Oecologia
DOI 10.1007/s00442-008-1057-3
PHYSIOLOGICAL ECOLOGY - ORIGINAL PAPER
Nutrient routing in omnivorous animals tracked by stable carbon
isotopes in tissue and exhaled breath
Christian C. Voigt Æ Katja Rex Æ Robert H. Michener Æ
John R. Speakman
Received: 12 September 2007 / Accepted: 16 April 2008
Ó Springer-Verlag 2008
Abstract Omnivorous animals feed on several food items
that often differ in macronutrient and isotopic composition.
Macronutrients can be used for either metabolism or body
tissue synthesis and, therefore, stable C isotope ratios of
exhaled breath (d13Cbreath) and tissue may differ. To study
nutrient routing in omnivorous animals, we measured
d13Cbreath in 20-g Carollia perspicillata that either ate an
isotopically homogeneous carbohydrate diet or an isotopically heterogenous protein-carbohydrate mixture. The
d13Cbreath converged to the d13C of the ingested carbohydrates irrespective of whether proteins had been added or
not. On average, d13Cbreath was depleted in 13C by only ca.
-2% in relation to the d13C of the dietary carbohydrates
and was enriched by +8.2% in relation to the dietary
proteins, suggesting that C. perspicillata may have routed
most ingested proteins to body synthesis and not to
metabolism. We next compared the d13Cbreath with that of
wing tissue (d13Ctissue) in 12 free-ranging, mostly omnivorous phyllostomid bat species. We predicted that species
with a more insect biased diet—as indicated by the N
isotope ratio in wing membrane tissue (d15Ntissue)—should
have higher d13Ctissue than d13Cbreath values, since we
expected body tissue to stem mostly from insect proteins
and exhaled CO2 to stem from the combustion of fruit
carbohydrates. Accordingly, d13Ctissue and d13Cbreath should
be more similar in species that feed predominantly on
plant products. The species-specific differences between
d13Ctissue and d13Cbreath increased with increasing d15Ntissue,
i.e. species with a plant-dominated diet had similar
d13Ctissue and d13Cbreath values, whereas species feeding at
a higher trophic level had higher d13Ctissue than d13Cbreath
values. Our study shows that d13Cbreath reflect the isotope
ratio of ingested carbohydrates, whereas d13C of body
tissue reflect the isotope ratio of ingested proteins, namely
insects, supporting the idea of isotopic routing in omnivorous animals.
Communicated by Carlos Martinez del Rio.
Introduction
C. C. Voigt (&) K. Rex
Research Group Evolutionary Ecology, Leibniz Institute for Zoo
and Wildlife Research, Alfred-Kowalke-Strasse 17,
10315 Berlin, Germany
e-mail: [email protected]
R. H. Michener
Department of Biology, Boston University,
Cummington Street, Boston 02215, MA, USA
J. R. Speakman
Aberdeen Centre for Energy Regulation and Obesity,
School of Biological Sciences, University of Aberdeen,
Tillydrone Avenue, Aberdeen AB24 2TZ, UK
Keywords Exhaled carbon dioxide Carollia perspicillata Stable carbon isotopes Infra-red stable isotope analyser Omnivory
Stable isotope analysis has become a widespread tool in
ecological studies (summarized in Fry 2006). This technique is based on the premise that the stable isotope ratio of
an animal closely matches that of its diet (DeNiro and
Epstein 1978, 1981). Isotopic differences between bulk diet
and animal body composition may occur owing to fractionation effects, i.e. the preferential turnover of molecules
labelled with light isotopes leading to an increase in the
enrichment of heavy isotopes in an animal’s body (but see
DeNiro and Epstein 1977). Animals with a narrow dietary
spectrum feed on food items that are similar in their
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Oecologia
macronutrient composition, such as seabirds feeding on
different species of fish or lions hunting or scavenging
larger mammals. In such cases, the basic assumption of an
isotopic match between bulk diet and consumer tissue may
not be violated. However, many animals consume a combination of two or several food items that differ not only in
macronutrient composition (relative proportions of carbohydrates, proteins and fat), but also in the macronutrients’
isotopic signatures. After ingestion, these substrates may be
routed to different destinations in the body, e.g. to
metabolism or tissue synthesis (Martı́nez del Rı́o and Wolf
2005). Carbohydrates, on the one hand, are likely to be
used preferentially to fuel metabolism, e.g. nectar in nectar-feeding bats (Voigt and Speakman 2007) and
hummingbirds (Welch et al. 2006, Carleton et al. 2006).
Proteins and fat, on the other hand, are more likely to be
allocated to organ and tissue synthesis (Tieszen and Fagre
1993). This phenomenon is referred to as ‘‘nutrient routing’’ (Ambrose and Norr 1993; Tieszen and Fagre 1993),
‘‘isotopic routing’’ (Schwarcz and Schoeninger 1991) or
‘‘metabolic routing’’ (Podlesak and McWilliams 2006).
Some discussion arose over the question of which type
of sample reflects the isotopic signature of the bulk diet
(Tieszen et al. 1983; Tieszen and Fagre 1993; Hatch et al.
2002a, b; Martı́nez del Rı́o and Wolf 2005). Some authors
suggested that the stable C isotope ratio of exhaled CO2
(d13Cbreath) best matches the d13C of the bulk diet
(d13Cdiet), since protein and fat synthesis involves larger
fractionation of stable C isotopes (Hatch et al. 2002a b), i.e.
enrichment of 13C in relation to 12C in protein-rich tissues
(Tieszen et al. 1983) and depletion of 13C in relation to 12C
in fat stores (DeNiro and Epstein 1977). This is supported
by several field studies in which animals fed on homogeneous food items (frugivorous birds: Podlesak et al.
