Role of the Feeding Current in O2 Uptake in Daphnia Magna

The Journal of Experimental Biology 202, 553–562 (1999) 553 Printed in Great Britain © The Company of Biologists Limited 1999 JEB1669

THE IMPORTANCE OF THE FEEDING CURRENT FOR OXYGEN UPTAKE IN THE WATER FLEA DAPHNIA MAGNA

R. PIROW*, F. WOLLINGER AND R. J. PAUL Institut für Zoophysiologie, Westfälische Wilhelms-Universität, Hindenburgplatz 55, D-48143 Münster, Germany *e-mail: [email protected]

Accepted 30 November 1998; published on WWW 3 February 1999

Summary

In the planktonic crustacean Daphnia magna from 2.3 to 2.7 mm at 20 °C. External PO∑ measurement (Branchiopoda, Cladocera), different views exist on the revealed oxygen depletion in the ventral body region but mechanism of respiratory gas exchange, ranging from gill essentially no change at posterior, lateral and dorsal breathing to general integumentary respiration. The positions. The PO∑ difference between the inflow and presumed structures for specialized gas exchange are outflow of the feeding current was 13.0 mmHg (1.73 kPa). located ventrally within the filter chamber, which is The flow rate of the feeding current ranged from 1.2 to continuously perfused with the ambient medium for food 5.2 ml h−1 and showed a close correlation with appendage gathering. To localize respiratory gas exchange in D. beat rate, which varied from 310.4 to 460.7 beats min−1. magna, we determined the contribution of the feeding Model calculations based on the Fick principle suggest that current to total oxygen transport. Combining microscopy oxygen extraction from the feeding current satisfies most with special optical techniques, we used a phosphorescent of the total of oxygen requirement of D. magna. oxygen-sensitive dye for oxygen partial pressure (PO∑) measurements and applied fluorescent microspheres for flow-rate analysis. Appendage beat rate was determined by Key words: Crustacea, Daphnia magna, feeding current, flow digital image-processing. All experiments were carried out imaging, oxygen extraction, oxygen phosphorimetry, particle on hypoxia-adapted animals with a body length ranging fluorimetry, zooplankton.

