3089
The Journal of Experimental Biology 202, 3089–3099 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JEB2272
THE SITES OF RESPIRATORY GAS EXCHANGE IN THE PLANKTONIC
CRUSTACEAN DAPHNIA MAGNA: AN IN VIVO STUDY EMPLOYING BLOOD
HAEMOGLOBIN AS AN INTERNAL OXYGEN PROBE
R. PIROW*, F. WOLLINGER AND R. J. PAUL
Institut für Zoophysiologie, Universität Münster, Hindenburgplatz 55, 48143 Münster, Germany
*e-mail: [email protected]
Accepted 13 September; published on WWW 28 October 1999
Summary
6 mmHg (0.8 kPa) for the rostrum. Although not all parts
Recent studies on Daphnia magna have revealed that the
of the circulatory system could be analyzed using this
feeding current is important for uptake of oxygen from the
technique, the data obtained from the accessible regions
ambient medium. Respiratory gas exchange should
suggest that the inner wall of the carapace is a major site
therefore mainly occur within the filtering chamber, whose
of respiratory gas exchange. Taking the circulatory pattern
boundaries are formed by the trunk and the extended
and the flow pattern of the medium in the filtering chamber
carapace shell valves. The precise site of gas exchange in
into consideration, it becomes clear that the haemolymph,
the genus Daphnia is, however, a matter of conjecture. We
after passing from the limbs to the carapace lacuna,
have developed a method of imaging the haemoglobin
becomes oxygenated while flowing through the ventral part
oxygen-saturation in the circulatory system of transparent
of the double-walled carapace in a posterior direction. The
animals, which provides an opportunity to localize oxygen
laterally flattened rostral region, where sensory and central
uptake from the environment and oxygen release to the
nervous system structures are located, seems to have direct
tissues. Experiments were carried out at 20 °C on
diffusive access to ambient oxygen, which could be
2.8–3.0 mm long parthenogenetic females maintained in
especially advantageous during severe hypoxia when the
hypoxic culturing conditions, which had resulted in an
convective transport systems fail to supply enough oxygen
increased haemoglobin content in the haemolymph. In
to that region.
lateral views of D. magna, the highest values of
haemoglobin oxygen-saturation occurred near the
posterior margin of the carapace and, surprisingly, in the
rostral part of the head. The ambient oxygen partial
Key words: Crustacea, Branchiopoda, Cladocera, Daphnia magna,
gas exchange, respiratory protein, haemoglobin, oxygen-saturation,
pressures at which haemoglobin was half-oxygenated were
spectral imaging, zooplankton.
15 mmHg (2.0 kPa) for the posterior carapace region and
Introduction
Aerobic energy production depends on the continuous
exchange of oxygen and carbon dioxide between the cellular
combustion sites of an organism and its environment. With
increasing body size, the metazoans of the higher phyla have
had to evolve dedicated organs with surface-enlarged thin
epithelia mediating the transfer of respiratory gases between
the ventilatory and the circulatory systems. Such extensively
elaborate structures are usually not present in animals smaller
than a few millimetres in length (Graham, 1988; Rombough
and Ure, 1991; Rombough, 1998), which is not particularly
surprising given their larger surface-to-volume ratios (Krogh,
1941). Thus, it is sometimes difficult to ascertain precisely
what the respiratory organ is or, if it is lacking, to determine
whether the whole body surface or only parts of it are
employed for integumentary respiration.
The present study aims to determine the favoured sites of
respiratory gas exchange in the water flea Daphnia magna. The
literature on crustacean biology (Gerstaecker, 1866–1879;
Giesbrecht, 1921; Storch, 1925; Krumbach, 1926–1927;
Flößner, 1972; Villee et al., 1979; Gruner, 1993) presents
different views about the sites of respiratory gas exchange in
the genus Daphnia, including suggestions of (i) gill breathing,
(ii) intestinal respiration and (iii) integumentary respiration.
Assigned to the class Branchiopoda, the genus Daphnia
possesses vesicle-like epipodites on its thoracic limbs, which
have been repeatedly regarded as gills and have sometimes
been termed branchial sacs (e.g. Claus, 1876). This assumption
is, so far, consistent with the morphological organization of a
typical crustacean, in which the gills derive primarily from
evaginations of the limb integument (Barnes, 1969; Gruner,
1993). In contrast to the gills of the advanced crustaceans,
however, the epipodites of the genus Daphnia are in no way
3090 R. PIROW, F. WOLLINGER AND R. J. PAUL
elaborated with respect to surface area and epithelial thickness
(Bernecker, 1909; Fryer, 1991) to enhance the rate of transfer
of respiratory gases. Although overlain by a cuticle that is very
thin (0.2–0.5 µm) relative to that of the rest of the leg (1–3 µm),
the epipodites are lined with an epithelium that is considerably
thicker (15–20 µm) than ordinary epithelium (3–5 µm;
Kikuchi, 1983). The selective stainability of the epipodites by
silver salts or vital stains (Fischel, 1908; Gicklhorn, 1925;
Gicklhorn and Keller, 1925a), formerly misinterpreted as
characteristic of respiratory epithelia (e.g. Gicklhorn and
Süllman, 1931), points in D. magna, as it does in other
crustaceans (Panikkar, 1941; Croghan, 1958), to an
osmoregulatory function, which has more recently been
confirmed ultrastructurally (Kikuchi, 1983). The role of the
neck or nuchal organ, in D. magna, a morphological feature
restricted to the first instar juvenile (Halcrow, 1982), has to be
seen in the same functional context (Potts and Durning, 1980;
Halcrow, 1982) rather than linked to respiratory gas exchange
(Gicklhorn and Keller, 1925b; Dejdar, 1930).
The striking phenomenon of anal water intake prompted
Lereboullet (1850) and later Weismann (1877) to assume that
intestinal respiration occurred in the daphniids. Anal water
uptake, caused by antiperistaltic movements of the hindgut
(Hardy and MacDougall, 1895), was later related to turgor
restoration (Fox, 1952; Fryer, 1970). It is also thought to
improve the efficiency of food utilization in the intestine
(Fryer, 1970).