2005; blood-licking vampires: Voigt et al. 2008). Other
authors argued that d13Cbreath will be biased towards the
d13C of the protein-poor dietary items when the diet varies
in protein enrichment and isotopic signature (Martı́nez del
Rı́o and Wolf 2005; Tieszen and Fagre 1993).
We examined nutrient routing in captive and freeranging omnivorous animals, namely bats of the family
Phyllostomidae. Members of this family, especially those
of the subfamilies Carolliinae, Glossophaginae and
Stenodermatinae are ideal candidates for such a study,
since species include various proportions of insects in their
diet (Gardner 1976; Herrera et al. 1998, 2001, 2002). In an
initial experiment with 20-g captive Carollia perspicillata,
we studied whether d13Cbreath converges on the isotopic
composition of the carbohydrate portion of the diet when
animals are fed a mixed solution of sugar and protein with
contrasting isotopic signatures. Prior to this experiment, we
fed two groups of C. perspicillata a constant diet originating from C3 plant products (low d13C) until d13Cbreath
123
matched the C3 origin of their diet. Then, we fed fasting
animals of group 1 a sugar solution with a high d13C and
monitored the rate at which d13Cbreath converged on the
isotopic signature of the diet. In group 2, we fed fasting
animals a mixed solution of sugar from CAM plants (high
d13C) and egg yolk with a low d13C. We hypothesized that
animals should fuel their metabolism preferentially with
exogenous carbohydrates, even in the presence of dietary
proteins. Therefore, we predicted that d13Cbreath should
level off at the same plateau in both experiments.
We then captured free-ranging phyllostomid bats and
collected breath samples and wing tissue biopsies for isotopic analysis. Omnivorous phyllostomid bats mainly feed
on two dietary components that differ in the macronutrient
and isotopic composition. Fruit and nectar are rich in carbohydrates, but poor in protein and fat, whereas insects are
rich in protein and fat, but poor in carbohydrates. Since
insects are usually primary consumers of plant products,
such as leaves and fruit, their tissue is enriched in 13C in
relation to their diet. This trophic enrichment equals 2–3%
between plants and insects (e.g. Herrera et al. 1998; Voigt
et al. 2006). We used the N isotope enrichment as an
indicator of the trophic level of the bat species (Herrera
et al. 1998; Schondube et al. 2001) and tested two
hypotheses. First, when macronutrients of dietary items are
used randomly in the bats’ bodies for metabolism (combustion) or anabolism (synthesis), we expected a constant
relationship between the d13C of wing tissue and exhaled
breath, irrespective of a species’ trophic position. In this
case, d13Cbreath should reflect the isotopic composition of
the bulk diet. Second, if carbohydrates are routed to
metabolism and proteins to anabolism, we expected the
difference between the d13C of wing tissue and exhaled
breath to increase with increasing trophic position of the
bat, since C atoms from combusted carbohydrates should
be found in the exhaled CO2 and those from assimilated
proteins in the wing tissue, i.e. bat species with a more
insect-biased diet should have higher d13C in wing tissue
caused by the trophic enrichment of 13C between plants
and insects than bat species with a more plant-biased diet.
Materials and methods
We conducted the following experiments to study the
routing of exogenous carbohydrates to metabolism. During
experiment 1, we monitored changes in d13Cbreath in fasting
Carollia perspicillata (Carolliinae, Phyllostomidae) and in
individuals that were recently fed with a hexose solution
derived from a CAM plant (Agave). We analysed d13Cbreath
at the field site using a portable infra-red stable isotope
analyser (IR-SIA). During experiment 2, we fed C. perspicillata a fructose solution derived from CAM plants and
Oecologia
then monitored changes in d13Cbreath using a conventional
isotope ratio mass spectrometer (IRMS). During experiment 3, we fed C. perspicillata a mixture of protein and
fructose with contrasting stable isotope signature and then
monitored changes in d13Cbreath using an IRMS. Prior to the
experiments, all animals were weighed to the nearest 0.1 g
using a handheld balance (Pesola, Baar, Switzerland).
Experiment 1: fasting individuals and individuals
fed hexose (IR-SIA analyses)
In September 2006, we captured seven male C. perspicillata close to a daytime roost in the rainforest of La Selva
Biological Station (Organization for Tropical Studies,
Costa Rica; 10°250 5200 N, 84°000 1200 W) using two polyester
ground mist nets (6 9 2.5 m; 70 dernier/2 ply, 16 mm
mesh, five shelves; R. Vohwinkel, Velbert, Germany). All
bats were marked with numbered plastic bands (Hughes,
London) on the right forearm and transferred to a flight
cage (6 m 9 3 m 9 3 m), where they were fed over a
period of 20 days with a mixture of honey water
(d13C = -25.9%) and banana (d13C = -23.7%) offered
ad libitum. The d13C of the honey-banana mesh equalled
-25.6% and the C:N ratio (C:N) 48:1. Since animals were
kept in an outdoor flight cage, animals had access to
additional protein sources such as insects. Animals were
exposed to ambient temperature, humidity and photo
regime.
The experiments started on the 21st day after capture
and were performed over 7 consecutive days between ca.
