Crater landscape: two-dimensional oxygen gradients in the circulatory system of the microcrustacean Daphnia magna
Institut für Zoophysiologie, Westfälische Wilhelms-Universität, Hindenburgplatz 55, 48143 Münster, Germany
* Author for correspondence (e-mail: pirow{at}uni-muenster.de)
Accepted 29 September 2004
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Summary |
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Key words: Crustacea, Branchiopoda, Cladocera, Daphnia, zooplankton, oxygen transport, ventilation, circulatory system, diffusion, convection, haemoglobin, hypoxia, phosphorescence lifetime imaging
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Introduction |
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Diffusion is effective for short distances only, so its relevance is
usually restricted to transport processes across thin physical barriers such
as the respiratory surfaces and within the tissues. Convection, in contrast,
often dominates the transport of oxygen in moving respiratory media and
circulating body fluids. Ventilatory and circulatory convection serve to
bridge long transport distances that may exist between the ambient medium and
the respiratory surfaces and between the respiratory surfaces and the tissues.
The mutually exclusive dominance of diffusion and convection within the
individual steps of the oxygen transport cascade has made it possible to
describe oxygen transport in large, physiologically advanced animals by
straightforward mathematical relationships
(Piiper, 1982;
Weibel, 1984
;
Shelton, 1992
) with only a few
parameters and variables, which are accessible by morphometric techniques
(e.g. Weibel,1979
,
1980
) and classical
physiological methods.
This concept of oxygen transport, however, appears to be too simple when
body sizes shrink to the millimetre scale. In these tiny organisms, transport
of oxygen from the respiratory surfaces to the tissues does not exclusively
follow the pathway predetermined by the circulating fluid. In addition, oxygen
may be moved simultaneously to the target tissues along different paths by
diffusion. The increasing influence of diffusion therefore complicates the
mathematical description of circulatory oxygen transport. Moreover, small body
size is an experimental obstacle that hampers attempts to obtain adequate
physiological information. Fortunately, recent advances in optical techniques
and digital image processing have expanded the methodical versatility of
microscopy, so that this information can be obtained in animals that are
highly transparent (Colmorgen and Paul,
1995; Burggren and Fritsche,
1995
; Paul et al.,
1997
,
1998
;
Schwerte and Fritsche,
2003
).
The transparent microcrustacean Daphnia magna (Branchiopoda,
Cladocera) is a valuable model organism that has stimulated the development
and adaptation of innovative optical techniques for studying oxygen transport
processes in millimetre-sized animals. These methods have made it possible to
obtain information on physiological key parameters such as ventilation and
perfusion rates, the in vivo oxygen saturation of haemoglobin, oxygen
partial pressure and tissue oxygenation state
(Paul et al., 1997; Pirow et
al.,
1999a
,b
,
2001
;
Bäumer, 2001
;
Bäumer et al., 2002
;
Seidl et al., 2002
). The
availability of physiological information has initiated attempts to push
conceptual barriers and to make use of the increasing computational power for
modelling and simulation of oxygen transport in millimetre-sized animals
(Pirow, 2003
;
Pirow and Buchen, 2004
).
These in vivo and in silico approaches were largely
motivated by the intention to comprehend the physiological implications of the
presence of haemoglobin (Hb) in D. magna. In contrast to other
crustaceans, which use haemocyanin as the respiratory protein, branchiopod
crustaceans including D. magna have a Hb that is freely dissolved in
the haemolymph (e.g. Weber and Vinogradov,
2001). When challenged by a reduction in ambient oxygen partial
pressure (PO2amb), D. magna shows a
striking increase in Hb concentration (e.g.
Kobayashi and Hoshi, 1982
;
Zeis et al.,
2003a
,b
).
The rise in concentration is accompanied by an increase in oxygen affinity
(e.g. Kobayashi et al., 1988
)
resulting from an altered subunit composition of the multimeric protein (e.g.
Kimura et al., 1999
; Zeis et
al.,
2003a
,b
).
Variations in the concentration and oxygen affinity of Hb should affect the
gradients of oxygen partial pressure (PO2) in
the circulatory system, causing alterations in the relative contributions of
convection and diffusion to circulatory oxygen transport. So far, nothing is
known about the internal oxygen milieu except some indirect information from
studies on the in vivo oxygenation state of Hb
(Hoshi and Yahagi, 1975;
Kobayashi and Tanaka, 1991
;
Pirow et al., 1999b
,
2001
). Direct measurement of
the internal PO2 would facilitate elucidation
of the effect of Hb on circulatory oxygen transport. In providing information
about the in vivo PO2 experienced by the
tissues, such measurements would also be of interest for studies on
oxygen-dependent gene expression. Finally, we would be able to learn more
about the homeostatic abilities of an animal whose internal milieu, owing to
its small body size, is much more affected by environmental disturbances than
that of larger animals.
In the present study, we therefore analysed the internal oxygen
distribution in different sized Hb-poor and Hb-rich animals of D.
magna under varying ambient oxygen conditions. To profile the
PO2 in the circulatory system, we made use of
an oxygen-sensitive phosphorescence probe introduced into the circulatory
system by microinjection. The method employed is based on the oxygen-dependent
quenching of phosphorescence and has the great advantage that it is
non-invasive, except for the addition of the phosphorence probe, and its
calibration is absolute (Dunphy et al.,
2002).
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Materials and methods |
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For the experiments, the animals were separated into three size groups
(small, medium, large) according to their body length (BL), which was
measured from the base of the posterior apical spine to the head. Small,
medium and large Hb-poor animals had BL=1.41.7 mm
(1.5±0.1 mm, N=6; means ± S.D.),
2.52.7 mm (2.6±0.1 mm, N=4) and 3.13.5
(3.3±0.1 mm, N=6). Hb-rich animals were generally smaller than
Hb-poor animals as a consequence of hypoxic incubation
(Kobayashi, 1982), so only
small and medium-sized animals with BL=1.41.8 mm
(1.6±0.1 mm, N=5) and 2.62.9 mm (2.7±0.1 mm,
N=7) were studied. Those animals that had already attained sexual
maturity (i.e. the medium and large-sized animals) had at most ten
parthenogenetic embryos of developmental stages 14 (see table 3 of
Green, 1956
) in the brood
chamber.
