·8 ± 1·2 (n = 24) after having fasted over at least 5 h during the daytime. The baseline value of δ13Cbreath of unfed C4 bats was not significantly different from the δ13C of the C4/CAM based diet (one sample Student's t-test: t23 = 0·93, P = 0·36). Animals that were maintained on a diet based on C3 plant products (C3 bats) had a δ13Cbreath of –26·5 ± 1·2 after having fasted over at least 5 h during the daytime. Similar to the stable carbon isotope signature of exhaled CO2 in unfed C4 bats, the δ13Cbreath of unfed C3 bats was not significantly different to that of the their diet (one sample Student's t-test: t23 = 1·96, P = 0·062). The difference in δ13Cbreath between C3 and C4 bats equalled 14·7 (Student's t-test: t47 = 43, P < 0·001).

fractional incorporation of sugars into the pool of metabolized substrate

Baseline measurements of δ13Cbreath in unfed animals did not change during the 60 min of an experimental run, neither in unfed C3 bats (repeated measures anova: F4,29 = 1·70, P = 0·67) nor in unfed C4 bats (repeated measures anova: F4,29 = 62, P = 0·08). We estimated the fractional turnover of stable carbon isotopes in exhaled CO2 of nectar-feeding bats after ingestion and combustion of different sugars, that is, fructose, glucose and sucrose (Fig. 1a–f). A few minutes after ingestion of naturally labelled sugar, the δ13Cbreath of the bats became enriched in 13C in C3 bats and depleted in 13C in C4 bats (Fig. 1). Equation coefficients for the single-pool exponential models of the six experiments are provided in Table 2 for each type of sugar. The average t50 value for the fractional turnover of fructose was not significantly different between C3 and C4 bats (Student's t-test: t12 = 0·26, P = 0·80) and equalled 8·7 ± 3·9 min. Similarly, average t50 values were not significantly different between C3 and C4 bats for glucose (mean 12·8 ± 5·9 min; Student's t-test: t13 = 1·62, P = 0·13) and sucrose (mean 14·0 ± 5·6 min; Student's t-test: t13 = 1·38, P = 0·19). Fructose was turned over at the fastest rate followed by glucose and sucrose. We estimated the expected asymptotic plateau of the δ13Cbreath after bats were completely equilibrated to the stable carbon isotope signature of the recently ingested sugar water using a single-pool exponential model. The expected plateaus deviated in five of six cases from the dietary stable carbon isotope signature (Fig. 1). Only in C3 glucose, δ13Cbreath was identical to the δ13C of the sugar water.

Figure 1.

δ13Cbreath (‰) in exhaled CO2 of G. soricina that were fed a 30% (m/m) sugar solution of either fructose (a, n = 8; b, n = 7), glucose (c, n = 8; d, n = 8) or sucrose (e, n = 8; f, n = 8). Bats of the experiments shown in (a), (c) and (e) were first equilibrated to a diet derived from C3 plants and then fed naturally labelled sugar water from C4 or CAM plants and bats of the experiments shown in (b), (d) and (f) were first equilibrated to a diet derived from C4 or CAM plants and then fed naturally labelled sugar water from C3 plants. Solid circles represent mean values of δ13Cbreath for a given time interval since the bats were fed with sugar water for the first time. The T-mark depicts ± 1 standard deviation, the dashed line the δ13C of the diet, and the curve exponential saturation curve fitted to the mean values. The Δdiet-breath value indicates the mean difference between δ13Cbreath(∞) and δ13Cdiet and t50 the time required to exchange 50% of the carbon in exhaled CO2 with those from recently ingested sugar.

Table 2.  Types of sugars used in the fractional sugar incorporation experiments, and regression coefficients (mean ± 1 standard deviation) of the single-pool experimental model averaged across all individuals within each treatment group
Sugarδ13Cbreath(∞) (‰)δ13Cbreath(0) –13Cbreath(∞) (‰)k (days−1)
C3 fructose−22·73 ± 1·0111·70 ± 1·280·0964 ± 0·0456
C4 fructose−12·47 ± 1·49−14·43 ± 2·300·0955 ± 0·0420
C3 glucose−26·06 ± 1·4814·66 ± 1·840·0952 ± 0·0570
C4 glucose−12·86 ± 1·24−14·14 ± 1·890·0477 ± 0·0095
C3 sucrose−23·92 ± 1·3811·83 ± 1·750·0652 ± 0·0222
C4 sucrose−16·74 ± 2·14−9·70 ± 2·470·0538 ± 0·0331