1300 and 1800 hours. Prior to the experiments, we diluted
agave syrup to a 30% (mass/mass) sugar solution (95%
fructose and 5% glucose) with an average d13C of -11.3%
by adding tap water. Breath collections were performed
twice for each individual. During the first run, animals
were randomly assigned to the group of individuals fed
with agave syrup water or to the group of fasting individuals. During the second run, we assigned the bats to the
opposite treatment.
We followed Welch et al. (2006), Carleton et al. (2006)
and Voigt and Speakman (2007) for breath collection in
small vertebrates. A single bat was transferred into a linen
bag (10 cm 9 20 cm) that was placed in a plastic bag
(15 9 30 9 1 cm3; Ziplock). CO2 was washed from the
bag by flushing air first through NaOH and then through the
bag via a plastic tube (diameter 3 mm) at a flow rate of
1.4l min-1 for about 8 min. The outlet of the plastic bag
consisted of a small slit of 4 cm length and 0.2 cm width.
Subsequently, we sealed the plastic bag temporarily for
1.5 min to let CO2 of the bat’s breath accumulate in the
bag. For breath collection, we sucked the air from close to
the position of the bat in the linen bag via a second plastic
tube (diameter 1 mm, length 4 cm) into the IR-SIA
(FANCi; Fischer Analysen Technik, Leipzig, Germany).
After each breath collection the plastic bag was unsealed
and CO2-free air was flushed through the bag again. Breath
collection was repeated every 10 min for a total of 1.5 h
following the first feeding event, since we expected an
exchange of stable C isotopes in exhaled CO2 during this
time period (Voigt and Speakman 2007). In the experiment
with fed bats, we tried to feed bats as often as possible
during the experiment runs to ensure that the bats’ breath
was equilibrated isotopically to the recently ingested food.
After the experiment, we released all bats into the flight
cage where they were offered their normal diet ad libitum.
Bats were released at the site of capture after we finished
breath collection from fed and fasting animals. All 13C/12C
ratios were expressed relative to the international standard
Pee Dee Belemnite (PDB) using the d notation (d13C) in
parts per thousand (%; see below). Precision was measured
each day and was on average better than ±0.3% (1r).
Experiments 2 and 3: individuals fed carbohydrates
or a mixed diet (IRMS analyses)
Experiments 2 and 3 were performed in November 2007.
We captured 13 male C. perspicillata at the same sites as
before using the same methods as during the previous field
season. Again, all bats were marked with numbered plastic
bands (Hughes) on the right forearm and transferred to a
flight cage (6 m 9 3 m 9 3 m), where they were maintained under the same conditions and fed the same food as
the animals of the first experiment.
The experiments started on the 21st day after capture
and were performed over 8 consecutive days between ca.
1400 and 1800 hours. Prior to the experiments, we mixed
one of two diets: diet A was used for the experiment 2 and
consisted of a 30% (mass/mass) fructose water solution
with an average d13C of -10.2%. Diet B was used for
experiments 3 and consisted of the 30% fructose water
stock solution and powdered egg yolk with an average d13C
of -22.1%. Diet B consisted of equal mass portions of
powdered egg yolk and fructose.
We used the same setup for collecting breath as before.
Approximately 10 ml of air was sucked from close to the
position of the bat in the 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 an evacuated Vacutainer (Labco,
Buckinghamshire, UK). 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.
Bats were fed repeatedly after approximately 20 and
40 min following the first feeding event to ensure that the
bats’ breath was equilibrated isotopically to the newly
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ingested diet. Bats were released at the site of capture after
the experiments.
Collection of breath samples in free-ranging bats
Between February and March 2005, we captured bats in the
forest of La Selva Biological Station. On 20 occasions, we
set up a combination of 6 m 9 2.5-m and 9 m 9 2.5-m
ground mist nets (see above for net types) between 1800
and 2200 hours. Bats were identified according to Timm
and LaVal (1998). Breath collection was performed
immediately after capture. For breath collection, bats were
mechanically restrained by wrapping gauze bandage
around their bodies, excluding their head and legs. Afterwards, bats were transferred singly into a 500-ml plastic
container to which a needle was hermetically fused with
the tip facing the outside and the base the inside of the
container (Fig. 1). The head of the bat faced the basal part
of the needle. After sealing the container (except for the
needle as an air outlet), container air was washed of CO2 by
flushing ambient air through NaOH and subsequently
through the container via a plastic tube (diameter 3 mm)
for 5 min at a flow-through rate of 1.8l min-1. Then we
sealed the plastic container for 3 min to let the CO2 of the
bat’s breath accumulate. For breath collection, we penetrated the Teflon membrane of an evacuated Vacutainer
(Labco) with the needle tip attached to the plastic container. Thus, approximately 10 ml of air was sucked from
the area close to the head of the restrained bat via the
needle into the Vacutainer. Bats were immediately taken
out of the container after the collection of the breath
sample and released at the site of capture. d13Cbreath was
measured using an IRMS.
Isotopic analysis of breath, wing tissue and dietary
samples using a conventional IRMS
Breath samples were automatically flushed from the Vacutainers in a stream of chemically pure He, after which a gas
chromatograph separated the CO2 gas from the other gases
before admitting it into the IRMS in a continuous flow.
Breath samples together with internal standards that had
been previously characterized relative to an international
13
C standard (IAEA-CO-1) were analysed in duplicate. All
13 12
C/ C were expressed relative to the international standard using the d notation (%) and the following Eq. 1:
Rsample
1 1; 000
ð1Þ
dX ¼
Rstandard
where Rsample and Rstandard represent the ratio between the
heavy and light isotope of element X of the sample and
standard, respectively. Precision was better than ±0.01%
(1r). All samples were analysed using a blind experimental
protocol.
