Rüdiger J. Paul

Phototaxis in water fleas (Daphnia magna) is differently influenced by visible and UV light

Abstract The effects of vertical illumination with monochromatic lights on phototaxis of Daphnia magna in a test chamber were determined at five levels of equal quantal flux density (between 188 and 6.42 · 10−5 nEinstein). Visible adaptation light (500 nm) and subsequent spectral test light had the same quantal flux density. The animals reacted to ultraviolet light (260–380 nm) with negative phototaxis, whereas visible light (420–600 nm) caused positive phototaxis. Action spectra were determined, based on the evaluation of different parameters of phototactic behavior. The maximum spectral sensitivity in the ultraviolet was found at 340 nm. The maximum spectral efficiency in the visible varied in dependence on light intensity. Ecological consequences of the results are discussed.

Anaerobiosis in the nematode Caenorhabditis elegans

In Caenorhabditis elegans, mortality rates and changes in concentrations of carbohydrate stores and anaerobic end products were determined in anoxic (test) and normoxic (control) animals at two different temperatures (10 and 20 degrees C). The anoxic tolerance of the free-living nematode proved to be well-developed: at 10 degrees C, about 50% of animals had survived a period of 50 h of anoxia. The carbohydrate stores (approximately 30 mmol glycosyl units kg-1 freshweight (FW)) were reduced by two-thirds within 24 h of anoxia at both temperatures. L-lactate, acetate, succinate, and propionate were identified as the main anaerobic end products. The amounts and proportions of the end products were dependent on temperature. They did not accumulate very much in the tissues, but were mainly excreted. During anoxia, the metabolism of C. elegans was depressed to 3-4% of the aerobic value. The food-source Escherichia coli was found to be at least partly alive in the gut of the animals. To separate between anaerobiosis in animals and bacteria, cleaning procedures were applied, and additional control measurements were made: anaerobic end products produced either by E. coli alone or by bacteria-free (axenic) bred nematodes were quantified at identical incubation conditions.

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

BackgroundTemperature 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.ResultsSpecific sets of proteins were found to be differentially expressed in 10°C or 20°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.ConclusionSpecific 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°C and 20°C acclimated animals reflect the increased amino-acids demand and the reduced protein reserves in the animals 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.

Circulation and respiratory control in millimetre-sized animals (Daphnia magna, Folsomia candida ) studied by optical methods

Abstract During hypoxia, oxyregulating water-breathers usually control O2 uptake by changing ventilatory convection. Using optical techniques we studied ventilation, circulation and respiratory control in small animals, a millimetre in size, which were more or less pronounced oxyregulators (Daphnia magna, Folsomia candida). In Daphnia we found no adaptive changes in the ventilatory water flow rate during hypoxia. Frequency and amplitude of the movements of the thoracic limbs remained constant during this environmental condition. During anoxia there was a reduction in both. In contrast to ventilatory convection, the circulatory blood flow rate adapted to hypoxia. At low oxygen partial pressures, the heart frequency strongly increased (compensatory tachycardia) in Daphnia, whereas the stroke volume remained constant. Accordingly, there was an increase in cardiac output during hypoxia. Folsomia also showed a marked increase of heart frequency during severe hypoxia. The adaptive changes in blood flow rate should help to maintain sufficient partial pressure differences between medium, blood and tissues and should help to avoid anoxic zones in the animal. During anoxia, the heart continued to beat in Daphnia (at a rate more or less similar to normoxia, but with a reduced stroke volume) for periods of many hours. The heart frequency showed typical courses during anoxia and subsequent normoxia, which are probably related to energy metabolism.

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

BackgroundFreshwater 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 [P o2]: 20 kPa) and hypoxia (P o2: 3 kPa), respectively.ResultsThe 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.ConclusionAcclimatory 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.

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.

Differential haemoglobin gene expression in the crustacean Daphnia magna exposed to different oxygen partial pressures.

Abstract 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 longterm 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.

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°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). J. Cell. Physiol. 214: 721–729, 2008.

Systemic and metabolic responses in Daphnia magna to anoxia

The anoxic tolerance of the water flea Daphnia magna proved to be well-developed. During the first 1 or 2 h of anoxia, perfusion rate was not essentially impeded: admittedly, there was a 30–40% reduction of the amplitude of heart contractions, but the heart beat at a rate either similar to normoxia (150 min−1 at T=15°C) or markedly higher (in more than half of the experiments). During that period, energy was provided by a high rate of anaerobic metabolism: l-lactate partly accumulated in the animals (0.36 μmol lactate g−1 FW min−1), partly it was excreted into the medium (0.08 μmol lactate g−1 FW min−1). The anaerobiosis was accompanied by a decrease of intracellular as well as extracellular pH (metabolic acidosis). A metabolic depression, starting after about 1 or 2 h of anoxia, helped to prolong survival time during long-term anoxia (up to 24 h): in many experiments, heart and perfusion rate sharply declined to a much lower level (well below the normoxic rate), which was maintained during the next hours of anoxia. The accumulation of lactate stopped, while lactate excretion into the medium continued at an unchanged rate. If the anoxic period exceeded 20 min, long-term recovery processes, lasting up to 4 h, were found during normoxia: the heart rate increased and reached a maximum frequently after 40–50 min of recovery. There was also a restoration of pre-anoxic intra- and extracellular pH values. Additional experiments on the effects of altered ambient PCO2/pH on heart rate and blood pH revealed pH to be a possible control factor of heart rate.

The process of hypoxic induction of Daphnia magna hemoglobin: subunit composition and functional properties

The process of oxygen-dependent hemoglobin induction in Daphnia magna was studied over an 11-day period of hypoxia (ambient oxygen partial pressure: 3 kPa). Along with the increase of hemoglobin concentration in the hemolymph, hemoglobin became the dominant protein fraction in gel filtration experiments using extracts of whole animals. The size of the native aggregates was constant. However, subunit composition depended on the duration of hypoxia: the pattern of predominantly expressed subunits under hypoxia deviated from that of normoxic individuals. The varying degree of hypoxic induction for different hemoglobin subunits was confirmed by autoradiography. Along with changes in hemoglobin subunit composition, oxygen affinity of the respiratory protein increased. The dynamics of the hemoglobin induction process was analysed. Newly synthesized hemoglobin can be detected within 18 h after the onset of hypoxia. A marked increase in hemoglobin concentration is evident from the third day of hypoxia, and a steady state of hemoglobin concentration is reached within 11 days. The changes of hemoglobin subunit expression in response to hypoxia form the structural basis for the observed adjustments of hemoglobin function leading to enhanced oxygen transport at low ambient oxygen concentrations.