The oxygen-sensitive phosphorescence probe
The oxygen-sensitive probe Oxyphor R2, a polyglutamic dendrimer containing
Pd-meso-tetra-(4-carboxyphenyl)porphyrin
(Dunphy et al., 2002),
purchased from Oxygen Enterprises (Philadelphia, PA, USA). Oxyphor R2, of
molecular mass of 2794 Da, possesses two absorption maxima at 415 and 524 nm
and shows a phosphorescence emission near 700 nm. The extinction coefficient
at 524 nm is 19 mmol l1 cm1 and the
quantum efficiency of phosphorescence is approximately 10%
(Dunphy et al., 2002
).
The method for measuring oxygen is based on the suppression of
phosphorescence by molecular oxygen
(Vanderkooi and Wilson, 1986).
When excited by light, the probe molecules enter into the triplet state. The
phosphorescence arises when the excited triplet state molecule returns to the
ground state with emission of a photon. In the presence of molecular oxygen,
the excited molecules may transfer their energy to oxygen and return to the
ground state without phosphorescence emission.
Because the rate of decay of the triplet state is proportional to the
concentration of excited triplet state molecules, which in turn is
proportional to phosphorescence intensity (It), the decay
of It after an excitation flash given at time t=0
follows an exponential law (Vinogradov and
Wilson, 1994):
![]() | (1) |
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Microscopic arrangement for the optical measurement of PO2
The phosphorescence imaging system consisted of an inverted microscope
(Zeiss Axiovert 100, Carl Zeiss, Oberkochen, Germany) equipped with a pulsed
Xenon microsecond flashlamp (µF900; Edinburgh Analytical Instruments,
Edinburgh, UK) for phosphorescence excitation
(Fig. 1). The flashlamp
produced flashes of 3 µs duration (full width at half-maximum pulse height)
at a rate of 100 Hz. The reflector slider of the microscope contained a
bandpass excitation filter with a transmittance wavelength of 535±35 nm
(peak wavelength ± full width at half-maximum transmission bandwidth),
a dichroic mirror with a cut-off wavelength of 580 nm and a long-pass emission
filter with cut-off wavelength of 590 nm. The phosphorescence of the oxygen
probe was imaged by a 16-bit liquid-nitrogen-cooled slow-scan CCD camera
(576x384 pixels; LN/CCD-576E, Princeton Instruments, Trenton, NJ, USA),
which was coupled to a gatable image intensifier (Princeton Instruments). Gate
pulses and high voltage were provided to the intensifier by a programmable
pulse gate generator (PG200 with MCP-100 option; Princeton Instruments). Two
computers served to control the activity of all system components and
synchronized image acquisition with phosphorescence excitation and gating of
the image intensifier.
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For image acquisition, we used the imaging software WinView 1.6.1.1 (Princeton Instruments), whose built-in Macro language made it possible to automate image-acquisition sequences and to control the activities of all system components. The acquired images had a reduced resolution of 288x192 pixels resulting from a hardware binning of 2 x2 pixels. Before analysis, images were smoothed by a 3 x3 low pass filter to remove random noise.
To monitor the circulatory activity of the animal during the experiments,
the heart rate was determined (Paul et
al., 1997) in parallel to PO2 by
trans-illuminating the animal with infrared light (
>830 nm). To
avoid optical interferences on the PO2
measurement, the infrared illumination was blocked by a mechanical shutter
during the acquisition of phosphorescence images.
Collection of phosphorescence intensity images
Image acquisition followed the procedure developed by Pawlowski and Wilson
(1994). To collect a single
phosphorescence intensity image, the imaging system triggered the flashlamp at
t=0 to excite the probe molecules. After waiting a certain delay time
td (s), the image intensifier was turned on to integrate
the phosphorescence light emitted during the time interval from
t=td to
t=td+
T, where
T
(=240 µs) is the gating pulse duration
(Fig. 2). Depending on signal
intensity, the flash lamp was triggered 8001600 times at 10 ms
intervals (with appropriate gating of the intensifier at a given
td) while the CCD camera integrated the collected
phosphorescence light from the flash sequence in a single exposure.
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To follow the exponential decay of phosphorescence intensity, a set of phosphorescence intensity images was taken at seven different td (10, 20, 40, 80, 160, 300, 600 µs; Fig. 3). From these images, a background image taken at td=3000 µs was subtracted to remove the influence of light arising from non-phosphorescence sources. The collection of eight phosphorescence intensity images, required to obtain a single PO2 image, took 24 min depending on the number of excitation flashes (800 or 1600) per phosphorescence intensity image.
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Calculation of phosphorescence lifetime and oxygen partial pressure
A set of phosphorescence intensity images can be regarded as a
three-dimensional data package of intensity values
Z(x,y,td) comprising the integrated intensity
(Z) for each pixel coordinate (x,y) as a function of delay
time td (Fig.
3). For each pixel coordinate, a single-exponential decay was
fitted to the data Z(td) based on the following
equation (Pawlowski and Wilson,
1994):
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This equation was linearized by taking the logarithms of both sides and a
linear regression analysis then yielded the phosphorescence lifetime and
the initial phosphorescence intensity I0 immediately after
the excitation flash. Information on
was employed to calculated the
PO2 according to
Equation 2 on the basis of the
calibration parameters
0 and kq. A pixel
coordinate was regarded to contain valid information on
PO2, when the following conditions were
fulfilled. (1) The intensity value Z at td=10
µs exceeded a threshold intensity measured at the boundary of the object
containing the oxygen-sensitive dye. (2) The squared correlation coefficient
r2 was greater than 0.95. (3) The phosphorescence lifetime
was within the range of 0700 µs. (4) The
PO2 was lower than
PO2amb+0.7 kPa.
After analyzing the intensity decay curves at all x,y positions, the results were depicted in an image in which pixel intensity encoded the PO2 in pseudo-colour presentation (Fig. 4). For those x,y coordinates where the four plausibility criteria were not met, the pixel position was masked out by setting pixel intensity to black colour. The PO2 analysis was performed by a program module written in Microsoft Visual C++, which made use of the functionality of the imaging software WinView/32 (Princeton Instruments).