Introduction Like other zooplankters, the water flea genus Daphnia epipodites as well as on the ventilatory function of the feeding (Branchiopoda, Cladocera) occupies an important position in current. The epipodites in D. magna have been found to be freshwater food webs by converting phytoplankton food into lined with an epithelium 15–20 µm thick (Kikuchi, 1983; biomass that then becomes available for higher trophic levels. Peters, 1987), which is 4–5 times thicker than over the rest of Specialized thoracic appendages equipped with fine setae the limb. Accordingly, respiratory gas transfer via the so-called enable Daphnia spp. to filter out suspended food particles from branchial sacs would be impeded by a large diffusion barrier. the surrounding water. Encased by the carapace, these Physiological data on D. magna, a species exhibiting a appendages beat rhythmically to pump water through a distinct oxyregulatory behaviour (Heisey and Porter, 1977; virtually closed filtering chamber (see Fig. 121 of Fryer, 1991), Kobayashi and Hoshi, 1984; Paul et al., 1997), have yielded which separates the feeding current into an inflow and outflow no indications to corroborate the ventilatory function of the section. The water current produced by the movements of these feeding current. The beating rate of the thoracic limbs stays thoracic limbs not only serves for feeding but is also thought constant when the oxygen availability decreases (Heisey and to be important in gas exchange (e.g. Gruner, 1993). Possible Porter, 1977; Colmorgen and Paul, 1995). In contrast, other sites for gas exchange are the inner walls of the carapace and oxyregulating water breathers such as fish or crabs increase the epipodites (Gruner, 1993), both structures lying in the their ventilation rate under hypoxic conditions to ensure a ventral region within the filtering chamber. The epipodites are constant rate of oxygen uptake. vesicular protuberances of the thoracic limbs, sometimes These findings suggest that the feeding current in Daphnia termed branchial sacs because of their presumed gill function. magna does not have an important role in respiratory gas The proposed gill function of the epipodites is a typical transport and that the entire body surface is involved in gas characteristic of branchiopod crustaceans (Barnes, 1969; exchange. The surface-to-volume ratio in this millimetre-sized Gruner, 1993). zooplankter is sufficiently large to permit integumentary However, morphological and physiological studies on respiration (Graham, 1988). The aim of the present study is to Daphnia spp. have cast doubt on the respiratory function of the assess the contribution of the feeding current to oxygen uptake 554 R. PIROW, F. WOLLINGER AND R. J. PAUL in the filter-feeder D. magna. We chose this cladoceran species tethered by its apical spine onto a coverslip using a small drop because Daphnia spp. have long been the focus of ecological of wax, allowing a lateral view of the animal. For flow-rate research (see, for example, Lampert, 1987) so that analysis, the apical spine was glued to a glass capillary tube comprehensive information about the biology of this genus is (1 mm o.d.) attached to a plastic cube (8 mm side length), available (e.g. Peters and DeBernardi, 1987). This report is part which allowed lateral, ventral and anterior views of the of a broader study addressing both the external and internal animal. The second antennae were immobilized using a small mechanisms of respiratory gas transport and the importance of drop of wax. This was necessary to prevent any flow currents haemoglobin in D. magna (R. Pirow, F. Wollinger and R. J. from the second antennae that could disturb the PO∑ Paul, in preparation). We studied ‘hypoxia-adapted’ animals, measurements or the flow-profile acquisition. The second which typically have an increased haemoglobin content antennae are important in locomotion, and the jerky (Kobayashi and Hoshi, 1982; Kobayashi et al., 1990). It has swimming movements they produce are likely to affect the been shown that D. magna, a common inhabitant of ponds and feeding and respiration processes by changing the catchment ditches, is physiologically well adapted to cope with area of fluid surrounding the animal. We did not explore these constraints such as oxygen deficiency (Kobayashi and Hoshi, effects further in this study. Tethered animals in the 1984; Paul et al., 1998), which could occur naturally in experimental chamber were always perfused with medium eutrophic habitats during the summer season. Haemoglobin- from the anterior and were allowed to acclimate to the rich D. magna are able to keep their rate of oxygen experimental conditions for 30 min. consumption constant down to an ambient oxygen tension of The inlet of the experimental chamber was connected using 8 mmHg (1.07 kPa; Kobayashi and Hoshi, 1984). polyvinyl chloride tubing to thermostatted glass vessels filled To clarify the role of the feeding current in oxygen uptake, with normoxic and anoxic culture medium. Two peristaltic we measured the oxygen partial pressure (PO∑) within the pumps perfused the chamber at variable PO∑ by mixing both feeding current and determined the PO∑ difference between the media at different ratios. The anoxic medium was produced by inflow and outflow. Furthermore, we recorded the flow pattern bubbling with normocapnic nitrogen using gas-mixing pumps of the feeding current and estimated the total rate of flow for (Wösthoff, Bochum, Germany). individual animals. A description of the flow rate was While performing optical