fat tissue turnover experiment

Bats in group E used for the fat tissue turnover experiment weighed on average 9·4 ± 0·8 g before the diet switch and 9·6 ± 0·7 g after the 4-day experiment. The change in body mass was not significant (paired t-test: t7 = 1·7, P = 0·16). The average δ13Cbreath of bats of group E at day 0 equalled –25·3 ± 1·2. After day 0, the δ13Cbreath of starved animals became enriched in 13C over the 4 days of the experiment, indicating the isotopic change of the diet from C3 to C4/CAM plant products (Fig. 2). We estimated a single-pool exponential regression equation for each individual bat and calculated a mean regression curve over all individuals. The regression equation was: y(‰) = –10·6 – 14·7 (±1·5)e −0·690 (±0·125)t(days) (regression coefficients given as mean ± 1 standard deviation). The fractional incorporation rate at which 50% of carbon atoms in fat tissue were exchanged by carbon atoms from dietary sugars equalled t50 = 1·0 ± 0·2 days. Therefore, a carbon atom remains in the fat deposits of G. soricina for on average 1·4 days.

Figure 2.

δ13Cbreath (‰) in exhaled CO2 of nectar-feeding bats (group E; n = 8) that fasted for 10 h since dawn on each experimental day. The diet was changed from a C3 to a C4/CAM based origin at day 0. Closed symbols indicate means and T-marks depict ± 1 standard deviation and the dashed line the asymptotic plateau (mean δ13Cbreath of individuals of the C4 group) used in the single-pool exponential decay model fitted to the mean values (solid line).

Discussion

fractional incorportation rates of dietary sugars into the pool of metabolized substrates

Nectar-feeding G. soricina metabolized exogenous carbohydrates within minutes after feeding to sustain their high metabolic rate. The fractional incorporation of dietary sugars into the pool of combusted substrates was high in all three types of sugars, irrespective of whether they originated from C3 or C4/CAM plants. Most mammals studied thus far with respect to the rate of sugar ingestion and metabolism are much larger than the 10 g bats of our study, and since larger digestive tracts probably cause delays in the substrate intake, processing and combustion, the fast fractional turnover rates of dietary sugars in nectarivorous bats may be partly due to the relatively short time required to process the sugars in the alimentary tract. In mice, 40% of carbon isotopes were exchanged in exhaled CO2 within 15 min when mice were fed corn (Perkins & Speakman 2001), suggesting that fractional turnover rates of exogenous substrate into the pool of metabolized substrates may be similarly high in other small mammals.

Hummingbirds make use of dietary carbohydrates at similarly fast rates. Welch et al. (2006) showed that starved broad-tailed hummingbirds (S. platycercus) fed with sucrose sustained c. 74% of their flight metabolism by sucrose combustion 20 min after drinking nectar. The fractional incorporation of dietary sugars into the pool of combusted substrates was slightly slower in G. soricina than in hummingbirds, although sucrase is abundantly present in both taxa (G. soricina: Hernandez & Martínez del Rio 1992; hummingbirds: Martínez del Rio & Karasov 1990; Martínez del Rio, Baker & Baker 1992). But given the threefold larger size of nectarivorous bats compared to hummingbirds, the mass-specific rates are even faster in nectarivorous bats than in hummingbirds. Yellow-rumped warblers (Dendroica coronata) that fed on a mixed diet of carbohydrates, proteins and fats, had mean t50 values of 4·4 h (Podlesak, McWilliams & Hatch 2005), indicating that fractional incorporation rates of exogenous substrates into the pool of metabolized substrates may be slower when birds are dietary generalists or when feeding on a mixed diet instead of on a pure carbohydrate diet.

Monosaccharides such as fructose and glucose were metabolized at a similar rate as the disaccharide sucrose, probably since sucrase – the enzyme that hydrolyses the disaccharide into its monosaccharides – is abundantly present in the digestive tract of G. soricina (Hernandez & Martínez del Rio 1992). Previous preference tests in fruit-eating and nectar-feeding phyllostomid bats revealed that members of both feeding ensembles preferred sucrose to the monosaccharides glucose and fructose (Herrera 1999). This preference behaviour is not explained by sugar specific fractional incorporation rates at which bats allocated sucrose and hexose sugars into the pool of metabolized substrates.

