Mitochondrial metabolism and respiration adjustments following temperature acclimation in Daphnia magna.

Seasonal temperature changes and local variations in the water column challenge lentic zooplankton in their habitat. At the cellular level, exposure to varying temperatures affects the mitochondrial functional properties of poikilothermic organisms. Metabolic enzymes that supply reduced substrates to the electron transport chain and elements of the oxidative phosphorylation system must therefore adjust their activity and flux rates to the altered temperature conditions. In the present study, Daphnia magna respiration was analyzed in response to acute and chronic changes in ambient temperature. Oxygen consumption as well as substrate and electron flux rates were measured at the animals' acclimation temperature and at two additional acute temperatures. High activity of citrate synthase (CS) in cold-acclimated animals (10 °C) may have resulted from mitochondrial quantitative adjustments. However, thermal sensitivity of the functional properties of mitochondrial enzymes was greater in warm-acclimated animals (30 °C). In whole animals, temperature-induced changes were partly compensated by acclimation, but these changes were promoted by acclimation in the case of the mitochondrial electron transport chain. Thus, respiration realised in whole animals was limited by the provision of reduced substrates in the tricarboxylic acid cycle rather than by restrictions of the respiratory chain complexes. This may minimize production of reactive oxygen species and resulting damage and reduce waste of substrates from the animals' energy reserves. Still, the integrated biomarker response indicated increased defense against oxidative stress at elevated temperatures.

Hemoglobin in Arthropods-Daphnia as a Model.

Hemoglobin is the respiratory protein of many arthropods, enhancing the oxygen transport capacity of the hemolymph. One example, that has been subject of extensive studies, is the hemoglobin of the crustacean genus Daphnia. Here the characteristics of this oxygen binding protein are reviewed. The genetic structure is the result of repeated duplication events in the evolution, leading to a variety of di-domain isoforms. Adjustments to environmental changes thus result from differential expression of these paralogs. The biochemical properties, including spectral characteristics, concentration ranges, molecular mass of monomers and native oligomers, are compared. Structural differences between isoforms can be correlated to functional properties of oxygen binding characteristics. The mechanism of hemoglobin induction via hypoxia-inducible factor 1 allows the response to altered oxygen and temperature conditions. Changes of the hemoglobin suite in quantity and functional quality can be linked to their benefits for the animals' physiological performance. However, there is a large inter- and intra-specific variability of this induction potential. The consequences of altered hemoglobin characteristics for the animals' success within their habitat are discussed.

The transcriptomic and proteomic responses of Daphnia pulex to changes in temperature and food supply comprise environment-specific and clone-specific elements.

BACKGROUND: Regulatory adjustments to acute and chronic temperature changes are highly important for aquatic ectotherms because temperature affects their metabolic rate as well as the already low oxygen concentration in water, which can upset their energy balance. This also applies to severe changes in food supply. Thus, we studied on a molecular level (transcriptomics and/or proteomics) the immediate responses to heat stress and starvation and the acclimation to different temperatures in two clonal isolates of the model microcrustacean Daphnia pulex from more or less stressful environments, which showed a higher (clone M) or lower (clone G) tolerance to heat and starvation. RESULTS: The transcriptomic responses of clone G to acute heat stress (from 20 °C to 30 °C) and temperature acclimation (10 °C, 20 °C, and 24 °C) and the proteomic responses of both clones to acute heat, starvation, and heat-and-starvation stress comprised environment-specific and clone-specific elements. Acute stress (in particular heat stress) led to an early upregulation of stress genes and proteins (e.g., molecular chaperones) and a downregulation of metabolic genes and proteins (e.g., hydrolases). The transcriptomic responses to temperature acclimation differed clearly. They also varied depending on the temperature level. Acclimation to higher temperatures comprised an upregulation of metabolic genes and, in case of 24 °C acclimation, a downregulation of genes for translational processes and collagens. The proteomic responses of the clones M and G differed at any type of stress. Clone M showed markedly stronger and less stress-specific proteomic responses than clone G, which included the consistent expression of a specific heat shock protein (HSP60) and vitellogenin (VTG-SOD). CONCLUSIONS: The expression changes under acute stress can be interpreted as a switch from standard products of gene expression to stress-specific products. The expression changes under temperature acclimation probably served for an increase in energy intake (via digestion) and, if necessary, a decrease in energy expenditures (e.g, for translational processes). The stronger and less stress-specific proteomic responses of clone M indicate a lower degree of cell damage and an active preservation of the energy balance, which allowed adequate proteomic responses under stress, including the initiation of resting egg production (VTG-SOD expression) as an emergency reaction.

