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The planktonic crustacean Daphnia magna synthesizes haemoglobin (Hb) macromolecules of variant subunit composition and oxygen affinity. This is one of the strategies by which the animals cope with variations in environmental conditions such as ambient oxygen tension. The enrichment of high-affinity Hb molecules in the haemolymph of hypoxia-exposed animals is thought to reduce Hb synthesis costs due to an enhanced transport efficiency of these molecules in comparison to the low-affinity Hb molecules. How great this economic advantage is, and under which conditions this benefit disappears, is still not fully understood. Here we implemented a rigorously simplified model of the daphnid body and described the transport of oxygen from the environment via the haemolymph to the tissues in terms of the convection-diffusion-reaction equation. The model was validated by comparing various model predictions with experimental data. A sensitivity analysis was used to evaluate the influence of parameter uncertainties on the model predictions. Cost-benefit analysis revealed in which way at the system's level the increase in Hb oxygen affinity improves the oxygen loading at the respiratory surfaces and impairs the release of oxygen to the tissues. The benefit arising from the improved oxygen loading exceeds the disadvantage of impaired unloading only under conditions where the ambient oxygen tension is critically low and the Hb concentration is high. The low-affinity Hb, on the other hand, provides an advantage given that the Hb concentration is low and the ambient oxygen tension is well above the critical level. Computer-aided modelling and simulation therefore provide valuable mechanistic insights into the driving forces that could have shaped the evolution of globin genes in daphnids.

Background: Freshwater planktonic crustaceans of the genus Daphnia show a remarkable plasticity to cope with environmental changes in oxygen concentration and temperature. One of the key proteins of adaptive gene control in Daphnia pulex under hypoxia is hemoglobin (Hb), which increases in hemolymph concentration by an order of magnitude and shows an enhanced oxygen affinity due to changes in subunit composition. To explore the full spectrum of adaptive protein expression in response to low-oxygen conditions, two-dimensional gel electrophoresis and mass spectrometry were used to analyze the proteome composition of animals acclimated to normoxia (oxygen partial pressure [Po2]: 20 kPa) and hypoxia (Po2: 3 kPa), respectively., Results: The comparative proteome analysis showed an up-regulation of more than 50 protein spots under hypoxia. Identification of a major share of these spots revealed acclimatory changes for Hb, glycolytic enzymes (enolase), and enzymes involved in the degradation of storage and structural carbohydrates (e.g. cellubiohydrolase). Proteolytic enzymes remained constitutively expressed on a high level., Conclusion: Acclimatory adjustments of the D. pulex proteome to hypoxia included a strong induction of Hb and carbohydrate-degrading enzymes. The scenario of adaptive protein expression under environmental hypoxia can be interpreted as a process to improve oxygen transport and carbohydrate provision for the maintenance of ATP production, even during short episodes of tissue hypoxia requiring support from anaerobic metabolism.

Branchiopod crustaceans are endowed with extracellular, high-molecular-mass hemoglobins (Hbs), the functional and allosteric properties of which have largely remained obscure. The Hb of the phylogenetically ancient Triops cancriformis (Notostraca) revealed moderate oxygen affinity, cooperativity and pH dependence (Bohr effect) coefficients: P(50) = 13.3 mmHg, n(50) = 2.3, and Phi = -0.18, at 20 degrees C and pH 7.44 in Tris buffer. The in vivo hemolymph pH was 7.52. Bivalent cations increased oxygen affinity, Mg(2+) exerting a greater effect than Ca(2+). Analysis of cooperative oxygen binding in terms of the nested Monod-Wyman-Changeux (MWC) model revealed an allosteric unit of four oxygen-binding sites and functional coupling of two to three allosteric units. The predicted 2 x 4 and 3 x 4 nested structures are in accord with stoichiometric models of the quarternary structure. The allosteric control mechanism of protons comprises a left shift of the upper asymptote of extended Hill plots which is ascribable to the displacement of the equilibrium between (at least) two high-affinity (relaxed) states, similar to that found in extracellular annelid and pulmonate molluscan Hbs. Remarkably, Mg(2+) ions increased oxygen affinity solely by displacing the equilibrium between the tense and relaxed conformations towards the relaxed states, which accords with the original MWC concept, but appears to be unique among Hbs. This effect is distinctly different from those of ionic effectors (bivalent cations, protons and organic phosphates) on annelid, pulmonate and vertebrate Hbs, which involve changes in the oxygen affinity of the tense and/or relaxed conformations.