General integumentary respiration seems plausible because
of the large surface-to-volume ratio of this millimetre-sized
animal and because of its delicate thin-walled integument
(Halcrow, 1976; Dahm, 1977). This hypothesis was
supported by the finding that the beating rate of the thoracic
limbs stays constant in D. magna (Heisey and Porter, 1977;
Paul et al., 1997) when the ambient oxygen concentration
decreases. If the limb movements serve for ventilation, then
the expected response to hypoxia in a water-breather with
oxyregulatory capacities would be an enhanced limb beating
rate (Randall et al., 1997). In the oxyregulating D. magna,
however, systemic responses differ from those of a typical
water-breather (Paul et al., 1997). The fact that there is no
increase in the limb beating rate need not be regarded as
negative proof of ventilatory function. In an attempt to filter
out as much food as possible from the ambient medium, when
there is little or no food available, planktonic filter feeders
such as D. magna exhibit close to maximum limb beating
rates (Porter et al., 1982). Elevated food concentrations lower
limb beating rate in D. magna and, surprisingly, the expected
‘hyperventilatory’ response can then be evoked by reducing
ambient oxygen concentration (R. Pirow and I. Buchen, in
preparation), indicating that the limb movements do indeed
have a ventilatory function. Respiratory gas exchange should
therefore occur within the animal’s filtering chamber,
because this region is well irrigated with fresh ambient water
during the steady process of filter feeding. Moreover, the
oxygen partial pressure was found to be lowered in the
medium leaving the filtering chamber (Pirow et al., 1999),
which indicates that oxygen is extracted from the feeding
current.
It has repeatedly been suggested that the inner wall of the
carapace is a major seat of respiratory exchange in the genus
Daphnia (Leydig, 1860; Fryer, 1991). Deriving from an
integumental fold of the maxillary region (Fryer, 1996), the
double-walled carapace consists of two shell valves, which
encase the thorax, abdomen and limbs, thus forming the lateral
boundaries of the filtering chamber. Taking into consideration
the water flow within the filtering chamber (Westheide and
Rieger, 1996) and the complex circulatory pattern (Hérouard,
1905; Storch, 1925), it seems very likely that the haemolymph
enters into intensive gas exchange with the medium when
circulating through the spaces between the inner and outer
walls of the carapace shell valves. Utilizing the presence of
blood haemoglobin (Hb), a respiratory protein with useful
oxygen-sensitive spectral characteristics, a newly developed
spectroscopic imaging technique enabled us to test this
hypothesis experimentally.
Materials and methods
Animals
Female water fleas Daphnia magna Straus were cultured
under the conditions described previously (Pirow et al., 1999).
To induce an increased blood haemoglobin (Hb) concentration,
parthenogenetic offspring were raised under conditions of
moderate hypoxia (30–40 % air saturation) produced by
bubbling nitrogen through the culture medium. According to
Kobayashi and Hoshi (1982), such hypoxic conditions result in
a sevenfold elevation of blood Hb concentration (basic level
1 g l−1 or 0.06 mmol O2 l−1) in 2.5 mm long adult females. The
animals used in our experiments had a body length ranging from
2.8 to 3.0 mm, measured from the anterior part of the head to
the posterior edge of the carapace at the base of the apical spine.
Preparation of animals for experiments
The experiments were carried out at 20 °C in a thermostatted
perfusion chamber (see Paul et al., 1997) that allowed
microscopic observation of single animals. To analyze its
spectral characteristics, the animal was immobilized by glueing
its apical spine to a 1 cm long synthetic brush-hair (histoacryl
adhesive; B. Braun Melsungen AG, Melsungen, Germany;
Cowles and Strickler, 1983). The animal was positioned
lateral-side down with the opposite side of the brush-hair and
the distal part of the ipsilateral second antenna glued onto a
coverslip. Owing to the curvature of the carapace shell valve,
the carapace came into contact with the coverslip at the level
of the base of the middle limbs pairs. The flow of medium
around the animal was consequently blocked only at this
contact site and was somewhat reduced at points surrounding
this area. The coverslip with the tethered animal was placed
onto the glass bottom of the perfusion chamber, which was
then sealed with a transparent screw-top without touching the
animal contralaterally. Experimental animals were perfused
from the anterior end with medium of variable oxygen partial
Respiratory gas exchange in Daphnia magna 3091
A
B
y
Camera controller
CCD camera
Image
data
Ix,y(λ)
...
Computer
x
Acquisition
interface
400
401
...
436
437
I0(λ)
λ (nm)
Specimen
stage
D/A
converter
Wavelength
control
Light guide
Monochromator
Absorbance, Ax,y(λ)
Objective lens
400
I0(λ)
Ax,y(λ) = log10 ——
Ix,y(λ)
Wavelength λ (nm)
437
Fig. 1. (A) Schematic diagram of the spectrophotometric microscope used for haemoglobin imaging. Monochromatic light supplied by a
computer-driven monochromator was used for illumination in the transmission mode. The microscopic image was digitized using a 16-bit
slow-scan CCD camera and transferred to a computer. (B) A stack of images was taken as the illumination wavelength was increased gradually
from 400 to 437 nm in steps of 1 nm. The absorption spectrum Ax,y(λ) of a selected x,y position of the image of the specimen was determined by
scanning through the image stack along the wavelength axis. Ax,y(λ) was derived from the spectrum Ix,y(λ) and the reference spectrum I0(λ),
which were obtained from a position inside and outside the image of the specimen, respectively.
pressure (PO∑) (see Pirow et al., 1999). Taking into account the
specific systemic adjustments of D. magna in response to
changes in PO∑ (Paul et al., 1997), an acclimation period of at
least 10 min preceded the data acquisition at each PO∑ level,
and this was found to be sufficient for the animal to attain a
new stable heart rate and Hb oxygen-saturation (R. Pirow,
C. Bäumer and R. J. Paul, unpublished data).
Experimental arrangement for spectral imaging
For spectral imaging, a series of gray-scale images of the
specimen was acquired while changing the wavelength of
monochromatic illumination. The apparatus (Fig. 1A)
consisted of an inverted microscope (Zeiss Axiovert 100, Carl
Zeiss, Oberkochen, Germany) combined with a computerdriven scanning-grating monochromator (T.I.L.L. Photonics,
Planegg, Germany; 75 W xenon arc lamp, spectral range
260–680 nm, spectral bandwidth 13 nm, response time <2 ms)
as an illumination system. Collimated monochromatic light
was guided to the microscope via a quartz fibre-optic light
guide (1.5 mm in diameter). The illumination wavelength was
set by a computer equipped with a D/A converter (DAS1602,
Keithley Metrabyte, Taunton, MA, USA). A 16-bit liquidnitrogen-cooled slow-scan CCD camera (576×384 pixels;
LN/CCD-576E, Princeton Instruments, Trenton, NJ, USA) was
mounted on the camera adapter of the microscope for image
acquisition. Images were digitized by a CCD controller (ST-
138, Princeton Instruments) and were transferred to the
computer via a high-speed serial interface (430 kHz maximum
pixel rate, Princeton Instruments). The low noise and the large
dynamic range of the CCD camera allowed the resolution of
minor differences in light absorption.