Samples of wing tissue and dietary items of the three
experiments were weighed on a microbalance (Sartorius,
Göttingen, Germany) and loaded into tin capsules. All
samples were combusted and analysed using a elemental
analyser (Eurovector Euro) and a continuous-flow system
(GV Instruments), coupled to an isotope ratio mass spectrometer (IsoPrime) at Boston University’s Stable Isotope
Laboratory. Atmospheric N was used as the standard for N
isotope analyses and PDB for stable C isotope analyses.
Stable C and N isotope ratios are given in the d notation
(%) following Eq. 1. Precision of isotope measurements
was ±0.01% (1r).
Exponential model and statistics
Fig. 1 Method of breath collection in small mammals such as bats.
Bats were mechanically restrained by gauze bandage and put into a
plastic container. CO2-free air was constantly flushed through the
container. For CO2 accumulation the air inlets were closed for 3 min.
To extract the breath from the container, the Teflon membrane of an
evacuated Vacutainer was penetrated by the needle attached to the
plastic container. Then, approximately 10 ml air was sucked from the
inside into the Vacutainer. After, CO2-free air was again flushed
through the container
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We expected that changes in isotopic composition of the
bats’ breath would follow a single-pool exponential model
after bats were fed (e.g. Tieszen et al. 1983; Voigt et al.
2003; Voigt and Speakman 2007). Therefore, we calculated equations of the following type for each fed C.
perspicillata according to Carleton and Martinez del Rio
(2005):
d13 CðtÞ ¼ dCð1Þ þ ðd13 Cð0Þ d13 Cð1ÞÞ ekt
13
13
13
ð2Þ
where d C(t) is d Cbreath at time t, d C(?) the asymptotic d13Cbreath when animals are equilibrated to the stable
C isotope signature of their diet, d13C(0) the d13Cbreath at
time 0 of the experiment, and k the fractional rate of
Oecologia
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 for all individual values. We calculated the time at which 50% of C isotopes are exchanged
in the animals’ breath (t50) according to Voigt et al. (2007).
We tested whether d13Cbreath(?) of the individual
regressions deviated from the expected values for exclusive
sugar combustion, mixed protein–sugar combustion and
exclusive protein combustion by using a Student t-test with
a 5% level of significance. We assumed that d13Cbreath(?)
would level off at the isotopic signature of the sugar or
protein portion of the diet when testing for an exclusive
sugar and protein combustion, respectively. We also
assumed that d13Cbreath(?) would level off at an intermediate value when animals fuel their metabolism on a
mixture of dietary carbohydrates and protein. We calculated this expected d13Cbreath(?) of mixed combustion by
assuming: (1) no diet-consumer offset, and (2) a 50%
contribution of protein and carbohydrates to metabolism
using Eq. 3:
d13 Cð1Þ ¼ 0:5ðd13 Csugar þ d13 Cprotein Þ
13
ð3Þ
In Eq. 3, d Csugar is the stable C isotope signature of the
sugar (-11.3% in experiment 1 and -10.2% in
experiment 2 and 3) and d13Cprotein is the d13C of the
protein portion of the diet (-22.1%).
Group-specific means are given ±1 SD and all statistical
tests were performed two-tailed at a significance level of
5% if not stated otherwise. To test for differences in the
three regression parameters among the three experimental
groups and whether they were influenced by individual
body mass, we performed an analysis of covariance. We
used a Friedman test to test whether d13Cbreath changed
during the course of the experiment in fasting animals.
To compare the performance of the IR-SIA with that of
a conventional IRMS, we measured d13Cbreath with the IRSIA as described above and collected additional breath
samples for later analysis using an IRMS. For this experiment we used seven C. perspicillata from the previous
experiment and two additional male C. perspicillata captured shortly after dusk at the same site as the other
individuals. We fed these individuals sugar water that
consisted of different ratios of sugars originating from C3
and C4/CAM plants to cover the whole range of d13C
values that can be expected from free-ranging bats feeding
on an exclusive or a mixed diet of C3 and/or C4/CAM plant
products. We assessed whether d13Cbreath values of the IRSIA deviated significantly from those of the IRMS by
testing whether the slope of a linear regression calculated
after the least squares method deviated significantly from 1
and whether the intercept with the y-axis deviated significantly from zero.
Results
Comparison of d13Cbreath measured with IR-SIA
and IRMS
A linear regression calculated after the least squares
method for the relationship between d13C measured with
IR-SIA and IRMS showed no deviation from 1 in the slope
[slope = 1.019 ± 0.44 (mean ± SE); Student’s t-test:
t8 = 0.041, P [ 0.05] and a significant overestimate of the
IR-SIA measurements by, on average, 1.6 ± 0.2%
(mean ± SE; one-tailed Student’s t-test: t8 = 2.66,
P \ 0.05). Therefore, we corrected d13Cbreath of the IR-SIA
by subtracting 1.6%. Overestimation was independent
of 13C enrichment in the breath samples, i.e. we found
no correlation between the isotopic difference between
IR-SIA and IRMS measurements and d13Cbreath measured
with the IRMS (Rs = 0.009, P = 0.99).
Experiment 1: fasting individuals and individuals
fed hexose (IR-SIA analyses)
During the experiment with fasting animals, d13Cbreath of
C. perspicillata did not change over time (Friedman test:
Ff = 9.3, P = 0.40). The average d13Cbreath in fasting
animals calculated over individual means equalled
-28.5% (mean -28.6 ± 0.4%), which was significantly
depleted in 13C in relation to the d13C of diet 1 by -3.0%
(Wilcoxon signed rank test: T+ = 0, T- = -553; n = 7,
P = 0.002).