The mechanisms, dynamics and effects of hypoxia acclimation were studied in the water flea Daphnia magna over three successive generations (parental, first and second filial generation: P, F1 and F2). The P generation was raised under normoxic conditions at 20 degrees C and became exposed to environmental hypoxia (10-19% air saturation) at maturity. Their progenies (F1 and F2) experienced hypoxia from birth onwards. Controls were kept under normoxic conditions. Individuals were successively sampled in a 3-day interval from each acclimation group to determine morpho-physiological parameters relevant in oxygen transport and regulation. Hypoxia acclimation induced adjustments at the haemoglobin (Hb) and metabolic level (within 3 days) but none at the systemic level. The convective performance and oxygen-sensitive control of the ventilatory and circulatory systems were the same in both acclimation groups. The Hb concentration and oxygen affinity increased by 266% and 32%, respectively. The 22% decrease in mass-specific oxygen consumption rate reduced the energy allocation to somatic growth without greatly affecting reproduction. The onset and duration of hypoxic exposure during ontogenesis have had a significant influence on Hb oxygen affinity and body size. Transgenerational effects of hypoxia acclimation could not be observed. The adjustments at the Hb and metabolic levels in combination with the smaller body size, which is advantageous to diffusive oxygen transport, reduced the critical ambient oxygen tension by approximately 50%.

Oxygen transport processes in millimetre-sized animals can be very complex, because oxygen molecules do not exclusively follow the pathway predetermined by the circulating fluid but may also simultaneously move from the respiratory surfaces to the tissues along different paths by diffusion. The present study made use of the oxygen-sensitive phosphorescence probe Oxyphor R2 to analyze the internal oxygen pathway in the transparent microcrustacean Daphnia magna. Oxyphor R2 was injected into the circulatory system and the distribution of oxygen partial pressure (P(O(2))) in the haemolymph was measured by phosphorescence lifetime imaging in the P(O(2)) range 0-6 kPa (0-30% air saturation). There were substantial differences in the shape of the two-dimensional P(O(2)) profiles depending on the concentration of haemoglobin (Hb) in the haemolymph. A steep global gradient, from posterior to anterior, occurred in animals with low concentrations of Hb (90-167 micromol l(-1) haem). In contrast, animals with a five- to sixfold higher concentration of Hb showed flat internal P(O(2)) gradients which, however, were only present under reduced ambient oxygen tensions (P(O(2)amb)=3-1 kPa), when Hb was maximally involved in oxygen transport. Under these conditions, the presence of Hb at high concentrations stabilized the unloading P(O(2)) in the central body to 0.9-0.4 kPa. Independent of Hb concentration and body size, the loading P(O(2)) was always 0.5 kPa below the P(O(2)amb). From these P(O(2)) profiles, it was possible (i) to follow the track of oxygen within the animal, and (ii) to visualize the shift from a diffusion-dominated to a convection-dominated transport as a result of increased Hb concentration.

The pathway for oxygen, the control of oxygen transport and the role of haemoglobin expression for the physiological adaptation to different oxygen and temperature conditions were studied in the ecological model organism Daphnia magna. Ventilation of the inner walls of the carapace as the main gas exchange area as well as of the embryos in the brood pouch are controlled, oxygen-dependent processes. The P(O2)-dependent increase of heart rate as well as perfusion rate during short-term, progressive hypoxia improves the circulatory oxygen transport within the body. The regulation of haemoglobin (Hb) expression is the central mechanism for a medium-term adaptation to hypoxia. Genetic control elements and oxygen conditions near the two Hb synthesis sites (fat cells, epipodite epithelial cells) determine, which types of Hb subunits and, accordingly, hetero-multimeric Hb macromolecules are produced. One synthesis site may respond mainly to internal, the other one to external oxygen conditions. Depending on environmental condition, either higher quantities of macromolecules of unchanged functionality (P50) or increasing amounts of macromolecules with higher oxygen affinity are synthesized. The Hb subunit DmHbA is probably of considerable importance for this functional change. The physiological benefits of haemoglobin in Daphnia are discussed. Physiological adaptation of Daphnia to different temperatures is also related to the control of oxygen transport processes with the regulation of haemoglobin expression again as a central mechanism.