PO∑ measurements, a special O2- necessary for further calculations using a model based on the sensitive dye (see below) was added to medium equilibrated Fick principle. PO∑ was determined using a phosphorimetric with different normocapnic O2/N2 gas mixtures. Two different method, whereas feeding current profiles were acquired using experiments were performed. In the first experiment, aimed at particle fluorimetry and digital image-processing. assessing qualitatively the sites of respiratory gas exchange in D. magna, PO∑ was measured at various positions close to the body surface after the perfusion had been stopped. A second Materials and methods experiment was performed to quantify oxygen extraction from Animals the feeding current. For this, the PO∑ difference between the Water fleas, D. magna Straus, were obtained from the inflow and outflow was measured immediately after the Staatliches Umweltamt, Nordrhein-Westfalen, Münster, perfusion of the chamber had been stopped. Germany. The animals were kept in 1 l glass beakers at approximately 20 °C under a 14 h:10 h L:D photoperiod using Arrangement for optical PO∑ measurements daylight fluorescent lamps. Experimental animals developed For the optical determination of PO∑, we used a special an increased blood haemoglobin content (Kobayashi and palladium–porphyrin compound [albumin-bound palladium- Hoshi, 1982; Kobayashi et al., 1990) induced by moderate meso-tetra (4-carboxyphenyl) porphine; Medical System hypoxia (30–40 % air saturation) resulting from bubbling Corp., Greenvale, NY, USA]. This compound phosphoresces nitrogen through the culture medium. The synthetic culture at 687 nm when excited at 416 or 523 nm (Lo et al., 1996), and medium (see Paul et al., 1997) had a pH of approximately 7.5 the phosphorescence can be quenched by molecular oxygen. and a conductivity of 3300 µScm−1. The animals were fed with This quenching results from collisions between the excited yeast and algae (Scenedesmus subspicatus) once daily. The phosphor and oxygen. The collisional or dynamic quenching animals used in experiments had a body length ranging from decreases the phosphorescence lifetime (Lakowicz, 1983), and 2.3 to 2.7 mm measured from the anterior part of the head to this characteristic is utilized for optical oxygen sensing the posterior edge of the carapace at the base of the apical (Vanderkooi and Wilson, 1986). The relationship between spine. phosphorescence lifetime τ (s) and oxygen tension PO∑ (mmHg) is described by the Stern–Volmer equation: Experimental conditions τ 0 τ All experiments were conducted at 20 °C in a special ÐÐτ = 1 + Kq 0PO2 , (1) thermostatted perfusion chamber (for details, see Paul et al., 1997) that allowed microscopic observation of single where τ0 is the lifetime in the absence of oxygen and Kq is the animals. The animals had to be tethered, which was achieved quenching constant (mmHg−1 s−1) (Pawlowski and Wilson, in two different ways. For PO∑ measurements, the animal was 1992). We used a phase-modulation method to determine the Role of the feeding current in O2 uptake in Daphnia magna 555 phosphorescence lifetime by measuring the phase relationship maximum pulse height) at a rate of 90 Hz, was mounted on between phosphorescence emission and repetitively pulsed the epifluorescence lamp adapter of an inverted microscope excitation. For a homogeneous sample, the phosphorescence (Zeiss Axiovert 100; Carl Zeiss, Oberkochen, Germany). The decay follows a single-exponential path, and the following reflector slider contained an excitation interference filter with equation can be applied to calculate the lifetime τ (s) from the a transmittance wavelength of 540±40 nm (peak wavelength phase-shift angle ϕ (degrees): ± full width at half-maximum transmission bandwidth), a dichroic mirror with a cut-off wavelength of 580 nm and a tanϕ =2πfτ , (2) long-pass emission filter with a cut-off wavelength of 590 nm. where f is the repetition frequency of the excitation light pulses The emitted phosphorescent light was collected by a (s−1). The derivation of equation 2 is given in the Appendix. photomultiplier (H5783-01; Hamamatsu Photonics, Japan). A phosphorescence microscope was used to measure PO∑ To avoid excitation light reaching the detector, a chopper optically (Fig. 1). A pulsed Xenon microsecond flashlamp (model 230; HMS Elektronik, Leverkusen, Germany) with a (F900; Edinburgh Analytical Instruments, Edinburgh, UK), 50 % open duty cycle was placed in front of the producing flashes with a duration of 3 µs (full width at half- photomultiplier. We reduced the entrance aperture of the photomultiplier to 1 mm in diameter to analyze only a small region of the microscopic phosphorescence image. A Phase Computer computer with a multi-function data-acquisition board Lock-in amplifier A/D info converter equipped with a timer circuit (DAS1602; Keithley Metrabyte, Taunton, MA, USA) checked the chopper status and triggered Timer PMT signal the flash lamp. The excitation flash occurred immediately circuit before the chopper opened the light path to the photomultiplier. The photomultiplier signal and the reference Reference signal signal supplied by the chopper were fed into a DSP lock-in amplifier (SR830; Stanford Research Systems, Sunnyvale, Photomultiplier CA, USA), which calculated the phase difference between the two signals. The phase-shift angle was recorded by the Chopper Chopper controller computer, and PO∑ was calculated using the constants τ0 and Emission Kq, which were determined in the calibration experiment pinhole described below.

