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.

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.

ROS-mediated relationships between metabolism and DAF-16 subcellular localization in Caenorhabditis elegans revealed by a novel fluorometric method.

Signalling pathways provide a fine-tuned control network for catabolic and anabolic cellular processes under changing environmental conditions (e.g. changes in oxygen partial pressure, Po2). These pathways frequently activate or deactivate transcription factors (TFs) in the cytoplasm, with the subsequent nuclear translocation of activated TFs constituting a prerequisite for gene control and expression. This study introduces a newly developed fluorometric method for the quantification of relationships between environmental factors and the subcellular localization of reporter-coupled TFs in Caenorhabditis elegans (and possibly other transparent organisms). We applied this method to determine and analyse the relationship between Po2 and the subcellular localization of the GFP-coupled transcription factor DAF-16 (FoxO) of the DAF-2 (insulin/IGF-1) signalling pathway via the DAF-16::GFP fluorescence intensity of whole worms (Po2 characteristic). The Po2 characteristic resembled the Po2-specific metabolic rate of C. elegans, with a critical Po2 (Pco2) of 3.6 kPa separating two Po2 ranges, where either anaerobic metabolism and DAF-16::GFP nuclear occupancy strongly increased (i.e. decreasing DAF-16::GFP fluorescence intensity) (Po2 < Pco2) or aerobic metabolism and DAF-16::GFP cytoplasmic localization prevailed (Po2 > Pco2). These results and other data, which included the Po2-specific mitochondrial oxidation-reduction state of whole worms (as determined using the endogenous NADH fluorescence) and the effects of higher levels of reactive oxygen species (ROS) or RNAi-mediated knockdowns of catabolic or anabolic control genes (aak-2 or let-363) on the Po2 characteristic, suggest that ROS play a decisive role for DAF-16 nuclear translocation due to tissue hypoxia or higher anabolic activity induced by aak-2(RNAi). As DAF-16 and its target genes are of central importance for the cellular stress resistance, ROS-mediated relationships between metabolism and DAF-16 subcellular (i.e. nuclear) localization provide protection of the cell machinery against elevated ROS formation under challenging metabolic conditions.

Bacterial diet and weak cadmium stress affect the survivability of Caenorhabditis elegans and its resistance to severe stress.

Stress may have negative or positive effects in dependence of its intensity (hormesis). We studied this phenomenon in Caenorhabditis elegans by applying weak or severe abiotic (cadmium, CdCl2) and/or biotic stress (different bacterial diets) during cultivation/breeding of the worms and determining their developmental speed or survival and performing transcriptome profiling and RT-qPCR analyses to explore the genetic basis of the detected phenotypic differences. To specify weak or severe stress, developmental speed was measured at different cadmium concentrations, and survival assays were carried out on different bacterial species as feed for the worms. These studies showed that 0.1 µmol/L or 10 mmol/L of CdCl2 were weak or severe abiotic stressors, and that E. coli HT115 or Chitinophaga arvensicola feeding can be considered as weak or severe biotic stress. Extensive phenotypic studies on wild type (WT) and different signaling mutants (e.g., kgb-1Δ and pmk-1Δ) and genetic studies on WT revealed, inter alia, the following results. WT worms bred on E. coli OP50, which is a known cause of high lipid levels in the worms, showed high resistance to severe abiotic stress and elevated gene expression for protein biosynthesis. WT worms bred under weak biotic stress (E. coli HT115 feeding which causes lower lipid levels) showed an elevated resistance to severe biotic stress, elevated gene expression for the innate immune response and signaling but reduced gene expression for protein biosynthesis. WT worms bred under weak biotic and abiotic stress (E. coli HT115 feeding plus 0.1 µmol/L of CdCl2) showed high resistance to severe biotic stress, elevated expression of DAF-16 target genes (e.g., genes for small heat shock proteins) but further reduced gene expression for protein biosynthesis. WT worms bred under weak biotic but higher abiotic stress (E. coli HT115 feeding plus 10 µmol/L of CdCl2) showed re-intensified gene expression for the innate immune response, signaling, and protein biosynthesis, which, however, did not caused a higher resistance to severe biotic stress. E. coli OP50 feeding as well as weak abiotic and biotic stress during incubations also improved the age-specific survival probability of adult WT worms. Thus, this study showed that a bacterial diet resulting in higher levels of energy resources in the worms (E. coli OP50 feeding) or weak abiotic and biotic stress promote the resistance to severe abiotic or biotic stress and the age-specific survival probability of WT.