The dual function of appendage movement (food acquisition, ventilation) proved to be the key to explaining the peculiar oxyregulatory repertoire of the planktonic filter feeder Daphnia magna. Short-term hypoxic exposure experiments with normoxia-acclimated animals under varying food concentrations revealed a dichotomous response pattern with a compensatory tachycardia under food-free conditions and a ventilatory compensation prevailing under food-rich conditions. Food-free, normoxic conditions resulted in maximum appendage beating rates (fA) and half-maximum heart rates (fH), which restricted the scope for oxyregulation to the circulatory system. Food-rich conditions (10(5) algal cells ml(-1)), on the contrary, had a depressing effect on fA whereas fH increased to 83% of the maximum. In this physiological state, D. magna was able to respond to progressive hypoxia with a compensatory increase in ventilation. A conceptual and mathematical model was developed to analyse the efficiency of ventilatory and circulatory adjustments in improving oxygen transport to tissue. Model predictions showed that an increase in perfusion rate was most effective under both food-free and food-rich conditions in reducing the critical ambient oxygen tension (PO2crit) at which oxygen supply to the tissue started to become impeded. By contrast, a hypothetical increase in ventilation rate had almost no effect on PO2crit under food-free conditions, indicating that appendage movement is driven by nutritive rather than respiratory requirements. However, the model predicted a moderate reduction of PO2crit by hyperventilation under food-rich conditions. Since the regulatory scope for an adjustment in fH was found to be limited in D. magna under these conditions, the increase in ventilation rate is the means of choice for a fed animal to cope with short-term, moderate reductions in ambient oxygen availability. Under long-term and more severe hypoxic conditions, however, the increase in the concentration and oxygen affinity of haemoglobin represents the one and only measure for improving the transport of oxygen from environment to cells.

The quantity and quality of the haemoglobin (Hb) of Daphnia magna is related to oxygen partial pressure in the water. Both the dynamics of hypoxia-induced Hb gene transcription, as well as Hb properties in animals incubated long-term at hyperoxia, normoxia and hypoxia, were investigated. Examination of Hb gene (dhb1-dhb3) transcription showed the expression of dhb2 and especially dhb3 to increase markedly approximately one hour after the onset of hypoxia, whereas dhb1 was expressed more or less constitutively. At an incubation close to anoxia, an onset of dhb3 transcription was found already after two minutes. In long-term incubated animals, concentration and oxygen affinity of Hb were lower at higher oxygen partial pressures. With decreasing oxygen availability, the subunit composition of Hb macromolecules changed. The share of the dhb2-encoded subunit, DHbF, increased already during moderate hypoxia. The increase of dhb3 mRNA (encoding DHbC) may be related to a transient increase of DHbC in the first days of hypoxia and/or to an additional coding of dhb3 for DHbD. The rise of DHbD, and particularly DHbA, only at severe hypoxia coincided with the increase of Hb oxygen affinity. The dhb1-encoded subunits DHbB and DHbE showed either a relatively moderate increase or even a decrease in concentration at hypoxia. In small animals with restricted homeostasis capabilities such as Daphnia, adaptation of the protein equipment seems to be a more effective strategy than allosteric modulator control.