Calibration of the phosphorescence microscope for PO∑ Emission measurements Excitation Flashlamp filter pinhole trigger signal To test the oxygen-sensitive dye, a calibration experiment Dichroic was carried out under the same conditions as in the mirror experiments involving Daphnia magna. The animal chamber was filled with dye-loaded medium (5 mg ml−1) equilibrated Xenon Excitation at different oxygen tensions. The measured phase response of flashlamp filter the dye solution showed a non-linear relationship with the oxygen tension (Fig. 2). To calculate values for the constants Animal chamber τ0 and Kq (see equation 1), we used a non-linear regression analysis (SigmaPlot; Jandel, San Rafael, CA, USA) yielding µ µ τ −1 −1 Fig. 1. Schematic diagram of the phosphorescence microscope used 561.0 s and 620.1 s for 0, and 327.0 mmHg s and −1 −1 for PO∑ measurements, showing the major components, the light path 340.5 mmHg s for Kq (2 independent calibrations). The and the electronic signal pathways. A microsecond Xenon flashlamp mean error (Gellert et al., 1965) in the measured phase-shift excites the oxygen-sensitive dye dissolved in the medium with light angle was ±0.46 °. Owing to the non-linear relationship, the pulses at 540±40 nm (peak wavelength ± full width at half-maximum accuracy of the PO∑ determination decreased at higher transmission bandwidth). The induced phosphorescent light passes oxygen tensions. The mean error increased from ±0.50 to through an emission filter with a cut-off wavelength of 590 nm ±4.41 mmHg (from ±0.07 to ±0.59 kPa) for calculated PO∑ before reaching a 1 mm diameter pinhole placed in the image plane. values of 5 and 25 mmHg (0.67 kPa and 3.33 kPa), × Thus, using an objective with a magnification of 10 , only light respectively. Above 25 mmHg (3.33 kPa), reliable P ∑ emanating from a 100 µm diameter region is imaged onto the O determination was not possible, because the pulse rate of the photomultiplier (PMT). The chopper in front of the photomultiplier is necessary to reject excitation light. The chopper status is checked flash lamp could not be increased up to the optimum pulse πτ −1 by a computer equipped with a timer card in order to trigger the flash frequency fopt given by fopt=(2 ) (Berndt and Lakowicz, lamp. The lock-in amplifier compares the photomultiplier signal with 1992). This is in contrast to the general applicability of the the reference signal supplied by the chopper controller and calculates oxygen-sensitive dye, which works at PO∑ values higher than the phase-shift angle used for PO∑ determination. 100 mmHg (13.33 kPa; Lo et al., 1996). 556 R. PIROW, F. WOLLINGER AND R. J. PAUL

20 board (DT2867; Data Translation, Marlboro, MA, USA). A two-step algorithm was implemented in the digital image- processing software (Colmorgen and Paul, 1995): binary contrast enhancement followed by a Boolean OR operation 15 which combined a given number of successive binary image frames. S-VHS video-tape recorders (AG-7355, Panasonic) were used to store the digitally processed track images as well as the original video signal generated by the SIT camera. (degrees) ϕ 10 Knowledge of the velocity distribution was essential for flow-rate determination. Track images (Fig. 3A) were acquired for lateral and anterior views of the animals and were further analyzed to obtain individual maps of velocity vectors using

Phase angle the image-analysis software package Optimas (Optimas Corp., 5 Seattle, WA, USA). For each track of the pattern, we determined velocity, direction and position. The velocity was calculated from the track length divided by the elapsed time. The coordinates of individual velocity vectors were converted 0 0510 15 20 25 30