The imaging software WinView and WinSpec (Princeton
Instruments) were used for image acquisition and analysis. The
built-in C-like programming language was employed to
generate macros, which automated image-operation sequences
and synchronized image acquisition and the selection of
illumination wavelength.
Details of image acquisition and image analysis
For image acquisition, we used an exposure time of 20 ms
and binning in the range 2×2 to 3×3 pixels. Binning had the
advantage of reducing acquisition time and saving storage
capacity at the expense of spatial resolution. To compensate
for CCD dark charge, a background image, taken with the
camera shutter closed, was automatically subtracted from the
incoming image data. Inhomogeneous illumination of the
microscopic field was automatically corrected by the imageacquisition programme. Prior to the wavelength scan, the
experimental chamber with the animal inside was placed under
the microscope. While changing the illumination wavelength
gradually from 400 to 437 nm in 1 nm steps, a stack of 38
images was taken within 4–11 s.
3092 R. PIROW, F. WOLLINGER AND R. J. PAUL
B
Absorbance, A(λ)
A
0.9
Ho(λ)
Hd(λ)
0.8
0.7
0.6
0.5
A(λ)=aHx(λ)+b
0.4
Hx(λ)=nHo(λ)+(1−n)Hd(λ)
Gauss fit
λ0 = 418.8 nm
c = 0.339
d = 13.60
g = 0.432
r2 = 0.995
0.8
0.7
0.6
0.5
1.0
Absorbance, A(λ)
0.9
Absorbance, A
Hb fit
n = 0.40
a = 0.910
b = 0.022
r2 = 0.994
0.4
400
λ−λ0
2
A(λ)=ce−0.5 
d  +g
410
420
430
Wavelength, λ (nm)
Fig. 2. Identification of haemoglobin (Hb) spectra and determination of Hb oxygen-saturation. (A) As an example, an area (18×19 pixel) of the
head region of the animal was selected, and the absorbance spectra in the wavelength range 400–437 nm were determined. Non-Hb spectra can
easily be distinguished from Hb spectra, which feature the Soret band with peak wavelengths ranging from 414 to 427 nm. (B) Two regression
equations, Hb fit and Gauss fit, were applied to identify Hb-characteristic absorbance spectra. On the basis of a weighted summation of
oxyhaemoglobin (oxy-Hb) and deoxyhaemoglobin (deoxy-Hb) reference spectra, Ho(λ) and Hd(λ), the Hb fit yielded the oxygen-saturation
coefficient n, the concentration/path length parameter a and the light-scattering parameter b. The Gauss fit was used to determine the peak
wavelength λ0 (arrows). A spectrum was identified as being Hb-characteristic if a, b, λ0 and both correlation coefficients r2 contained plausible
values (see Table 1); otherwise, data were omitted. Oxy- and deoxy-Hb reference spectra, Ho(λ) and Hd(λ), were obtained from a diluted
solution of Daphnia magna Hb.
The image stack could be regarded as a three-dimensional
data package of intensity values I(x,y,λ) comprising the
intensity (I) for each pixel (x,y) of the image as a function of
the wavelength (λ). To retrieve the intensity spectrum Ix,y(λ)
for a selected x,y position, the image stack was scanned along
the wavelength axis (Fig. 1B). The corresponding absorption
spectrum Ax,y(λ) was calculated according to Lambert–Beer’s
law by taking log10{[I0(λ)]/[Ix,y(λ)]}, where I0(λ) is the
reference spectrum (Fig. 1B). The value for I0(λ) was retrieved
from a region outside the image of the animal and represented
light that had not interacted with the specimen.
Identification of Hb spectra and generation of Hb oxygensaturation images
Although D. magna is highly transparent, the presence of
light-absorbing compounds other than Hb must be taken into
consideration. The main light absorber in biological fluids and
tissues in the violet and blue parts of the spectrum is the
porphyrin system, which is a constitutional part of Hb and
cytochromes. Further relevant chromophores in D. magna are
algal chlorophylls (light absorption in the range 400–500 nm;
Libbert, 1987) in the gut lumen and ingested carotenoids
(absorption maxima in the range 450–500 nm; Herring 1968),
which can accumulate in the gut wall, the fat cells and the
ovaries (Green, 1957). Although carotenoids are transported in
the circulatory system, the haemolymph colour in Hb-rich
D. magna results from Hb (Herring, 1968). When
spectroscopically analyzing haemolymph spaces not
obstructed by the organs mentioned above, haemolymph Hb is
identifiable if present at sufficiently high concentration
(2.5 g l−1; Kobayashi and Takahashi, 1994). This has been
successfully achieved in several studies (Fox, 1948; Green,
1956; Hoshi and Yahagi, 1975; Kobayashi and Takahashi,
1994).
Advanced theories describe the spectral behaviour of
chromophores in turbid tissues (Cheong et al., 1990; Seiyama
et al., 1994). However, the transparency of D. magna allowed
us to choose a less complex approach. Identification of Hb was
based on a spectral comparison of in vivo spectra with
reference spectra of oxygenated (oxy-Hb) and deoxygenated
(deoxy-Hb) Hb (see below). The regression equation (Fig. 2B;
Hb fit) was derived from Beer’s law, which describes a two-
Respiratory gas exchange in Daphnia magna 3093
Table 1. Empirical data range of regression-analysis
parameters used for the identification of haemoglobin spectra
Parameter
a
Valid range
0–1.0
b
λ0
r2 (Hb fit)
r2 (Gauss fit)
0–1.0
413–428
>0.90
>0.90
Description
Related to haemoglobin
concentration and optical
path length
Related to light scattering
Peak wavelength
Correlation of determination
(correlation coefficient)
Correlation of determination
(correlation coefficient)
See Fig. 2 for regression equations.
component system assuming oxy-Hb and deoxy-Hb to be the
only absorbing substances. Incident light can be scattered in D.
magna by floating haemolymph cells, muscle bundles or
supporting structures such as the carapace or endoskeletal
sheets. This effect was taken into account by the scattering
factor b (Fig. 2B; Hb fit), which was assumed to be
wavelength-independent within the narrow wavelength range
selected. A second regression equation (Fig. 2B; Gauss fit) was
used to determine the peak wavelength. Regression parameters
and correlation coefficients were checked for plausibility
(Table 1) to distinguish Hb-characteristic spectra from non-Hb
spectra.
