Low turnover rates of carbon isotopes in tissues of two nectar-feeding bat species
1 Institute for Zoo and Wildlife Research, Alfred-Kowalke-Str. 17, 10315
Berlin, Germany
2 Zoologisches Institut II, Universität Erlangen-Nürnberg,
Staudtstr. 5, 91058 Erlangen, Germany
3 Stable Isotope Laboratory, Department of Biology, Boston University,
Boston, MA 02215, USA
4 Center for Ecology and Conservation Biology, Department of Biology, Boston
University, Boston, MA 02215, USA
* Author for correspondence (e-mail: voigt{at}izw-berlin.de)
Accepted 29 January 2003
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Summary |
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Key words: metabolism, carbon isotope, bat, Leptonycteris curasoae, Glossophaga soricina, fractionation
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Introduction |
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Tieszen et al. (1983)
showed that the isotope turnover rates in tissues are related to the
tissue-specific turnover rates. On a whole-animal basis, it could be argued
that animals with a high mass-specific energy turnover rate should have a high
isotope turnover rate. Nectar-feeding bats (Glossophaginae, Phyllostomidae)
have mass-specific metabolic rates that exceed those of most other eutherians
by a factor of two (von Helversen and
Winter, in press
). Thus, we were interested to determine at what
rate stable carbon isotopes are exchanged in tissues of nectar-feeding bats.
We postulated that the isotope turnover of nectar-feeding bats should be
higher than in other eutherian mammals.
Stable isotopes have been used in the study of bats and especially in
nectar-feeding bats (Herrera et al.,
1993,
1998
,
2001
;
Fleming et al., 1993
;
Fleming, 1995
). Fleming et al.
(1993
) showed that migratory
Leptonycteris curasoae, within their northern distribution range,
feed extensively or, in some cases even exclusively, on the nectar of
CAM-plants (Cactaceae and Agavaceae) with a carbon isotope ratio of
approximately -10
. By contrast, in the southern parts of their
distributional range in Mexico, which corresponds to the wintering habitat of
northern populations, Leptonycteris feeds primarily on nectar of
C3 plants that secrete sugar with a carbon isotope ratio of
-25
. By analyzing the carbon isotope abundance in tissues of L.
curasoae from different geographical regions, Fleming et al.
(1993
) tracked the seasonal
patterns of dietary preferences and migratory movements of this species, and
concluded that L. curasoae depends on a spatiotemporal nectar
corridor of cacti and agave flowers during migration.
We performed a diet-switching experiment with two glossophagine bat species
(Leptonycteris curasoae and Glossophaga soricina), during
which we changed the carbon isotope ratio of the diet by almost 14.
Diet-switching experiments are facilitated in nectar-feeding animals because
the isotope ratio of the diet can be changed without changing the overall
composition of the diet, as both diets are based on sugar water. We collected
three types of tissue (hair, wing membrane and blood) that caused minimal harm
to the animals. We selected blood as our standard target tissue, assuming that
it would show the highest turnover rate. We chose small pieces of wing
membrane because we expected it to exhibit an intermediate turnover rate
relative to blood and hair. Biopsies of wing membrane are routinely taken for
DNA extraction during genetic studies (Worthington Willmer and Barratt, 1996)
and, based on our experience, punctured wing membranes regenerate within 3-4
weeks. We chose hair because of its expected low turnover rate
(Tieszen et al., 1983
). As a
metabolically inert tissue at maturity, isotope ratios in hair should reflect
the isotope composition of food consumed only during the time of hair
growth.
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Materials and methods |
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After 7 days on the initial diet, we switched the diet of plants to one with a carbon isotope composition of the C4 and CAM photosynthetic pathways (Table 2), referred to as day 1 of the experiment. As before, the stock solutions were diluted to a sugar concentration of 18% (mass/mass). To complement the sugary solution of the bats, we added several mg of vitamins and mineral supplements to the diet each day.
Before and after each night, the sugar water was weighed to an accuracy of 1 g and we refer to ingested food as the difference between the two measurements. The concentration of 13C in the ingested food was estimated by calculating the proportion of each of the three main food sources that were ingested each day and by multiplying this value with the corresponding concentration of 13C in the food items (Tables 1 and 2).