The initial d13Cbreath did not deviate between runs with
fed and fasting bats (mean difference, 0.5 ± 4.1%; Wilcoxon matched pairs test: T+ = 15, T- = -13; n = 7
pairs, P = 0.94). On average animals ingested
1.8 ± 0.7 ml of agave syrup water per individual. The
d13Cbreath changed immediately after ingestion of the sugar
water in all seven animals and remained constant in five of
the seven individuals. In two animals, d13Cbreath dropped
after an initial increase back to lower values
(d13Cbreath \ -18%). In these animals, d13Cbreath was
probably not in isotopic equilibrium with the hexose water,
since both animals consumed less than 1 ml of hexose
water. We excluded the values of these two animals from
further analyses. In the other animals, the mean time
required to exchange 50% of the C atoms in the exhaled
CO2 with C atoms from the fed hexose water equalled
9.5 ± 6.1 min (Fig. 2; Table 1). The median of individual
d13Cbreath(?) equalled -12.7% and was not significantly
different to the d13C of the hexose diet (Wilcoxon signed
rank test: T+ = 3, T- = -12, n = 5, P = 0.313). Individual d13C(?) values were significantly depleted in 13C in
relation to the dietary sugars in two out of five individuals
(Table 1).
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-10
δ13Csugar
-15
-20
-25
-30
(a)
sugar diet (IR-SIA)
Experiment 3: individuals fed protein and fructose
(IRMS analyses)
-35
δ13Csugar
-10
In this experiment, protein was added that was depleted in
C by ca. 12% in relation to the d13C of 30% fructose (m/
m) sugar solution of experiment 2. Bats fed on average
2.7 ± 1.1 ml of the mixed solution during the 1-h time
period of the experiment. On average, it took
13.8 ± 9.4 min to exchange 50% of C atoms in the pool of
metabolised substrate with exogenous substrate (Table 1).
After 1 h, the average d13Cbreath levelled off at -13.9 ±
4.4%, which did not significantly deviate from the d13C of
the fructose in the sugar solution (Wilcoxon signed rank
test: T+ = 3, T- = -18, n = 6, P = 0.156), but was
significantly more enriched (by 8.2%) in 13C in relation to
the protein portion of the diet (Wilcoxon signed rank test:
T+ = 21, T- = 0, n = 6, P = 0.0313). Individual
d13C(?) values were significantly depleted in 13C in relation to the dietary sugars in one out of six individuals
(Table 1), and significantly enriched in 13C in relation to the
dietary proteins in four out of six individuals. None of the
individual d13C(?) values deviated from a mixed combustion model.
13
-15
δ13Cbreath (‰)
plateau value of -16.0% (Table 1), which was significantly lower (by 5.8%) than the d13C of the fructose
solution (Wilcoxon signed rank test: T+ = 3, T- = -12;
n = 7, P = 0.0016). Individual d13C(?) values were
significantly depleted in 13C in relation to the dietary
sugars in two out of seven individuals (Table 1).
-20
-25
(b)
-30
Sugar diet (IRMS)
-35
δ13Csugar
-10
-15
-20
δ13Cprotein
-25
-30
(c)
Mixed diet (IRMS)
-35
0
10
20
30
40
50
60
Time elapsed since initial feeding (min)
Fig. 2 a–c Stable C isotope ratios of exhaled breath (d13Cbreath) (%)
in Carollia perspicillata that were equilibrated to a diet based on
banana and honey water (d13C = -25.6%) and then fed hexose sugar
solutions at time 0, 20, and 40 min. An exponential regression model
was calculated for each individual (curved lines). a Animals were fed
a 30% (m/m) hexose water (straight solid line, d13C = -11.3%) and
d13Cbreath was measured using an infra-red stable isotope analyser
(IR-SIA; experiment 1: n = 5). b Animals were fed a 30% (m/m)
fructose water (solid straight line, d13C = -10.2%) and d13Cbreath
was measured using an isotope ratio mass spectrometer (IRMS;
experiment 2: n = 7). The six animals of experiment 3 (c) were fed a
mixture of protein (straight dotted line, d13C = -22.1%) and 30%
(m/m) fructose water (straight solid line, d13C = -10.2%) and
d13Cbreath was measured using an IRMS. Box plots show the variation
of estimated individual plateaus of the exponential regression models:
lines within the boxes depict the median, the boxes represent the 25
and 75% percentile
Experiment 2: individuals fed fructose (IRMS analyses)
In this experiment, C. perspicillata was fed a 30% (m/m)
fructose solution and the isotopic signature of exhaled
CO2 was analysed using a conventional IRMS. The
amount of ingested food averaged 2.4 ± 0.9 ml per
individual. The average time required to exchange 50% of
metabolised substrate with newly ingested sugar equalled
9.5 ± 7.0 min, and the d13Cbreath converged to a median
123
Synopsis of experiments with captive animals
Animals of the three experimental groups fed on similar
amounts of sugar water (one-way ANOVA: F(2,14) = 1.35,
P = 0.289). Neither k nor d13C(?) differed significantly
among the animals of the three experiments or were
affected by body mass (k; experiment, F(2,14) = 0.53,
P = 0.60; body mass, F(1,14) = 0.58, P = 0.46; d13C(?);
experiment, F(2,14) = 1.84, P = 0.20; body mass,
F(1,14) = 0.06, P = 0.82). The average t50 for all animals
was 10.9 ± 7.5 min. On average, d13Cbreath converged on a
value of -14.2 ± 3.3% in the 18 animals of the experiment. This plateau value was significantly depleted in 13C
(by -2.0%) in relation to the stable C isotope signature of
the hexose portion of the diet (Wilcoxon signed rank test:
T+ = 11, T- = -149, n = 18, P = 0.0004).
