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First published online November 19, 2004
Journal of Experimental Biology 207, 4393-4405 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01318

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Crater landscape: two-dimensional oxygen gradients in the circulatory system of the microcrustacean Daphnia magna

R. Pirow^*,, C. Bäumer and R. J. Paul

Institut für Zoophysiologie, Westfälische Wilhelms-Universität, Hindenburgplatz 55, 48143 Münster, Germany

View larger version (10K): [in a new window]   Fig. 1. Schematic diagram of the microscopic set-up used for PO2 imaging, showing the major components, the light path (dotted lines) and the pathways for image data (broken arrows) and signal data (solid arrows).

View larger version (36K): [in a new window]   Fig. 2. Timing of phosphorescence image acquisition. The curve shows the exponential decay of phosphorescence intensity It after an excitation flash at time t=0. The image intensifier is turned on at delay time td for the gate period T. The grey-shaded area indicates that portion of phosphorescence light (Z) that is integrated on the CCD array during T. I0, initial phosphorescence intensity immediately after the excitation flash.

View larger version (46K): [in a new window]   Fig. 3. (A) Sets of phosphorescence intensity images of a 1.4 mm Hb-poor Daphnia magna taken at seven different delay times (td) under 0.5% (top) and 19.6% air saturation (bottom), respectively. From all phosphorescence images, a background image acquired at td=3000 µs has been subtracted. For orientation, an image of the animal taken under transmission illumination is shown at the left. In the top sequence, the limbs appear in sharp contours because the animal had stopped its limb beating activity owing to the low oxygen conditions. Note the strong phosphorescence signal in the region of the shell gland (arrow). (B) For the heart region, i.e. the image areas marked by white ellipses in A, the integrated phosphorescence intensity (Z) was plotted against td. Exponential decay curves (solid lines) were fitted according to Equation 3 to the data that were measured at 0.5% (filled circles) and 19.6% air saturation (open circles). a.u., arbitrary units.

View larger version (28K): [in a new window]   Fig. 4. Example of a calibration experiment of Oxyphor R2 in the haemolymph of a Hb-poor Daphnia magna. A circular drop of the dye-loaded haemolymph sample was equilibrated at seven different ambient oxygen tensions (PO2amb=0, 1.02, 2.05, 3.07, 4.09, 5.12, 6.14 kPa; 0–30% air saturation). The phosphorescence lifetime () of the sample was imaged and is depicted in pseudo-colour presentation (top row). Pixel positions outside the sample image were masked out by setting pixel intensity to black colour. The relationship between the reciprocal of the mean of each image and PO2amb (open circles) was then analyzed by linear regression analysis using the Stern–Volmer equation (–1=0–1 + kqPO2amb), which yielded the estimates of the lifetime at zero-oxygen concentration (0) and of the quenching constant (kq). 0 and kq were used to transform phosphorescence lifetime images (top row) into oxygen partial pressure (PO2) images (bottom row). The numbers below the gallery indicate the mean value of each PO2 image.

View larger version (14K): [in a new window]   Fig. 5. Calibration of Oxyphor R2 in the haemolymph of D. magna. The data show the reciprocal of the phosphorescence lifetime plotted against oxygen partial pressure PO2. (A) Comparison of individual calibrations in Hb-poor (open circles, N=3) and Hb-rich haemolymph (filled triangles, N=3). Individual calibrations were analyzed by linear regression analysis using the Stern–Volmer equation (Equation 2) and the regression parameters averaged to obtain a mean regression line (broken line). (B) Pooled data (means ± S.D., N=7) of further calibrations with Hb-poor and Hb-rich haemolymph. The linear regression line (broken line) was used to convert in vivo phosphorescence lifetime images into PO2 images.

View larger version (65K): [in a new window]   Fig. 6. Two-dimensional distributions of oxygen partial pressure (PO2) in the haemolymph of a 1.4 mm Hb-poor (top) and a 1.7 mm Hb-rich Daphnia magna (bottom), respectively, under different ambient oxygen partial pressures (PO2amb). The PO2amb values are indicated below each gallery. Note the different scaling of PO2 in the pseudo-colour images as indicated by the colour bars at the top. During image acquisition, the optical focus was set to the median plane of the animal. For orientation, an image of the animal taken under transmission illumination is shown at the left side of each gallery.

View larger version (96K): [in a new window]   Fig. 7. Oxygen partial pressure (PO2) distribution in the haemolymph in the median plane of Daphnia magna. Left: 1.7 mm Hb-rich animal at an ambient oxygen partial pressure (PO2amb) of 1.7 kPa. Right: 1.4 mm Hb-poor animal at a PO2amb of 5.1 kPa. Note the marked differences in the steepness of the PO2 gradients between both animals. The orientation of the animal with respect to the viewer is indicated by the positional information and by the small sketch in front left.

View larger version (43K): [in a new window]   Fig. 8. Responses in internal oxygen partial pressure (PO2) and heart rate (fH, open squares) of differently sized (small, medium, large: 1.5±0.12, 2.5±0.3, 3.3±0.13 mm) Hb-poor and Hb-rich Daphnia magna to decreasing ambient oxygen partial pressures (PO2amb). The haemolymph PO2 is given for the carapace lacuna (loading PO2, open circles), the dorsal lacuna (open diamonds), the heart region (open triangles), and the central body region (unloading PO2, closed triangles). The dotted lines represent lines of identity where PO2=PO2amb.

View larger version (26K): [in a new window]   Fig. 9. Involvement of Hb in circulatory oxygen transport. (A) The range between the loading and unloading oxygen partial pressure (PO2; bold solid lines) of medium-sized Hb-poor and Hb-rich animals under different ambient oxygen tensions (PO2amb) was mapped onto the oxygen equilibrium curve of Hb (thin solid lines). The ordinate shows the concentration of chemically bound oxygen ([O2]), which is the product of the fractional oxygen saturation of Hb (S) and haem concentration ([Haem]; Hb-poor: 115 µmol l–1, Hb-rich: 666 µmol l–1). Oxygen equilibrium curves (S vs. haemolymph PO2) were calculated according to the Hill equation assuming a cooperativity coefficient of 1.6 (Kobayashi et al., 1988) and half-saturation oxygen tensions of 1.0 kPa (Hb-poor) and 0.5 kPa (Hb-poor; Zeis et al., 2003a), respectively. (B) Differences between the loading and unloading [O2] in relation to PO2amb for Hb-poor (top) and Hb-poor animals (bottom). Black and white bars refer to the physically dissolved and chemically bound portions of transported oxygen, respectively. A value of 12.3 µmol l–1 kPa–1 was assumed as solubility coefficient for oxygen in haemolymph (Pirow and Buchen, 2004).