Blood-sucking bugs as a gentle method for blood-collection in water budget studies using doubly labelled water.

Abstract

During doubly-labelled water (DLW) experiments, blood collection by venous puncture may traumatize animals and consequently affect the animals' behaviour and energy budget. Recent studies have shown that blood-sucking bugs (Triatominae; Heteroptera) can be used instead of conventional needles to obtain blood from animals. In this paper, we validate the bug method in captive nectar-feeding bats, Glossophaga soricina, for water budget analysis by comparing the daily water flux estimated with the DLW method with values measured by an energy balance method. As the mean daily water flux of the DLW method was not significantly deviating from the expected value, blood-sucking bugs may substitute more invasive methods of blood collection in DLW experiments. Based on the DLW estimates, daily energy and water intake rates were calculated and compared to values measured with the energy balance method. The DLW method and the energy balance method yielded on average similar results regarding the daily energy intake (DLW method: 48.8+/-14.2 kJ d(-1) versus energy balance method: 48.1+/-9.9 kJ d(-1)) and daily water intake (DLW method: 13.7+/-2.4 mL d(-1) versus energy balance method: 14.7+/-3.0 mL d(-1)). Based on the calculated water and sugar intake per day, we estimated the sugar concentration of ingested nectar to equal on average 16.2+/-2.4% (mass/mass), which fell close to the measured sugar concentration of 17% (mass/mass) bats fed on during the experiment. We conclude that it is possible to extrapolate mean daily energy and water intake for animal groups, populations and species based on DLW estimates, but due to the large variance of results (low accuracy), it seems inadequate to calculate values for single individuals.

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Blood-sucking bugs as a gentle method for blood-collection in water
budget studies using doubly labelled water
Christian C. Voigta,*, Robert Michenerb, Gudrun Wibbelta,
Thomas H. Kunzb, Otto von Helversenc
aInstitute for Zoo and Wildlife Research, PF 601103, 10252 Berlin, Germany
bStable Isotope Laboratory, Department of Biology, Boston University, 5 Cummington St., 02215 Boston, MA, USA
cInstitute of Zoology II, University of Erlangen-Nuremberg, Staudtstr. 5, 91053 Erlangen, Germany
Received 7 December 2004; received in revised form 13 July 2005; accepted 31 July 2005
Available online 16 September 2005
Abstract
During doubly-labelled water (DLW) experiments, blood collection by venous puncture may traumatize animals and consequently affect
the animals’ behaviour and energy budget. Recent studies have shown that blood-sucking bugs (Triatominae; Heteroptera) can be used
instead of conventional needles to obtain blood from animals. In this paper, we validate the bug method in captive nectar-feeding bats,
Glossophaga soricina, for water budget analysis by comparing the daily water flux estimated with the DLW method with values measured by
an energy balance method. As the mean daily water flux of the DLW method was not significantly deviating from the expected value, blood-
sucking bugs may substitute more invasive methods of blood collection in DLWexperiments. Based on the DLWestimates, daily energy and
water intake rates were calculated and compared to values measured with the energy balance method. The DLW method and the energy
balance method yielded on average similar results regarding the daily energy intake (DLW method: 48.8T14.2 kJ d?1versus energy balance
method: 48.1T9.9 kJ d?1) and daily water intake (DLW method: 13.7T2.4 mL d?1versus energy balance method: 14.7T3.0 mL d?1).
Based on the calculated water and sugar intake per day, we estimated the sugar concentration of ingested nectar to equal on average
16.2T2.4% (mass/mass), which fell close to the measured sugar concentration of 17% (mass/mass) bats fed on during the experiment. We
conclude that it is possible to extrapolate mean daily energy and water intake for animal groups, populations and species based on DLW
estimates, but due to the large variance of results (low accuracy), it seems inadequate to calculate values for single individuals.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Doubly-labelled water method; Glossophaga soricina; Energy budget; Water budget; Blood-sucking bugs; Minimally-invasive; Dipetalogaster
maximus; Sugar concentration
1. Introduction
Since the ground-breaking studies of Lifson and co-
workers about the doubly-labelled water (DLW) method
(e.g. Lifson and McClintock, 1966), this technique has been
used in numerous studies and provided useful insights into
the water and energy budgets of free-ranging animals,
including humans (summarized in Speakman, 1997). During
DLWexperiments, animals are given a water solution that is
enriched with heavy isotopes of hydrogen and oxygen. On
the basis of differential washout rates of deuterium (or
tritium) and oxygen-18, it is possible to estimate the daily
water flux and daily carbon dioxide production rates. Using
standard conversion factors, daily carbon dioxide produc-
tion rate is then converted into daily energy expenditure.