Oxygen tension PO2 (mmHg) Fig. 2. Calibration of the oxygen-sensitive dye. The dye solution was equilibrated at different oxygen tensions (PO∑) and introduced into the gas-tight experimental chamber. The phase response of the dye was measured using the phosphorescence microscope with repetitively pulsed light at a pulse rate f of 90 Hz. The phase-shift angle ϕ showed a non-linear relationship with PO∑. Two independent calibrations (filled circles, open triangles) were performed, and the calibration constants were determined using a non-linear regression −1 analysis and the equation ϕ=tan [2πfτ0/(1+Kqτ0PO∑)] (see equations 1 and 2 for definition of symbols). The solid line was calculated from the averaged calibration values with τ0=581.6 µs and −1 −1 Kq=333.8 mmHg s (1 mmHg=0.133 kPa).

Flow-pattern analysis and flow-rate estimation B The water current produced by the activity of the thoracic limbs was quantified from the mean flow velocity multiplied by the flow cross-sectional area (Rouse, 1978). First, it was necessary to acquire and analyse the flow pattern. To image the flow, we added fluorescent microspheres (Fluoresbrite plain YG Microspheres; Polysciences, Eppelheim, Germany) with a particle diameter of 10 µm to the medium. These fluorescein-labelled latex particles showed a green fluorescence at 540 nm when excited at 458 nm. The stock solution, supplied as a suspension of 2.5 % solids in water, was diluted with medium by a factor of 3000, yielding a concentration of approximately 16×103 particles ml−1. The fluorescence image was scanned using a light-sensitive silicon- intensified target (SIT) camera (C2400; Hamamatsu, Japan) Fig. 3. Flow visualization and flow analysis. (A) The track image attached to the camera port of an inverted fluorescence showing the tracks of the fluorescent microspheres for the median microscope (Axiovert 10; Carl Zeiss, Oberkochen, Germany). plane of a Daphnia magna was generated by digital image- processing using an integration time of 2 s. Each track was analysed A mercury lamp was used for excitation, and the fluorescence to determine flow velocity and flow direction at the respective filter set consisted of an excitation filter with a transmittance position. (B) Mean velocity vectors were calculated for all area wavelength of 450–490 nm, a dichroic mirror with a cut-off elements arising from a partitioning of the median plane by an wavelength of 510 nm and a long-pass emission filter with a overlaid elliptical coordinate system. Each arrow indicates the mean cut-off wavelength of 520 nm. The tracks of the fluorescent direction of the flow as well as the mean distance covered in 1 s (data microparticles were imaged using an on-line image-processing from 16 track images). Scale bar, 1 mm. Role of the feeding current in O2 uptake in Daphnia magna 557 into an elliptical coordinate system (Fig. 3B), which facilitated within the inflow sector. Finally, this value was multiplied by the quantification of convergent flow patterns (see below). The the correction factor of 1.22. origin and shape of the coordinate system were defined by The geometrical model was further utilized to determine the fitting an ellipse to the ventral contour of each animal being flow cross-sectional area, which had a curved shape investigated. Adjacent radial lines enclosed an angle of 10 °, comparable with that of a piece of a shell (Fig. 4A). An and ellipses were spaced 0.15 mm apart. For each area element estimate of the cross-sectional area was calculated by of the partitioned plane, a mean velocity vector was calculated numerical integration taking the model variables described by vectorial computation (Fig. 3B). above and in Fig. 4B as well as a mean carapace aperture of The inflow section, selected for flow-rate determination, 0.40±0.03 mm (mean ± S.D., N=3), which was determined from showed a convergent laminar flow pattern, which was aligned frontal and ventral views of the animal. with the radial lines of the coordinate system. Owing to the In total, 76 track images were obtained from five animals, convergence of flow, the mean flow velocity had to be derived and 2805 particle tracks were analysed. Flow rates were from the velocity distribution across a curved section (Fig. 4A) determined separately for each animal at distances of perpendicular to all streamlines. Sophisticated techniques have 0.525 mm, 0.675 mm, 0.825 mm and 0.975 mm from the been developed to tackle the intricate task of analysing flow animal’s ventral edge. This guaranteed a fourfold flow fields generated by zooplankters with high spatial resolution in determination for each animal. the third dimension (Strickler, 1985; Fields and Yen, 1993; Stamhuis and Videler, 1995). However, the technical Determination of appendage beat rate prerequisites – simultaneous observation of two perpendicular The appendage beat rate (fA) of the experimental animals planes under laser sheet illumination – could not be easily was determined from video recordings using motion-analysis implemented in our experimental arrangement, which was software (Paul et al., 1997). designed primarily to allow simultaneous physiological measurements such as PO∑, pH, haemoglobin oxygen- Statistical analysis saturation and heart rate (Paul et al., 1997, 1998; R. Pirow, F. Statistical differences were assessed using a paired two- Wollinger and R. J. Paul, in preparation) using transmission or tailed t-test (P<0.05). Correlation between appendage beat rate epifluorescence illumination. and flow rate was assessed using linear regression analysis. We extracted the mean flow velocity from median-plane Data are expressed as means ± S.E.M., with N indicating the vector maps (Fig. 3B) taking into consideration how the three- number of animals examined unless stated otherwise. dimensional flow pattern was projected onto the image plane. Using an objective with a numerical aperture of 0.04 and a total magnification of 1.25, the depth of field was 3.0 mm, and most Results of the particle pathlines marking the inflow of the feeding Oxygen extraction from the feeding current current were therefore in focus. The two-dimensional To obtain initial indications of the sites of respiratory gas projection neither allowed the separation of pathlines lying in exchange in Daphnia magna, we measured the oxygen tension different object planes (z-planes) nor showed all tracks at real at ventral, posterior, dorsal and lateral positions close to the length. Converging pathlines not in parallel with the plane of body surface. There was little change in the PO∑ of the view were projected with shorter lengths. Accordingly, a single perfusion medium at posterior, dorsal or lateral positions mean velocity vector in the median-plane vector map (Fig. 3B) (Fig. 5). However, a pronounced reduction in PO∑ was found comprised the velocity information of all particle pathlines at the ventral side of the animal, indicating a significant oxygen located along the z direction. However, without further flux into the animal in this region. Medium which had passed correction, this vector could not be utilized as a measure of the through the ventral space enclosed by the carapace showed a mean flow velocity at its respective position along the flow reduced PO∑ (Fig. 5). direction. Further experiments were directed towards a quantitative The characteristics of the inflow pattern made it possible to analysis of the O2 uptake via the feeding current, which retrieve this essential information by using a simplified required the determination of the PO∑ difference between the geometrical model. This model, which described the flow inflow and outflow currents. To ensure a high level of accuracy pattern extending in the z direction, assumed a convergent V- in oxygen tension measurements, the PO∑ of the perfusion shaped inflow with a lateral opening angle α0 of 90 ° and a medium was reduced to 16.2 mmHg (2.16 kPa). It was uniform velocity distribution (Fig. 4B). Both assumptions necessary to check that the animals’s limb movements were were proved to be reasonable simplifications by analysing the not affected by exposure to this low ambient oxygen tension. flow patterns obtained from anterior views of the animals. During 10 min after switching from normoxia to hypoxia, the Using the calculations detailed in Fig. 4B yielded a correction mean fA did not change significantly (before factor of 1.22. On the basis of this model, an estimate of the 311.6±38.5 beats min−1; after 300.9±33.0 beats min−1; paired mean flow velocity could be derived from median-plane vector two-tailed t-test: t=0.928, d.f.=4, P=0.405, N=5). The inhalant maps (Fig. 3B). Initially, a mean flow velocity was calculated feeding current had a PO∑ of 16.2±0.4 mmHg (2.16±0.05 kPa, from those velocity vectors lying on the same elliptical arc N=5), whereas the exhalant PO∑ had decreased significantly 558 R. PIROW, F. WOLLINGER AND R. J. PAUL

