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Benefits of haemoglobin in the cladoceran crustacean Daphnia magna

R. Pirow^*, C. Bäumer and R. J. Paul

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

View larger version (45K): [in a new window]   Fig. 1. Microscopic arrangement for the simultaneous measurement of haemoglobin (Hb) oxygen-saturation, heart rate, appendage beating rate and NADH fluorescence. The heart region of the animal was transilluminated with green light in the wavelength range 504–608 nm. Light that passed through this region was analysed spectrally to determine Hb oxygen-saturation. Using infrared transillumination at wavelengths above 780 nm, the periodic movements of the heart and the thoracic appendages were monitored by a light-sensitive CCD camera, and digital motion analysis was used to calculate heart and appendage beating rate. For alignment purposes, a longpass filter (LP 520) in front of the sensitive CCD camera attenuated, but did not completely block, the bright detection light beam used for Hb measurements. The central part of the animal was epi-illuminated with ultraviolet light at 365 nm to excite the pyridine nucleotides in the tissues, and the fluorescence image in the wavelength range 450–490 nm was collected by a light-sensitive SIT camera. The optical paths are visualized by different grey shadings. Dashed lines indicate light reaching the respective detector without having effects on the respective measurement. BP, bandpass filter; BS, beam splitter; DBS, dichroic beam splitter; f, focal length; Hal, halogen lamp; LG, light guide; LP, longpass filter; and ß, absorption bands of oxy-Hb.

View larger version (47K): [in a new window]   Fig. 2. (A) A sequence of absorption spectra A() was obtained from the heart region of haemoglobin (Hb)-rich (above) and Hb-poor (below) animals during a normoxic/anoxic transition experiment. While progressing from the oxygenated to the deoxygenated state, Hb changed its absorbance characteristics, and this can be followed by the disappearance of the maxima at 540 and 576 nm and by the appearance of a broad peak around 560 nm. Note that, because of the difference in Hb concentration, the spectra of the two groups are scaled differently. In addition, the spectra have been displaced vertically by a constant factor to demonstrate better the changes in absorbance. Because of the lower Hb concentration, the spectra of Hb-poor animals appeared to be tilted to the right, which indicates the presence of non-Hb components with higher absorbances in the shorter wavelength range. Moreover, the unequal spacing of spectra reflects fluctuations in the optical properties of the body region analyzed, which were caused by slight body movements of the animal. These effects made it impossible to use reference spectra of purified Hb solutions for the determination of Hb saturation. Instead, in vivo spectra, Ho() and Hd() for oxy-Hb and deoxy-Hb, respectively, obtained under normoxic and anoxic conditions were used for spectral comparisons that permitted the determination of Hb oxygen-saturation and (B) the calculation of corrected absorption spectra A'() by applying equations 1 and 2 (see text for details).

View larger version (69K): [in a new window]   Fig. 3. Localization of NADH fluorescence in Daphnia magna. To obtain satisfactory fluorescence images, the excitation light was concentrated on the central portion of the animal. The body region in focus comprised the mandible (Mand), the base of the large antenna (A2M) and the bases of the thoracic limbs (1–5). Switching from normoxia (A) to anoxia (B) resulted in a strong increase in fluorescence intensity, indicating an accumulation of mitochondrial NADH. The difference image (C) reveals that this increase in intensity derived from various groups of muscles associated with the respective appendages (D).

View larger version (22K): [in a new window]   Fig. 4. Temporal organization of the experiment. The animal was exposed to a controlled, gradual transition from normoxia to anoxia (A) while haemoglobin (Hb) oxygen-saturation (B), heart rate (C), appendage beating rate (D) and NADH fluorescence intensity (E) were measured continuously. At very low oxygen tensions, the thoracic appendages showed an irregular rather than a periodic temporal pattern of movement. Therefore, appendage beating rate was counted manually every 30 s (as indicated by the filled circles). NADH fluorescence always showed a linear decline in intensity which, at a certain low level of oxygen tension, was superimposed by the hypoxia-induced increase in mitochondrial NADH content. The data were therefore trend-corrected by calculating a linear regression line (dashed line) for the initial 12–18 min of the experiment during which Hb oxygen-saturation remained unchanged.

View larger version (18K): [in a new window]   Fig. 5. (A) Absorption spectra Ar() and Ap() of haemolymph samples obtained from haemoglobin (Hb)-rich and Hb-poor animals (N=5 each), respectively, were measured under oxygenated conditions in a counting chamber with an optical pathlength of 0.02 cm using water as a reference. The absorption maxima at 540 nm and 576 nm indicate the presence of Hb in both haemolymph samples. Data are given as means ± S.E.M. (B) Plotting Ar() against Ap() showed that data pairs within the wavelength range 550–588 nm fall on a straight line (Ar=k1Ap+k0, with k0=-0.0526 and k1=7.015, r2=0.993, N=39). Data pairs in the lower wavelength range deviated from this line, indicating that components other than haemoglobin contributed to the absorbance of the haemolymph sample in a wavelength-dependent manner.