Different techniques have been employed to collect blood
from animals. Standard bleeding techniques involve con-
ventional needles that may cause undue stress to the animals
under investigation. Stress may bias the outcome of the
DLW experiment as it is known to affect the behaviour as
well as the metabolic rate of study animals (see discussion
in Speakman, 1997).
1095-6433/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpa.2005.07.011
* Corresponding author. Tel.: +49 30 5168 609; fax: +49 30 5126 104.
E-mail address: voigt@izw-berlin.de (C.C. Voigt).
Comparative Biochemistry and Physiology, Part A 142 (2005) 318 – 324
www.elsevier.com/locate/cbpa

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von Helversen and Reyer (1984) were the first to use
blood-sucking bugs (Triatominae; Reduviidae; Heteroptera)
in a DLWexperiment, arguing that these bugs are especially
well adapted to the quick and gentle ingestion of blood (von
Helversen et al., 1986). We recently showed that blood-
sucking bugs can be reliably used instead of conventional
needles for drawing blood from endothermic animals
without biasing the estimated daily energy expenditure in
DLW studies (Voigt et al., 2003). In another study, we
demonstrated that hydrocorticosterone levels are lower in
domestic rabbits when blood-sucking bugs are used instead
of conventional needles (Voigt et al., 2004). Thus, blood-
sucking bugs may indeed represent a gentle alternative for
blood collection in DLW studies.
In this current study, we validated the use of blood-
sucking bugs for estimating the daily water flux in DLW
experiments. We measured the water budget of 10 g nectar-
feeding bats in an experimental setup by quantifying the
ingested water and the amount of metabolic water produced
per day. Then we compared these values with the water
budget as estimated with the DLW method. Additionally, we
tested whether it is possible to extrapolate the sugar
concentration of the ingested sugar water based on DLW
estimates. This may provide important information in
ecophysiological studies of free-ranging nectarivorous
animals. We used nectar-feeding Glossophaga soricina
(Phyllostomidae) for this validation study, since the energy
budgets are particularly easy to measure in nectarivorous
animals in a laboratory experiment owing to a high
assimilation efficiency of the ingested sugar (Winter,
1998). In addition, blood-sucking bugs may be especially
suitable for DLW studies in small mammals, as bugs easily
acquire blood from diminutive veins that are otherwise
inaccessible for human experimenters.
2. Materials and methods
2.1. Energy balance method
The experimental procedure was described in detail in
Voigt et al. (2003). In brief, we randomly selected 3 male
and 3 female Glossophaga soricina, Rehn 1950, from a
captive colony of about 50 individuals maintained in
greenhouses at the University of Erlangen-Nu ¨rnberg. The
animals were transferred to an air-conditioned room (25
-C), where they were maintained under a light–dark
regime of 12 h:12 h, and ad libitum food (17% mass/
mass honey water, banana and pollen). During each
experiment, a single bat was released into a flight cage
(7 m long, 1 m wide, 2 m high), constructed using
polyurethane sheeting to prevent the bat from landing
anywhere other than on a piece of cork suspended from an
electronic balance (Mettler PM-100, accuracy 1 mg). This
balance was connected to a personal computer allowing
continuous recording of body mass and time spent in daily
flight. A ventilator was used to draw fresh air into the
flight cage. Thirty-seven milliliters of sugar solution was
dispensed into a feeder by a computer-controlled pump
when bats inserted their head into the feeder and
interrupted a light beam. During the experiment, bats were
sustained on a diet of 17.0% (mass/mass; Kru ¨ss refrac-
tometer, 0.1% accuracy) sugar solution with dry mass of
sugar consisting of 26% sucrose, 37% glucose and 37%
fructose (energy content: 15.91 kJ g?1; Wieser, 1986),
resulting in a food reward of 0.1 kJ per feeder visit. We
assumed an assimilation efficiency of sugar of 99%
(Winter, 1998).
