The stable carbon isotope ratio of exhaled CO2(δ13Cbreath) reflects the isotopic signature of the combusted substrate and is, therefore, suitable for the non-invasive collection of dietary information from free-ranging animals. However,δ 13Cbreath is sensitive to changes in ingested food items and the mixed combustion of exogenous and endogenous substrates. Therefore, experiments under controlled conditions are pivotal for the correct interpretation of δ13Cbreath of free-ranging animals. We measured δ13Cbreath in fasted and recently fed insectivorous Myotis myotis (Chiroptera) to assess the residence time of carbon isotopes in the pool of metabolized substrate, and whether δ13Cbreath in satiated individuals levels off at values similar to the dietary isotope signature(δ13Cdiet) in insect-feeding mammals. Meanδ 13Cbreath of fasted individuals was depleted by–5.8‰ (N=6) in relation toδ 13Cdiet. After feeding on insects, bats exchanged 50% of carbon atoms in the pool of metabolized substrates within 21.6±10.5 min, which was slower than bats ingesting simple carbohydrates. After 2 h, δ13Cbreath of satiated bats levelled off at –2.6‰ belowδ 13Cdiet, suggesting that bats combusted both exogenous and endogenous substrate at this time. A literature survey revealed that small birds and mammals metabolize complex macronutrients at slower rates than simple macronutrients. On average, δ13Cbreathof fasting birds and mammals was depleted in 13C by–3.2±2.0‰ in relation toδ 13Cdiet. δ13Cbreathof satiated animals differed by –0.6±2.3‰ fromδ 13Cdiet when endogenous substrates were not in isotopic equilibrium with exogenous substrates and by +0.5±1.8‰(N=6 species) after endogenous substrates were in isotopic equilibrium with exogenous substrates.
During the past decades, ecologists have increasingly used stable isotopes to study trophic interactions and dietary preferences in animal food webs(reviewed in Fry, 2006). Animals assimilate stable isotopes from the diet into their tissue or release them in their excreta and exhaled breath(DeNiro and Epstein, 1978; DeNiro and Epstein, 1981). Usually, excreta are depleted in 13C in relation to the stable carbon isotope signature of the diet, whereas animal tissues are enriched in 13C in relation to the diet by enzymatic action called isotopic fractionation (DeNiro and Epstein,1978; DeNiro and Epstein,1981). Isotopes are assimilated into animal tissue according to organ-specific tissue turnover rates (e.g. Tieszen et al., 1981). Thus, stable isotope signatures in organs with different turnover rates shed light on the diet consumed by the animal during the corresponding time periods. The stable carbon isotope composition (δ13C) of bone is considered to integrate the stable isotope ratio of the food consumed during an animal's lifetime (e.g. Tieszen and Fagre,1993). The stable carbon isotope ratio of other tissues, such as muscle, fat and liver, provides information on shorter time periods (Tieszen et al., 1981). The isotopic incorporation rates into these tissues have been found to be variable (Dalerum and Angerbörn, 2005) and possibly linked to the mass of the mammal (Carleton and Martínez del Rio, 2005). The stable carbon isotope signature of exhaled breath(δ13Cbreath) is considered to be the fastest recorder of dietary information, becauseδ 13Cbreath should match the isotopic signature of the ingested and combusted substrate(Perkins and Speakman, 2001; Hatch et al., 2002a; Hatch et al., 2002b).