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Calibration of Oxyphor R2 in the haemolymph
Oxyphor R2 was dissolved in 60 mmol l1 NaCl in a
concentration of 10 mg ml1 (i.e. 3.6 mmol
l1). The dissolved phosphorescence probe was 1:10 diluted
with buffered saline (60 mmol l1 NaCl, 20 mmol
l1 Hepes, pH 7.4 at 20°C) containing 6% bovine serum
albumin to obtain a stock solution for injection. The albumin was required for
establishing a stable microenvironment for the phosphorescence probe
(Lo et al., 1997). To avoid
blocking the micropipettes, the stock solution was sterile filtered using
cellulose acetate membrane filters (pore size 0.45 µm; Nalgene, Rochester,
New York, USA).
The stock solution of the dye was injected in Hb-poor and Hb-rich
individuals of BL 2.53.0 mm. After a 30 s period, which was
sufficient time to distribute the dye solution in the circulatory system, one
of the large antennae was cut off and the oozing haemolymph aspirated by a
pulled glass capillary. The haemolymph sample was then transferred to a
coverslip and covered by a layer of silicone oil (Wacker Siliconoel, AK350,
Drawin Vertriebs-GmbH, Ottobrunn, Germany) to avoid dehydration. The coverslip
was then transferred into a thermostatted perfusion chamber
(Paul et al., 1997), where the
sample was equilibrated with humidified, normocapnic gas mixtures of different
oxygen levels (030% air saturation;
Fig. 4) using a gas mixing-pump
(Wösthoff, Bochum, Germany). The equilibration time per oxygen step was
3045 min. The phosphorescence lifetime
was determined at the end
of each equilibration step. The relationship between
and the oxygen
partial pressure was then analyzed by linear regression analysis to obtain
estimates of the lifetime at zero-oxygen concentration
0 and
of the quenching constant kq
(Equation 2;
Fig. 4).
Microinjection and experimental conditions
Before the start of experiments, animals were transferred into
nutrient-free normoxic medium for 14 h. The selection of fasting
animals ensured that digestive processes did not affect oxygen consumption
rate and systemic functions involved in oxygen transport
(Pirow and Buchen, 2004).
Single animals were immobilized by gluing (with histoacryl; B. Braun AG,
Melsungen, Germany) their posterior apical spine to a bristle that was then
fixed to a cover glass using a small lump of plasticine.
The oxygen-sensitive dye was introduced into the circulatory system using micropipettes pulled (Puller 88; from Zaschka, Zoologisches Institut, Universität München, Germany) from standard borosilicate glass capillaries (1B100F-4; World Precision Instruments, Sarasota, FL, USA) and bevelled (Beveler 1300M; World Precision Instruments) to an angle of 30°, o.d. 1226 µm, using a 0.3 µm aluminium oxide-coated film (3 M, Neuss, Germany).
Depending on the animal's body size, a volume of 65200 nl of a dye stock solution was microinjected (Transjector 5246; Eppendorf, Hamburg, Germany) into the haemolymph space at a dorsal position directly downstream of the heart. The injection volume was chosen to obtain a sufficiently high phosphorescence signal without affecting the rhythmicity of the heart and the limbs. After microinjection, the animal fixed to the cover glass was transferred into a thermostatted perfusion chamber with its head orientated against the medium flow. The large antennae were freely moveable, and the animal did not contact the top and the bottom (cover glass) of the chamber. The flow rate of the medium was set to 8 ml min1. The chamber was initially perfused with air-saturated medium, and the animal was allowed to acclimate to these conditions for 30 min.
An adequate phosphorescence signal that allowed the imaging of internal PO2 could only be obtained under hypoxic conditions, because the phosphorescence intensity as well as the change in phosphorescence lifetime per unit change of PO2 decreased with increasing PO2. Images of internal PO2 were therefore taken at ambient oxygen levels of 30, 25, 20, 16, 12, 10, 8, 4, 2 and 0% air saturation. Each animal was exposed to the descending order of oxygen levels with a duration of 15 min for the first level and of 5 min for all other levels. At the end of each oxygen step, phosphorescence images were taken and the heart rate was measured.
Determination of haemolymph Hb concentration
Haemolymph samples were taken from Hb-poor and Hb-rich individuals of the
three different size groups, and Hb concentrations were measured according to
Becher (2002). For the
small-sized group, haemolymph samples of two individuals with
BL=1.82.0 mm (Hb-poor: 1.9±0.1 mm, N=5;
Hb-rich: 1.9±0.1 mm, N=6) were pooled to obtain a sufficiently
large volume for the measurement. It was not possible to examine individuals
smaller than 1.8 mm. The medium and large Hb-poor animals had
BL=2.6±0.1 mm (N=6) and 3.5±0.2 mm(N=5),
respectively, and the medium Hb-rich animals had BL=2.7±0.1
(N=5).
To draw a haemolymph sample, the animal was transferred to a microscope
slide and adhering water was gently removed with filter paper. Using a very
fine spring scissor (No. 15001-08; Fine Science Tools, North Vancouver,
Canada), the second antenna was proximally amputated
(Fritzsche, 1917) and the
oozing haemolymph was aspirated into a 2 µl capillary (minicaps; Hirschmann
Laborgeräte, Eberstadt, Germany). The capillary was then transferred into
the light path of a monolithic miniature spectrometer (MMS-UV/VIS, spectral
range: 194738 nm, 256 pixel photodiode array; Carl Zeiss
OEM-Spektralsensorik, Oberkochen, Germany) coupled via a Front End
Electronics and a PC interface board (14-bit resolution; tec5 AG, Oberursel,
Germany) to a computer. The software SDAS_32D (tec5 AG) was used for spectrum
acquisition. Absorption spectra in the range of 520590 nm were measured
under oxygenated conditions using water as reference sample. The haem-based Hb
concentration was determined as described elsewhere
(Pirow et al., 2001
). An
appropriate correction for the path length when using a cylindrical capillary
as microcuvette was taken into consideration
(Becher, 2002
).
Statistical analysis
Data were expressed as mean values ± standard deviation
(S.D.); N = number of animals examined. Statistical
differences in mean values were assessed using the t-test after
differences in variance had been checked using the F-test. A two-way
ANOVA (Zar, 1999) was applied
to test the effect of acclimation state and body size on a physiological
variable. Statistical differences were considered significant at
P<0.05.
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Results |
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Further calibrations with Hb-poor and Hb-rich haemolymph (N=7
each) were carried out during the in vivo PO2
measurements (Fig. 5B). As in
the first calibration experiment (Fig.