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.

PMK-1 p38 MAPK promotes cadmium stress resistance, the expression of SKN-1/Nrf and DAF-16 target genes, and protein biosynthesis in Caenorhabditis elegans.

The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.

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.

Detoxification and sensing mechanisms are of similar importance for Cd resistance in Caenorhabditis elegans.

The present study employed mass spectrometry (ICP-MS) to measure the internal cadmium concentrations (Cdint) in Caenorhabditis elegans to determine Cd uptake from a Cd-containing environment as well as Cd release under Cd-free conditions. To analyze the functional role of several ATP binding cassette (ABC) transporters (e.g., HMT-1 and MRP-1) and phytochelatin synthase (PCS), we compared wild-type (WT) and different mutant strains of C. elegans. As a pre-test on selected mutant strains, several time-resolved experiments were performed to determine the survival rate and avoidance behavior of C. elegans under Cd stress, which confirmed the already known Cd sensitivity of the deletion mutants mrp-1Δ, pcs-1Δ, and hmt-1Δ. In addition, these experiments revealed flight reactions under Cd stress to be almost completely absent in mrp-1Δ mutants. The ICP-MS studies showed Cd uptake to be significantly higher in mrp-1Δ and WT than in hmt-1Δ. As Cd is ingested with food, food refusal due to very early Cd stress and its perception was likely the reason for the reduced Cd uptake of hmt-1Δ. Cd release (detoxification) was found to be maximal in mrp-1Δ, minimal in hmt-1Δ, and intermediate in WT. High mortality under Cd stress, food refusal, and minimal Cd release in the case of hmt-1Δ suggest a vital importance of the HMT-1/PCS-1 detoxification system for the survival of C. elegans under Cd stress. High mortality under Cd stress, absence of an avoidance behavior, missing food refusal, and maximal Cd release in the case of mrp-1Δ indicate that MRP-1 is less important for Cd detoxification under severe stress, but is probably important for Cd perception. Accordingly, our results suggest that the survival of WT under Cd stress (or possibly other forms of metal stress) primarily depends on the function of the HMT-1/PCS-1 detoxification system and the presence of a sensing mechanism to control the uptake of Cd (or other metals), which keeps internal Cd (or metal) concentrations under control, to some extent, for the timely mobilization of protection and repair systems.

The JNK-like MAPK KGB-1 of Caenorhabditis elegans promotes reproduction, lifespan, and gene expressions for protein biosynthesis and germline homeostasis but interferes with hyperosmotic stress tolerance.

AIMS: This study focused on the role of the JNK-like MAPK (mitogen-activated protein kinase) KGB-1 (kinase, GLH-binding 1) for osmoprotection and other vital functions. METHODS: We mapped KGB-1 expression patterns and determined lifespan, reproduction and survival rates as well as changes in body volume, motility, and GPDH (glycerol-3-phosphate dehydrogenase) activity for glycerol production in wildtype (WT), different signaling mutants (including a kgb-1 deletion mutant, kgb-1∆) and RNAi-treated worms under control and hyperosmotic conditions. KGB-1-mediated gene expressions were studied, for instance, by RNA Sequencing, with the resulting transcriptome data analyzed using orthology-based approaches. RESULTS: Surprisingly, mutation/RNAi of kgb-1 and fos-1 (gene for an AP-1, activator protein 1, element) significantly promoted hyperosmotic resistance, even though hyperosmotic GPDH activity was higher in WT than in kgb-1∆. KGB-1 and moderate hyperosmolarity promoted and severe hyperosmolarity repressed kgb-1, fos-1, and jun-1 (gene for another AP-1 element) expression. Transcriptome profiling revealed, for instance, down-regulated genes for protein biosynthesis and up-regulated genes for membrane transporters in kgb-1∆ and up-regulated genes for GPDH-1 or detoxification in WT, with the latter indicating cellular damage and less effective osmoprotection in WT. CONCLUSION: KGB-1 promotes reproduction and lifespan and fosters gene expressions for AP-1 elements, protein biosynthesis, and balanced gametogenesis, but inhibits expressions for membrane transporters perhaps in order to control energy consumption. Reduced protein biosyntheses and enhanced membrane transports in kgb-1∆ most likely contribute to the high hyperosmotic tolerance of the mutant by easing the burden of the existing chaperone machinery and promoting regulatory volume increases upon hyperosmotic stress.