The earliest experiments conducted under controlled conditions supported the idea that δ13Cbreath closely matches the isotopic composition of the recently ingested food(δ13Cdiet)(DeNiro and Epstein, 1981; Klein et al., 1988; Tieszen and Fagre, 1993). Since δ13Cbreath seems to provide a snapshot of the stable isotope signature of the substrate currently metabolized, it is increasingly used by experimental physiologists and behavioural ecologists to study metabolic substrate use in captive (e.g. Carleton et al., 2006) and free-ranging animals (e.g. Podlesak et al., 2005). Diet-switching experiments during which the isotopic composition of the diet is drastically changed at the onset of the experiment– from e.g. plant products of C3 plant origin to those of C4/CAM plant origin – have provided important insights into substrate combustion in birds (e.g. Hatch et al.,2002a; Hatch et al.,2002b; Carleton et al.,2006) and mammals (e.g. Jeuckendrup and Jentjens,2000; Voigt and Speakman,2007). During diet-switch experiments, breath samples are collected at regular time intervals after animals have started to consume a meal differing in stable carbon isotope composition from their previous diet. Such experiments have revealed that simple sugars are routed to combustion within less than 10 min in small birds and mammals(Voigt and Speakman, 2007; Welch et al., 2006; Welch et al., 2008; Voigt et al., 2008a) and in approximately 30 min in exercising humans(Jeuckendrup and Jentjens,2000). Complex macronutrients such as cellulose, starch and proteins are probably more difficult to digest than simple sugars, since the number of enzymatic processes is likely to increase with increasing complexity of the catabolized macronutrient. Therefore, animals ingesting complex macronutrients may incorporate exogenous substrate at slower rates than animals ingesting simple sugars.
In addition to differences in the time lag at which macronutrient combustion is reflected in δ13Cbreath,δ 13Cbreath may be affected by other factors such as isotopic fractionation during enzymatic or physical processes, or by the combined use of exogenous and endogenous substrates. Endogenous substrates are usually depleted in 13C by –0.6 to –8.4‰ in relation to the diet at the time of lipogenesis (e.g. DeNiro and Epstein, 1977;Podlesak et al., 2006). Accordingly, δ13Cbreath of fasting animals is usually depleted in 13C in relation to the previous diet (e.g. Hatch et al.,2002a; Hatch et al.,2002b). In many animals, especially free-ranging animals, it may not be evident whether individuals combust exclusively endogenous substrates or a mixture of exogenous and endogenous substrates. In this case, the difference between δ13Cbreath andδ 13Cdiet, henceforth calledΔ diet–breath, may be affected by the isotopic composition of both substrates and the ratio at which they are used. Following this line of argument, if endogenous substrates are not in isotopic equilibration with the most recent diet and if animals metabolize both types of substrate, Δdiet–breath may largely deviate from the most recent δ13Cdiet (e.g. Podlesak et al., 2005; Carleton et al., 2006). Continuous ingestion of new food items with a δ13C signature differing from that of the previous diet will eventually cause an exchange of isotopes in endogenous reserves over time. Thus,Δ diet–breath should decrease with increasing equilibration of fat and glycogen to the newδ 13Cdiet(Carleton et al., 2006).
Considering all these factors that may have a large and short-term impact on δ13Cbreath and consequentlyΔ diet–breath, the aim of our study was (1) to generate baseline data of stable isotope turnover andΔ diet–breath for a protein-combusting animal and (2) to survey the literature for general patterns to improve our ability to interpret stable isotope breath data of free-ranging animals.
First, using small insectivorous greater mouse-eared bats (Myotis myotis, Vespertilionidae), we quantifiedΔ diet–breath in fasting individuals, the rate at which exogenous substrates are incorporated into the pool of metabolized substrates after ingestion of a protein-rich diet and the plateauδ 13Cbreath at which the animals' breath levelled off after equilibration to the new diet. We predicted that (1) the rate at which M. myotis would make use of dietary proteins for metabolism would be slower than in sugar-combusting bats of similar size, (2)δ 13Cbreath should be depleted in 13C in relation to δ13Cdiet in fasting animals, and (3)δ 13Cbreath should level off close toδ 13Cdiet in satiated animals.
Second, we reviewed the current knowledge on species-specificΔ diet–breath values and fractional rates at which exogenous substrates are incorporated into the pool of metabolized substrates for birds and mammals. We predicted that (1) animals ingesting complex macronutrients, such as proteins and complex carbohydrates, would have a slower fractional incorporation rate of exogenous substrate into the pool of metabolized substrate than animals feeding on simple carbohydrates such as hexose or sucrose, (2) fasting birds and mammals would have, in general,negative Δdiet–breath values, since endogenous substrates are usually depleted in 13C in relation to the diet, and(3) Δdiet–breath should decrease in satiated animals with increasing equilibration of endogenous substrate to the isotopic signature of the diet.