Automated measurement of upper thermal limits in small aquatic animals.

We present a method for automating the measurement of upper thermal limits in small aquatic organisms. Upper thermal limits are frequently defined by the cessation of movement at high temperature, with measurement being performed by manual observation. Consequently, estimates of upper thermal limits may be subject to error and bias, both within and among observers. Our method utilises video-based tracking software to record the movement of individuals when exposed to high, lethal temperatures. We develop an algorithm in the R computing language that can objectively identify the loss of locomotory function from tracking data. Using independent experimental data, we validate our approach by demonstrating the expected response in upper thermal limits to acclimation temperature.

Reactive oxygen species (ROS) and the heat stress response of Daphnia pulex: ROS-mediated activation of hypoxia-inducible factor 1 (HIF-1) and heat shock factor 1 (HSF-1) and the clustered expression of stress genes.

BACKGROUND INFORMATION: Heat stress in ectotherms involves direct (e.g. protein damage) and/or indirect effects (temperature-induced hypoxia and ROS formation), which cause activation of the transcription factors (TF) heat shock factor 1 (HSF-1) and/or hypoxia-inducible factor 1 (HIF-1). The present study focused on the links between stress (ROS) signals, nuclear (n) and cytoplasmic (c) HSF-1/HIF-1 levels, and stress gene expression on mRNA and protein levels (e.g. heat-shock protein 90, HSP90) upon acute heat and ROS (H2 O2 ) stress. RESULTS: Acute heat stress (30°C) evoked fluctuations in ROS level. Different feeding regimens, which affected the glutathione (GSH) level, allowed altering the frequency of ROS fluctuations. Other data showed fluctuation frequency to depend also on ROS production rate. The heat-induced slow or fast ROS fluctuations (at high or low GSH levels) evoked slow or fast fluctuations in the levels of nHIF-1α, nHSF-1 and gene products (mRNAs and protein), albeit after different time delays. Time delays to ROS fluctuations were, for example,shorter for nHIF-1α than for nHSF-1 fluctuations, and nHIF-1α fluctuations preceded and nHSF-1 fluctuations followed fluctuations in HSP90 mRNA level. Cytoplasmic TF levels either changed little (cHIF-1α) or showed a steady increase (cHSF-1). Applying acute H2 O2 stress (at 20°C) revealed effects on nHIF-1α and mRNA levels, but no significant effects on nHSF-1 level. Transcriptome data additionally showed coordinated fluctuations of mRNA levels upon acute heat stress, involving mRNAs for HSPs and other stress proteins, with all corresponding genes carrying DNA binding motifs for HIF-1 and HSF-1. CONCLUSIONS: This study provided evidence for promoting effects of ROS and HIF-1 on early haemoglobin, HIF-1α and HSP90 mRNA expressions upon heat or ROS stress. The increasing cHSF-1 level likely affected nHSF-1 level and later HSP90 mRNA expression. SIGNIFICANCE: Heat stress evoked ROS fluctuations, with this stress signal forwarded via nHIF-1 and nHSF-1 fluctuations to stress gene expression. The frequency of ROS fluctuations seemed to integrate information about ROS productionrate and GSH antioxidant buffer capacity, resulting in stress protein expression of different speed. Results of this study suggest ROS as early (pre-damage) and protein defects as later (post-damage) stress signals to trigger heat stress responses.

Conservation of the Notch antagonist Hairless in arthropods: functional analysis of the crustacean Daphnia pulex Hairless gene.

The Notch signaling pathway is highly conserved in all animal metazoa: upon Notch receptor activation, transcription of Notch target genes is turned on by an activator complex that centers on the transcription factor CSL. In the absence of signal, CSL assembles transcriptional repression complexes that display remarkable evolutionary diversity. The major antagonist of Notch signaling in insects named Hairless was originally identified in Drosophila melanogaster. It binds to the Drosophila CSL homologue Suppressor of Hairless [Su(H)] and recruits the two general co-repressors, Groucho and C-terminal binding protein. Whereas the majority of Notch signaling components is conserved between insects and vertebrates, Hairless is found only in insects. Here, we present the analysis of the Hairless gene from Daphnia pulex and, hence, for the first time from a crustacean. Daphnia and Drosophila Hairless protein sequences are highly diverged. Known functional domains, however, the Su(H), Groucho and the C-terminal binding protein interactions domains, are well conserved. Moreover, direct binding of the Daphnia Hairless protein and the respective Drosophila interaction partners was detected, demonstrating the conservation at the molecular level. In addition, interaction between Daphnia Hairless and Drosophila Su(H) was demonstrated in vivo, as co-overexpression of the respective genes during Drosophila development resulted in the expected downregulation of Notch activity in the fly. Structural models show that the Hairless-Su(H) repressor complexes from Daphnia and Drosophila are almost indistinguishable from one another. Amino acid residues in direct contact within the Hairless-Su(H) complex are at absolutely identical positions in the two homologues.