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.

The p38 MAPK PMK-1 shows heat-induced nuclear translocation, supports chaperone expression, and affects the heat tolerance of Caenorhabditis elegans.

The p38 mitogen-activated protein kinase PMK-1 of Caenorhabditis elegans has been associated with heavy metal, oxidative and pathogen stress. Pmk-1 is part of an operon comprising three p38 homologues, with pmk-1 expression suggested to be regulated by the operon promoter. There are contradictory reports about the cellular localization of PMK-1. We were interested to study principles of pmk-1 expression and to analyze the role of PMK-1 under heat stress. Using a translational GFP reporter, we found pmk-1 expression to be driven by a promoter in front of pmk-1. PMK-1 was detected in intestinal cells and neurons, with a cytoplasmic localization at moderate temperature. Increasing temperature above 32 °C, however, induced a nuclear translocation of PMK-1 as well as PMK-1 accumulation near to apical membranes. Testing survival rates revealed 34-35 °C as critical temperature range, where short-term survival severely decreased. Mutants of the PMK-1 pathway (pmk-1Δ, sek-1Δ, mek-1Δ) as well as a mutant of JNK pathway (jnk-1Δ) showed significantly lower survival rates than wild-type or mutants of other pathways (kgb-1Δ, daf-2Δ). Rescue and overexpression experiments verified the negative effects of pmk-1Δ on heat tolerance. Studying gene expression by RNA-seq and semi-quantitative reverse transcriptase polymerase chain reaction revealed positive effects of the PMK-1 pathway on the expression of genes for chaperones, protein biosynthesis, protein degradation, and other functional categories. Thus, the PMK-1 pathway is involved in the heat stress responses of C. elegans, possibly by a PMK-1-mediated activation of the transcription factor SKN-1 and/or an indirect or direct PMK-1-dependent activation (hyperphosphorylation) of heat-shock factor 1.

A phenomenological approach shows a high coherence of warming patterns in dimictic aquatic systems across latitude.

To predict the coherence in local responses to large-scale climatic forcing among aquatic systems, we developed a generalized approach to compare long-term data of dimictic water bodies based on phenomenologically defined hydrographic events. These climate-sensitive phases (inverse stratification, spring overturn, early thermal stratification, summer stagnation) were classified in a dual code (cold/warm) based on threshold temperatures. Accounting for a latitudinal gradient in seasonal timing of phases derived from gradients in cumulative irradiation (2.2 days per degree latitude), we found a high spatial and temporal coherence in warm-cold patterns for six lakes (84 %) and the Baltic Sea (78 %), even when using the same thresholds for all sites. Similarity to CW-codes for the North Sea still was up to 72 %. The approach allows prediction of phase-specific warming trends and resulting instantaneous or time-delayed ecological responses. Exemplarily, we show that warming during early thermal stratification controls food-web-mediated effects on key species during summer.

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.

Secondary metabolites of Pseudomonas fluorescens CHA0 drive complex non-trophic interactions with bacterivorous nematodes.

Non-trophic interactions are increasingly recognised as a key parameter of predator-prey interactions. In soil, predation by bacterivorous nematodes is a major selective pressure shaping soil bacterial communities, and many bacteria have evolved defence mechanisms such as toxicity. In this study, we show that extracellular secondary metabolites produced by the model soil bacterium Pseudomonas fluorescens CHA0 function as a complex defence strategy against bacterivorous nematodes. Using a collection of functional mutants lacking genes for the biosynthesis of one or several extracellular metabolites, we evaluated the impact of bacterial secondary metabolites on the survival and chemotactic behaviour of the nematode Caenorhabditis elegans. Additionally, we followed up the stress status of the nematodes by measuring the activation of the abnormal DAuer Formation (DAF) stress cascade. All studied secondary metabolites contributed to the toxicity of the bacteria, with hydrogen cyanide efficiently repelling the nematodes, and both hydrogen cyanide and 2,4-DAPG functioning as nematicides. Moreover, these metabolites elicited the DAF stress response cascade of C. elegans, showing that they affect nematode physiology already at sublethal concentrations. The results suggest that bacterial secondary metabolites responsible for the suppression of plant pathogens strongly inhibit bacterivorous nematodes and thus likely contribute to the resistance of bacteria against predators in soil.