2.2. Doubly-labeled water method
During a DLW experiment, a bat was captured at
approximately 15:30 h, weighed, and injected subcutane-
ously with 100 AL water that was enriched with isotopes of
oxygen-18 (30 at.%) and deuterium (10 at.%). The mean
duration of equilibration was 1.5 hT13 min (SD), which
was sufficient for complete equilibration of isotopes in body
water (Voigt et al., 2003). An initial blood sample was
obtained from a bat at 17:00 h, one hour before the onset of
darkness. We used starved blood-sucking bugs (Heteroptera;
Reduviidae: Dipetalogaster maximus, larval instar 3 and
Triatoma infestans, larval instar 4) for collecting blood (see
details in Voigt et al., 2003, 2004). After the bug ingested
approximately 80 to 100 AL of blood, we removed and
decapitated it, perforated its abdomen with a microcapillary
tube and collected the blood from the bug’s abdomen. All
tubes were immediately flame-sealed and stored at 0 -C
until analysis. After taking the initial blood sample, the bat
was returned to the flight cage (ca. 17:20 h). The following
afternoon we repeated the blood collection at 17:00 h. Each
time, bats were weighed to the nearest 1 mg and differences
in the two measurements were considered daily changes in
body mass (Dbm).
As described in Voigt et al. (2003) we used the mean
body water content of 5 desiccated animals to estimate the
total body water pool of the bats during DLW experiments,
because the bug’s haemolymph contaminates to a certain
extent the blood samples (Voigt et al., 2003). Body mass at
the time of initial blood sample (ca. 17:00 h) was multiplied
by the mean relative percent body water content (66.8%;
mean value for 5 desiccated specimens). Blood samples for
the analysis of background concentration of oxygen-18 and
deuterium were taken from 8 G. soricina at 17:00 h
following the procedure described above. Isotope enrich-
ments in these background samples are reported in Voigt et
al. (2003).
Blood was distilled following Nagy (1983), and oxygen
isotopes in the water samples were analysed with the
guanidine-hydrochloride method and deuterium isotopes
with the zinc-reduction method (see Voigt et al., 2003). All
samples were analysed at least in duplicate using a Finnigan
Delta-S isotope ratio mass spectrometer at the Boston
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319

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University Stable Isotope Laboratory. A laboratory standard
was analysed in duplicate after every sixth sample.
2.3. Measurement of water budgets with the DLW method
The water budgets of the study animals were measured
with the DLW method by determining the fractional
turnover rate (kD) of deuterium following Eq. (1):
kD¼ln ci? cb
ð Þ ? ln cf? cb
t
ðÞ
ð1Þ
with cbrepresenting the basal deuterium enrichment (atomic
percent), ci the initial deuterium enrichment (atomic
percent), cfthe final deuterium enrichment (atomic percent)
and t the time (h) that elapsed between the initial and final
sample. Daily water flux (rH20; mL d?1) was then calculated
with Eq. (2) assuming that 25% of water loss fractionated
(Speakman, 1997):
rH2O¼ 1:018TNTkD
with N representing the size of the body water pool (mL).
The daily water intake (DWI; mL d?1) was calculated by
subtracting the metabolic water (rH20metabol; mL d?1) from the
daily water flux (rH20; mL d?1)
ð2Þ
DWI ¼ rH2O? rH2Ometabol
The amount of metabolic water produced each day by
catabolism of sugar and fat was calculated after Eq. (4):
ð3Þ
rH2Ometabol¼ sTWSþ f TWfat
with s representing the amount of sugar (g d?1), Wsthe
equivalent of water production per gram catabolized sugar
(0.56 mL g?1; Schmidt-Nielsen, 1990), f the amount of
catabolized fat and Wfatthe equivalent of water production
per gram catabolized fat (1.07; mL g?1; Schmidt-Nielsen,
1990). The amount of catabolized fat was estimated
according to Eq. (5):
ð4Þ
f ¼DbmTEDbm
Efat
ð5Þ
with Dbm equalling body mass changes between the initial
and final blood sample (g d?1), EDbmthe energy equivalent
of body mass changes (31.2 kJ g?1, Winter and von
Helversen, 1998; Voigt, 2000) and Efatthe energy equiv-
alent of one gram fat (39.4 kJ g?1, Wieser, 1986).