At the end of the first week and during each subsequent sampling event, bats were weighed to the nearest 0.01 g on an electronic balance (Mettler PM-100, Columbus, OH, USA) and three types of samples were taken from each bat. We took two tissue samples from the wing membrane using a 3 mm diameter biopsy punch. Next, we drew approximately 30 µl of blood from the propatagial vein using a small sterile needle. Finally, we removed hair from an area of approximately 0.25 cm2 from the back of each bat. The hair was cut with scissors at the base close to the skin and from varying regions of the back but from the same spot at a given day. All samples were placed into Eppendorf tubes, labelled and transferred immediately into a drying oven, where they were dried to constant mass at 60°C. Subsequently, samples were stored in a freezer below 0°C. After changing the diets, we took blood, wing membrane and hair samples at the end of the second, fourth, sixth and eighth weeks.
To remove external contaminants from skin and hair samples, we washed the
samples with a chloroform/methanol solvent (1:1). To test for any differences
between washed and unwashed samples, we collected samples of hairs from eight
Leptonycteris and divided each sample into two parts. The first part
was washed in the solvent and the second part remained untreated. We then
measured the carbon (13C,
) and nitrogen isotopes
(
15N,
) in the hair samples following the procedure
described below and performed pair-wise comparisons between washed and
unwashed samples. We found a significant carbon enrichment of 1
in
treated versus untreated samples but no difference in
15N (Table
3).
|
Sample analysis and conversion to the notation
Samples were combusted and the resultant gases (N2 and
CO2) were sequentially measured in a CE 1110 elemental analyzer
connected via a continuous flow system to a Thermo Finnigan Delta
Plus isotope ratio mass spectrometer (Thermo Finnigan, Bremen, Germany). The
sample isotope ratios were compared with international gas standards (USGS-24
and IAEA-N1). Precision was better than ±0.1 for both nitrogen
and carbon. Isotope ratios are expressed in the
notation in parts per
thousand (
) using the following equation for carbon isotopes:
![]() | (1) |
![]() | (2) |
We used the carbon isotope ratio of Vienna Pee Dee Belemnite limestone and the nitrogen isotope ratio of air as standards.
Statistical analysis and curve estimation
To test for differences in mean isotope enrichment between (1) the tissues
and (2) the diets, we performed one-way analysis of variance (ANOVA). We ran
post-hoc Tukey HSD tests for pair-wise comparisons to evaluate
differences in mean values.
We calculated mean isotopic values for all sample periods. In theory,
changes in isotopic composition should follow an exponential curve (e.g.
Tieszen et al., 1983). Hence,
equations of the type y=a+bect were fitted to the
13C data from each tissue and each bat species. In this
equation, a represents the asymptotic
13C value for
the tissue equilibrated on a C4/CAM-diet, b equals the
overall change in isotope ratio, c is the turnover rate of carbon
isotopes in the tissues, and y the mean carbon isotope ratio in the
tissue at the time t. For the reasons of simplicity, we refer to
c as the regression coefficient in the exponential model.
We assumed that the different tissues equilibrate to an isotope ratio close
to the value measured for the C4/CAM-diet plus the average
fractionation value found for that specific tissue. Thus, a equalled
the average isotope ratio of carbon isotopes in the C4/CAM-diet
plus the difference between 13C of the C3 diet
and the tissue caused by fractionation. Additionally, we assumed that
b equalled the overall change in isotopic composition in the two
diets, which was 13.7
in Leptonycteris curasoae and
13.6
in Glossophaga soricina. Estimation of c was
performed on an iterative basis starting with a value of 0.05. The iteration
was stopped after changes in the sum of squares were smaller than
1.0x10-8. To estimate the half time of the carbon isotope
exchange in the different tissues, we calculated t50 using
the following equation:
t50=loge(0.5)/c, where
t50 is the time in days in which half of the carbon
isotopes were exchanged in the corresponding tissue, and 0.5 represents the
exchange of 50% isotopes. For reasons of simplicity we will describe the half
life of carbon isotopes (=t50) in hair, although we are
aware that the isotope composition of hair reflects only the period of growth,
as hair is an isotopically inert tissue.