Carbon atoms from naturally labelled glucose were not traceable in the δ13Cbreath before 30 min after sugar uptake in humans (Péronnet 2003) or even several hours after the meal in larger mammals such as horses (Ayliffe et al. 2004). Unfortunately, exact t50 values cannot be calculated from data of Ayliffe and colleagues (2004) or Péronnet (2003) since the subjects did not even come close to the expected asymptotic plateau of equilibration. In horses, a maximum enrichment of 13C in δ13Cbreath was reached after 5 h (4 enrichment of 13C above baseline) which suggests that t50 equals more than 5 h. In llamas t50 equalled 2·8 h (Sponheimer et al. 2006). Summarizing, fractional incorporation rates of sugars into the pool of metabolized substrates of G. soricina are the fastest ever measured rates among mammals.

combined use of exogenous and endogenous substrates

Non-exercising G. soricina metabolized exogenous carbohydrates immediately after ingestion and after an hour δ13Cbreath converged to the isotopic signature of the ingested sugars. Since δ13Cbreath was enriched in 13C when animals fed sugars derived from C3 plants but depleted when sugars originated from C4 or CAM plants, and since differences caused by enzymatic fractionation should have the same sign for a given type of sugar, we conclude that differences between the asymptotic value of our regression equations and the dietary δ13C are most likely caused by fuelling metabolism partly by endogenous substrate, such as fat or glycogen. We calculated the proportion (P) at which nectarivorous bats used endogenous substrates by using a linear mixing model (eqn 3) for two pools of metabolized substrates, that is, endogenous and exogenous substrates:

image( eqn 3)

Bats used a significant portion of endogenous substrate in addition to the oxidation of exogenous carbohydrates: The mean proportion of endogenous substrates in the pool of metabolized substrates equalled 23 ± 17% for bats fed on sucrose water (one-sample Student's t-test: t14 = 6·6, P < 0·0001), 18 ± 11% for bats fed on fructose water (one-sample Student's t-test: t15 = 5·5 , P < 0·0001) and only 5 ± 10% for bats fed on glucose water (one-sample Student's t-test: t15 = 2·2, P = 0·041). Accordingly, nectarivorous bats used a small, but significant proportion of endogenous substrates after ingestion and digestion of a sugary diet. Since enzymatic activity may follow a diurnal pattern (Stevenson et al. 1975; Saito, Murakami, Suda 1976) and since metabolism of dietary carbohydrates usually occurs at night in free-ranging bats, it seems plausible that nectarivorous bats fuel their metabolism completely with exogenous substrate when foraging for nectar at night. Most other mammals maintain their aerobic metabolism predominantly by mobilising fat and glycogen (Roberts et al. 1996); for example, endogenous substrates support 70%–85% of the metabolic rate in humans, with a higher contribution of exogenous substrates in exercising subjects (Adopo et al. 1994; Jeukendrup & Jentjens 2000; Péronnet 2003).

Non-exercising hummingbirds, fuelled 90% of their metabolism with exogenous carbohydrates (Carleton et al. 2006), and exercising hummingbirds switch to an almost exclusive combustion of exogenous carbohydrates to maintain their costly flight activities (Welch et al. 2006). Our findings in non-exercising nectarivorous bats are in line with those from Carleton et al. (2006), indicating a predominant but not exclusive use of exogenous substrate when animals are resting.

turnover of fat tissue in vertebrate pollinators

Glossophaga soricina turned over fat tissue 15 times faster than, for example, Mongolian gerbils: t50 = 1·0 days in nectarivorous bats compared to t50 = 15·6 days in gerbils, but turnover rates of fat tissues were almost identical to those reported for hummingbirds (t50 = 1·5 and 0·8 days in broad-tailed hummingbirds; Carleton et al. 2006). This high turnover of fat stores in both pollinator taxa is most likely caused by their relatively small fat deposits and their relatively high mass-specific metabolic rate (Voigt et al. 2006) and it underlines that nectar specialists must balance lipogenesis and fat catabolism very accurately if they are to remain in energy balance.