5A), we did not find any significant differences (t-test:
P>0.44 for all cases) in the experimentally determined at 0
kPa (482±32 vs 486±35 µs) nor in the derived
regression parameters
0 (474±50 vs
457±19 µs) and kq (1441±213 vs
1457±174 kPa1 s1) between Hb-poor
(PO2amb range: 04 kPa) and Hb-rich
haemolymph (PO2amb range: 06 kPa). From
these individual calibration data, a mean calibration curve
(Fig. 5B) was calculated for
the whole PO2amb range of 06 kPa. The
derived regression parameters of
0=484 µs and
kq=1364 kPa1 s1 were
used to convert the in vivo phosphorescence lifetime images of all
animals into PO2 images. Since the variability
of
increased with increasing PO2amb
(Fig. 5B),
PO2 determination was more affected by error at
higher than at lower PO2amb values. For
example, at PO2amb of 6.1 kPa, the individual
calibration values ranged from 83 to 133 µs (mean=96 µs), which
would yield calculated PO2 values of
4.07.4 kPa (mean=6.3 kPa). At the lower
PO2amb of 2.0 kPa, individual
values of
178231 µs (mean=195 µs) would translate into
PO2 values of 1.72.6 kPa (mean= 2.3
kPa).
Two-dimensional distribution of oxygen partial pressure in the haemolymph
Images showing the PO2 distribution within
the circulatory system were obtained from lateral views of the animals. The
optical focus was always set in a manner to obtain a sharp image of the heart
wall. The phosphorescence signal therefore came predominantly from the
animal's median plane and included information from the different haemolymph
spaces (lacunae) of the trunk and the limbs, the parts of the carapace lacuna
at ventral, posterior and dorsal positions, and the haemolymph space in the
head region. Depending on body size, either the whole animal or the dorsal
body region only was imaged. A total acquisition time of 24 min was
needed to obtain the data for one single PO2
image. As a consequence of this long acquisition time, sharp contours of
moving structures such as the limbs disappeared in the phosphorescence
intensity images (Fig. 3) and,
consequently, in the PO2 images
(Fig. 6).
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Owing to the large haemolymph volume, which in D. magna comprises
about 60% of total body volume (Kobayashi,
1983), more or less the whole image of the animal contained valid
information on PO2
(Fig. 6). The extent of valid
PO2 information was lower at higher ambient
oxygen partial pressures owing to the reduced phosphorescence intensity.
Inspection of data obtained from small animals (1.5±0.12 mm long) revealed large internal PO2 gradients in Hb-poor and Hb-rich animals at PO2amb=6.16.3 kPa (30% air saturation; Fig. 6). The highest PO2 values mainly occurred in the posterior and dorsal regions of the animals. In Hb-rich animals, high PO2 values extended into the head region including the rostral region. The lowest values were found in the central part of the animals. In Hb-poor animals, this low-oxygen zone was shifted anteriorly. The lowest values occurred in the centre of the anterior half of the animals around the bases of the second antennae.
Lowering the PO2amb from 6.1 to 1.7 kPa resulted in a flattening of internal PO2 gradients in Hb-rich animals (Fig. 6). These flat profiles contrasted with the steep profiles observed in Hb-poor animals at higher PO2amb (Fig. 7). In both groups, further reductions of PO2amb resulted in the formation of anoxic zones, which extended progressively from the central body region to the periphery (Fig. 6). The critical PO2amb resulting in the incipient formation of anoxic zones was higher in Hb-poor than in Hb-rich animals.
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Heart rate
Heart rate (fH) was monitored in parallel to haemolymph
PO2 as an indicator of circulatory activity
during the hypoxic exposure experiments, and to assess the influence of the
injection procedure on the physiological state of the animals, by comparing
the data of our injected animals with those of non-injected animals from the
literature. Under normoxic conditions
(PO2amb=20 kPa), the small, medium-sized and
large Hb-poor animals had mean fH of 149±47
(N=6), 147±25 (N=4) and 148±36 beats
min1 (N=6), respectively. The normoxic
fH of small and medium-sized Hb-rich animals were somewhat
lower at 131±5 (N=5) and 136±20 beats
min1 (N=7). Neither oxygen acclimation (two-way
ANOVA: F=1.26, d.f.=1, P=0.274) nor body size
(F=0.01, d.f.=2, P=0.990) had a significant effect on
normoxic fH. In response to a reduction in
PO2amb from normoxia (20 kPa) to 6 kPa
hypoxia, all groups showed a compensatory tachycardia followed by a plateau in
the range of PO2amb of 16 kPa
(Fig. 8). The averages of the
maximum individual fH of small, medium-sized and large
Hb-poor animals were 293±55, 285±16 and 310±30 beats
min1, respectively. The respective maximum values of Hb-rich
animals (small and medium: 226±29 and 246±52 beats
min1) were 1423% lower than those of Hb-poor animals.
The two-way ANOVA showed a significant effect of the acclimation state
(F=8.77, d.f.=1, P=0.007) but no significant influence of
body size (F=0.53, d.f.=2, P=0.594) on maximum
fH.
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Hb concentration in the haemolymph
The haem-based Hb concentrations of small, medium and large Hb-poor animals
were 90±8 µmol l1 (N=5), 115±25
µmol l1 (N=6) and 167±38 µmol
l1 (N=5), respectively. Hb-rich animals had a five-
to sixfold higher concentration with mean values of 450±129 µmol
l1 (N=6; small) and 666±132 µmol
l1 (N=5; medium).
Influence of Hb concentration and body size on internal PO2
To compare the internal PO2 of animals of
different body size and different Hb concentration, three peripheral positions
in the circulatory system of D. magna were selected
(Fig. 8F): (i) the dorsal
lacuna of the trunk, (ii) the carapace lacuna near the median dorsal ridge and
(iii) the heart region. Both lacunae conduct the haemolymph to the
pericardium, from where it is aspirated by the heart and expelled into the
dorsal head region (Pirow et al.,
1999b). For small and medium-sized animals, the central body
region with the lowest PO2 was additionally
analyzed to obtain information on the unloading
PO2.