After analyzing the absorption spectra at all x,y positions,
the results were depicted as an image (see Fig. 3) in which
pixel intensity encoded Hb oxygen-saturation (pseudo-colour
presentation). Blue represented deoxygenated Hb and red
represented fully oxygenated Hb. For those x,y coordinates
where no Hb spectra were detectable, the pixel intensity was
set to black.
Preparation of a diluted Hb solution for spectral comparisons
A diluted Hb solution was prepared from haemolymph
samples taken from 20 Hb-rich female adults. After amputating
the distal half of the second antenna, the oozing haemolymph
was aspirated into a pulled-glass capillary tube (0.58 mm i.d.),
which was then emptied into 380 µl of ice-cold phosphate
buffer (20 mmol l−1, pH 7.0) containing 2.63 mmol l−1
ascorbate. After centrifugation at 10 000 g (10 min, 4 °C), the
supernatant was transferred to a flow-through cuvette (138-OS,
Hellma, Müllheim/Baden, Germany; 5 mm path length), and
absorbance spectra were acquired under normoxic and anoxic
conditions at 20 °C using the apparatus described above
(Fig. 1A). Oxygen was exhausted enzymatically (Lo et al.,
1996) by injecting 10 µl of ascorbate oxidase (10 units; Sigma
Chemical Co.) into the cuvette. The absorbance maxima at
414 nm and 427 nm were identified as those of the Soret bands
of oxy-Hb and deoxy-Hb, which corresponded to the values of
Sugano and Hoshi (1971) for oxy-Hb (414 nm) but not for
deoxy-Hb (423 nm). However, comparison with the
absorbance spectrum of purified, deoxygenated D. magna Hb
(B. Zeis, personal communication) revealed that the peak
wavelength of 427 nm was correct. Both the oxy-Hb and
deoxy-Hb spectra were employed as templates with which in
vivo Hb spectra were compared (Fig. 2B).
Statistical analyses
Data are expressed as mean values ± standard deviation
(S.D.), with N indicating the number of animals examined.
Absorption spectra were fitted by linear regression analysis
(Hb fit, see Fig. 2B for regression equation) with stepwise
variation of the oxygen-saturation coefficient n from 0 to 1.0.
To determine peak wavelength, absorption spectra were fitted
with a four-parameter Gaussian equation (Gauss fit, Fig. 2B)
utilizing the Levenberg–Marquardt algorithm (Press et al.,
1992). All regression equations were programmed in the C-like
macro language of WinSpec software, which allowed fast offline analysis of spectral images.
Haemoglobin oxygen-saturation images were analysed
further to obtain in vivo oxygen-binding curves for different
regions of the circulatory system. Regions of interest were
selected, each comprising 200–400 valid Hb oxygen-saturation
values. For each region of interest, a frequency distribution was
calculated (class width 2 % Hb oxygen-saturation), and the
median value was determined. Statistical differences in Hb
oxygen-saturation between different haemolymph regions
were assessed using a paired one-sided t-test (P<0.05).
Results
Images of Hb oxygen-saturation (SO∑) were taken in lateral
views of D. magna at different ambient PO∑ levels (Fig. 3).
Determination of SO∑ was possible in those haemolymph
spaces that were not obstructed by the large antennae, the gut,
the eggs or the beating limbs. The rostral head region and the
posterior parts of the carapace shell valves showed generally
higher SO∑ values than the other haemolymph spaces under
normoxic and hypoxic conditions. Because of posture
variations in the abdominal region, SO∑ could not always be
determined in the ventral posterior carapace region
(Fig. 3A,B).
Five positions in the circulatory system of D. magna were
selected for a quantitative comparison. The corresponding in
vivo oxygen-binding curves showing SO∑ as a function of
ambient PO∑ were constructed to make the regional differences
in Hb oxygen-saturation more apparent (Fig. 4). At normoxia
(155.7±2.3 mmHg, 20.75±0.31 kPa, N=5), the highest SO∑
values were found in the rostral region (position 1, 90.2±4.8 %)
and in the carapace lacuna near the posterior part of the shell
valves at the base of the apical spine (position 3, 80.4±4.4 %).
Position 5 yielded the lowest SO∑ (24.1±19.6 %), which
represented, in principle, the mean value of the Hb oxygensaturations of two types of overlying haemolymph space at that
position: the dorsal lacuna and the carapace lacuna (see Fig. 5).
However, owing to differences in lateral extension and
therefore in optical path length, the dorsal lacuna may have
made a greater contribution to this mixed SO∑ value. The
3094 R. PIROW, F. WOLLINGER AND R. J. PAUL
Fig. 3. Images of haemoglobin (Hb) oxygen-saturation of an individual Daphnia magna at various
ambient PO∑ levels (A, 153.2 mmHg; B, 15.2 mmHg; C, 5.5 mmHg; D, anoxia). The upper images
show the posture of the body within the carapace. Posture variations in the abdominal region were
responsible for the different image areas for which valid SO∑ values were obtained
(1 mmHg=0.133 kPa).
oxygenation of the haemolymph in the unobstructed carapace
lacuna at the level of the brood chamber positioned to the right
and dorsally from position 5 was generally higher than that
measured at position 5. It is therefore reasonable to assume that
the SO∑ of the dorsal lacuna, which guides the haemolymph
coming from the post-abdomen (see Fig. 5), was actually lower
than that measured at position 5.
When entering the extended carapace lacuna, the
haemolymph spreads out radially along curved paths that
become confluent at the median dorsal ridge, where blood flow
is directed anteriorly to the heart. Located at the dorsal
carapace ridge half-way between position 3 and the heart,
position 4 showed a lower SO∑ (62.8±3.6 %) than position 3.