The stable C isotope signature in tissue and exhaled
breath in relation to omnivory
In wing tissues of free-ranging phyllostomid bats, median
d13C equalled -26.4% (mean -26.3 ± 1.6%; Fig. 3) and
Oecologia
Table 1 Individual regression coefficients (mean ± 1 SE) for the single-pool exponential regression model calculated for the fractional
incorporation rates of ingested sugar solution (hexose sugars and mix of protein and fructose) into the pool of metabolised substrates
Exp.a
Ind.
Body mass
(g)
1
A
22.3
2
3
d13Cbreath(?)
(%)
d13Cbreath(0) –
d13Cbreath(?)
(%)
k (min-1)
Sugar
combustion
Mixed
combustion
Protein
combustion
-9.7 ± 1.2
-18.9 ± 1.1
0.0348 ± 0.005
0.5 n.s.
n.a.
n.a.
B
21.4
-12.7 ± 0.4
-13.5 ± 1.2
0.183 ± 0.055
1.3 n.s.
n.a.
n.a.
C
20.5
-13.8 ± 0.3
-12 ± 0.8
0.094 ± 0.016
3.0*
n.a.
n.a.
D
22.6
-15.1 ± 0.3
-15.1 ± 0.6
0.079 ± 0.008
5.7*
n.a.
n.a.
E
18.5
-12.6 ± 0.5
-17.7 ± 0.1
0.093 ± 0.016
1.1 n.s.
n.a.
n.a.
F
22.1
-18.4 ± 2.5
-9.6 ± 0.4
0.100 ± 0.094
1.4 n.s.
n.a.
n.a.
G
20.3
-10.6 ± 0.9
-20.7 ± 2.0
0.028 ± 0.025
0.2 n.s.
n.a.
n.a.
H
24.2
-16.0 ± 1.1
-15.0 ± 1.7
0.095 ± 0.027
2.1*
n.a.
n.a.
I
21.6
-15.5 ± 2.1
-10.8 ± 3.7
0.161 ± 0.128
1.1 n.s.
n.a.
n.a.
J
23.5
-16.4 ± 2.2
-6.1 ± 2.5
0.065 ± 0.071
1.2 n.s.
n.a.
n.a.
K
20.5
-18.5 ± 0.9
-16.7 ± 1.5
0.143 ± 0.03
4.0*
n.a.
n.a.
L
23.3
-12.2 ± 0.8
-13.7 ± 1.1
0.091 ± 0.017
1.0 n.s.
n.a.
n.a.
M
22.3
-19.1 ± 2.8
-10.0 ± 3.2
0.064 ± 0.057
1.3 n.s.
0.4 n.s.
0.4 n.s.
N
O
23.9
21.8
-16.5 ± 1.5
-14.7 ± 3.9
-10.5 ± 1.9
-9.7 ± 5.0
0.076 ± 0.036
0.108 ± 0.137
1.8 n.s.
0.5 n.s.
0.1 n.s.
0.2 n.s.
1.6*
0.8 n.s.
P
23.7
-7.3 ± 2.8
-21.8 ± 2.6
0.023 ± 0.006
0.4 n.s.
1.3 n.s.
2.2*
Q
19.0
-16.1 ± 0.9
-10.6 ± 1.4
0.109 ± 0.034
2.7*
0.0 n.s.
2.8*
R
20.1
-9.9 ± 3.4
-19.2 ± 3.1
0.035 ± 0.014
0.0 n.s.
0.8 n.s.
1.5*
In experiment 1 five Carollia perspicillata were fed a hexose sugar solution, in experiment 2 seven C. perspicillata were fed a fructose sugar
solution and in experiment 3 six C. perspicillata were fed a mixed solution of protein and fructose. d13Cbreath values were measured using an
infra-red stable isotope analyzer in experiment 1 and an isotope ratio mass spectrometer in experiments 2 and 3. Deviation from the assumption
of exclusive sugar combustion, mixed combustion and exclusive protein combustion are indicated by t-values
n.s. no significant deviation, n.a. not applicable
* Significant deviation from the assumption at a 5% level of significance
18
-28.3% (mean -28.0 ± 2.0%; Fig. 3). Four bats showed
relatively high d13Cbreath values suggesting that they
had recently ingested a mixed diet of fruit from C3 and
C4/CAM plants. The d13Cbreath of three Artibeus watsoni
averaged -15.8 ± 2.3% and that of one Carollia castanea
equalled -15.7%. The plant sources for these high d13C
values were unknown. In the following analysis, we have
only included individuals with a distinct C3 isotopic signature in their exhaled CO2, i.e. species with d13Cbreath
lower than -19%. The Dtissue – breath value of species
increased with increasing d15 N values of species
(rS = 0.87, n = 11, P = 0.0004; Fig. 4).