The highest PO2 values always occurred in the carapace lacuna (Fig. 8; open circles). Since this compartment receives haemolymph that has passed the respiratory surfaces (i.e. the inner walls of the carapace), the PO2 at this position may be regarded as loading PO2. In all size groups of Hb-poor and Hb-rich animals, the loading PO2 decreased almost linearly with decreasing PO2amb of 16 kPa. Moreover, PO2ambPO2 remained nearly constant, with similar mean values in Hb-poor (small: 0.78, medium: 0.49, large: 0.41 kPa) and Hb-rich animals (small: 0.46, medium: 0.48 kPa). The two-way ANOVA revealed significant effects of oxygen acclimation (F=9.25, d.f.=1, P=0.004) and body size (F=8.40, d.f.=2, P=0.001) on the difference of PO2ambPO2.
The dorsal lacuna generally had the lowest PO2 (Fig. 8; open diamonds) in comparison to the other two peripheral positions, with the exception of the small Hb-poor animals (Fig. 8A). At the PO2amb of 6 kPa, the mean PO2ambPO2 was 3.13.7 kPa in the medium and large animals of both acclimation groups, 1.9 kPa in the small Hb-poor animals, and 1.6 kPa in the small Hb-rich animals. Similar to situation in the carapace lacuna, the PO2 in the dorsal lacuna decreased more or less linearly with decreasing PO2amb in the range of 62 kPa. However, PO2ambPO2 did not remain constant but became smaller with decreasing PO2amb. At PO2amb<2 kPa, the PO2 deviated from the trend extrapolated from the linear relationship within the PO2amb of 62 kPa.
The heart region, which receives haemolymph from the carapace lacuna and the dorsal lacuna, had PO2 values (Fig. 8; open triangles) that were a little higher (Hb-poor animals) than or almost identical (Hb-rich animals) to those in the dorsal lacuna. The only exception were the small-sized Hb-poor animals (Fig. 8A), in which the heart region had a PO2 lower than that at the two other peripheral positions.
In comparison to the three peripheral positions, the unloading PO2 in the central body region (Fig. 8; closed triangles) showed the lowest change per unity change in PO2amb, indicating a certain degree of oxygen homeostasis. This stabilization was most pronounced in the medium-sized Hb-rich animals, which experienced only a slight decrease in the unloading PO2 from 0.9 to 0.4 kPa upon reduction of PO2amb from 3 to 1 kPa. Under the same conditions, the unloading PO2 of the medium-sized Hb-poor animals decreased much more markedly from 0.8 to 0.1 kPa. Similar differences in the degree of stabilization were found between the small-sized Hb-rich and Hb-poor animals within the range of PO2amb of 12 kPa.
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Discussion |
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To calibrate Oxyphor R2 in the haemolymph of D. magna, a stock
solution (pH 7.4) of 360 µmol l1 Oxyphor R2 containing
5.4% bovine serum albumin was injected into the circulatory system, and
haemolymph samples were then drawn and measured at 20°C. The
concentrations of Oxyphor R2 and albumin resulted in a sufficiently large
phosphorescence signal and still permitted the microinjection of the highly
viscous stock solution. The proportion of 0.17 g albumin per µmol Oxyphor
R2 used in the present study was similar to that used by Dunphy et al.
(2002). Despite similar
physicochemical conditions, however, the ranges of our calibration
constants (
0=451486 µs;
kq=13641535 kPa1
s1) were somewhat lower than those reported by Dunphy et al.
(2002
).
Calibration of Oxyphor R2 in Hb-poor and Hb-rich haemolymph at
PO2amb within the range 04 kPa yielded
almost identical 0 and kq values. Only at
values of PO2amb >4 kPa did the measured
of Hb-poor and Hb-rich haemolymph samples differ from each other,
suggesting that a variation in Hb concentration might have an influence on the
oxygen-dependent properties of the probe at higher
PO2amb.
During the experimental series on Daphnia, further calibrations in
Hb-poor and Hb-rich haemolymph were carried out to obtain parameters for
converting in vivo lifetime images into
PO2 images. The data of these calibration
measurements were pooled and yielded a kq of 1364
kPa1 s1 and a 0 of 484
µs for the PO2amb range 06 kPa
(030% air saturation). Since the variability of the measured
of
the different calibrations increased with increasing
PO2amb (Fig.
5B), the PO2 determination was more
affected by error at higher than at lower
PO2amb values, with deviations up to 30%. These
deviations resulted from the large variability of individual calibrations and
might be caused by variations in the microenvironment of the dye in the
circulatory system of animals. For future experiments, separate calibrations
for each animal would improve the precision of the measurement.
Influence of the injection procedure on heart beat activity
To measure the internal PO2 optically, the
oxygen-sensitive dye had to be introduced into the circulatory system of
D. magna by microinjection. The injection volume was chosen so as to
obtain a sufficiently high phosphorescence signal without affecting the
rhythmicity of the heart and the limbs. All injected animals showed the
expected compensatory tachycardia (Paul et
al., 1997) in response to the reduction in
PO2amb, indicating that control of
fH was not affected by the injection procedure. However,
the hypoxia-induced maxima of mean fH (226310
min1) of our 1.43.5 mm animals appeared to be
somewhat lower than those reported for non-injected animals. Reported values
obtained under comparable experimental conditions are 342364
min1 (2.42.8 mm animals;
Pirow et al., 2001
) and
355379 min1 (2.24.0 mm; M. Seidl, R. J. Paul
and R. Pirow, manuscript in preparation), respectively. A depression of
fH by the injection procedure therefore cannot be
excluded. A plausible explanation for this effect might be the increase in
haemolymph viscosity by the introduction of albumin. However, we regard the
differences in the maximum fH between injected and
non-injected animals to be of minor consequence for the measured internal
PO2 distributions of Hb-poor and Hb-rich
animals.
As an animal with a body size in the millimetre range, D. magna
relies on a mixed diffusiveconvective oxygen transport in the
circulatory system (Pirow,
2003; Pirow and Buchen,
2004
). Consequently, a moderate depression in the convective
transport share would be compensated by an increase in the diffusive transport
share. This shift from convection to diffusion would steepen internal oxygen
gradients (the driving force for diffusion) without affecting the general
shape of the oxygen profiles. The effect of a reduced circulatory convection
becomes negligible under those ambient oxygen tensions at which Hb is
maximally involved in oxygen transport. When Hb is present at high
concentration and oxygen is reversibly loaded and unloaded along the steep
part of the oxygen equilibrium curve, then Hb can act as an oxygen buffer that
damps the effect of fluctuations in perfusion rate on internal
PO2. Finally, further information on
non-injected animals is available that supports the validity of the internal
PO2 data (see below).