2.4. Measurement of energy budgets with the DLW method
As described in Voigt et al. (2003), the rate of carbon
dioxide production (rco2; mol h?1) was calculated with Eq.
7.17 from Speakman (1997), controlling for 25% evapo-
rative water loss to total water flux at a body temperature 37
-C. The rate of carbon dioxide production (rco2; mol h?1)
was converted to hourly energy expenditure (kcal h?1),
following Lusk (1976), assuming a respiratory quotient of 1
(see Voigt et al., 2003 for equations). Hourly energy
expenditure was converted into daily energy expenditure
(DEE; kJ d?1), multiplying by 4.184 (J cal?1), over the 24 h
duration of the experiment (24 h d?1). Daily energy intake
(DEI; kJ d?1) was then calculated using Eq. (6), compen-
sating for the energy gain through fat catabolism.
DEI ¼ DEE ? DbmTEDbm
The amount of sugar that was consumed each day by the
bats was calculated by dividing the DEI through the energy
equivalent of sugar (15.91 kJ per gram sugar, Wieser, 1986).
ð6Þ
2.5. Sugar concentration of ingested nectar
The sugar concentration (%) of the imbibed nectar was
calculated after:
Cnectar¼
with s (g d?1) representing the amount of sugar ingested per
day and DWI the daily water intake (mL g?1).
s
s þ DWI
ð7Þ
2.6. Statistics
Before using parametric statistics, we tested if underlying
assumptions, such as normal distribution, were not violated.
We then performed pair-wise comparisons using Student t-
tests. The level of significance was Bonferroni-corrected to
0.01 as five comparisons were performed with the same
dataset. In addition, we calculated least squares regressions
for the comparison of values between the DLW and energy
balance method. The individual means were weighted
according to the number of data points included. The latter
analysis was performed to test for a systematic deviation
with increasing variable size, e.g. whether the regression
slope deviates significantly from one. All values are given
as meansT1 SD. If not otherwise stated, statistical tests were
performed two-tailed using SPSS (SPSS Inc., 1998).
3. Results
In general, blood-sucking bugs approached the bats
within a minute after release from the vial in which they
were kept and immediately started to search for a site to
puncture the bat’s skin. After piercing the focal animal with
the tip of their proboscis, which is more than 30-times
smaller in diameter than a needle (0.60?30 mm; Fig. 1),
bugs filled their crop with approximately 100 AL of blood
within 2 to 12 min. We obtained approximately 70 to 80 AL
of blood from the bugs by puncturing the abdomen and by
withdrawing the blood using conventional microcapillary
tubes.
Experimental individuals of Glossophaga soricina
weighed on average 9.6T0.9 g and lost on average
0.20T0.02 g during the experiment, which was not
significant according to a Student t-test (t5=2.1, p=0.084;
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320

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Table 1). The mean daily water flux equalled 16.6T3.3 mL
d?1when measured with the energy balance method and
15.6T2.8 mL d?1when estimated with the DLW method
(Table 2). The difference was not significant (paired t-test:
t5=1.36, p=0.23), indicating no bias of the bug method
(Fig. 2). The slope of a linear regression line calculated after
the least squares method was not significantly deviating
from unity (slope=0.71, t5=0.84, p>0.05). The signed
percentage difference in daily water flux between the two
methods averaged 5.5T9.5%, whereas the unsigned per-
centage difference averaged 9.1T5.1% (see Table 2).