16
Number of individuals
14
12
10
8
6
4
2
0
-35
-30
-25
-20
-15
-10
δ13Cbreath (‰)
Discussion
13
Fig. 3 Frequency distribution of d Cbreath (%) in 52 free-ranging
omnivorous bats captured in a rainforest dominated by C3 plants. The
d13Cbreath of four individuals indicated the consumption of plant
products that originated from C4/CAM plants (d13Cbreath [ -19%)
median d15 N 10.4% (mean 10.1 ± 2.6%; see Table 2 for
species-specific values). Median d13Cbreath of free-ranging
omnivorous bats captured in the rainforest equalled
Metabolised substrate in C. perspicillata
C. perspicillata metabolised exogenous carbohydrates
immediately after ingestion. After approximately 11 min,
50% of C atoms in the pool of metabolised substrates was
exchanged with C atoms of newly ingested food. This fast
123
Oecologia
Table 2 Stable carbon isotope ratio (mean d13 ± 1 SD; %) in wing
tissue and exhaled breath and N isotope ratio (mean d15N ± 1 SD; %)
in wing tissue of 12 phyllostomid bat species (sorted according to
increasing 15N enrichment
Species (n)
Wing tissue
d13C (%)
Exhaled
breath
d13C (%)
5.3
-25.9
-24.2
Artibeus jamaicensis (1)
7.5
-25.8
-25.0
Artibeus watsoni (7)
7.8 ± 2.4 -27.3 ± 2.2 -27.9 ± 1.9
Artibeus lituratus (2)
8.0
Carollia perspicillata (6)
9.3 ± 1.4 -26.9 ± 0.4 -26.9 ± 0.9
d15N (%)
Vampyressa nymphaea (1)
-25.6
-31.2
Artibeus phaotis (6)
Carollia castanea (10)
9.8 ± 2.7 -28.0 ± 2.1 -28.1 ± 1.9
10.9 ± 1.6 -27.6 ± 1.1 -26.5 ± 1.3
Carollia sowelli (7)
11.1 ± 1.7 -27.6 ± 1.0 -29.3 ± 1.0
Glossophaga commissarisi 12.0 ± 0.6 -25.0 ± 1.5 -28.5 ± 3.1
(4)
Hylonycteris underwoodi
(1)
12.4
-25.0
-27.5
Ectophylla alba (2)
12.7
-28.1
-29.8
Phyllostomus discolor (1)
14.4
-22.5
-25.1
Numbers in brackets depict sample size
6
δ13Ctissue (‰) - δ13Cbreath (‰)
4
2
0
6
8
10
12
14
-2
-4
δ15Ntissue (‰)
Fig. 4 Relationship between the isotopic difference of wing membrane tissue (d13Ctissue) and breath samples (d13Cbreath) in relation to
d15Ntissue (%) of omnivorous bat species. One species (Artibeus
lituratus) exhibited much lower d13C in exhaled breath than in solid
tissue, suggesting that this species recently switched to a new diet
with a much lower d13C. We considered this single data point as an
outlier and calculated a linear regression line following the least
squares method for the remaining 11 bat species (solid line). The
regression indicated that the discrepancy between d13Cbreath and
d13Ctissue increased with increasing trophic level, i.e. bat species with
a higher proportion of insects in their diet had lower d13Cbreath values
in relation to d13Ctissue
123
allocation of exogenous substrates to the pool of metabolised substrates is similar to that observed in Glossophaga
soricina, a 10-g nectar-feeding bat (Voigt and Speakman
2007), suggesting that phytophagous bats, in general, may
quickly mobilize exogenous carbohydrates for metabolism.
Fractional incorporation rates of exogenous substrates into
the pool of metabolised substrates are slower in similar-sized
mammals feeding on complex diets (t50 = ca. 30 min in
blood-licking vampire bats, Voigt et al. 2008; t50 = ca.
20 min in corn-feeding mice, Perkins and Speakman 2001).
In large mammals, t50 values are even higher, e.g. more than
30 min in humans drinking a sugar solution (Péronnet
2003), several hours in horses (Equus ferus; Ayliffe et al.
2004), and several days in alpacas (Lama pacos; Sponheimer et al. 2006) feeding both on hay.
In 18 out of 20 C. perspicillata, d13Cbreath converged on
the stable C isotope signature of the ingested hexose sugars. In two animals that consumed less than 1 ml of sugar
solution, d13Cbreath dropped to lower values after an initial
increase shortly after their meal, indicating that they
switched back to the combustion of endogenous substrates
after most of their sugar meal was metabolized. In the
remaining 18 animals, plateau values of d13Cbreath were, on
average, depleted by ca. -2% in relation to the carbohydrate portion of the diet. In a similar experiment with 10-g
G. soricina, d13Cbreath was also depleted by ca. -2% in
relation to the d13C of the fructose diet (Voigt and
Speakman 2007). This difference implies that satiated bats
of both experiments used a combination of exogenous and
endogenous substrate to fuel their metabolism. However,
we assume that active C. perspicillata would fuel their
metabolism exclusively with exogenous food at night,
since enzymatic activity is known to increase at times of
activity (Stevenson et al. 1975; Saito et al. 1976), i.e.
presumably at night in the case of nocturnal bats. Larger
mammals, including humans, fuel only a minor portion of
their metabolism with exogenous substrate, even during
periods of moderate exercise (Adopo et al. 1994; Roberts
et al. 1996; Péronnet 2003).
We hypothesized that C. perspicillata would fuel their
metabolism predominantly with simple sugars when given
the choice between dietary proteins and carbohydrates.
