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

<p>Abstract</p> <p>Background</p> <p>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 <it>Daphnia pulex</it>.</p> <p>Results</p> <p>Specific sets of proteins were found to be differentially expressed in 10°C or 20°C acclimated <it>D. pulex</it>. 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.</p> <p>Conclusion</p> <p>Specific sets of cold-repressed and cold-induced proteins in <it>D. pulex </it>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 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 <it>D. pulex </it>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.</p

Respiratory plasticity in response to changes in oxygen supply and demand

Aerobic organisms maintain O2 homeostasis by responding to changes in O2 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 O2 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 O2 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 O2 supply and deman

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

Cambronero_etal_microsatellite_genotyping

Cambronero_etal_microsatellite_genotyping: microsatellite genotyping results for the genotypes used in the study obtained from 13 microsatellite loci arranged in two multiplexes. The genotype IDs are as in Orsini et al, 2016 Molecular Ecology. The microsatellite loci were first published in Jansen et al, 2010 Molecular Ecology Resources as part of a panel of 84 microsatellites. -9 indicates missing data

Cambronero_etal_microsatellite_genotyping

Cambronero_etal_microsatellite_genotyping: microsatellite genotyping results for the genotypes used in the study obtained from 13 microsatellite loci arranged in two multiplexes. The genotype IDs are as in Orsini et al, 2016 Molecular Ecology. The microsatellite loci were first published in Jansen et al, 2010 Molecular Ecology Resources as part of a panel of 84 microsatellites. -9 indicates missing data

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. (C) 2016 Elsevier Inc. All rights reserved

Target analysis for the adipokinetic hormone of 'Daphnia'

Members of the red pigment-concentrating hormone (RPCH)/adipokinetic hormone (AKH) peptide family are involved in many physiological, developmental and behavioral processes in insects, and pigment translocation in decapod crustaceans. In the branchiopod Daphnia pulex, a preprohormone for RPCH/AKH is encoded in the genome and the mature peptide, Dappu-RPCH (pQVNFSTSW-amide) is predicted from the preprohormone. Here, we have developed a method to measure the peptide in hemolymph of Daphnia. It uses reversed-phase liquid chromatography and high-resolution mass spectrometry of the singly- and the doubly-charged peptide ions, respectively, and their gas phase fragmentation for target identification. Pilot studies were conducted with synthetic Dappu-RPCH and the method was sensitive at the low fmol level without biological matrix. When hemolymph samples were however spiked with known amounts of the synthetic peptide, much less peptide was recovered and detected. Thus far, with our method of peptide purification, the endogenous peptide was not detectable in hemolymph of Daphnia where AKH titres of 1 fmol/µl hemolymph are expected.Die Mitglieder der Familie des rotes-Pigment-konzentrierenden (RPCH) / adipokinetischen Hormons sind in viele physiologische und Verhaltens-Prozesse von Insekten sowie die Pigment-Translokation in Zehnfußkrebsen involviert. Im Genom des Kiemenfußkrebses Daphnia pulex ist ein Preprohormon für RPCH/AKH kodiert, nach dem das voll ausgebildete Peptid, Dappu-RPCH (pQVNFSTSW-amide), vorausgesagt wird. Wir haben eine Methode entwickelt, um das Peptid in der Hämolymphe von Daphnia nachzuweisen. Sie verwendet Umkehrphasen-Flüssigchromatographie und hochauflösende Massenspektrometrie des einfach und des zweifach geladenen Peptidions und deren Gasphasenfragmentierung zur Target-Identifizierung. Pilotstudien wurden mit synthetischem Dappu-RPCH durchgeführt. Die Methode war empfindlich auf dem niedrigen fmol-Level ohne biologische Matrix. Wurde jedoch die Hämolymphe mit bekannten Mengen des synthetischen Peptids versetzt, wurde viel weniger Peptid gemessen. Mit unserer bisherigen Methode zur Peptidreinigung konnte daher noch kein endogenes Peptid in der Hämolymphe von Daphnia bestimmt werden, wo AKH-Titer von 1 fmol/µl erwartet werden

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

<p>Abstract</p> <p>Background</p> <p>Freshwater planktonic crustaceans of the genus <it>Daphnia </it>show a remarkable plasticity to cope with environmental changes in oxygen concentration and temperature. One of the key proteins of adaptive gene control in <it>Daphnia pulex </it>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 [<it>P</it>o₂]: 20 kPa) and hypoxia (<it>P</it>o₂: 3 kPa), respectively.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>Acclimatory adjustments of the <it>D. pulex </it>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.</p