Possible effect of body mass changes on regression coefficients
In a preliminary experiment with five Leptonycteris curasoae, we
observed that within 22 days following the diet switch bats had lost some body
mass. To evaluate a possible bias in our experiment due to loss of body mass,
we calculated exponential exchange curves on an individual basis. We then
tested whether the rate of increase c of the exponential functions
was related to the change in body mass. We predicted that the regression
coefficient c decreased with increasing loss of body mass if carbon
isotopes of catabolized fat or protein of C3 origin mixed with the
ingested carbon isotopes of C4/CAM origin. We expected such effects
to be most apparent in tissues with a relatively high turnover and thus
performed the statistical analysis only for the data set of wing membrane and
blood. The level of significance was Bonferroni-corrected to 2.5%, because two
data sets were tested for each individual.
Values are expressed as means ± 1 S.D. In general, two-tailed tests were performed. We used SPSS (version 9.0) for all statistical analysis and regression models.
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Results |
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Nutritional intake and body mass changes
On average, individuals of L. curasoae ingested more sugar water
per day (20.3±2.9 ml day-1) than did individuals of G.
soricina (19.2±1.8 ml day-1) (Student t-test,
t110=2.6, P=0.014). Daily rate of food intake did
not vary between diets 1 and 2 (L. curasoae: Student t-test:
t61=1.95, P=0.06, G. soricina: Student
t-test, t61=0.23, P=0.82). In addition,
the mean nutritional intake rate remained constant throughout the experiment
(ANCOVA: L. curasoae: F4,58=1.4, P=0.25;
G. soricina: F4,58=0.97, P=0.43). In
both species, mean body mass decreased significantly during the course of the
experiment (ANOVA for repeated measures: L. curasoae, interval:
F4,39=37, P<0.001; G. soricina,
interval: F4,39=63, P<0.001). In addition,
mean body mass was significantly different between individuals (L.
curasoae, F9,39=26, P<0.001; G. soricina,
individual, F9,39=43, P=0<0.001). On day 1 of
the experiment, the mean body mass of L. curasoae was 23.6±2.1
g and G. soricina, 10.2±0.7 g. At the end of the experiment,
mean body mass of L. curasoae had decreased to 21.7±2.3 g and
of G. soricina to 9.4±1.0 g. Thus, both species lost on
average 8% of their initial body mass during the course of the experiment.
Body mass changes of individuals of both species are plotted in
Fig. 2A,B.
|
Changes of isotope abundance during the experiment
After switching the diet to plant products of C4/CAM-origin, the
enrichment of heavy carbon isotopes increased in all three types of tissues
sampled in both species (Fig.
3A,C,E for Leptonycteris curasoae and
Fig. 3B,D,F for Glossophaga
soricina). At the end of the experiment, at day 60, none of the tissues
had equilibrated to the expected point of carbon isotopic enrichment (first
numerical value in the exponential regression equations plotted in the
graphs). Based on the calculated exponential equations, we estimated the half
life of the carbon isotopes in tissues (see t50 values in
Figs 3 and
4). In both species, wing
membrane and blood had very similar turnover rates of approximately 116 days
(range: 102-134 days, Table 4).
Based on a paired t-test, mean regression coefficients of wing
membrane and blood were not significantly different within species (L.
curasoae: t9=1.0, P=0.32, G.
soricina: t9=1.3, P=0.22). In addition,
neither mean regression coefficients of blood nor those of wing membrane were
different between species (Student t-test; blood:
t18=0.1, P=0.91; wing membrane:
t18=1.4, P=0.17). Compared to blood and wing
membrane, estimated t50 values for hair were higher by a
factor of five, averaging 537 days in L. curasoae
(Table 4). We could not detect
a significant change in 13C in hairs of G.
soricina.
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Effect of body mass loss on isotope turnover rates
On an individual basis, we tested whether body mass loss was related to the
regression coefficient of the exchange curve of carbon isotopes in
Leptonycteris curasoae (Fig.