Validation of internal PO2 data
Information on internal PO2 values was
obtained from the haemolymph spaces in the median plane of the animal. To
check the accuracy our internal PO2 data, we
used indirect information derived from the oxygenation state of Hb in the
heart region of D. magna. According to previous studies using
medium-sized animals (Pirow et al.,
2001; Bäumer et al.,
2002
), Hb was found to be half-saturated with oxygen at a
PO2amb of 2.93.2 kPa (Hb-poor animals)
and 1.62.0 kPa (Hb-rich animals), respectively. Since the oxygen
affinities of Hb in whole blood samples of Hb-poor and Hb-rich animals are
known (1.0 vs 0.5 kPa; Zeis et
al., 2003a
), it is possible to estimate the difference in oxygen
tension between the ambient medium and the heart region
(PO2ambPO2heart),
and to use this figure for validation purposes. For Hb-poor animals, this
estimation yields a difference of
(PO2ambPO2heart)=1.92.2
kPa, which agrees well with the difference of 1.72.0 kPa determined in
the present study at PO2amb=2.93.2 kPa.
The same correspondence with no more than 10% deviation was found in small and
large Hb-poor animals (cf. Bäumer et
al., 2002
). For medium-sized Hb-rich animals, the difference of
(PO2ambPO2heart)=1.11.5
kPa estimated from the Hb oxygenation state was a little higher than the
0.70.9 kPa value determined in the present study. A similar deviation
was found for small Hb-rich animals (cf.
Bäumer et al., 2002
). This
discrepancy is, however, not particularly surprising since the half-saturation
oxygen tension of Hb in our Hb-rich animals could have been a little higher
than 0.5 kPa, because this value was the lowest one reported by Zeis et al.
(2003a
) for whole blood of
hypoxia-acclimated Hb-rich animals.
The shape of the two-dimensional oxygen profiles
Imaging of internal PO2 distribution in
small-sized animals of D. magna revealed interesting information
concerning the internal pathway of oxygen. In Hb-poor animals, there was a
steep overall anterior to posterior gradient, containing a localized region of
much lower PO2 (Figs
6,
top, 7). The lowest values
occurred in the centre of the anterior half of the animal, a region densely
packed with the large, active muscles of the second antennae and the
mandibles. A localized oxygen sink in this region suggests that these body
structures are the first ones to suffer from an undersupply of oxygen in
situations of progressive environmental hypoxia. Indeed these tissues,
including the bases of the limb muscles, proved to be sensitive, early
indicators (via NADH fluorescence) of the incipient impediment of
tissue oxygen supply in D. magna
(Pirow et al., 2001). The
global shape of the two-dimensional oxygen profiles is well in line with model
predictions (Pirow and Buchen,
2004
) as well as with results of an experimental study that used
Hb as an internal oxygen probe (Pirow et
al., 1999b
), and the general description of the internal pathway
for oxygen in D. magna is corroborated by the present study.
When flowing through the limbs and subsequently along the inner walls of the carapace valves, the haemolymph takes up oxygen from the ambient medium (see Fig. 8F). During this oxygenation process, haemolymph moves in the posterior direction, which explains the high PO2 values found in the posterior body region of the animals (Fig. 6, top). The oxygenated haemolymph in the carapace eventually reaches the median dorsal ridge of the carapace (carapace lacuna in Fig. 8F) before returning to the pericardium. Along the way to the heart, the haemolymph may lose oxygen by centripetal diffusion, which explains the posterioranterior gradient in PO2 (Fig. 7, Hb-poor case). In the pericardium, the haemolymph from the carapace lacuna becomes mixed with that from the dorsal lacuna. The dorsal lacuna receives haemolymph that has passed the intestinal lacuna, the fifth limb pair, and the post-abdomen. From the pericardium, the haemolymph is aspirated by the heart and expelled into the dorsal head region, from where it moves to the central nervous system before entering the ventral and intestinal lacunae of the trunk.
Keeping the haemolymph and medium flow pattern (Fig. 8F) in mind, the formation of a global gradient directed posterior-to-anterior can be easily explained by (i) uptake of oxygen into the haemolymph that moves along the ventral body side in a posterior direction, and (ii) centripetally directed diffusive loss of oxygen from the haemolymph that flows along the dorsal side in an anterior direction.
The sixfold higher Hb concentration in Hb-rich animals resulted in a
flattening of internal oxygen profiles under hypoxic conditions (Figs
6, bottom,
7). The large
anteriorposterior gradient, characteristic of Hb-poor animals,
disappeared and the high PO2 values extended
into the head region. This pattern with high oxygenation values in the rostral
region has already been reported (Pirow et
al., 1999b) and shows that the head region, with its sensory and
central nervous structures, still receives an adequate supply when ambient
oxygen becomes less available. This observation indicates that Hb-rich animals
have a striking physiological advantage over Hb-poor animals. The increase in
Hb concentration also caused a shift of the low-oxygen zone from the anterior
to the central body region (Fig.
6, bottom). Both these observations, the smoothing of oxygen
gradients and the positional shift of the low-oxygen zone, indicate that the
extra Hb buffers the haemolymph PO2 and
enhances the convective oxygen transport in the circulatory system. The
presence of steep PO2 gradients in Hb-poor
animals, by contrast, suggests that diffusion is more important for internal
transport processes.
Importance of body size and Hb concentration on internal PO2
Based on the quantitative information available for the three peripheral
and one central body region (Fig.