The haemolymph currents in the dorsal lacuna and the carapace
lacuna become mixed in the pericardium, from where the
haemolymph is aspirated and expelled into the dorsal head
region, where an even lower SO∑ of 57.3±17.6 % was
determined (position 2). This hierarchy of SO∑ levels found
among the five selected haemolymph regions persisted at lower
ambient oxygen concentrations. A statistical comparison
(paired one-sided t-test) at two PO∑ levels (155.7 and
14.1 mmHg, 20.75 and 1.88 kPa) showed that the SO∑ in the
rostral head region (position 1) was significantly higher than
at all other positions (t>3.57, d.f.=4, P<0.05, N=5). Moreover,
the SO∑ in the carapace lacuna near the base of the apical spine
(position 3) was significantly higher than at positions 4 and 5
(t>4.5, d.f.=4, P<0.05, N=5).
The in vivo oxygen-binding curves were further used to
estimate the ambient oxygen partial pressures (P50) at which
the Hb was half-oxygenated using linear interpolation. For
positions 1 and 3, which we regarded as regions containing
oxygenated haemolymph (see Discussion), P50 was 6 mmHg
(0.8 kPa) at position 1 and 15 mmHg (2.0 kPa) at position 3.
Discussion
The oxygen-saturation of Hb was measured in the
circulatory system of the planktonic crustacean Daphnia
magna with two-dimensional spatial resolution. In contrast to
previous in vivo studies (Fox, 1945; Kobayashi and Tanaka,
Respiratory gas exchange in Daphnia magna 3095
A
100
Hb saturation (%)
1
80
60
2
40
20
0
B
Hb saturation (%)
100
3
80
4
60
40
5
20
0
0
10
20
30
150
160
Oxygen tension (mmHg)
1
2
4
5
3
Fig. 4. In vivo haemoglobin (Hb) oxygen-binding curves at five
positions in Daphnia magna. The PO∑ of the perfusion medium was 0
mmHg (N=4), 4.3±1.7 mmHg (N=2), 14.1±0.7 mmHg (N=5),
19.2±0.5 mmHg (N=2) or 155.7±2.3 mmHg (N=5). Hb oxygensaturation is given as a mean value and, if N>2, standard deviation.
1 mmHg=0.133 kPa.
1991), this imaging technique provided a view of the level of
haemolymph oxygenation of various body regions, which can
be used to localize the uptake of oxygen from the environment
and the release of oxygen to the tissues. This technique holds
further potential for the visualization of rapidly changing
oxygen distributions in small transparent animals with a time
Fig. 5. Schematic representation of the main haemolymph spaces and
currents in Daphnia magna. The simplified ventral view (A) shows
the second and third limb pair; the blood flow of the first and fourth
limb pair is only partially indicated. In the dorsal view (B), a piece of
the left shell valve at the level of the brood chamber was removed.
The ventral (green) and dorsal membranes (yellow) separate the
trunk, thereby forming three main blood spaces: the ventral,
intestinal and dorsal lacunae. The ventral integument is folded back
medianly to the ventral membrane, thus dividing the ventral lacuna
symmetrically. In addition, two vertical membranes (blue; A) divide
the two resulting ventral lacunae and the limbs into medial and
lateral compartments. Leaving the head region in a posterior
direction, haemolymph enters either the intestinal lacuna or the
medial ventral lacunae. From the medial ventral lacunae, currents
project into the five limbs pairs and then enter the lateral ventral
lacunae. The haemolymph of the first four limb pairs then passes via
the so-called pedicles into the carapace lacuna. Some remaining
blood from the medial ventral lacunae and that from the fifth limb
pair (not shown) joins with that of the intestinal lacuna to perfuse the
abdominal tissues before returning via the dorsal lacuna to the
pericardium. In the carapace lacuna, the haemolymph spreads out
radially along curved paths and then becomes confluent at the
median dorsal ridge of the carapace before returning to the
pericardium, where the haemolymph is mixed with that of the dorsal
lacuna. The two branches of the feeding current inside the filtering
chamber, the subcarapace flow and the median filter flow emerging
from the two posterior interlimb spaces, are indicated in light blue
(combined from Hérouard, 1905; Kohlhage, 1994, cited in
Westheide and Rieger, 1996).
3096 R. PIROW, F. WOLLINGER AND R. J. PAUL
resolution within the hundred millisecond range (R. Pirow, F.
Wollinger, U. Baumeister and R. J. Paul, in preparation).
The level of Hb oxygen-saturation in a section of the
circulatory compartment is, in principle, determined by (i) the
immediate diffusive loss of oxygen to the tissues, (ii) the
immediate diffusive influx of oxygen from the ambient
medium, and (iii) the oxygenation level and the flow rate of
the haemolymph entering that section. Because the different
sections of the open circulatory system are directionally linked
by the blood flow pattern, the corresponding Hb oxygensaturation values can be related to each other to characterize
the diffusive exchange processes for an individual section.
Although the trunk, the limbs and the ventral half of the
carapace in D. magna were only partly accessible using our
technique, the data obtained from the remaining body regions
can provide indications of potential sites of oxygen uptake
when the haemolymph and flow patterns in the medium are
taken into consideration. The haemolymph of the first four
limb pairs passes into the carapace lacuna (Hérouard, 1905;
Fig. 5), where it radiates along curved paths. In the ventral half
of the carapace valves, the haemolymph moves in a posterior
direction while coming into close contact with the two
branches of flow of medium inside the filtering chamber (M.
Gophen, personal communication; Kohlhage, 1994: cited in
Westheide and Rieger, 1996). The flow of medium and
haemolymph are partly concurrent and partly in cross-current
orientation to each other. Following the curvature of the
ventral-posterior carapace margin, the haemolymph reaches
the median dorsal ridge of the carapace at the base of the apical
spine (position 3, Fig. 4), where we found higher Hb oxygensaturation values than at positions located downstream of that
region (see Figs 4, 5). The 50 % oxygenation of Hb occurred
at an ambient oxygen partial pressure (P50) of 15 mmHg
(2.0 kPa). Kobayashi and Tanaka (1991) and Kobayashi and
Takahashi (1994) measured Hb oxygen-saturation in the dorsal
carapace region of hypoxia-acclimated 2.5–2.8 mm long D.
magna and reported a P50 of 15–17 mmHg (2.0–2.3 kPa),
which is consistent with our data.
The high oxygenation level in the posterior part of the
carapace suggests that influx of oxygen into the haemolymph
occurred during flow through the ventral half of the carapace.