Fig. 4. (A) A three-dimensional graphical representation showing the A outline of a 2.5 mm long Daphnia magna plotted in the median plane and the inflow of the feeding current. As the feeding current was characterized by convergent flow boundaries, flow cross sections perpendicular to all stream lines had a curved shape (shell segments). 90° The flow rate was estimated separately for each of these shell segments from the product of mean flow velocity and flow cross- Flow 0.4 mm y sectional area. Shell segments were spaced 0.15 mm apart with a z 1 distance of 0.525 mm between the inner shell and the ventral edge of 2 the animal. Mean flow velocities were determined from median- 1 mm 3 Shell segment 4 number plane images (see Fig. 3), whereas shell segment areas were x calculated numerically by integration, assuming a carapace aperture Median plane of 0.4 mm and a V-shaped inflow with a lateral opening angle α0 of 90 °. (B) Derivation of the velocity correction factor. The plane of view is at 90 ° to the animal’s median plane, indicated as a dashed line in A. Converging streamlines show the inflow pattern, whereas B circular arcs represent the shell segments penetrated perpendicularly by the streamlines. Underestimation of flow velocity occurs when flow vectors of magnitude vi, originating from different circular arcs at various lengths and orientations, are projected as vi′ onto the same - Opening angle position in the image plane, resulting in an average flow velocity vi′. v α0 Flow 2 v1 The real velocity v0 in the median plane, required for flow - velocity v1l1=v2l2=constant estimation, can be inferred from vi′ as shown. Integration yielded a correction factor of 1.22, which was based on the following Carapace assumptions: flow vectors originating from the same circular arcs are l1 of equal length; the product of flow velocity and arc length (vi×li) is constant; and lateral opening angle α0 is 90 °. l2 Arc length ranged from 4.34±0.23 to 2.04±0.11 mm2 (paired two-tailed t- Real Correction factor test: t=16.450, d.f.=4, P<0.0001, N=5) for the outer and inner length, vi for α0=90° shell segments, which were 0.975 and 0.525 mm away from the n animal’s ventral edge, respectively. The interindividual ′ 1 ′ v = Σvi n i=l variations in cross-sectional area were small in comparison with π ′ 2v0 4 2α α those of the flow velocity data (Fig. 6B). The area variations v = π π ∫ cos d α 4 must have resulted exclusively from differences in the median v 1 0 v′=v0(π+0.5) inflow angle and/or the animal’s shape since other geometrical Projected factors, such as the lateral inflow angle and the carapace ′ v =1.22v′ length, vi 0 aperture, were entered into the computation as constants. ′ 2α vi=v0cos From the outer to the inner shell segment, the flow was slightly accelerated from 226.0±58.4 to 277.4±85.1 µms−1 (paired two-tailed t-test: t=–1.377, d.f.=4, P=0.241, N=5). The (paired two-tailed t-test: t=21.162, d.f.=4, P<0.0001, N=5) to variability among individuals was large (Fig. 6B). The flow rate a value of 3.2±0.4 mmHg (0.43±0.05 kPa, N=5). Daphnia across different shell segments varied from 0.9 to 6.5 ml h−1 (Fig. magna was obviously able to withdraw a considerable amount 6C). This wide range was largely due to the differences in flow of oxygen from the feeding current despite the hypoxic velocity among individuals. Within an individual, variation in ∑ conditions imposed by the measuring procedure. The PO flow rates was much smaller. An interindividual comparison difference between the inflow and outflow currents was revealed a positive correlation between appendage beat rate (fA) 13.0±0.6 mmHg (1.73±0.08 kPa, N=5) at an ambient oxygen and flow rate (Fig. 7). At almost the lowest mean individual fA tension of 16.2±0.4 mmHg (2.16±0.05 kPa). of 310.4 beats min−1, the flow rate was 1.2 ml h−1; at higher values of f of 393.0 beats min−1 and 460.7 beats min−1, flow Estimation of the flow-rate of the feeding current A rates were 4.5 ml h−1 and 5.2 ml h−1, respectively. The flow rate of the feeding current was quantified for individual animals from the product of mean flow velocity and flow cross-sectional area. Both variables were determined at Discussion four different distances from the body along the inflow current. Main region for oxygen uptake The flow cross-sectional area of the shell segments decreased In the filter-feeder D. magna, we found that the water current with decreasing distance to the ventral edge of the animal, produced by the activity of the thoracic limbs is involved in which reflected the convergence of flow (Fig. 6A). Values gas exchange in addition to food uptake. External PO∑ Role of the feeding current in O2 uptake in Daphnia magna 559 measurements in the ambient medium revealed O2 depletion in the dorsal region. Further studies are needed to determine the the ventral body region but essentially no change at posterior, respiratory structures responsible for gas exchange in this lateral and dorsal positions. This finding indicates an oxygen crustacean species. flux into the animal across the ventral body surface. Hoshi and Takahashi (1981) found, in reoxygenation experiments Flow rate of the feeding current utilizing the blood haemoglobin of Daphnia magna as an The flow rate of the feeding current in D. magna was internal oxygen probe, that haemoglobin was recharged fastest quantified to determine whether oxygen extraction from the in the dorsal vessel. However, they made no measurements in feeding current is sufficient to supply the total oxygen the ventral or posterior regions. In the genus Daphnia, the requirement of the body. The method used in the present study haemolymph moves from anterior to posterior in the ventral allowed flow-rate estimations to be made for single animals on part of the trunk, as well as within the double-walled carapace, the basis of flow-velocity and flow-geometry measurements. and then enters the dorsal vessel to flow in the opposite For a 2.5 mm long D. magna, our estimated flow rate was in direction (see Hérouard, 1905). Recent studies in D. magna, the range 1.2–5.2 ml h−1, which agrees with results from utilizing a method to image haemoglobin oxygen-saturation ecological studies of the clearance rates of feeding D. magna. (R. Pirow, F. Wollinger and R. J. Paul, in preparation), showed The clearance rate is the flow rate multiplied by the food that the haemolymph becomes oxygenated while passing retention factor. At maximum retention factors, close to 1.0, through the ventral body region, before it subsequently enters Heisey and Porter (1977) reported clearance rates of 3.5– 4.5 ml h−1 for a 2.34 mm D. magna, Porter et al. (1982) reported 4mlh−1 for a 2.7 mm animal, and Brendelberger (1991) gave A )