Hypoxia-inducible haemoglobins of Daphnia pulex and their role in the response to acute and chronic temperature increase.

Daphnia pulex is challenged by severe oxygen and temperature changes in its habitat. In response to hypoxia, the equipment of oxygen transport proteins is adjusted in quantity and quality by differential expression of haemoglobin isoforms. This study focuses on the response of 20°C acclimated animals to elevated temperature using transcriptomic and proteomic approaches. Acute temperature stress (30°C) induced the hypoxia-inducible Hb isoforms most strongly, resulting in an increase of the haemoglobin mRNA pool by 70% within 8h. Long-term-acclimation to moderately elevated temperature (24°C) only evoked minor changes of the Hb mRNA suite. Nevertheless, the concentration of the hemolymph pool of haemoglobin was elevated by 80%. In this case, the constitutive Hb isoforms showed the strongest increase, with Hb01 and Hb02 contributing by 64% to the total amount of respiratory protein. The regulation patterns upon acute temperature stress likely reflect temperature-induced tissue hypoxia, whereas in case of persisting exposure to moderately elevated temperature, acclimation processes enabled the successful return to oxygen homeostasis. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Limits of the thermal tolerance of two lentic crustaceans: The role of lactate dehydrogenase.

Limits of thermal tolerance in animal life is dependent on energy supply. Accordingly, the lowered ATP production capacity in ectotherms at high temperatures, which arises from a mismatch between oxygen supply and demand and the consequent switch from aerobic to anaerobic metabolism, affects the thermal resistance of these animals. The anaerobic ATP production capacity depends on the functional properties of the enzymes that reduce pyruvate. Thus, the present study focused on the role of the lactate dehydrogenase (LDH) of two daphnid species for anaerobic energy production at warm temperatures and the implications for their specific heat tolerances. Daphnia magna showed a higher thermal limit (indicated by immobilization time at 37°C) than Daphnia pulex, and in both species, this limit increased with rising acclimation temperature. In contrast to D. pulex, D. magna accumulated significant amounts of lactate at higher ambient temperatures. The intensity of anaerobic metabolism was also affected by acclimation temperature. Studying the functional enzyme properties revealed altered maximal reaction rates and substrate inhibitions of the LDH suites of the two daphnid species. D. magna LDH showed a significantly lower substrate inhibition than D. pulex LDH. The LDH isoform composition and the temperature-induced changes differed between both species. The detected qualitative modulations of the LDH suites may have resulted from differential isoform expression and different maturation processes. The species-specific LDH characteristics imply a higher anaerobic energy production at warm temperatures in D. magna, which likely contributes to the higher heat tolerance of this species.

Acute changes in temperature or oxygen availability induce ROS fluctuations in Daphnia magna linked with fluctuations of reduced and oxidized glutathione, catalase activity and gene (haemoglobin) expression.

BACKGROUND INFORMATION: ROS (reactive oxygen species) as well as components of the antioxidant redox systems may act as signals. To link acute environmental change with gene expression, changes in ROS and GSH/GSSG (reduced/oxidized glutathione) level were measured upon acute changes in temperature or oxygen availability in the aquatic key species Daphnia magna together with HIF-1 (hypoxia-inducible factor 1)-mediated Hb (haemoglobin) expression. RESULTS: Acute exposures to 30°C or hypoxia, which induced tissue hypoxia (and possibly elevated mitochondrial ROS production), caused resembling fluctuations of ROS and GSH levels, with frequency and number of peaks increasing and their delay decreasing with the magnitude of environmental change (size of tissue hypoxia). Acute hyperoxia induced an initial decrease in ROS level. Evidence is also provided for the promoting effects of ROS on catalase activity. A signalling function of the ROS fluctuations upon acute changes in temperature was found in the case of Hb, the expression of which is known to respond to temperature changes, by detecting corresponding time courses of both transcription and protein formation. CONCLUSION: ROS-dependent signalling was affected by changes in temperature or oxygen availability. Feedback interactions between ROS and the glutathione redox system, possibly driven by elevated mitochondrial ROS production, likely contributed to the appearance of the ROS and GSH fluctuations upon acute environmental change. Fluctuating ROS levels, which reflect for the magnitude of environmental change, could be a way to transfer information on ROS production to subsequent processes (gene expression) while avoiding too-high and damaging ROS levels.