MATERIALS AND METHODS
Study animals and isotopic signature of experimental food types
We studied metabolic substrate use in six male adult Myotis myotisBorkhausen 1797 in a captive population housed in facilities at the Max-Planck-Institute for Ornithology in Seewiesen. M. myotis is an insectivorous bat that forages on carabid beetles in temperate zone forests(Arlettaz et al., 1997). Our study animals were kept under an inverted dark–light cycle. The dark phase, i.e. the activity period, started at 09:00 h and lasted until 17:00 h. As exclusive bat food for the duration of the experiment, we raised mealworms(larval stages of Tenebrio molitor) on corn from either Hungary or Ecuador for 7 weeks. Ten days before the breath sampling, bats were fed mealworms enriched in 13C with corn from Hungary. Previous experiments in other bats demonstrated that this time period is sufficient to equilibrate the endogenous reserves of bats isotopically to a new diet(Voigt and Speakman, 2007). During the experiment, bats were fed with mealworms that were enriched in 13C with corn from Ecuador. We collected several mealworms from each diet for later stable isotope analyses. Dead mealworms were dried to constant mass at 40°C in a drying oven and then a subsample from the tissue of inner organs was taken for stable carbon isotope analyses. The mealworms raised for 7 weeks on Hungarian corn had a δ13C of–24.1±0.8‰ (diet 1), while those raised on the Ecuadorian corn had a δ13C of –22.0±0.3‰ (diet 2),indicating that the isotopic signature of the mealworms was slightly more enriched in 13C than before.
Incorporation rate of exogenous substrate into the pool of metabolized substrates
We assessed the rate at which exogenous substrate was incorporated into the pool of metabolized substrates in a diet-switch experiment, i.e. we fed fasted bats that had been maintained over 6 or 7 days ad libitum on diet 1 with diet 2. Prior to the diet-switch experiment, bats fasted for at least 13 h, which is fully congruent with the bats' natural diurnal pattern of food intake. All experiments were performed during the activity period of the bats.
Before offering mealworms, we collected an initial breath sample from each bat. Following the ingestion of the first mealworm we collected breath samples after 10, 20, 40, 60, 80, 100 and 120 min (see below for the technique of breath collection). Bats were weighed to the nearest 0.1 g before and after the experiment using a hand-held balance (Pesola, Baar, Switzerland). We lost body mass data from two individuals for the time after the experiment and,therefore, can only provide body mass change data for four individuals.
For breath sampling, bats were transferred singly into cotton bags (17 cm×25 cm) that were individually put into a larger plastic bag (volume 0.5 ml; Ziplock™, Racine, WI, USA). Ambient air was washed of CO2 using NaOH and flushed through the bag via a plastic tube (diameter 3 mm) at a minimal flow through rate of 1.4 l min–1. The outlet of the plastic bag consisted of a small slit of 4 cm (width 0.2 cm). A small tube was positioned with one end close to the bat's head inside the bag (diameter 1 mm, length 4 cm). We fused a needle hermetically to the other end of the tube outside the bag. For CO2accumulation, we sealed the plastic bag for 1 min. M. myotis have a resting metabolic rate of approximately 25 ml O2h–1 (Hanu,1959). Therefore, we expected CO2 to accumulate to approximately 0.5% during this time span. We then sucked air from the bag including the bat's breath into a vacutainer (Labco, Buckinghamshire, UK) by penetrating the Teflon membrane of the vacutainer with the needle. After each breath collection the plastic bag was unsealed again and CO2-free air was flushed through the bag. Breath collection was repeated after 10, 20,40, 60, 80, 100 and 120 min following the first feeding event, since we expected an exchange of stable carbon isotopes in exhaled CO2during this time period (Voigt &Speakman, 2007; Voigt et al.,2008a). Bats were fed repeatedly after 20, 40 and 60 min following the first feeding event to ensure that the bat's breath was equilibrated isotopically to the new diet. We define isotopic equilibration of an animal's tissue or breath as the status in which the isotopic composition of animal tissue or breath does not change any more with continuous consumption of the same food items.