This conclusion is supported by the recent finding that D. magna
extracts ambient oxygen from the feeding current (Pirow et al.,
1999): hypoxia-acclimated females showed a PO∑ of 3.2 mmHg
(0.43 kPa) in the exhalant part of the feeding current under
hypoxic conditions (16.2 mmHg or 2.16 kPa). Similar hypoxic
conditions (15 mmHg or 2.0 kPa) were found to effect 50 %
oxygenation of Hb in the posterior part of the carapace (this
study). As the haemolymph PO∑ in that body region must, in
principle, be lower than the PO∑ of the exhalant part of the
feeding current, thus ensuring the diffusive transfer of oxygen
from the medium to the blood, 50 % oxygenation of Hb should
occur at a haemolymph PO∑ below 3.2 mmHg (0.43 kPa). This
inference is reasonable when referring to the data of Kobayashi
et al. (1988), who analyzed the in vitro oxygen-binding
characteristics of purified D. magna Hb (0.1 mol l−1 phosphate
buffer, 20 °C) and reported 50 % oxygenation of Hb at
1.1 mmHg (0.15 kPa) for hypoxia-acclimated (Hb-rich) animals.
Taking into account Fick’s first law of diffusion, it becomes
clear that the conditions for gas exchange across the inner
carapace wall are indeed favourable. The arthropod integument
consists of an outer cuticular and an inner epithelial layer
(Gruner, 1993), whose diffusional resistances to oxygen differ
by a factor of approximately 10 (Krogh, 1919). The cuticular
layer therefore represents a crucial diffusional barrier. D.
magna shows no marked cuticular thickening (Halcrow, 1976;
Dahm, 1977), and the whole integumental covering has been
found to be permeable to oxygen (R. Pirow, F. Wollinger, U.
Baumeister and R. J. Paul, in preparation). The inner wall of
the double-walled carapace is covered by a delicate cuticle
(<1.0 µm), which is several times thinner than the cuticle on
other parts of the body (Dahm, 1977) thus facilitating gas
transfer across the inner carapace wall.
In addition to the solid integumental barrier, fluid boundary
layers represent another obstacle for diffusive gas transport.
Millimetre-sized animals live in an environment in which their
fluid dynamics can be in the range of low Reynolds numbers
where viscous forces are dominant (Koehl and Strickler, 1981;
Gerritsen et al., 1988). As a consequence, these organisms are
barely able to shed the surrounding viscous water layers. The
thickness of the boundary layer depends on the velocity of the
medium relative to the body surface. In Daphnia, reduced
boundary layers should occur inside the filtering chamber,
where the medium drains through in a jerky manner
accelerated to velocities of 10–15 mm s−1 (2–3 mm D.
magna/pulex; Gerritsen et al., 1988). However, similar
velocities can occur during swimming, which directly affect
body surfaces other than those inside the filtering chamber. The
range of variation is large, from mean swimming speeds of
9–15 mm s−1 (2.8 mm for D. magna, 20 °C: Kobayashi and
Gonoi, 1985; Fryer, 1991) to sinking speeds of 3 mm s−1
(1.5 mm D. pulex at 25 °C; Gorski and Dodson, 1996), to more
stagnant conditions when the animal rests on the bottom with
the carapace contacting the substratum. The two latter
situations, as well as conditions in which the animal maintains
its position in the water column using strokes of the large
antennae, are comparable with our experimental situation.
The maintenance of a large difference in PO∑ is decisive for
rapid diffusion of oxygen from the medium to the blood. In the
filtering chamber, this requirement is fulfilled by the rapid
renewal rate of the medium (27–87 µl min−1; Pirow et al.,
1999), which is several times higher than the perfusion rate
(3–4 µl min−1; Paul et al., 1997). The high ventilation-toperfusion ratio favours the oxygenation of the haemolymph. In
addition, the presence of Hb enhances oxygen transfer from
medium to haemolymph. As long as the Hb is not fully loaded
with oxygen, there is only a slight change in blood PO∑ during
oxygenation, so that the driving force for diffusion can be
largely maintained.
The presence of the highest oxygenation levels in the rostral
region, with a P50 of 6 mmHg (0.8 kPa), was surprising and
implies a thin-walled rostral integument with a low diffusive
Respiratory gas exchange in Daphnia magna 3097
resistance. More important in this context, however, is the shape
of the rostrum, which is flattened laterally. The diffusion
distance from the integument to the centre of the haemolymph
space is consequently very short, thus permitting more rapid
penetration of oxygen by diffusion. External diffusive boundary
layers adjacent to the rostral integument should be reduced in
free-moving animals, which are propelled forward in a jerky
manner by powerful strokes of the second antennae. The
conditions in our experiment were comparable insofar as
tethered animals were perfused from the front. Whether there
is a substantial contribution to total oxygen uptake is
questionable. Nevertheless, this bypass would provide
additional oxygen for sensory and central nervous structures
located in the rostral head region (Claus, 1876), which could be
of advantage during severe hypoxia when the convective
transport system fails to supply enough oxygen to that location.
The water flea Daphnia magna shows a variety of
adaptations that have arisen from a filter-feeding life in a
variable aquatic environment. In addition to what is known
about the adjustments occurring at the level of the respiratory
pigment (Kobayashi et al., 1988, 1990) and at the metabolic
and systemic levels (Paul et al., 1997, 1998), this study
discloses additional adaptations relevant to respiratory gas
transport. The enlarged shell valves of the carapace are not
only a constitutional part of the filter apparatus, forming the
lateral boundaries of the filtering chamber, but their special
structure allows the thin inner wall to be employed for oxygen
uptake while the thickened outer wall provides stability for the
carapace. The special shape of the rostrum can make ambient
oxygen easily accessible to the central nervous structures,
which could be important in sustaining vital control functions
during hypoxic conditions.