8F), we found no obvious influence of body size on internal
PO2 except in small Hb-poor animals. In these
animals, the PO2 in the carapace lacuna (i.e.
the loading PO2;
Fig. 8F) was 0.8 kPa below the
PO2amb, whereas in all other groups, the
difference between PO2amb and loading
PO2 was only 0.40.5 kPa. Moreover, of
the three peripheral positions analyzed, the heart region generally assumed an
intermediate PO2 value, except again in small
Hb-poor animals, where the heart PO2 was lowest
(Fig. 8A). These distinguishing
characteristics of the small Hb-poor animals suggest a greater diffusive loss
of oxygen from the haemolymph to the tissues when flowing (i) from the
respiratory surfaces to the carapace lacuna, as well as (ii) from the carapace
lacuna and the dorsal lacuna to the heart. Internal oxygen transport in the
smaller Hb-poor animals therefore appears to be effected more by diffusion
than in larger Hb-poor counterparts. Short transport distances from the
peripheral to central body regions, in combination with the internal oxygen
gradient, are prerequisites for a high diffusive flux and may explain this
effect of body size. However, we cannot exclude the possibility that the
injection of the viscous dye solution overproportionally slowed circulation in
the smallest animals, thereby contributing to the shift from convective to
diffusive oxygen transport. The absence of this body-size/injection effect
within the Hb-rich animals can be explained by the presence of a greater
concentration of circulating Hb, which stabilizes internal
PO2 and smooths oxygen gradients.
This smoothing and stabilizing effect of Hb was most striking in
medium-sized Hb-rich animals, in which the PO2
values of the monitored body regions converged with a progressive reduction of
PO2amb from 3 to 1 kPa
(Fig. 8E). Under these
conditions, the unloading PO2 decreased only
slightly from 0.40.9 kPa, which indicated an oxygen homeostasis in the
central body region. The PO2 at this position
was well above zero required to drive the diffusion of oxygen from the
haemolymph into the tissues. Medium-sized Hb-poor animals, by contrast,
experienced a much stronger decrease of the unloading
PO2 from 0.8 to 0.1 kPa
(Fig. 8B) under the same
ambient conditions. The strong decrease of unloading
PO2 in these animals very likely reflects the
inability of the circulatory system to sustain the oxygen supply to the
centrally located tissues. This suggestion is corroborated by respiration
measurements, which showed that 2.5 mm Hb-poor animals of D. magna
are unable to sustain their oxygen uptake rate at a
PO2amb lower than 4 kPa at 20°C
(Kobayashi and Hoshi, 1984).
The shut-down of aerobic metabolism might also explain the strong convergence
of the monitored PO2 values that occurred in
all groups at PO2amb <1 kPa.
Direct measurement of internal PO2 is ideal
for quantitative assessment of the contribution of Hb to circulatory oxygen
transport. Since most experimental data and model calculations are available
for D. magna 2.5 mm long (for references, see
Pirow, 2003;
Pirow and Buchen, 2004
), we
restricted our assessment to medium-sized animals. By making reasonable
assumptions about the oxygen binding characteristics of Hb, it was possible to
determine from the loading and unloading PO2
curves (Fig. 8B,E) those parts
of the oxygen equilibrium curve that were used for reversible oxygen binding
under different PO2amb conditions
(Fig. 9A). This analysis
indicated that Hb-rich animals reversibly load and unload oxygen over a
progressively smaller part of the oxygen equilibrium curve with decreasing
PO2amb. A similar tendency was present in
Hb-poor animals.
|
Information on the operation range of the oxygen equilibrium curve was
further employed to determine the difference between the loading and unloading
oxygen concentration ([O2]) in the haemolymph
(Fig. 9B). The fact that
[O2] was about twice as high in Hb-rich animals compared to
Hb-poor animals strongly points towards a greater contribution of convection
to circulatory oxygen transport. To estimate this convective contribution, we
initially ignored the role of diffusion and assumed that all oxygen is
transported by circulatory convection. If this assumption is correct, then the
Fick principle of convection should yield an oxygen transport rate (= stroke
volume x heart rate x
[O2]) that is equal to the
oxygen consumption rate of the animal. From the BL of Hb-poor and
Hb-rich animals (2.6 vs 2.7 mm), the stroke volume (8.2 vs
9.0 nl) and the oxygen consumption rate (24 vs 26 nmol
h1) were calculated according to Bäumer et al.
(2002
). For maximum
[O2] (86 vs 162 µmol l1;
Fig. 9B) and maximum heart
rates (285 vs 246 min1;
Fig. 8B,E), we obtained
convective transport rates that accounted for 50% and 85% of the oxygen
consumption rates of Hb-poor and Hb-rich animals, respectively. While these
results showed the assumption of an exclusive convective transport to be
invalid, they can nevertheless be regarded as a measure of relative
contribution of convection to circulatory oxygen transport. The remaining 50%
and 15% are then accounted for by a diffusional transport occurring in
parallel to convection.
List of symbols
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Baumeister, U. (1999).Säure-Basen-Regulation und Respiration bei Zooplankton-Organismen . Diploma thesis, University of Münster, Germany.
Bäumer, C. (2001). Optische Analyse des zirkulatorischen Sauerstofftransports im großen Wasserfloh, Daphnia magna. PhD thesis, University of Münster, Germany.
Bäumer, C., Pirow, R. and Paul, R. J. (2002). Circulatory oxygen transport in the water flea Daphnia magna. J. Comp. Physiol. B 172,275 -285.[Medline]
Becher, B. (2002). Untersuchungen zur hypoxie-induzierten Hämoglobin-Synthese des großen Wasserflohs Daphnia magna. PhD thesis, University of Münster, Germany.
Burggren, W. and Fritsche, R. (1995). Cardiovascular measurements in animals in the milligram range. Brazil. J. Med. Biol. Res. 28,1291 -1305.[Medline]
Colmorgen, M. and Paul, R. J. (1995). Imaging of physiological functions in transparent animals (Agonus cataphractus, Daphnia magna, Pholcus phalangioides) by video microscopy and digital image processing. Comp. Biochem. Physiol. A 111,583 -595.[CrossRef]
Dunphy, I., Vinogradov, S. A. and Wilson, D. F. (2002). Oxyphor R2 and G2: phosphors for measuring oxygen by oxygen-dependent quenching of phosphorescence. Anal. Biochem. 310,191 -198.[CrossRef][Medline]
Elendt, B.-P. and Bias, W.-R. (1990). Trace nutrient deficiency in Daphnia magna cultured in standard medium for toxicity testing. Effects of the optimization of culture conditions on life history parameters of D. magna. Wat. Res. Biol. 245,1157 -1167.
Fritzsche, H. (1917). Studien über Schwankungen des osmotischen Druckes der Körperflüssigkeiten bei Daphnia magna. Int. Rev. ges. Hydrob. Hydrog. 8, 22-80.