Daily water intake, measured with the energy balance
method, averaged 14.7T3.0 mL d?1(see Table 2). A pair-
wise comparison of the measured water intake rates and the
daily water intake (13.7T2.4 mL d?1) calculated on the
basis of DLW estimates and metabolic water production,
indicates no significant deviation (paired t-test t5=1.31,
p=0.25: see Table 1, Fig. 3). The slope of a linear
regression line calculated after the least squares method
was not significantly deviating from unity (slope=0.613,
t5=1.02, p>0.05). The signed percentage difference in daily
water intake between the two methods averaged 6.1T11.0%,
whereas the unsigned percentage difference averaged
10.2T6.3% (see Table 2).
The daily energy intake rate measured with the energy
balance method equalled 48.1T9.9 kJ d?1. The daily energy
intake (DEI=48.8T14.2 kJ d?1) derived with Eq. (6) from
the daily energy expenditure (DLW) and the daily energy
intake rate measured with the energy balance method were
not significantly different (paired t-test t5=0.36, p=0.73).
The slope of a linear regression line calculated after the least
squares method was not significantly deviating from unity
(slope=1.379, t5=1.72, p>0.05). The signed percentage
difference in daily energy intake between the two methods
averaged 0.1T11.0%, whereas the unsigned percentage
difference averaged 9.0T4.9% (see Table 2).
The sugar concentration was estimated on the basis of the
daily water and energy intake and the energy equivalent of
Table 1
Measures of the energy balance (EB) method and the doubly-labelled water (DLW) method in addition to estimates derived from DLW data (M = male, F =
female, bm = body mass, Dbm = daily change in body mass)
Energy balance method DLW methodCalculated values based on the DLW method
Ind. bm (g)
Dbm
(g d?1)
Water intake
(mL d?1)
Energy intake
(kJ d?1)
Water flux
(mL d?1)
Water flux
(mL d?1)
CO2-
Production
(mol h?1)
Energy
expenditure
(kJ d?1)
Energy
intake
(kJ d?1)
Sugar
intake
(g d?1)
Water
intake
(mL d?1)
Sugar
concentration
(% m/m)
M1
M1
M1
M1
M1
M1
M2
M2
F2
F2
F1
M3
F3
10.6
10.1
10.9
10.7
10.4
11.4
10.4
9.8
8.6
8.3
10.3
9.3
8.6
?0.62
?0.20
0.07
?0.76
?0.34
?0.61
?0.14
?0.37
?0.28
?0.41
0.16
?0.25
?0.10
11.1
14.1
20.2
15.4
12.8
14.1
14.6
12.2
15.1
13.2
17.4
10.1
18.7
36.3
45.8
65.7
50.3
41.7
45.8
47.4
40.1
49.2
43.1
56.8
33.1
60.9
12.9
15.9
22.5
17.8
14.6
16.2
16.4
13.9
17.0
15.0
19.3
11.5
20.9
14.9
16.1
20.8
20.3
16.6
18.8
16.1
15.2
17.1
13.3
17.3
10.4
17.1
0.00413
0.00530
0.00574
0.00588
0.00439
0.00492
0.00526
0.00437
0.00488
0.00485
0.00516
0.00309
0.00606
46.9
60.2
65.1
66.7
49.9
55.9
59.8
49.6
55.4
55.0
58.6
35.1
68.7
27.6
53.9
67.4
43.1
39.2
36.8
55.5
38.2
46.6
42.3
63.5
27.3
65.8
1.7
3.4
4.2
2.7
2.5
2.3
3.5
2.4
2.9
2.7
4.0
1.7
4.1
13.4
14.0
18.5
18.2
15.0
17.0
14.0
13.6
15.2
11.4
15.2
9.2
14.7
11.5%
19.4%
18.6%
13.0%
14.1%
12.0%
19.9%
15.0%
16.1%
18.9%
20.8%
15.7%
21.9%
Fig. 1. Electron microscope image of a 0.60?30 mm needle (A) and the proboscis of a Dipetalogaster maximus (B). The inlet photos C and D show the tip of
the needle and the tip of the bug’s proboscis. Photos A and B were taken with a magnification factor of 12 and the smaller inlet pictures C and D with a factor of
200. The bug is puncturing the skin only with the saw blade like tip of the proboscis shown in the picture D, whereas the whole diameter of the needle as seen
in picture A is inserted into the skin when using a conventional needle.