4A) and Glossophaga soricina
(Fig. 4B). In blood samples of
G. soricina, the regression coefficients increased significantly with
increasing loss of body mass of the corresponding individual
(Table 5). Thus, contrary to
our expectation, turnover rates were faster in those animals that lost body
mass than those with a constant body mass, at least in blood of G.
soricina. When controlling for the effect of loss of body mass in G.
soricina, t50 of blood was 126 days (see legend of
Fig. 4).
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Discussion |
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We found a low rate of growth in hair of Leptonycteris curasoae
and no significant change in the isotope composition of Glossophaga
soricina hair. Because hair is a metabolically inert tissue, only the
basal, growing part should reflect the isotope ratio of the current diet. Thus
far, all bats studied with respect to hair growth and molt showed a seasonal
pattern (Constantine, 1957,
1958
;
Kunz, 1974
; Mazak,
1963
,
1965
). Thus, if both study
species were in a phase of reduced hair growth, only negligible amounts of
carbon with a C4 isotope signature would be included. Since the
patterns of hair growth are unknown for glossophagine bats, we cannot evaluate
the effects of seasonal hair growth in our study species.
In the wing membrane of bats, the estimated half life of carbon isotopes
was 100 and 130 days. In contrast to this finding, biopsies of wing membranes
heal at a remarkably rapid rate (see also
Worthington Wilmer and Barratt,
1996). In nectar-feeding bats, punctured wing membranes caused by
a biopsy punch heal and completely close within 3-4 weeks. However, the
regeneration time of damaged bat wings is probably not representative of the
regeneration time of the whole tissue. Quite likely, special biochemical
processes are triggered after a wing membrane is damaged and, as a
consequence, regeneration time of wounds in the membrane is higher than the
overall turnover time in that tissue. Wing membranes comprise an extremely
reduced dermis that is sandwiched between the dorsal and ventral layer of the
epidermis (Quay, 1970
). To
increase elasticity and stiffness, bat wings consist of a two-dimensional
network of large, macroscopic collagenelastin fibre bundles
(Holbrook and Odland, 1978
).
Palaeontologists use bone collagen as an indicator of yearly or lifetime diets
of organisms because collagen is known to regenerate at a low rate (e.g.
t50=173 days in quails:
Hobson and Clark, 1992a
). High
concentrations of collagen and elastin in wing membranes and their possible
low rate of regeneration could explain our findings of a low carbon isotope
turnover rate in the wing membrane. Bat wings are also cooler than the core
body temperature of bats. Lancaster et al.
(1997
) showed that the
temperature of the wing membrane is approximately 1-2°C above the ambient
temperature. Thus, in the present study, biochemical processes in the wing
membrane probably took place at a temperature of 24-25°C. The specific
molecular composition and the relatively low temperature could explain the low
turnover rates of carbon isotopes in bat wings.
Blood was estimated to have a t50 value of 120 and 126
days. Because this is the first measurement of blood turnover in bats, we
compared our data with those from mammals other than bats. In humans,
erythrocytes have a t50 value of 60 days
(Schmidt and Thews, 1995). In
avian blood, the carbon isotope half life was 11 days in Japanese quails
(Coturnix japonica; Hobson and
Clark, 1992a
) and approximately 30 days in young, growing American
crows (Corvus brachyrhynchos;
Hobson and Clark, 1993
). Our
results indicate that the estimated isotope half-life of approximately 120
days in bat blood is higher than corresponding values from other animals,
including humans, and is also in contrast to our prediction.
We discuss this unexpected result in the context of three different scenarios. (1) The rate of isotope turnover was slowed down because animals were torpid or lethargic. (2) The bats accumulated a nutritional deficit, which contributed to a loss of body mass. In the latter situation, the mobilization of fat and/or body proteins would affect the estimate of stable isotope turnover rates, so we evaluated the possibility that carbon from sugar and carbon from body tissues are used for different purposes: energy versus tissue synthesis. Finally, we consider the possibility that the measured turnover rates of carbon isotopes are representative for the actual tissue turnover despite the presence of body mass loss.