2 6 A 5 4 3 2 1

Cross-sectional area (mm 0 0.6 )

-1 B 0.5 0.4 0.3 B 0.2 0.1 Mean velocity (mm s Mean velocity 0 7

) C

-1 6 5 4 3 2 Flow rate (ml h Flow 1 0 0.525 0.675 0.825 0.975 Distance to the animal (mm) Fig. 5. Example of measured oxygen tension (PO∑, values in mmHg) at various positions close to a specimen of Daphnia magna observed Fig. 6. Flow characteristics of the feeding current. Flow cross- in lateral (A) and ventral (B) views. The perfusion medium had a PO∑ sectional area (A), flow velocity (B) and flow rate (C) were of 25 mmHg (3.33 kPa). Oxygen depletion from the medium determined at four different positions along the inflow of the feeding occurred in the ventral region in the outflow of the feeding current. current, at distances of 0.525 mm, 0.675 mm, 0.825 mm and No changes in PO∑ could be detected within measurement accuracy 0.975 mm from the animal’s ventral edge. Each symbol represents a at the posterior, dorsal and lateral positions. Scale bars, 1 mm. value for a single animal. 560 R. PIROW, F. WOLLINGER AND R. J. PAUL

7 To assess the importance of the feeding current to whole- animal oxygen uptake in a quantitative manner, the measured ) 6 -1 PO∑ difference can be compared with that predicted from a 5 model calculation. For example, this could establish whether (ml h

w 4 ¥ the oxygen demands of the animal are met exclusively from V 3 the oxygen withdrawn from the feeding current. In a converted 2 form, the Fick principle of convection (Piiper et al., 1971) allows an estimation of the difference in oxygen partial

Flow rate, Flow 1 ∆ ∑ (mmHg) from the total rate of oxygen pressure PO . 0 consumption M ∑ (mol h−1), the oxygen solubility β of the 280 300 320 340 360 380 400 420 440 460 480 500 O . medium (mol l−1 mmHg−1) and the flow rate V (ml h−1): Appendage beat rate, f (min-1) w A . . M Fig. 7. Relationship between inflow current flow rate (V ) and ∆P = ÐÐÐÐO. 2 . (3) w O2 βV appendage beat rate (fA). Linear regression analysis yielded a w positive correlation (y=0.026x−6.313, r2=0.86, P=0.024, N=5). Each circle represents data from one animal. Appendage beat rate was In normoxic conditions and at 20 °C, the rate of oxygen monitored continuously during flow-pattern acquisition. Flow rates consumption of a 2.5 mm D. magna is in the range . −1 and appendage beat rates are given as means ± S.D. (Vw, fourfold 30–40 nmol h (see Bohrer and Lampert, 1988; Paul et al., determination, see Fig. 6C; fA, N=75 fA values). 1997; dry mass versus body length relationship from Porter et −1 −1 al., 1982). Taking an O2 solubility of 1.81 µmol l mmHg (at 20 °C and 2 ‰ salinity; Gnaiger and Forstner, 1983) and a flow rate ranging from 1.2 to 5.2 ml h−1 (this study), then the Fick 4.4 ml h−1 for a 2.5 mm D. magna feeding on Scenedesmus principle yields a PO∑ difference of 3.2–18.4 mmHg (0.43– acutus. 2.45 kPa). The experimental PO∑ difference of 13.0 mmHg Oxygen extraction from the feeding current (1.73 kPa), measured during hypoxic rather than normoxic conditions, lies within the upper half of the estimated ∆PO∑ Oxygen uptake via the feeding current was studied in range. We are aware that the ambient conditions at which the hypoxia-adapted D. magna at 10.5 % air saturation (16.2 mmHg compared data (experimental ∆PO∑ and model calculation) or 2.16 kPa). Under these conditions, where the rate of oxygen were obtained are not the same. However, a low ambient PO∑ uptake is still constant in haemoglobin-rich animals (Kobayashi . of 16.2 mmHg (2.16 kPa) was found to reduce neither MO∑ and Hoshi, 1984) and where we found fA to be unchanged, we (Kobayashi and Hoshi, 1984) nor fA (this study) and therefore found a PO∑ difference of 13.0 mmHg (1.73 kPa) between the . Vw in hypoxia-adapted D. magna. Thus, the comparison inhalant and exhalant part of the feeding current. The extraction suggests that (hypoxia-adapted) D. magna obtained most of coefficient, i.e. the ratio of the amount of O removed to the 2 their required oxygen from the feeding current. amount of O available (Dejours, 1981), had a value of 0.80. 2 This finding raises the question of whether zooplankters of This value indicates that oxygen extraction from the feeding comparable body size really need convective processes for current was highly efficient under these hypoxic conditions. oxygen supply. Animals with a body size in the single- Furthermore, the results imply that the diffusion resistance via millimetre range are distinguished from their larger the ventral integument must be low, and that the P ∑ in the O companions by a larger surface-area-to-volume ratio, and it is arterialized haemolymph is lower than that of the exhalant part generally assumed that diffusion alone is sufficient to satisfy of the feeding current (3.2 mmHg or 0.43 kPa). At these very their oxygen requirements. Paul et al. (1997), however, showed low haemolymph P ∑ values, internal oxygen transport can be O that internal convection is of vital importance in D. magna sustained only by using a respiratory pigment with a strong under hypoxic conditions. To assess the role of external oxygen-binding affinity. Indeed, it has been found that the convection, a simple geometrical model can be utilized. On the haemoglobin of hypoxia-adapted D. magna satisfies this basis of Fick’s first law of diffusion, the following equation requirement. In vivo studies by Kobayashi and Tanaka (1991) permits a calculation of the PO∑ gradient that exists in stagnant revealed that haemoglobin from the more venous dorsal water surrounding an oxygen-consuming spherical body haemolymph region was still half-oxygenated at an ambient P ∑ O (Seymour and Bradford, 1995): of 15 mmHg (2.00 kPa), which is very similar to the ambient . oxygen tension used in our experiments. Kobayashi et al. ∆ MO2 1 −1 PO = ÐÐÐÐÐ ÐÐ ÐÐ , (4) (1988) studied the in vitro characteristics of D. magna 2 4πDβ ΂ rs rx ΂ haemoglobin at 20 °C and reported a half-saturation value (P50) of 1.6 mmHg (0.21 kPa), which is lower than the exhalant PO∑ where rs is the radius of the sphere (m), rx is the distance from of the feeding current. Both findings suggest that D. magna the sphere centre to a position outside the sphere (m), β is the haemoglobin becomes more than half-saturated during severe oxygen solubility of the medium (mol l−1 mmHg−1; see above), hypoxia and can thus contribute to internal oxygen transport and D is the diffusion constant for oxygen (2.04×10−5 cm2 s−1 even at very low oxygen tensions in the haemolymph. at 20 °C and for pure water: Himmelblau, 1964). Taking a rate Role of the feeding current in O2 uptake in Daphnia magna 561