C.C. Voigt et al. / Comparative Biochemistry and Physiology, Part A 142 (2005) 318–324
321

Page 5

sugar. The mean value of 16.2T2.4% did not differ
significantly from the expected value of 17.0% (t-test:
t5=0.77, p=0.47).
4. Discussion
4.1. Validation of blood-sucking bugs for measuring daily
water flux in DLW experiments
Blood-sucking bugs are highly adapted to the fast and
minimal-invasive acquisition of blood from their endotherm
victims. In the 1980s, J.A. Nu ´n ˜ez suggested to use bugs for
blood-sampling when study animals may be easily stressed
or when veins are almost inaccessible (von Helversen et al.,
1986). The first DLW study with blood-sucking bugs
(Rhodnius prolixus) was performed by von Helversen and
Reyer (1984) in 11 g free-ranging Anoura caudifer. Voigt et
al. (2003) validated the bug-bleeding method for the
measurement of daily energy expenditure in DLW experi-
ments. They found a significant bias in the estimation of
total body water possibly due to the contamination of blood
with bug heamolymph or intestinal liquids. Thus, alternative
methods, such as desiccation of animals, have to be
employed to gain data on total body water. Notwithstanding
daily energy expenditure did not significantly deviate from
expected values when bugs were used in DLW experiments
(Voigt et al., 2003). The present study yielded the same
result for daily water flux. The lack of bias is probably due
to the dilution of the ingested blood with almost constant
amounts of bug liquids in the initial and final samples. As
the calculation of daily energy expenditure and water flux
depends on relative washout rates, contamination effects
caused by the bug do not compromise the DLW method.
Recent measurements of hydrocorticosterone in rabbits
that were treated with conventional needles and with blood-
sucking bugs showed a greater stress response when
needles were used instead of bugs (Voigt et al., 2004). In
some DLW studies, experimental animals routinely loose
more than 10% of their body mass (e.g. in hummingbirds
Calypte anna; Powers and Nagy, 1988). This mass loss
may be caused by stress owing to the blood sampling
routine. Experimental animals of the current study lost on
average only 2% of their body mass. von Helversen and
Reyer (1984) used bugs to study field metabolic rates of
Anoura caudifer in Venezuela and their study animals lost
on average only 1%. Possibly, bugs are helpful in
preventing stress-induced body mass losses in experimental
animals. Bugs as a gentle method of blood sampling could
as well substitute the one-sample method (Webster and
Weathers, 1989) that is occasionally applied to reduce the
trauma of study animals (e.g. Powers and Conley, 1994).
The one-sampling method is more sensitive to error
propagation as the initial isotope concentration is extrapo-
lated (Speakman, 1997). In summary, blood-sucking bugs,
such as Triatoma infestans, Dipetalogaster maximus or
Rhodnius prolixus (all Reduviidae) may be ideal for
accessing blood vessels in endothermic animals that are
easily stressed or in mammals or birds that are small or
have almost inaccessible veins owing to a thick layer of
subcutaneous fat tissue.
Daily water flux (ml d-1) (energy balance method)
05 1015 20 25
Daily water flux (ml d-1) (DLW)
0
5
10
15
20
25
Fig. 2. Daily water flux (mL d?1) measured with the energy balance
method in relation to the daily water flux (mL d?1) as estimated with the
doubly-labelled water method (DLW). Filled symbols indicate mean values
of individuals for which several data points were collected or single data
points of individuals for which only one data point was collected. Open
symbols indicate all data points of an individual for which a mean value
was calculated. The dashed line indicates equivalence. The solid line
represents a regression line calculated after the least squares method on the
basis of individual means and weighted according to the number of
individual data points.