After feeding C. perspicillata a mixed diet of carbohydrates and proteins with contrasting stable C isotope
signatures, d13Cbreath levelled off above the d13C of the
protein portion of the diet at values close to the d13C of the
carbohydrate portion. However, based on the analysis of
individual regression parameters, we cannot rule out the
possibility that bats combusted a mixture of ingested proteins and carbohydrates. The protein enrichment in the
ingested sugar solution may have been above the minimum
protein requirements of our study bats and, therefore, bats
of our experiment may have routed some of the ingested
Oecologia
proteins to metabolism. Body mass as an indicator of
nutritional status did not influence k or d13C(?). We
assume that free-ranging C. perspicillata would route most
of their protein to tissue synthesis instead of metabolism,
since fruits eaten by phyllostomid bats contain, on average,
4 times as much water-soluble carbohydrate and 6 times as
much complex carbohydrate as protein (Wendeln et al.
2000). Thus, free-ranging fruit-eating C. perspicillata may
save the few proteins in fruits for body synthesis and
allocate the large amounts of carbohydrates to metabolism.
Stable C isotope signature in tissue and exhaled breath
of omnivorous animals
Based on the results of our experiments, we hypothesized
that d13Cbreath of free-ranging omnivorous phyllostomid
bats would reflect the d13C of the carbohydrate portion of
the diet, whereas d13Ctissue would reflect the d13C of the
non-carbohydrate portion of the diet. We expected species
with an insect-dominated diet to have a higher d13Ctissue
than predominantly fruit-eating bats, since proteins originate from plants in obligate phytophagous bats or from
insects in species with a mixed diet (Herrera et al. 2001,
2002). The isotopic signature of exhaled CO2, however,
should be similar in obligate fruit-eating and omnivorous
species, as it reflects the stable C isotope signature of the
carbohydrate source of the animals, namely plant products
such as fruits or nectar. Thus, the difference between
d13Ctissue and d13Cbreath should increase with increasing
trophic level i.e. increasing d15N.
Nitrogen isotope ratios in wing tissue indicated that bats
of our study area ingested varying portions of insects and
plant products such as fruits or nectar (see also Herrera
et al. 2001, 2002). Phyllostomus discolor exhibited the
highest d15Ntissue, as high as in tropical insectivorous bats
(Herrera et al. 1998; Voigt et al. 2007). Ectophylla alba, a
4-g stenodermatine bat that is known to be a specialist for
fig fruits (Bernal and LaVal 2002), also had high d15N
values, suggesting that they may also include insects in
their diet. According to their d15N, the two nectar-feeding
bat species Hylonycteris underwoodi and Glossophaga
commissarisi and bats of the genera Carollia included a
large portion of insects in their diet. Thus, members of the
subfamilies Carolliinae and Glossophaginae, at least those
that we looked at in our study, could be considered as
dietary generalists (Herrera et al. 2001, 2002). By contrast,
most stenodermatines of our study, such as Vampyressa
nymphaea and members of the genus Artibeus, showed low
d15N values, indicating that these species met their protein
requirements by consuming fruits (Herrera et al. 2002;
Voigt et al. 2007). Overall, species-specific d15N values
ranged over 9%. Since 15N is enriched in relation to 14N by
ca. 3.5% across trophic levels in bats (Herrera et al. 1998;
Voigt et al. 2003; Voigt and Matt 2004), phyllostomid bat
species of this study most likely covered more than two
tropic levels.
According to their d13Cbreath, most bats of our field study
could be assigned to a food web based on C3 plant products. However, some individuals exhibited d13Cbreath values
with an isotopic signature of a C4/CAM plant-based food
web, whereas the d13C values of wing tissue of these animals suggested that they belonged to a C3 plant-based food
web. This finding highlights the usefulness of d13Cbreath
measurements for dietary studies, since short-term changes
in dietary preferences are easy to detect in d13Cbreath, when
dietary sources are isotopically distinct.
In our field study, we hypothesized that macronutrients
are routed differentially to either combustion or tissue
synthesis in species that feed on food items varying in
macronutrient composition and stable C isotope signature.
We observed that the difference between d13Ctissue and
d13Cbreath increased with increasing d15N of the species,
and we found the largest difference between these parameters ([2%) in species with a presumably high
percentage of animal protein in their diet. A difference of
2% is similar to the trophic enrichment of 13C between
plants and insects (Herrera et al. 1998; Voigt et al. 2006),
and could, therefore, be expected for the isotopic difference
between breath and tissue of predominantly insect-feeding
phyllostomids.
The results of our field study are in line with those of our
laboratory experiment supporting, in general, the idea of
macronutrient routing in omnivorous animals. Our experiments with captive C. perspicillata showed that the
isotopic signature of exhaled CO2 converged on the d13C of
the recently ingested carbohydrates, irrespective of whether
proteins were additionally ingested or not. Therefore, we
assume that Carollia allocated most dietary sugars to
metabolism and most dietary proteins to body synthesis.
We speculate that nutrient routing could be monitored in
other free-ranging mammals as well using stable C isotope
values of breath and tissue samples. A combined isotopic
analyses of exhaled breath and tissues may yield a more
accurate picture of the diet in omnivorous mammals, and
most likely also in omnivorous birds.
Acknowledgements Peter Thompson and Dr Paula Redman analysed the samples at the Aberdeen Centre for Energy Regulation and
Obesity. We thank Carolin Werres for help during the field work at La
Selva Biological Station and OTS for providing support and infrastructure during the course of this study. We acknowledge the
generous support of the Costa Rican authorities, especially Javier
Guevara at SINAC. The experiments complied with the current laws
of Costa Rica. We thank Dr Detlev Kelm for commenting on an
earlier version of this manuscript. This work was financed by a grant
from the Deutsche Forschungsgemeinschaft to CCV (Vo890/7).
123
Oecologia
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