. −1 of oxygen consumption MO∑ of 35 nmol h (see above), a PO∑ oxygen partial pressure (mmHg) sphere of radius (rs) 1 mm and rx→∞, equation 4 yields a PO∑ rs radius of a sphere (m) difference of 209.5 mmHg (27.9 kPa) between the surface of rx distance from the centre of a sphere to a the sphere and the ambient medium. Accordingly, pure position x (m) diffusion would not cover the oxygen requirement even under t time (s) normoxic conditions (155.2 mmHg or 20.69 kPa). An T pulse interval (s) . −1 immobile, spherical body and a stagnant environment are, of Vw flow rate (ml h ) −1 course, assumptions far removed from natural conditions, but v0 real flow velocity (m s ) this calculation suggests that external convection becomes v1, v2 velocities of flow vectors originating from the increasingly necessary under conditions of progressive circular arcs 1 and 2 (m s−1) −1 hypoxia (see also Daykin, 1965). vi velocity of a flow vector (m s ) v′i velocity of a flow vector projected onto the animal’s median plane (m s−1) - −1 Appendix vi mean velocity of projected flow vectors (m s ) Derivation of the equation tanϕ=2fτ x, y, z Cartesian coordinates (non-dimensional) The oxygen-sensitive dye is repetitively excited with light α angle between a streamline and the animal’s pulses at the pulse interval T (s). The periodic properties of the median plane (degrees) phosphorescence signal allow the application of a Fourier α0 lateral inflow angle (degrees) − − analysis. The information concerning the phase-shift angle ϕ β oxygen solubility of the medium (mol l 1 mmHg 1) is derived from the phase value of the fundamental τ phosphorescence lifetime (s) (modulation) frequency according to tanϕ=b/a, where a and b τ0 phosphorescence lifetime in the absence of are the Fourier coefficients (Marmarelis and Marmarelis, oxygen (s) 1978): ϕ phase-shift angle (degrees) ω circular frequency (s−1) T a = ÐÐ2 ͐ I(t)cos(ωt)dt , (A1) T 0 The technical assistance of Ina Buchen is gratefully acknowledged. We thank Martina Fasel for drawing the T Daphnia magna pictures. The study was supported by the b = ÐÐ2 ͐ I(t)sin(ωt)dt , (A2) T 0 Deutsche Forschungsgemeinschaft (Pa 308/4-2). where −t/τ References I(t)=I0e , (A3) Barnes, R. D. (1969). Invertebrate Zoology. Philadelphia: Saunders. π Berndt, K. W. and Lakowicz, J. R. (1992). Electroluminescent ω = ÐÐ2 = 2πf . (A4) T lamp-based phase fluorometer and oxygen sensor. Analyt. Chem. Physiol. 201, 319–325. Equation A3 describes the decay of phosphorescence intensity Bohrer, R. N. and Lampert, W. (1988). Simultaneous measurements I (arbitrary units) at time t (s), and equation A4 gives the of the effect of food concentration on assimilation and respiration circular frequency ω (s−1). Both Fourier coefficients are in Daphnia magna Straus. Funct. Ecol. 2, 463–471. Brendelberger, H. (1991). Filter mesh size of cladocerans predicts calculated by integration over the pulse time interval (0...T), ϕ π τ retention efficiency for bacteria. Limnol. Oceanogr. 36, 884–894. yielding the equation tan =2 f . 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 List of symbols image processing. Comp. Biochem. Physiol. 111A, 583–595. a, b Fourier coefficients (non-dimensional) Daykin, P. N. (1965). Application of mass transfer theory to the D diffusion constant for oxygen (cm2 s−1) problem of respiration of fish eggs. J. Fish. Res. Bd Can. 22, f repetition frequency of excitation light pulses (s−1) 159–171. −1 fA appendage beat rate (beats min ) Dejours, P. (1981). Principles of Comparative Respiratory −1 Physiology. Amsterdam, New York, Oxford: Elsevier/North- fopt optimum pulse frequency (s ) i index Holland Biomedical Press. Fields, D. and Yen, J. (1993). Outer limits and inner structure: the I(t) decay of phosphorescence intensity (arbitrary 3-dimensional flow field of Pleuromamma xiphias (Calanoida: units) at time t Metrinidae). Bull. Mar. Sci. 53, 84–95. −1 −1 Kq quenching constant (mmHg s ) Fryer, G. (1991). Functional morphology and the adaptive radiation l1, l2 circular arc length (m) of the Daphniidae (Branchiopoda: Anomopoda). Phil. Trans. R. . −1 MO∑ rate of oxygen consumption (mol h ) Soc. Lond. B 331, 1–99. n number of flow-velocity values Gellert, W., Küstner, H., Hellwich, M. and Kästner, H. (1965). 562 R. PIROW, F. WOLLINGER AND R. J. PAUL

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