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.

Reproductive fitness and dietary choice behavior of the genetic model organism Caenorhabditis elegans under semi-natural conditions.

Laboratory breeding conditions of the model organism C. elegans do not correspond with the conditions in its natural soil habitat. To assess the consequences of the differences in environmental conditions, the effects of air composition, medium and bacterial food on reproductive fitness and/or dietary-choice behavior of C. elegans were investigated. The reproductive fitness of C. elegans was maximal under oxygen deficiency and not influenced by a high fractional share of carbon dioxide. In media approximating natural soil structure, reproductive fitness was much lower than in standard laboratory media. In seminatural media, the reproductive fitness of C. elegans was low with the standard laboratory food bacterium E. coli (γ-Proteobacteria), but significantly higher with C. arvensicola (Bacteroidetes) and B. tropica (ß-Proteobacteria) as food. Dietary-choice experiments in semi-natural media revealed a low preference of C. elegans for E. coli but significantly higher preferences for C. arvensicola and B. tropica (among other bacteria). Dietary-choice experiments under quasi-natural conditions, which were feasible by fluorescence in situ hybridization (FISH) of bacteria, showed a high preference of C. elegans for Cytophaga-Flexibacter-Bacteroides, Firmicutes, and ß-Proteobacteria, but a low preference for γ-Proteobacteria. The results show that data on C. elegans under standard laboratory conditions have to be carefully interpreted with respect to their biological significance.

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.

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.

The MAP kinase JNK-1 of Caenorhabditis elegans: location, activation, and influences over temperature-dependent insulin-like signaling, stress responses, and fitness.

The mitogen-activated protein kinase (MAPK) pathways and insulin-like signaling play pivotal roles in cellular stress response. Using an anti-phospho-SAPK/JNK antibody and a daf-16::GFP-based reporter assay, the present study shows in Caenorhabditis elegans that ambient temperature (1-37 degrees C) specifically influences the activation (phosphorylation) of the MAP kinase JNK-1 as well as the nuclear translocation of DAF-16, the main downstream target of insulin-like signaling. Activated JNK-1 was detected only in neuronal cells, and JNK-1 was found to be controlled by the MAPK JKK-1 under heat stress. Comparative analyses on the wildtype and a jnk-1 deletion mutant revealed a promoting influence of JNK-1 on both nuclear DAF-16 translocations and DAF-16 target gene (superoxide dismutase 3, sod-3) expressions within peripheral, non-neuronal tissue. Consequently, the mutant exhibited a reduced thermal tolerance and reproductive fitness at higher temperatures. These results provide evidence of indirect interactions between neuronal MAPK and peripheral insulin-like signaling in response to environmental stimuli (temperature, H2O2).

Respiratory plasticity in response to changes in oxygen supply and demand.

Aerobic organisms maintain O(2) homeostasis by responding to changes in O(2) supply and demand in both short and long time domains. In this review, we introduce several specific examples of respiratory plasticity induced by chronic changes in O(2) supply (environmental hypoxia or hyperoxia) and demand (exercise-induced and temperature-induced changes in aerobic metabolism). These studies reveal that plasticity occurs throughout the respiratory system, including modifications to the gas exchanger, respiratory pigments, respiratory muscles, and the neural control systems responsible for ventilating the gas exchanger. While some of these responses appear appropriate (e.g., increases in lung surface area, blood O(2) capacity, and pulmonary ventilation in hypoxia), other responses are potentially harmful (e.g., increased muscle fatigability). Thus, it may be difficult to predict whole-animal performance based on the plasticity of a single system. Moreover, plastic responses may differ quantitatively and qualitatively at different developmental stages. Much of the current research in this field is focused on identifying the cellular and molecular mechanisms underlying respiratory plasticity. These studies suggest that a few key molecules, such as hypoxia inducible factor (HIF) and erythropoietin, may be involved in the expression of diverse forms of plasticity within and across species. Studying the various ways in which animals respond to respiratory challenges will enable a better understanding of the integrative response to chronic changes in O(2) supply and demand.