List of symbols
absorption-related coefficient (haemoglobin
concentration, optical path length) (Hb fit)
b
scattering coefficient (Hb fit)
c, d, g
Gauss fit coefficients
A
absorbance
absorption spectrum at x,y position
Ax,y(λ)
Ho(λ),
absorption spectra of oxy-haemoglobin and
Hd(λ)
deoxy-haemoglobin
Hx(λ)
haemoglobin absorption spectra of unknown
oxygen saturation
I
intensity
I0(λ)
reference spectrum
Ix,y(λ)
intensity spectrum at the x,y position of the image
n
haemoglobin oxygen-saturation coefficient
P50
oxygen partial pressure at which haemoglobin is
half-oxygenated (mmHg, kPa)
PO∑
oxygen partial pressure (mmHg, kPa)
SO∑
haemoglobin oxygen-saturation (%)
x, y
Cartesian coordinates
λ
wavelength (nm)
λ0
peak wavelength (nm)
a
The technical assistance of Ina Buchen is gratefully
acknowledged. We thank Martina Fasel for the excellent
three-dimensional drawings and G. Sundermann and J. Lange
for allowing us to inspect their scanning electron microscope
images of water fleas. We are especially grateful to
Alan Rietman Knauth for the linguistic and stylistic
improvements to the manuscript. Supported by the Deutsche
Forschungsgemeinschaft (Pa 308/7-1).
References
Barnes, R. D. (1969). Invertebrate Zoology. Philadelphia: Saunders.
Bernecker, A. (1909). Zur Histologie der Respirationsorgane bei
Crustaceen. Zool. Jahrb. 27, 583–630.
Cheong, W.-F., Prahl, S. A. and Welch, A. J. (1990). A review of
the optical properties of biological tissues. IEEE J. Quantum
Electron. 26, 2166–2185.
Claus, C. (1876). Zur Kenntnis der Organisation und des feineren
Baues der Daphniden und verwandter Cladoceren. Z. Wiss. Zool.
27, 362–402.
Cowles, T. J. and Strickler, J. R. (1983). Characterization of feeding
activity patterns in the planktonic copepod Centropages typicus
Kroyer under various food conditions. Limnol. Oceanogr. 28,
106–115.
Croghan, P. C. (1958). The osmotic and ionic regulation in Artemia
salina (L.). J. Exp. Biol. 35, 219–233.
Dahm, E. (1977). Morphologische Untersuchungen an Cladoceren
unter besonderer Berücksichtigung der Ultrastruktur des Carapax.
Zool. Jb. Anat. 97, 68–126.
Dejdar, E. (1930). Die Korrelationen zwischen Kiemensäckchen und
Nackenschild bei Phyllopoden. Z. Wiss. Zool. 136, 422–452.
Fischel, A. (1908). Untersuchungen über vitale Färbung an
Süßwassertieren, insbesondere bei Cladoceren. Int. Rev. Ges.
Hydrob. Hydrog. 1, 73–141.
Flößner, D. (1972). Krebstiere, Crustacea: Kiemen- und Blattfüßer,
Branchiopoda; Fischläuse, Branchiura. In Die Tierwelt
Deutschlands (ed. M. Dahl and F. Peus), p. 60. Teil, Jena: Gustav
Fischer.
Fox, H. M. (1945). The oxygen affinities of certain invertebrate
haemoglobins. J. Exp. Biol. 21, 161–165.
Fox, H. M. (1948). The haemoglobin of Daphnia. Proc. R. Soc. Lond.
B 135, 195–212.
Fox, H. M. (1952). Anal and oral intake of water by Crustacea. J.
Exp. Biol. 29, 583–599.
Fryer, G. (1970). Defaecation in some macrothricid and chydorid
cladocerans and some problems of water intake and digestion in the
Anomopoda. Zool. J. Linn. Soc. 49, 255–270.
Fryer, G. (1991). Functional morphology and the adaptive radiation
of the Daphniidae (Branchiopoda: Anomopoda). Phil. Trans. R.
Soc. Lond. B 331, 1–99.
Fryer, G. (1996). The carapace of the branchiopod Crustacea. Phil.
Trans. R. Soc. Lond. B 351, 1703–1712.
Gerritsen, J., Porter, K. G. and Strickler, J. R. (1988). Not by
sieving alone: Observations of suspension feeding in Daphnia.
Mar. Sci. 43, 366–376.
Gerstaecker, A. (1866–1879). Die Klassen und Ordnungen der
Arthropoden, 5. Band, 1. Abt., Crustacea. Leipzig, Heidelberg: C.
F. Winter’sche Verlagshandlung.
Gicklhorn, J. (1925). Über spezifische und lokale Reduktion von
3098 R. PIROW, F. WOLLINGER AND R. J. PAUL
Silber- und anderen Metallsalzen in den Kiemensäckchen von
Daphnia M. Lotos 73, 83–96.
Gicklhorn, J. and Keller, R. (1925a). Über elektive Vitalfärbungen
der Kiemensäckchen von Daphnia magna Müller als Beispiel
organ- und zellspezifischer Differenzierung. Z. Zellforsch. 2,
515–537.
Gicklhorn, J. and Keller, R. (1925b). Bau und Funktion des
‘Haftorgans’ von Daphnia bzw. des ‘Kopfschildes’ von Leptodora
und Polyphemus auf Grund vitaler Elektivfärbung. Zool. Anz. 64,
217–234.
Gicklhorn, J. and Süllman, H. (1931). Die Permeabilität der
Kiemensäckchen von Daphnia magna M. Protoplasma 13,
617–636.
Giesbrecht, W. (1921). Crustacea. In Handbuch der Morphologie der
Wirbellosen Tiere, Band 4 (ed. A. Lang), pp. 9–252. Jena: Gustav
Fischer.
Gorski, P. R. and Dodson, S. I. (1996). Free-swimming Daphnia
pulex can avoid following Stokes’ law. Limnol. Oceanogr. 41,
1815–1821.
Graham, J. B. (1988). Ecological and evolutionary aspects of
integumentary respiration: body size, diffusion and the
Invertebrata. Am. Zool. 28, 1031–1045.
Green, J. (1956). Variation in the haemoglobin content of Daphnia.
Proc. R. Soc. Lond. B 145, 214–232.
Green, J. (1957). Carotenoids in Daphnia. Proc. R. Soc. Lond. B 147,
392–401.
Gruner, H.-E. (1993). Lehrbuch der speziellen Zoologie (begr. durch
A. Kaestner), Band 1, 4. Teil, Wirbellose Tiere: Arthropoda. Jena,
Stuttgart, New York: Gustav Fischer.
Halcrow, K. (1976). The fine structure of the carapace integument of
Daphnia magna Straus (Crustacea Branchiopoda). Cell. Tissue Res.
169, 267–276.
Halcrow, K. (1982). Some ultrastructural features of the nuchal organ
of Daphnia magna Straus (Crustacea: Branchiopoda). Can. J. Zool.