Adaptive haemoglobin gene control in Daphnia pulex at different oxygen and temperature conditions.

Hypoxia-induced haemoglobin (Hb) expression is a central regulatory mechanism in Daphnia in response to environmental hypoxia or warm temperatures. Changes in Hb concentration as well as Hb subunit composition, which modulate Hb oxygen affinity, guarantee the oxygen supply of tissues under these environmental conditions. Based on the sequenced D. pulex genome, Hb genes were related to the properties of haemolymph Hb, which included its concentration and oxygen affinity (both measured by spectrophotometry) as well as the Hb subunit composition (determined by 2-D gel electrophoresis and ESI-MS analysis). Permanent cultures of D. pulex acclimated to different oxygen conditions (normoxia and hypoxia) and temperatures (10°C, 20°C, and 24°C), showed characteristic changes in Hb concentration, subunit composition and oxygen affinity. Several subunits (Hb4, Hb7, Hb8, and Hb10) were obviously responsible for changes in oxygen affinity including those, which carry a number of hypoxia-responsive elements (HREs) upstream of the respective gene (hb4 and hb10). Analysing the effects of different oxygen- or temperature-acclimations on Hb subunit expression in D. pulex and D. magna on a common basis (Hb concentration or oxygen affinity) revealed a general pattern of oxygen and temperature effects on Hb, which implies that Hb quantity and quality are mostly influenced by the degree of tissue hypoxia. Differences between both species in the onset of hypoxia-induced differential Hb expression and Hb oxygen affinity, which are probably related to different HRE patterns and functionally important differences in the amino acid sequence of only a few subunits, cause a reduced ability of D. pulex to adjust Hb function to temperature changes in comparison to D. magna.

Metabolic adjustments during starvation in Daphnia pulex.

Zooplankton organisms face a variable food supply in their habitat. Metabolic adjustments during periods of starvation were analysed from changes in metabolite level to gene expression in the microcrustacean Daphnia pulex during starvation. The animals exploited their carbohydrate stores first, but their lipid and protein reserves were also degraded, albeit more slowly. Glycogenolysis and probably gluconeogenesis led to hyperglycaemia after 16 h of starvation. The concentration of α-ketoglutarate and the rate of oxygen consumption also reached maxima during this period. Nuclear HIF-1α levels and α-ketoglutarate concentration showed inverse correlation. Effects of this 2-oxoacid on prolyl hydroxylase activity, HIF-1α stability and the role of this transcription factor in the changes of the expression level of several putatively HIF-1-mediated metabolic genes are discussed. Transcriptome profiling via RNA-Seq revealed a downregulation of genes for protein biosynthesis and an upregulation of genes for carbohydrate metabolism during starvation. Thus, the adjustments of energy metabolism in response to food deprivation were quantified from the level of metabolites, signal transduction and gene expression, and possible connections of the respective dynamics of observed changes were analysed.

Microplastic burden in Daphnia is aggravated by elevated temperatures.

Contamination of freshwater habitats with microplastic is threatening particularly filter-feeders within the aquatic community. Using Daphnia magna and Daphnia pulex as models, the effects of food supply and temperature on the ingestion of polystyrene spheres (diameter 1 µm, concentration of 200 ng*ml-1) was analysed. The ingestion rates of microplastic beads were increased in conditions of low food and high temperatures, reflecting the complex regulation patterns of the water current generated by the animals' thoracic limbs. Maximal enrichment of 1160 times the concentration in the ambient medium was observed within one hour. Analyses of the impact of microplastic ingestion on physiological parameters used the carbohydrate concentration as an indicator for the animals' metabolic state. Exposure to the microplastic beads for three days in the presence or absence of Desmodesmus subspicatus did not affect the animals' glycogen reserves beyond the response to the prevailing food and temperature conditions. Projecting the insights from laboratory experiments to the habitat situation, increased burdens of microplastic particles can be expected in filtering zooplankton organisms in warm water and scarce supply of food, like the clear-water phase of lakes in the summer.