Many bat species are known for their propensity to enter torpor under low
ambient temperatures or unfavourable food regimes. Torpor or lethargic
conditions have been mostly studied in bats of temperate zones, namely
vespertilionid species (see review by
Speakman and Thomas, in
press). Most tropical bats do not enter torpor on a regular basis,
although studies are scarce on this phenomenon in tropical species. Cruz-Neto
and Abé (1997
) found
that captive G. soricina reduced their body temperature when deprived
of food. Facultative torpor has also been observed in other nectar-feeding
bats, such as L. curasoae and Choeronycteris mexicana in
captivity (C. Voigt, personal observation). Thus, some phyllostomids do reduce
their body temperature under certain conditions. It could be argued that the
low turnover rates in tissues of the two species of nectar-feeding bats could
be due to prolonged periods of torpor, but two facts argue against this
hypothesis. First, the bats of this study were always ready to fly from their
roost when they were disturbed and torpid or lethargic bats are incapable of
flight. Second, the nectar-uptake rate did not change during the course of the
experiment, which would have been expected if bats used torpor after the
switch in diets. Thus, torpor is an unlikely explanation for the observed low
turnover rates.
Both G. soricina and L. curasoae lost approximately 8% of
their initial body mass during the experiment. Nutritional deficits due to
insufficient energy, mineral or nitrogen supplies, are possible causes for
such a trend. Both species ingested approximately 20 ml of sugar water each
day. As the bats were fed with 18% sugar water, this can be converted into a
daily energy intake rate of approximately 60 kJ day-1. This value
falls into the range of previously reported rates of energy expenditure for
both species (von Helversen and Winter, in
press). In addition, Horner et al.
(1998
) estimated the field
metabolic rate of L. curasoae as at least 40 kJ day-1 and
daily energy intake rates of non-reproductive G. soricina ranged from
7 to 68 kJ day-1 in a captive study
(Winter, 1993
;
Voigt, in press
). We find it
unlikely that the study bats suffered from an insufficient supply of energy.
Other forms of nutritional stress may have occurred despite our intention to
provide all essential nutrients such as vitamins and minerals.
In the present work, we simulated the extent to which fat mobilization
affects the overall isotope ratio of the carbon pool that is available to the
bat for homeostasis. In this simulation, we assumed that the isotopic
fractionation between exogenous foods and tissues is the same as that for the
metabolism of tissues and endogenous reserves. Assuming that (1) loss of body
mass was solely due to the mobilization of fat and (2) body fat contained
approximately 20% water, loss of 1.6 g body mass would be equivalent to
approximately 1.3 g dry fat in a L. curasoae weighing 20 g. Assuming
also that a mammalian fat molecule consists of two C16 chains and
one C14 chain, fat molecules would consist of 42 carbon, 100
hydrogen and 6 oxygen atoms. Taking the mass of carbon, hydrogen and oxygen
into account, 1.3 g fat consists then of approximately 0.94 g carbon
(12C), 0.18 g hydrogen (1H) and 0.18 g oxygen
(14O). Following this, 0.94 g carbon with an isotopic signature of
approximately -22
13C should have mixed with the
ingested sugar molecules. L. curasoae ingested on average 20.4 ml
sugar water per day, or 1.2 l sugar water during the 2-month period of the
experiment. For reasons of simplicity, we have calculated all following values
for glucose (C6H12O6), which was the
predominant sugar molecule in the diet. 1 ml of 18% sugar water contains 180
mg sugar. As nectarivorous bats absorb sugar in the intestine with an
efficiency of almost 100% (Winter,
1998
), each Leptonycteris is likely to have ingested 216
g sugar during the 2-month period. This is equivalent to approximately 86.4 g
carbon, 14.5 g hydrogen and 115.1 g oxygen, respectively. Following this
simulation, the ingestion of 216 g sugar results in the uptake of 86.4 g
carbon from sugar (
13C=-10
), in contrast to 0.94 g
carbon from fat (
13C=-22
). Thus, the overall isotope
ratio of the carbon pool available for homeostasis would be -10.02
,
compared to -10.00
without fat mobilization. Even if fat was more
depleted in heavy isotopes by approximately 3
, a fractionation factor
found in fat tissues of other animals, the overall
13C value
would change only slightly to -10.05
. Therefore, fat mobilization
alone cannot explain the unexpectedly low estimates of isotope turnover rates
seen when carbon isotopes are used for homeostasis, irrespective of their
origin.