Stable carbon isotope analysis
Subsamples from the bats' diet (inner organs of the mealworms, excluding the cuticula) were dried at 40°C in a drying oven to constant mass,weighed on a Sartorius microbalance (Satorius AG, Göttingen, Germany) and loaded into tin capsules. All samples were combusted and analysed with a Flash elemental analyser and a Conflo II, coupled to a Delta-Advantage isotope ratio mass spectrometer (FisherThermo, Bremen, Germany) at the Stable Isotope Laboratory of the Leibniz-Institute for Zoo and Wildlife Research, Berlin(Germany). Samples were analysed in combination with internal standards that had previously been characterized relative to an international 13C standard (NBS22). All 13C/12C data were expressed relative to the international standard in the δ notation (‰)using Eqn 1. Precision was better than ±0.03‰ (1 s.d.).
For comparative reasons, we calculated the time at which 50% of carbon isotopes were exchanged in the animal's 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% of isotopes. All values are given as means ± 1 s.d. and all statistical tests were performed two tailed unless otherwise stated.
We surveyed the literature for experimental studies that usedδ 13Cbreath as a predictor forδ 13Cdiet in birds and mammals. For each study, we obtained the following six parameters: (a) taxonomic group, (b) body mass in grams, (c) t50 (min), or the fractional incorporation rate at which 50% of carbon atoms in exhaled CO2 have been exchanged with carbon atoms from the exogenous substrate, (d)Δ diet–breath for animals having fasted over a prolonged time period, (e) Δdiet–breath for animals having ingested a diet that was more enriched in 13C in relation to the previous diet (exogenous substrate with a C4 signature and endogenous substrate with a C3 signature and, thus, endogenous substrate not in isotopic equilibrium with the new diet), and (f) Δdiet–breathfor animals having ingested a diet that was isotopically identical to the previous diet (endogenous substrate in isotopic equilibrium with the new diet). In cases where we obtained two or more values for one of these parameters, we calculated an arithmetic mean for these data to yield a single species-specific data point. We calculated a Wilcoxon matched-pairs signed-rank test for comparison of Δdiet–breath between scenarios d and e, and d and f, and a Mann-Whitney U-test for a comparison of Δdiet–breath between scenarios e and f. All three tests were performed one tailed, as we expected that the exclusive or additional combustion of endogenous substrates would cause a depletion of 13C in exhaled breath. We calculated a Mann–Whitney U-test for comparison of the t50 values in animals digesting complex substrates, such as starch- or protein-rich diets,and animals digesting simple substrates, such as hexose or sucrose. This analysis was only performed for mammals and birds weighing less than 1 kg, as we expected a large impact of passage rate and endosymbiontic fermentation on t50 values in large mammals (e.g. in ruminants).
Isotopic change of δ13Cbreath in insect-feeding Myotis myotis
The body mass of fasted M. myotis averaged 23.1±1.9 g 1 h after the onset of the dark cycle. The meanδ 13Cbreath of fasted M. myotis equalled–29.9±0.7‰, which was –5.8‰ lower than theδ 13C of the diet (–24.1‰) that the bats fed on during the 6–7 days preceding the day of breath collection; this difference was significant (Wilcoxon signed-rank test: T+=0, T–=–21, P=0.0156). After being fed continuously with mealworms (δ13C=–22.0‰),δ 13Cbreath became enriched in 13C(Fig. 1). The body mass of bats increased until the end of the feeding experiment by on average 4.4±1.6 g due to the ingestion of mealworms. The regression model calculated for the fractional incorporation rate of exogenous substrate into the pool of metabolized substrate reads as follows:δ 13Cbreath(t)=–24.6(±0.7)–5.5(±1.1)e–t/31.2(±15.2). The mean time required to exchange 50% of the carbon atoms in the exhaled CO2 with carbon atoms of the recently ingested protein source was 21.6±10.5 min (Table 1). The δ13Cbreath levelled off at–24.6±0.7‰ after 120 min, which was –2.6‰lower than the δ13C of the new diet (Wilcoxon signed-rank test: T+=0, T–=–21, P=0.0313).