To determine the contribution of circulatory convection to tissue oxygen supply in animals of Daphnia magna, heart rate ( f(H)), in-vivo Hb oxygen-saturation ( S(Hb)) and NADH fluorescence intensity ( I(NADH)) as a measure of the tissue oxygenation state were simultaneously measured using digital motion analysis, microabsorption spectroscopy and fluorescence microscopy. In addition, the relationship between stroke volume and body size was established. Groups of differently sized animals (small: 1.4-1.6 mm, medium: 2.7-2.9 mm, large: 3.3 mm) with either low (Hb-poor) or high Hb concentration (Hb-rich) in the hemolymph were exposed to a gradual decrease in ambient oxygen partial pressure ( P(O2amb)) between normoxia and anoxia. In all groups, f(H) increased in response to progressive hypoxia. The hypoxic maximum in f(H) was highest in medium-sized Hb-poor animals, whereas perfusion rate increased continuously with increasing body size in Hb-poor and Hb-rich animals. The P(O2amb) at which Hb in the heart region was half-saturated (in-vivo P(50)) was higher in medium-sized (Hb-poor: 3.2 kPa, Hb-rich: 2.0 kPa) than in small (Hb-poor: 2.1 kPa, Hb-rich: 1.5 kPa) and large animals (Hb-poor: 1.9 kPa). The in-vivo P(50) was always lower in Hb-rich than in Hb-poor animals. The I(NADH) indicated an impairment of tissue oxygenation starting at higher critical P(O2amb) with increasing body size and with lower Hb concentration. Model calculations suggest that at the respective critical P(O2amb), circulatory convection delivers less than half of the oxygen demand in Hb-poor animals. In contrast, in Hb-rich animals, the contribution of circulatory convection to tissue oxygen supply at respective critical P(O2amb) was much greater due to the higher concentration of Hb.

To determine the contribution of haemoglobin (Hb) to the hypoxia-tolerance of Daphnia magna, we exposed Hb-poor and Hb-rich individuals (2.4-2.8 mm long) to a stepwise decrease in ambient oxygen partial pressure (P(O(2)amb)) over a period of 51 min from normoxia (20.56 kPa) to anoxia (<0.27 kPa) and looked for differences in their physiological performance. The haem-based concentrations of Hb in the haemolymph were 49 micromol l(-1) in Hb-poor and 337 micromol l(-1) in Hb-rich animals, respectively. The experimental apparatus made simultaneous measurement of appendage beating rate (fA), NADH fluorescence intensity (I(NADH)) of the appendage muscles, heart rate (fH) and in vivo Hb oxygen-saturation possible. In response to progressive, moderate hypoxia, both groups showed pronounced tachycardia and a slight decrease in fA. The fA and fH of Hb-rich animals were generally 4-6 % lower than those of Hb-poor animals. In addition, Hb-rich animals showed a significant decrease in the P(O(2)amb) at which the Hb in the heart region was half-saturated and a striking reduction in the critical P(O(2)amb) of appendage-related variables. In Hb-poor animals, the I(NADH) signal indicated that the oxygen supply to the limb muscle tissue started to become impeded at a critical P(O(2)amb) of 4.75 kPa, although the high level of fA was largely maintained until 1.77 kPa. The obvious discrepancy between these two critical P(O(2)amb) values suggested an anaerobic supplementation of energy provision in the range 4.75-1.77 kPa. The fact that I(NADH) of Hb-rich animals did not rise until P(O(2)amb) fell below 1.32 kPa strongly suggests that the extra Hb available to Hb-rich animals ensured an adequate oxygen supply to the limb muscle tissue in the P(O(2)amb) range 4.75-1.32 kPa. This finding illustrates the physiological benefit of Hb in enabling the animal to sustain its aerobic metabolism as the energetically most efficient mode of fuel utilization under conditions of reduced oxygen availability.

Thermal acclimation in Daphnia magna was studied on various levels to test the recent “oxygen-limited thermal tolerance” hypothesis. Preference behaviour in a thermal gradient was determined by both, ambient temperature and corresponding oxygen concentration. Swimming activity depended on aerobic energy provision and reflected the match or mismatch of oxygen supply and energy demand at different ambient temperatures. Thermal acclimation modified both types of behaviour and more slightly heat tolerance. Perfusion and haemoglobin properties turned out to be central control variables to adapt oxygen transport to varying energy demands at different ambient temperatures. Exceptional advantages of Daphnia as an experimental model organism allowed to confirm on a behavioural, physiological and biochemical level that thermal acclimation is strongly based on the adaptation of oxygen transport allowing unidirectional shifts of the thermal tolerance range to warmer or colder temperatures. [Copyright &y& Elsevier]