Preferential use of carbon from fat or proteins for tissue synthesis and
carbon from sugar for energy synthesis could, however, change the above
picture. Hobson and Stirling
(1997) addressed this problem
of selective metabolic pathways or differential isotopic routing. In a dietary
study on polar bears, the authors found that carbon isotope ratios in blood
were not distinguishable between offshore populations feeding on seals and
inland populations feeding on berries, although seals as a fat- and
protein-rich food source and berries as a carbohydrate-rich food source
differed by approximately 9
in carbon isotope ratios. This led to the
conclusion that inland populations probably burned the carbohydrates from
berries directly instead of incorporating them into tissues. This is a likely
explanation in the specific case. In contrast to polar bears, nectar-feeding
bats are dietary specialists depending strongly and sometimes even exclusively
on nectar as their food source. Bats may supplement their diet with pollen,
fruits and to some extent with insects, but the main carbon source of
nectar-feeding bats is nectar. Therefore, nectar-feeding bats ultimately have
to incorporate nectar carbon into their tissue. However, it is likely that in
our study that carbon isotopes from mobilized body substrates were
incorporated into the blood, thus mixing with the carbon of assimilated sugar.
Obviously, this effect should be most apparent in starving animals and absent
in animals with constant body mass. Hatch et al.
(1995
) showed that the
catabolic states of rooster chicks and adult hens are indicated by increased
levels of
13C in haemoglobin. In both species of our study,
we find individuals that lost body mass and individuals that maintained an
almost constant body mass during the experiment
(Fig. 2). We expected that the
regression coefficient c of the estimated exchange curve would be
underestimated in catabolizing animals and that the coefficient should reflect
true values in individuals with constant body mass. We tested for a
significant positive relationship between the regression coefficients
c and body mass loss in four tissues, but found only a negative
correlation for G. soricina blood
(Fig. 4). Therefore, loss of
body mass did not result in an overestimate of the isotope turnover half life
in these specific cases. Possibly, additional fractionation effects obscured
the presumed positive correlation between loss of body mass and regression
coefficient, or may even have reversed this trend.
Bat blood is unusual in several ways. First, blood from bats contains more
erythrocytes per ml than in other mammals (26x106
erythrocytes ml-1 blood in bats versus approximately
18x106 erythrocytes ml-1 blood in Rodentia or
Insectivora; summarized by Neuweiler,
1993). Secondly, hemoglobin concentrations are higher in bats than
in other mammals or even birds (0.24 g ml-1 blood in a pipistrelle
bat versus 0.18 g ml-1 blood in hummingbirds; summarized by
Neuweiler, 1993
). These
extreme values might be an adaptation to the high energy demands of flight
(Voigt and Winter, 1999
;
Voigt, 2000
). In birds, the
oxygen capacity of blood is maximized by unidirectional lungs, resulting in an
optimal oxygen uptake. Assuming that similar sized bats and birds are
producing erythrocytes at similar rates, we hypothesize that bats reach a
similar oxygen capacity of blood by having larger amounts of smaller,
longer-lived erythrocytes. Such a mechanism could compensate for the less
efficient bidirectional lung of bats. Thus, long-lived erythrocytes may be an
adaptation of bats to the high energy demands of flight. By using the same
experimental setup and separating the cellular fraction of blood from plasma
by centrifugation (see also Hobson and
Clark, 1993
), it will be possible to test this idea.
In summary, the estimated turnover rates of isotopes in the target tissues were consistently low for both study species. In L. curasoae, we could trace small amounts of carbon isotopes from C4-sugar in the hair, but no significant amounts from C4-sugar could be found in hair of G. soricina. This finding is probably explained by the seasonal growth of hair. The rate of isotope turnover in wing membrane was low, with t50 values ranging from 100 to 130 days. The particular composition and design of the wing membrane and its relatively low temperature are likely explanations for this result. In blood, the turnover rate of carbon isotopes was unexpectedly low. The carbon isotope half life was 120-126 days. We suggest that long-lived erythroycytes are a special adaptation of bats for maintaining a high oxygen capacity of blood, which is a prerequisite for enduring, aerobic flight performance. Further investigations are needed in the field of bat blood physiology.
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Acknowledgments |
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