t50 and Δdiet–breath in birds and mammals
The fractional rates of incorporation of exogenous substrates into the pool of metabolized substrates ranged from a few minutes in nectar-feeding small birds and bats to almost 3 days in hay-ingesting alpacas (see Table 2). In species below 1 kg body mass, animals had higher t50 values when digesting complex substrates such as a starch- or protein-rich diet than when digesting simple substrates such as hexose or sucrose (Mann–Whitney U-test: U=0.0, U′=25, N1=5, N2=5, P=0.0079). In fasting birds and mammals,mean δ13Cbreath was depleted in relation toδ 13Cdiet by –3.2±2.0‰(N=4 bird and N=5 mammal species; Table 2, Fig. 2). In animals that had recently fed on a diet with a C4 signature but still carried endogenous reserves with a C3 signature, δ13Cbreath was depleted in 13C by –0.6±2.3‰ in relation to theδ 13C of the new diet (N=5 bird and N=7 mammal species). After continuously feeding on the new diet, the endogenous reserves (fat and glycogen) became isotopically equilibrated to the new diet.δ 13Cbreath was, on average, enriched in 13C by +0.5±1.8‰ in relation to the diet after isotopic equilibration of endogenous substrate with the diet (N=6 species; Fig. 2). Depletion of 13C in exhaled breath in relation toδ 13Cdiet was stronger in fasting animals than in animals that had recently fed on an isotopically contrasting diet (Wilcoxon matched-pairs signed-rank test: T+=7, T–=–38, N=9 pairs, P=0.0371; scenario d–e) and also more pronounced than in animals carrying endogenous substrates already isotopically equilibrated to the diet (Mann–Whitney U-test: U=2.0, U′=38, N1=9, N2=5, P=0.0062; Fig. 2;scenario d–f). On average, Δdiet–breath was not significantly different between satiated animals with endogenous substrates equilibrated to the new diet and satiated animals with endogenous substrates not equilibrated to the new diet (Mann–Whitney U-test: U=19, U′=36, N1=12, N2=5, P=0.377; Fig. 2; scenario e–f).
Fractional incorporation rates in mammals and birds
We fed fasting vespertilionid bats (Myotis myotis) mealworms and monitored changes in the bats' δ13Cbreath over a 2 h period. The δ13Cbreath of the bats converged quickly on the stable carbon isotope signature of the newly ingested food items. M. myotis required twice as much time to exchange 50% of carbon atoms in exhaled CO2 with those of the last meal as similar-sized Carollia perspicillata (family Phyllostomidae) feeding on simple sugars (Voigt et al.,2008a), but only 70% of the time similar-sized vampire bats need to metabolize recently ingested blood (Voigt et al., 2008). Mice (Mus musculus) feeding on corn, which includes mainly complex carbohydrates,i.e. starch, had almost identical t50 values to M. myotis (Perkins and Speakman,2001). The lower t50 values of sugar-ingesting bats may be caused by the faster enzymatic processing of small molecules, such as hexose or sucrose, than more complex molecules, such as starch and proteins. Our literature survey supports the notion that animals require more time to digest and combust complex macronutrients. However, t50 values may be affected not only by the type of combusted macronutrient but also by the animal's size and phylogenetic background (Carleton and Martínez del Rio, 2005). Currently, we lack sufficient data to shed light on the effects of phylogenetic inertia on t50 values. Data for t50 listed in Table 2 suggest that large animals have slower incorporation rates (t50 values) than small animals. Despite being large, alpacas and horses make use of complex carbohydrates such as cellulose by endosymbiontic fermentation. This mode of digestion may additionally slow down the speed at which exogenous substrates are available to fuel metabolism. Our survey also highlighted the fact that t50 values were lower when the focus organisms were exercising [see reports on humans (Jeukendrup and Jentjens, 2000) and bats(Welch et al., 2008)]. However, the effect of exercise may become negligible in small animals ingesting simple sugars, as t50 values were almost identical for resting and flying nectar-feeding bats feeding on a sugar water solution (Voigt and Speakman,2007; Welch et al.,2008).