Acclimatory responses of the Daphnia pulex proteome to environmental changes. II. Chronic exposure to different temperatures (10 and 20 degrees C) mainly affects protein metabolism.

BACKGROUND: Temperature affects essentially every aspect of the biology of poikilothermic animals including the energy and mass budgets, activity, growth, and reproduction. While thermal effects in ecologically important groups such as daphnids have been intensively studied at the ecosystem level and at least partly at the organismic level, much less is known about the molecular mechanisms underlying the acclimation to different temperatures. By using 2D gel electrophoresis and mass spectrometry, the present study identified the major elements of the temperature-induced subset of the proteome from differently acclimated Daphnia pulex. RESULTS: Specific sets of proteins were found to be differentially expressed in 10 degrees C or 20 degrees C acclimated D. pulex. Most cold-repressed proteins comprised secretory enzymes which are involved in protein digestion (trypsins, chymotrypsins, astacin, carboxypeptidases). The cold-induced sets of proteins included several vitellogenin and actin isoforms (cytoplasmic and muscle-specific), and an AAA+ ATPase. Carbohydrate-modifying enzymes were constitutively expressed or down-regulated in the cold. CONCLUSION: Specific sets of cold-repressed and cold-induced proteins in D. pulex can be related to changes in the cellular demand for amino acids or to the compensatory control of physiological processes. The increase of proteolytic enzyme concentration and the decrease of vitellogenin, actin and total protein concentration between 10 degrees C and 20 degrees C acclimated animals reflect the increased amino-acids demand and the reduced protein reserves in the animal's body. Conversely, the increase of actin concentration in cold-acclimated animals may contribute to a compensatory mechanism which ensures the relative constancy of muscular performance. The sheer number of peptidase genes (serine-peptidase-like: > 200, astacin-like: 36, carboxypeptidase-like: 30) in the D. pulex genome suggests large-scaled gene family expansions that might reflect specific adaptations to the lifestyle of a planktonic filter feeder in a highly variable aquatic environment.

Acclimatory responses of the Daphnia pulex proteome to environmental changes. I. Chronic exposure to hypoxia affects the oxygen transport system and carbohydrate metabolism.

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.

Temperature-induced changes in body lipid composition affect vulnerability to oxidative stress in Daphnia magna.

Optimizing physiological functions at different temperatures includes shifts in the lipid composition of ectothermic animals. These shifts may be associated with changes in lipid peroxidation in response to oxidative stress, because lipids differ in their susceptibility to oxidative damage. Polyunsaturated fatty acids (PUFA) are particular prone to peroxidation. Here, we analyzed changes in the fatty acid composition, cholesterol content and the level of oxidative damage as thiobarbituric reactive substances (TBARS) in Daphnia magna as a function of acclimation temperature. The total fatty acid content was highest in cold-acclimated animals. The relative share of most PUFA decreased with increasing acclimation temperature. In contrast, the contribution of saturated and monounsaturated fatty acids (SFA and MUFA) increased with acclimation temperature, although the latter to a lower extent. Cholesterol content remained unchanged. The level of oxidative damage was lowest in animals reared at warm temperatures, most likely reflecting their lowest content of PUFA. Heat exposure (1 h at 33 °C) caused the highest increase in lipid peroxidation in cold-acclimated animals, containing more PUFA. Our data suggest that cold-induced adjustments in the body lipid composition increase the vulnerability of zooplankton to heat-induced oxidative stress. In particular, animals performing diel vertical migration may be highly susceptible to temperature-induced lipid damage.

Haemoglobin-mediated response to hyper-thermal stress in the keystone species Daphnia magna.