allocation of substrates to metabolism, storage and tissue synthesis

This study suggests the hypothesis that ingested carbohydrates may be allocated preferentially to metabolism and not to tissue synthesis or storage. Consequently, stable carbon isotopes of the carbohydrate portion of a diet is detectable in the breath of an animal, but not necessarily in the solid tissue in which mostly proteins or fat are incorporated (Perkins & Speakman 2001). Previous isotopic studies in G. soricina showed that the t50 values of stable carbon isotopes was low for red blood cells when bats were sustained on a carbohydrate rich/low protein diet (Voigt & Matt 2002; Voigt et al. 2003) and that estimates of t50 increased with decreasing percentage of carbohydrates and increasing percentage of protein in the diet (Miron et al. 2006). In light of the current breath isotope study, it becomes clear that bats mostly metabolized the few available carbohydrates in the study by Miron et al. (2006), whereas carbohydrates were incorporated into blood cells in the study by Voigt et al. (2003). Therefore, the contrasting difference of t50 values in the range of 20–40 days in Miron et al. (2006) and more than 100 days in Voigt et al. (2003) for blood cells most likely resulted from different enzymatic pathways bats used. Bats that were sustained on a carbohydrate poor diet mostly metabolized sugars and assimilated proteins, whereas bats that were sustained on a carbohydrate rich diet metabolized carbohydrates, but in addition assimilated carbohydrates also into blood cells. Following this line of argument, a carbohydrate rich diet of free-ranging nectarivorous bats would cause a low and a protein-rich diet a fast turnover rate of blood cells.

the ecological context of substrate use

Nectar specialists live in the fast lane having metabolic rates higher than those of similar-sized animals with different feeding habits (Voigt et al. 2006). The underlying cause for this phenomenon is that nectar specialists are forced by flowering plants to continuously search for new nectar sources since nectar rewards are barely large enough to satisfy the energetic needs of pollinator-specific flower exploitation costs, making flower visitation marginally profitable, but sufficiently small to encourage pollinators to return to the flowers after nectar replenished (Klinkhammer & deJong 1993; Voigt et al. 2006). Since aerial locomotion is more costly for fat than for lean individuals (Voigt 2000), fat deposits should be regulated so that they are able to supply the energy requirements of diurnal inactivity, that is, in times when exogenous substrates are absent. Carbohydrates derived from flowers may be the more appropriate substrate for fuelling the pollinator's activities when foraging for nectar. This raises the question of whether fat deposits are turned over at sufficient rates to support the metabolism of resting nectarivorous bats in the daytime roost.

The body mass of free-ranging G. soricina contains c. 5% fat stores (McNab 1976). A t50 value of 1·0 days for fat tissue turnover suggests that 50% of body fat is exchanged over a day. Consequently, a 9·6-g nectarivorous bat mobilizes c. 0·24 g fat each day. Assuming that the energy equivalent of 1 g fat is 39 kJ (Wieser 1986), bats would derive 9·4 kJ of energy each day by catabolizing lipids. Given a field metabolic rate of 45·7 kJ d−1 for a glossophagine bat of similar mass (Voigt et al. 2006), 21% of energy turned over per day originates from the mobilization of fat stores. According to Cruz-Neto & Abe (1997), resting metabolic rate of our G. soricina equals 65·6 mL O2 h−1 at 25 °C. This adds up to 787 mL O2 for the 12 h period of daylight. Converting this value into an energy equivalent (19·81 J mL O2 at RQ 0·75; Wieser 1986) yields 15·6 kJ of energy turned over during the daytime. The fact that fat contributes only with 9·4 kJ to the daytime metabolism supports the observation by Kelm & von Helversen (2007) that bats use a mixture of fat and carbohydrates during the beginning of day and then increasingly metabolize endogenous substrate. This would, however, require that the nocturnal life of nectarivorous bats is completely or predominantly fuelled by exogenous carbohydrates, yielding a slight surplus by the end of the night. The current study suggests that nectarivorous bats mostly, if not exclusively, use exogenous sugars when available to fuel their metabolically expensive activities at night.

Acknowledgements

We would like to thank Otto von Helversen (University of Erlangen) for allowing us to use individuals of his captive colony of nectar-feeding bats. We are grateful to Monika Otter for feeding the study animals. Peter Thompson and Paula Redman most kindly analyzed the breath samples at the Aberdeen Centre for Energy Regulation and Obesity. We thank Raul Suarez, Carlos Martínez del Rio, Detlev Kelm, Dina Dechmann and Katja Rex for commenting on an earlier version of this manuscript. This work was financed by a grant from the ‘Deutsche Forschungsgemeinschaft’ to CCV (Vo890/7).

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