Isotopic differences between diet and exhaled CO2
The δ13Cbreath of M. myotis fasting for 13 h was depleted in 13C by almost –6‰ in relation to the δ13Cdiet of the animal's previous diet. In general, Δdiet–breath averaged–3.2±2.0‰ for nine bird and mammal species that had fasted over an extended period of time (Table 2, Fig. 2). Thus, a depletion of 13C in relation to the isotopic signature of the diet(Δdiet–breath) seems to be a common phenomenon in fasting animals and is probably caused by the strong fractionation of stable carbon isotopes during lipogenesis (DeNiro and Epstein, 1977).
After we fed fasting M. myotis with mealworms (diet 2) with a stable carbon isotope signature that was enriched by +2‰ in relation to the bats' previous food (diet 1), the bats'δ 13Cbreath converged to a new plateau, which was depleted by –2.6‰ in relation to the δ13C of the new diet. By contrast, δ13Cbreath of blood-feeding vampire bats (Desmodus rotundus) levelled off +2‰ above theδ 13C of their recently ingested blood meal(Voigt et al., 2008b). Given that both bat species process and metabolize protein, and assuming similar fractionation factors during protein combustion, M. myotis probably used more 13C-depleted endogenous substrate to fuel its metabolism than D. rotundus.
Many previous studies have looked at the relative level of the animals'δ 13Cbreath plateau values after a diet switch (see Table 2). During most diet-switch experiments animals used a combination of exogenous and endogenous substrates to fuel their metabolism and in most cases experimental animals were labelled with carbon atoms from a C3 food web (low δ13C)at the beginning of the experiment. Since fat and glycogen stores of these experimental animals were built from carbon atoms of a C3 food source,Δ diet–breath may deviate from zero throughout the experiment, because the exogenous substrate combustion in relation to the endogenous substrate combustion may vary over time (e.g. Carleton et al., 2006). On average, Δdiet–breath equalled–0.6±2.3‰ in 12 bird and mammal species that had recently ingested food with a C4 signature, but still carried endogenous reserves with a C3 signature (Table 2, Fig. 2). After isotopic equilibration of endogenous substrate with the new diet, averageΔ diet–breath equalled +0.5±1.8‰(N=6 species; Fig. 2). Thus, Δdiet–breath decreased with increasing equilibration of endogenous substrates with the stable carbon isotope signature of the new diet.
Our survey highlights the fact that δ13Cbreathmay be affected not only by the stable carbon isotope signature of the recently ingested food but also by the time elapsed since an isotopic shift in the diet, the turnover rate of endogenous substrates and the ratio at which endogenous and exogenous substrates are used for metabolism, e.g. when active or resting. Thus, animals that have recently changed to food items with an isotopic composition different from their previous diet may exhibit the largest range in Δdiet–breath. Interpretation ofδ 13Cbreath may prove difficult if no alternative measure of substrate combustion is available, e.g. the respiratory quotient of exhaled breath or metabolic products in blood plasma. We, therefore, advise caution in the interpretation of δ13Cbreath from free-ranging animals without further validation studies under controlled experimental conditions, preferably in the same animal species as investigated in the field. The sensitivity of δ13Cbreath to various factors such as fractionation, mixed combustion of exogenous and endogenous substrates and others may provide a powerful tool for physiologists and behavioural ecologists, once the underlying mechanisms are clearly understood.
Peter Thompson and Dr Paula Redman analysed the samples at the Aberdeen Centre for Energy Regulation and Obesity, and Karin Sörgel at the Stable Isotope Laboratory of the IZW. The experiments complied with the current laws of Germany; animals were caught and kept under licence of the responsible regulatory authorities; Regierungspräsidium Freiburg, licence no. 55-8852.44/1095, Veterinäramt Starnberg). We thank Carlos Martínez del Rio, Scott Carleton and Detlev H. Kelm for commenting on an earlier version of this manuscript.