Table 2
Signed and unsigned differences in measured daily water intake (DWI; mL d?1), daily water flux (DWF; mL d?1) and daily energy intake (DEI; kJ d?1)
between the energy balance (EB) method and the doubly-labelled water method (DLW) (SD = standard deviation)
EB DLWPercentage difference
DWIDWF DEIDWIDWFDEI Signed DWI Unsigned DWI Signed DWF Unsigned DWFSigned DEI Unsigned DEI
M1
M2
F2
F1
M3
F3
Mean
SD
14.6
13.4
14.1
17.4
10.1
18.7
14.7
3.0
16.6
15.2
16.0
19.3
11.5
20.9
16.6
3.3
47.6
43.8
46.1
56.8
33.1
60.9
48.1
9.9
16.0
13.8
13.3
15.2
9.2
14.7
13.7
2.4
17.9
15.7
15.2
17.3
10.4
17.1
15.6
2.8
44.7
46.8
44.5
63.5
27.3
65.8
48.8
14.2
?9.5%
?2.9%
5.7%
12.8%
9.3%
21.1%
6.1%
11.0%
9.5%
2.9%
5.7%
12.8%
9.3%
21.1%
10.2%
6.3%
?7.7%
?3.3%
5.4%
10.4%
9.9%
18.0%
5.5%
9.5%
7.7%
3.3%
5.4%
10.4%
9.9%
18.0%
9.1%
5.1%
6.2%
?7.0%
3.6%
?11.7%
17.4%
?8.0%
0.1%
11.0%
6.2%
7.0%
3.6%
11.7%
17.4%
8.0%
9.0%
4.9%
The percentage difference was calculated according to the following equation: (EB?DLW)/EB.
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4.2. Daily water intake and energy intake
Comparison of daily water intake measured with the
energy balance method and the daily water flux estimated
with the DLW method reveals that the daily water flux is not
equivalent to daily water intake as it does not include the
amount of water that is produced during substrate catabo-
lism. Based on the body mass change between the initial and
final blood sampling, it is possible to calculate the amount
of catabolized fat and consequently the amount of water
produced during fat catabolism. Similarly, it is possible to
calculate the amount of metabolic water produced by the
combustion of food, such as nectar. Subtracting the amount
of water produced metabolically yielded an estimate of daily
water intake that did not differ significantly from the
expected value (Fig. 3A).
Comparison of daily energy intake between the two
methods demonstrates that the average daily energy intake
of groups of animals can be estimated with the DLW
method. Thus, complete water and energy budgets can be
derived with the DLW method when daily body mass
changes are recorded and the catabolized substrate is
known. However, it is important to note that the variation
in the data set was large (Table 2) and that individual
estimates deviated considerably from the expected value
(Figs. 2 and 3). Therefore, this technique may be inadequate
for calculating values for individuals. Nonetheless, it may
be sufficiently accurate to yield mean values for populations
or species.
4.3. Nectar concentration
von Helversen and Reyer (1984) presented a method for
estimating the sugar concentration of nectar that mammals
(or birds) imbibe during DLW experiments. They calcu-
lated an expected mean sugar concentration of ingested
nectar of ca. 19.8% (mass/mass) for nectar-feeding
Anoura caudifer (Glossophaginae, Phyllostomidae) in
Venezuela. In slight modification of their approach, we
compensated for the metabolic water produced during the
Table 3
Doubly-labelled water experiments in free-ranging nectar-feeding bats and daily water intake, daily energy intake and sugar concentration of imbibed nectar
calculated after the procedure as described in the text (Anoura caudifer (AC) von Helversen and Reyer (1984); Syconycteris australis (SA) Geiser and Coburn
(1999); Glossophaga commissarisi (GC) Voigt et al. (submitted for publication); G. longirostris (GL) and G. soricina (GS) Voigt (1998) (bm = body mass;
Dbm = daily changes in body mass, SD = standard deviation)
Species DLW estimates Calculated values based on the DLW method
Sample
size
bm (g)
Dbm
(g d?1)
Water flux
(mL d?1)
FMR
(kJ d?1)
Water intake
(mL d?1)
Energy intake
(kJ d?1)
Nectar
(% m/m)
AC
SA
GLb
GS
GC
MeanTSD
7
7
5
1
13
11.6T0.2
17.3T1.4
12.7T0.9
10.5
8.7T0.6
12.2T3.2
0.0T0.3
0.0a
?0.2T0.5
?0.2
?0.1T0.2
?0.1T0.1
13.6T0.8
26.7T5.1
19.6T4.2
15.0
18.5T5.6
18.7T5.1
51.8T4.1
76.9T9.0
34.5T11.4
51.8
45.7T6.8
52.1T15.5
11.7T0.7
24.0T4.9
18.4T4.1
13.2
16.9T5.4
16.8T4.8
52.3T6.9
76.9T9.0
29.5T13.5
45.6
43.5T6.8
49.6T17.4
21.9T2.6
17.1T3.2
9.7T0.5
17.8
14.2T2.8
16.1T4.5
aDaily body mass changes were set zero as no information was available.
bIncluding lactating females.