60, 1257–1264.
Hardy, W. B. and MacDougall, W. (1895). On the structure and
function of the alimentary canal of Daphnia. Proc. Camb. Phil. Soc.
B 8, 41–50.
Heisey, D. and Porter, K. G. (1977). The effect of ambient oxygen
concentration on filtering rate and respiration rate of Daphnia
magna mendotae and Daphnia magna. Limnol. Oceanogr. 22,
839–845.
Hérouard, E. (1905). La circulation chez les Daphnies. Mem. Soc.
Zool. Fr. 18, 214–232.
Herring, P. J. (1968). The carotenoid pigments of Daphnia magna
Straus. I. The pigments of animals fed Chlorella pyrenoidosa and
pure carotinoids. Comp. Biochem. Physiol. 24, 187–203.
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.
Kikuchi, S. (1983). The fine structure of the gill epithelium of a
freshwater flea, Daphnia magna (Crustacea: Phyllopoda) and
changes associated with acclimation to various salinities. I. Normal
fine structure. Cell. Tissue Res. 229, 253–268.
Kobayashi, M., Fujiki, M. and Suzuki, T. (1988). Variation in and
oxygen-binding properties of Daphnia magna hemoglobin.
Physiol. Zool. 61, 415–419.
Kobayashi, M. and Gonoi, H. (1985). Horizontal movement of pale
and red Daphnia magna in low oxygen concentration. Comp.
Biochem. Physiol. 58, 190–196.
Kobayashi, M. and Hoshi, T. (1982). Relationship between the
haemoglobin concentration of Daphnia magna and the ambient
oxygen concentration. Comp. Biochem. Physiol. 72A, 247–249.
Kobayashi, M., Nezu, T. and Tanaka, Y. (1990). Hypoxic induction
of hemoglobin synthesis in Daphnia magna. Comp. Biochem.
Physiol. 97A, 513–517.
Kobayashi, M. and Takahashi, Y. (1994). In vivo oxygenation of
hemoglobin in early embryos of Daphnia magna. Comp. Biochem.
Physiol. 107A, 127–131.
Kobayashi, M. and Tanaka, Y. (1991). Oxygen-transporting function
of hemoglobin in Daphnia magna. Can. J. Zool. 69, 2968–2972.
Koehl, M. A. R. and Strickler, J. R. (1981). Copepod feeding
currents: food capture at low Reynolds number. Limnol. Oceanogr.
26, 1062–1073.
Krogh, A. (1919). The rate of diffusion of gases through animal
tissues, with some remarks on the coefficient of invasion. J.
Physiol., Lond. 52, 391–408.
Krogh, A. (1941). The Comparative Physiology of Respiratory
Mechanisms. New York: Dover Publications, Inc.
Krumbach, T. (1926–1927). Handbuch der Zoologie (begr. durch W.
Kükenthal), 3. Band, 1. Hälfte. Berlin, Leipzig: Walter de Gruyter
& Co.
Lereboullet, A. (1850). Observations anatomiques et physiologiques.
Mem. Soc. Mus. Hist. Nat. 4, 208–212.
Leydig, F. (1860). Naturgeschichte der Daphniden (Crustacea,
Cladocera). Tübingen: Laupp & Siebeck.
Libbert, E. (1987). Lehrbuch der Pflanzenphysiologie. Stuttgart, New
York: Gustav Fischer.
Lo, L.-W., Koch, C. J. and Wilson, D. F. (1996). Calibration of
oxygen-dependent quenching of the phosphorescence of Pd-mesotetra (4-carboxyphenyl) porphine: A phosphor with general
application for measuring oxygen concentration in biological
systems. Analyt. Biochem. 236, 153–160.
Panikkar, N. K. (1941). Osmoregulation in some palaemonid
prawns. J. Mar. Biol. Ass. U.K. 25, 317–259.
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. 120A, 519–530.
Pirow, R., Wollinger, F. and Paul, R. J. (1999). Importance of the
feeding current for oxygen uptake in the water flea Daphnia magna.
J. Exp. Biol. 202, 553–562.
Porter, K. G., Gerritsen, J. and Orcutt, J. D. (1982). The effect of
food concentration on swimming patterns, feeding behavior,
assimilation and respiration by Daphnia. Limnol. Oceanogr. 27,
935–949.
Potts, W. T. W. and Durning, C. T. (1980). Physiological evolution
in the branchiopods. Comp. Biochem. Physiol. 68A, 475–484.
Press, W. H., Teukolsky, S. A., Vetterling, W. T. and Flannery,
B. P. (1992). Numerical Recipes in C. Cambridge: Cambridge
University Press.
Randall, D., Burggren, W. and French, K. (1997). Eckert Animal
Physiology: Mechanisms and Adaptations. New York: W. H.
Freeman & Company.
Rombough, P. J. (1998). Partitioning of oxygen uptake between gills
and skin in fish larvae: a novel method for estimating cutaneous
oxygen uptake. J. Exp. Biol. 201, 1763–1769.
Rombough, P. J. and Ure, D. (1991). Partitioning of oxygen uptake
Respiratory gas exchange in Daphnia magna 3099
between cutaneous and branchial surfaces in larval and young
juvenile chinook salmon Oncorhynchus tshawytscha. Physiol.
Zool. 64, 717–727.
Seiyama, A., Chen, S.-S., Kosaka, H. and Shiga, T. (1994).
Microspectroscopic measurement of the optical properties of rat
liver in the visible region. J. Microsc. 175, 84–89.
Storch, O. (1925). Cladocera. In Biologie der Tiere Deutschlands (ed.
P. Schulze), Lief. 15, Teil 14. Berlin: Gebrüder Borntraeger.
Sugano, H. and Hoshi, T. (1971). Purification and properties of
blood hemoglobin from fresh-water cladocera, Moina macropa and
Daphnia magna. Biochim. Biophys. Acta 229, 349–358.
Vilee, C. A., Walker, W. F. and Barnes, R. D. (1979). Introduction
into Animal Biology. Philadelphia, London, Toronto: Saunders.
Weismann, A. (1877). Beiträge zur Naturgeschichte der Daphnoiden.
Z. Wiss. Zool. 28, 93–254.
Westheide, W. and Rieger, R. (1996). Spezielle Zoologie, 1. Teil,
Einzeller und Wirbellose Tiere. Jena, Stuttgart, New York: Gustav
Fischer.