Green, J. (1956). Variation in the haemoglobin content of Daphnia. Proc. R. Soc. B 145,214 -232.[Medline]
Hoshi, T. and Yahagi, N. (1975). Studies on physiology and ecology of plankton. XXIX. In vivo equilibrium of oxygen with blood haemoglobin of Daphnia magna. Sci. Rep. Niigata Univ. Ser. D (Biol.) 12,1 -7.
Kimura, S., Tokishita, S., Ohta, T., Kobayashi, M. and Yamagata,
H. (1999). Heterogeneity and differential expression under
hypoxia of two-domain hemoglobin chains in the water flea, Daphnia magna.J. Biol. Chem. 274,10649
-10653.
Kobayashi, M. (1982). Influence of body size on haemoglobin concentration and resistance to oxygen deficiency in Daphnia magna. Comp. Biochem. Physiol. A 72,599 -602.[CrossRef]
Kobayashi, M. (1983). Estimation of the haemolymph volume in Daphnia magna by haemoglobin determination. Comp. Biochem. Physiol. A 76,803 -805.[CrossRef]
Kobayashi, M. and Hoshi, T. (1982). Relationship between the hemoglobin concentration of Daphnia magna and the ambient oxygen concentration. Comp. Biochem. Physiol. A 72,247 -249.[CrossRef]
Kobayashi, M. and Hoshi, T. (1984). Analysis of respiratory role of haemoglobin in Daphnia magna. Zool. Sci. 1,524 -532.
Kobayashi, M. and Tanaka, Y. (1991). Oxygen-transporting function of hemoglobin in Daphnia magna. Can. J. Zool. 69,2968 -2972.
Kobayashi, M., Fujiki, M. and Suzuki, T. (1988). Variation in and oxygen-binding properties of Daphnia magna hemoglobin. Physiol. Zool. 61,415 -419.
Lo, L. W., Vinogradov, S. A., Koch, C. J. and Wilson, D. F. (1997). A new, water soluble, phosphor for oxygen measurements in vivo. Adv. Exp. Med. Biol. 428,651 -656.[Medline]
Paul, R. J., Colmorgen, M., Hüller, S., Tyroller, F. and Zinkler, D. (1997). Circulation and respiratory control in millimetre-sized animals (Daphnia magna, Folsomia candida) studied by optical methods. J. Comp. Physiol. B 167,399 -408.
Paul, R. J., Colmorgen, M., Pirow, R., Chen, Y. H. and Tsai, M. C. (1998). Systemic and metabolic responses in Daphnia magna to anoxia. Comp. Biochem. Physiol. A 120,519 -530.
Pawlowski, M. and Wilson, D. F. (1994). Imaging oxygen pressure in tissue in vivo by phosphorescence decay. Adv. Exp. Med. Biol. 361,83 -93.[Medline]
Piiper, J. (1982). Respiratory gas exchange at lungs, gills and tissues mechanisms and adjustments. J. Exp. Biol. 100,5 -22.[Abstract]
Pirow, R. (2003). The contribution of hemoglobin to oxygen transport in the microcrustacean Daphnia magna A conceptual approach. Adv. Exp. Med. Biol. 510,101 -107.[Medline]
Pirow, R. and Buchen, I. (2004). The
dichotomous oxyregulatory behaviour of the planktonic crustacean Daphnia
magna. J. Exp. Biol. 207,683
-696.
Pirow, R., Wollinger, F. and Paul, R. J.
(1999a). The importance of the feeding current for oxygen uptake
in the water flea Daphnia magna. J. Exp. Biol.
202,553
-562.
Pirow, R., Wollinger, F. and Paul, R. J.
(1999b). The sites of respiratory gas exchange in the planktonic
crustacean Daphnia magna: An in vivo study employing blood
haemoglobin as an internal oxygen probe. J. Exp. Biol.
202,3089
-3099.
Pirow, R., Bäumer, C. and Paul, R. J. (2001). Benefits of haemoglobin in the cladoceran crustacean Daphnia magna. J. Exp. Biol. 204,3425 -3441.[Medline]
Schwerte, T. and Fritsche, R. (2003). Understanding cardiovascular physiology in zebrafish and Xenopus larvae: the use of microtechniques. Comp. Biochem. Physiol. A 135,131 -145.
Seidl, M., Pirow, R. and Paul, R. J. (2002). Water fleas (Daphnia magna) provide a separate ventilatory mechanism for their brood. Zoology 105, 15-23.
Shelton, G. (1992). Model applications in respiratory physiology. In Oxygen Transport in Biological Systems, vol. 51, SEB Seminar Series (ed. S. Egginton and H. F. Ross), pp.1 -44. Cambridge: Cambridge University Press.
Urich, K. (1990). Vergleichende Biochemie der Tiere. Stuttgart, New York: Gustav Fischer Verlag.
Vanderkooi, J. M. and Wilson, D. F. (1986). A new method for measuring oxygen in biological tissues. Adv. Exp. Med. Biol. 200,189 -193.[Medline]
Vinogradov, S. A. and Wilson, D. F. (1994). Phosphorescence lifetime analysis with a quadratic C-programming algorithm for determining quencher distributions in heterogeneous systems. Biophys. J. 67,2048 -2059.[Abstract]
Weber, R. E. and Vinogradov, S. N. (2001).
Nonvertebrate hemoglobins: Functions and molecular adaptations.
Physiol. Rev. 81,569
-628.
Weibel, E. R. (1984). The Pathway for Oxygen. Cambridge, MA: Harvard University Press.
Weibel, E. R. (1979). Stereological Methods, vol. 1. London, UK: Academic Press.
Weibel, E. R. (1980). Stereological Methods, vol. 2. London, UK: Academic Press.
Zar, J. H. (1999). Biostatistical Analysis. Upper Saddle River, NJ: Prentice Hall.
Zeis, B., Becher, B., Goldmann, T., Clark, R., Vollmer, E., Bölke, B., Bredebusch, I., Lamkemeyer, T., Pinkhaus, O., Pirow, R. and Paul, R. J. (2003a). Differential haemoglobin gene expression in the crustacean Daphnia magna exposed to different oxygen partial pressures. Biol. Chem. 384,1133 -1145.[Medline]
Zeis, B., Becher, B., Lamkemeyer, T., Rolf, S., Pirow, R. and Paul, R. J. (2003b). The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties. Comp. Biochem. Physiol. B 134,243 -252.[CrossRef][Medline]
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