05 10 15 20
Daily water intake (ml d-1) (DLW)
0
5
10
15
20
Daily energy intake (kJ d-1)
(energy balance method)
020 406080
Daily energy intake (kJ d-1) (DLW)
0
20
40
60
80
AB
Daily water intake (ml d-1)
(energy balance method)
Fig. 3. Estimates of daily water intake ((A); solid circles; mL d?1) and daily energy intake ((B); open circles; kJ d?1) based on the energy balance method and
the doubly labelled water method (DLW). Filled symbols indicate mean values of individuals for which several data points were collected or single data points
of individuals for which only one data point was collected. Open symbols indicate all data points of an individual for which a mean value was calculated. The
dashed line indicates equivalence. The solid lines represent regression lines calculated after the least squares method on the basis of individual means and
weighted according to the number of individual data points.
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catabolism of fat and sugar. The calculation of expected
sugar concentration of the ingested nectar for the current
data set yielded 16.2%, which did not differ significantly
from the expected 17%, supporting the suggestion by von
Helversen and Reyer (1984). Recalculating the values of
their study yields a mean sugar concentration of
21.9T2.6% for A. caudifer (Table 3). This value falls in
the range of nectar concentrations found in bat-pollinated
plants of that region (range: 9–29%, von Helversen and
Reyer, 1984). Table 3 also gives estimates of sugar
concentrations of imbibed nectar for four other nectar-
feeding bat species: Syconycteris australis, Glossophaga
longirostris, G. commissarisi and G. soricina. The plants
visited by S. australis and G. soricina are unknown.
Individuals of G. longirostris took up nectar with a sugar
concentration of 11.1T5.4%, which falls close to the
range of sugar concentrations of bat-pollinated plants in
the foraging habitat of G. longirostris on the Antillean
Island of Grenada: 12% (Irlbachia allata, Gentianaceae),
13% (Lobelia flavescens, Lobeliaceae) and 20% (Cres-
centia cujete, Bignoniaceae) (Voigt, 1998). The estimated
sugar concentration of nectar that G. commissarisi ingested
in the lowland rainforests of Costa Rica (Voigt et al.,
submitted for publication) equalled 14.2T2.8%. Bat-polli-
nated plants that flowered during the study period provided
nectar with similar sugar concentration: The bromeliad
Vriesea gladioliflora (Bromeliaceae) offers nectar with
16.6% sugar, the balsa tree Ochroma pyramidale (Bom-
bacaceae) 11.9% and the epiphyte Markea neurantha
(Solanaceae) 14.5% (Tschapka, 2004).
5. Conclusions
In summary, blood-sucking bugs are a practical method
for obtaining blood in a gentle manner from focal animals
during DLW experiments. DLW derived values of daily
water flux are not biased owing to the possible contami-
nation with fluids from the bug. In the present study, it was
also shown that mean daily energy and water intake rates
can be reliably estimated for animal groups or species using
DLW values and information on changes in body mass. In
nectarivorous species, the calculation of mean daily water
and energy intake may even allow the approximation of the
sugar concentration of nectar imbibed by animals. This
could be an important additional information in ecophysio-
logical studies of nectarivorous animals. This said, it is
important to keep in mind that it is probably invalid to
extrapolate values for individuals, as the accuracy of DLW
derived estimates is insufficient.
Acknowledgments
We are most grateful to our colleagues Sylvia Ortmann,
Heribert Hofer for commenting on an earlier version of the
manuscript and three anonymous reviewers for helpful
suggestions. Dagmar Viertel kindly helped in producing the
electron microscope pictures.
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