Anthropogenic global warming has become a major geological and environmental force driving drastic changes in natural ecosystems. Due to the high thermal conductivity of water and the effects of temperature on metabolic processes, freshwater ecosystems are among the most impacted by these changes. The ability to tolerate changes in temperature may determine species long-term survival and fitness. Therefore, it is critical to identify coping mechanisms to thermal and hyper-thermal stress in aquatic organisms. A central regulatory element compensating for changes in oxygen supply and ambient temperature is the respiratory protein haemoglobin (Hb). Here, we quantify Hb plastic and evolutionary response in Daphnia magna subpopulations resurrected from the sedimentary archive of a lake with known history of increase in average temperature and recurrence of heat waves. By measuring constitutive changes in crude Hb protein content among subpopulations, we assessed evolution of the Hb gene family in response to temperature increase. To quantify the contribution of plasticity in the response of this gene family to hyper-thermal stress, we quantified changes in Hb content in all subpopulations under hyper-thermal stress as compared to nonstressful temperature. Further, we tested competitive abilities of genotypes as a function of their Hb content, constitutive and induced. We found that Hb-rich genotypes have superior competitive abilities as compared to Hb-poor genotypes under hyper-thermal stress after a period of acclimation. These findings suggest that whereas long-term adjustment to higher occurrence of heat waves may require a combination of plasticity and genetic adaptation, plasticity is most likely the coping mechanism to hyper-thermal stress in the short term. Our study suggests that with higher occurrence of heat waves, Hb-rich genotypes may be favoured with potential long-term impact on population genetic diversity.

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna.

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.

Adjustments of serine proteases of Daphnia pulex in response to temperature changes.

Elevated temperatures considerably challenge aquatic invertebrates, and enhanced energy metabolism and protein turnover require adjustments of digestion. In Daphnia, the serine proteases chymotrypsin and trypsin represent the major proteolytic enzymes. Daphnia pulex acclimated to different temperature conditions or subjected to acute heat stress showed increased expression level of serine proteases with rising temperatures. Transcripts of trypsin isoforms were always present in higher amounts than observed for chymotrypsin. Additionally, trypsin isoform transcripts were induced by elevated temperatures to a larger extent. Correspondingly, trypsin activity dominated in cold-acclimated animals. However, the enzymatic activity of chymotrypsin increased at elevated temperatures, whereas trypsin activity slightly decreased, resulting in a shift to dominating chymotrypsin activity in warm-acclimated animals. Zymograms revealed eight bands with proteolytic activity in the range of 20 to 86 kDa. The single bands were assigned to trypsin or chymotrypsin activity applying specific inhibitors or from casein cleavage products identified by mass spectrometric analysis. The total amount of proteolytic activity was elevated with acclimation temperature increase and showed a transient decrease under acute heat stress. The contribution of the different isoforms to protein digestion indicated induction of chymotrypsin with increasing acclimation temperature. For trypsin, the share of one isoform decreased with elevated temperature, while another isoform was enhanced. Thus differential expression of serine proteases was observed in response to chronic and acute temperature changes. The observed phenotypic plasticity adjusts the set of active proteases to the altered needs of protein metabolism optimizing protein digestion for the temperature conditions experienced in the habitat.

Oxygen preference of Daphnia magna is influenced by Po2 acclimation and biotic interactions.

Oxygen preference of the planktonic crustacean Daphnia magna was observed within an oxygen gradient. Long-term acclimation to specific oxygen conditions determined the preferred oxygen tension. Animals from hypoxic acclimation stayed in oxygen-poor water at first. They chose oxygen tensions above the critical partial pressure, thus maintaining aerobic energy supply, but they avoided higher oxygen concentrations. However, they gradually took more oxygen-rich positions within 2 d. Animals from severe hypoxic and hyperoxic acclimation showed stenoxic behavior. Specimens raised in moderately hypoxic or normoxic medium spread out more widely in the oxygen gradient (euryoxic behavior). The possible role of hemoglobin and antioxidant defense capacity limiting the animals' distribution at low or high oxygen concentrations, respectively, is discussed. In tests with mixed groups of D. magna from opposing oxygen acclimation conditions, the animals clustered at intermediate oxygen conditions. However, the acclimation condition of the major group was found to determine the position taken within the gradient to a greater extent. Selected oxygen tensions were closer to the preference values of the larger group, yet the influence of the minority prevented the majority in heterogeneous groups from taking their originally determined preference position. Thus, aggregation behavior interfered with the acclimation-dependent oxygen preference behavior.

Molecular adaptation of Daphnia magna hemoglobin.

Daphnia magna responds to changing environmental conditions impeding aerobic metabolism by synthesizing hemoglobin of adequate quantity and quality to maintain oxygen supply of the tissues. Hemoglobin subunit composition and its oxygen affinity were analyzed as a function of temperature as well as depending on the oxygen partial pressure of the medium. Additionally, the time course of acclimation to hypoxia was studied. Correlating structural and functional changes, the role of individual subunits for the increase in oxygen affinity is discussed.