View larger version (25K): [in a new window]   Fig. 6. Responses of appendage beating rate, NADH fluorescence intensity, heart rate and haemoglobin (Hb) oxygen-saturation of normoxia-acclimated Hb-poor (left) and hypoxia-acclimated Hb-rich Daphnia magna (right) to decreasing ambient oxygen partial pressures (PO2amb). Each data point represents the mean ± S.D. (N=6). Solid lines represent either plateaus in the physiological data or mean regression lines obtained by averaging the variables of individual regression lines. Dashed lines and the respective values represent critical PO2amb values, marking the significant decrease in appendage beating rate, the intersection of two solid lines or the PO2amb at which 50 % of the Hb was saturated. The only exception concerns the critical PO2amb in C (see Fig. 7). NADH fluorescence intensity was trend-corrected (see Fig. 4E) and normalized to 100 %. Sigmoidal curves were generated by nonlinear regression analysis using the Hill equation, which yielded the PO2amb at which 50 % of Hb was saturated, and the Hill coefficient n as a measure of sigmoidity (r2>0.990 for both curves). The two arrows on the abscissa indicate the PO2amb at which the animals were reared.

View larger version (20K): [in a new window]   Fig. 7. Detailed representation of the data of Fig. 6A–D. The appendage beating rate (fA) profiles (dotted lines) of haemoglobin (Hb)-poor and Hb-rich animals appeared to be aligned parallel to each other until the reduction in ambient oxygen partial pressure (PO2amb) from 3.3 to 2.6 kPa (mean 2.95 kPa) induced significantly diverging changes in fA (Hb-poor, fA=+10.23±12.70 min-1; Hb-rich, fA= -6.19±5.06 min-1; unpaired two-tailed t-test: t=2.94, d.f.=10, P=0.01; means ± S.D., N=6). Similar diverging changes were initiated in NADH fluorescence intensity INADH (solid lines) when the PO2amb was reduced from 5.3 to 4.2 kPa (mean 4.75 kPa) (Hb-poor INADH=+1.30±0.80 min-1 versus Hb-rich INADH=+0.42±0.44 min-1; unpaired two-tailed t-test: t=2.36, d.f.=10, P=0.04; means ± S.D., N=6). This divergence in INADH extended over the PO2amb range from 4.75 to 1.32 kPa (shaded area), where INADH of Hb-rich animals appeared to stabilize at 103 %, whereas that of Hb-poor animals rose from 104 to 123 %. Note that, in Hb-poor animals, the increase in fA at 2 kPa coincided with the increase in INADH. Arrows indicate significantly diverging responses in the respective variables. The standard deviations of the curves have been omitted for clarity.

View larger version (31K): [in a new window]   Fig. 8. Comparison of tissue-specific (solid line, NADH fluorescence intensity of the limb muscles; this study) and whole-animal (dotted line, lactate content; dashed line, rate of oxygen consumption) responses of haemoglobin (Hb)-poor (A) and Hb-rich Daphnia magna (B) to different ambient oxygen partial pressures (PO2amb). Lactate determination was performed using animals 1.7–2.0 mm long incubated at different values of PO2amb for 1 h at 22°C (Usuki and Yamagushi, 1979). The rate of oxygen consumption (dashed line) was measured in animals 2.5 mm long at 20°C using a closed respirometry system (Kobayashi and Hoshi, 1984).

View larger version (15K): [in a new window]   Fig. 9. The in vivo saturation curve describes the relationship between haemoglobin (Hb) oxygen-saturation, measured in the circulatory system of an animal, and the oxygen partial pressure of the ambient medium (PO2amb). The shape and position of the in vivo saturation curve are highly variable because they depend not only on the intrinsic oxygen-binding properties of Hb but also on the respective position in the circulatory system, on the haemolymph concentration of Hb and on the systemic and metabolic characteristics of the animal. A simple model of the circulatory system (A) demonstrates the positional effect and the influence of Hb concentration. Oxygenated haemolymph leaves the respiratory organ and enters the tissue compartment where, at the proximate position, the in vivo saturation curve is measured (solid lines in B and C). Because oxygen is released from Hb and transferred to the metabolizing tissue, the in vivo saturation curve measured at the distant position appears to be shifted to the right (dotted lines). This rightward shift is more pronounced at lower (B) than at higher (C) Hb concentrations.

View larger version (22K): [in a new window]   Fig. 10. Knowing their dependencies on ambient oxygen partial pressure (PO2amb) made it possible to correlate limb beating activity with haemolymph oxygen concentration ([O2]). In haemoglobin (Hb)-rich animals, the physiological effect of a sevenfold higher Hb concentration manifested itself in the range of PO2amb from 4.75 to 1.32 kPa, within which the extra Hb could still provide enough oxygen to sustain aerobic energy provision in the highly active limb muscles. Within almost the same range of PO2amb, Hb-poor animals were also able to maintain a high limb beating activity, but energy provision was already supported by anaerobic mechanisms (see Fig. 8). Note that, in the two groups, anaerobic energy provision was initiated at different levels of [O2] (horizontal arrows). The open circles indicate the PO2amb and the corresponding [O2] that caused an impairment of cardiac activity. The breadth of the graphs reflects the range of uncertainty in determining the proportion of [O2] that is physically dissolved in the haemolymph. The red arrow indicates the PO2amb at which Hb-rich animals were raised.

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