Non-invasive MRI Studies of Ventilatory and Cardiovascular Performance in Edible Crabs Cancer pagurus During Warming Under Elevated CO2 Levels.

The thermal tolerance of marine decapod crustacea is defined through their capacities for oxygen uptake and distribution. High ambient CO2 levels were previously shown to reduce hemolymph oxygen levels at enhanced cardiac performance during warming. This study investigated the impacts of warming under two CO2 levels on ventilation and hemolymph circulation in edible crabs Cancer pagurus. It also highlights changes in the ventilatory and cardiac pauses displayed by Decapoda under routine metabolism. Animals were exposed to step-wise, sub-critical warming (12-20°C over 5 days) under control (470 µatm) and high (1,350 µatm) water PCO2. Flow-through respirometry was combined with magnetic resonance imaging and infra-red photoplethysmography to allow for simultaneous, non-invasive measurements of metabolic rates ( M ˙ O 2 ), ventilation and cardiovascular performance. Crabs spent significantly more time in a low M ˙ O 2 state (metabolic pause), when experiencing high CO2 conditions above 16°C, compared to normocapnic warming. Heart rates leveled off beyond 18°C at any CO2 level. Cardiac output continued to increase with high-CO2-warming, due to elevated cardiac stroke volumes. Consequently, temperature-dependent branchial hemolymph flow remained unaffected by CO2. Instead, a suppressing effect of CO2 on ventilation was found beyond 16°C. These results indicate constrained oxygen uptake at stable cardiovascular performance in a decapod crustacean. Cancer pagurus: urn:lsid:zoobank.org:act:B750F89A-84B5-448B-8D80-EBD724A1C9D4.

Studying the cardiovascular system of a marine crustacean with magnetic resonance imaging at 9.4 T.

OBJECTIVES: An approach is presented for high-field MRI studies of the cardiovascular system (CVS) of a marine crustacean, the edible crab Cancer pagurus, submerged in highly conductive seawater. MATERIALS AND METHODS: Structure and function of the CVS were investigated at 9.4 T. Cardiac motion was studied using self-gated CINE MRI. Imaging protocols and radio-frequency coil arrangements were tested for anatomical imaging. Haemolymph flow was quantified using phase-contrast angiography. Signal-to-noise-ratios and flow velocities in afferent and efferent branchial veins were compared with Student's t test (n = 5). RESULTS: Seawater induced signal losses were dependent on imaging protocols and RF coil setup. Internal cardiac structures could be visualized with high spatial resolution within 8 min using a gradient-echo technique. Variations in haemolymph flow in different vessels could be determined over time. Maximum flow was similar within individual vessels and corresponded to literature values from Doppler measurements. Heart contractions were more pronounced in lateral and dorso-ventral directions than in the anterior-posterior direction. DISCUSSION: Choosing adequate imaging protocols in combination with a specific RF coil arrangement allows to monitor various parts of the crustacean CVS with exceptionally high spatial resolution despite the adverse effects of seawater at 9.4 T.

STC1 and PTHrP Modify Carbohydrate and Lipid Metabolism in Liver of a Teleost Fish.

Stanniocalcin 1 (STC1) and parathyroid hormone-related protein (PTHrP) are calciotropic hormones in vertebrates. Here, a recently hypothesized metabolic role for these hormones is tested on European sea bass treated with: (i) teleost PTHrP(1-34), (ii) PTHrP(1-34) and anti-STC1 serum (pro-PTHrP groups), (iii) a PTHrP antagonist PTHrP(7-34) or (iv) PTHrP(7-34) and STC1 (pro-STC1 groups). Livers were analysed using untargeted metabolic profiling based on proton nuclear magnetic resonance (1H-NMR) spectroscopy. Concentrations of branched-chain amino acid (BCAA), alanine, glutamine and glutamate increased in pro-STC1 groups suggesting their mobilization from the muscle to the liver for degradation and gluconeogenesis from alanine and glutamine. In addition, only STC1 treatment decreased the concentrations of succinate, fumarate and acetate, indicating slowing of the citric acid cycle. In the pro-PTHrP groups the concentrations of glucose, erythritol and lactate decreased, indicative of gluconeogenesis from lactate. Taurine, trimethylamine, trimethylamine N-oxide and carnitine changed in opposite directions in the pro-STC1 versus the pro-PTHrP groups, suggesting opposite effects, with STC1 stimulating lipogenesis and PTHrP activating lipolysis/ß-oxidation of fatty acids. These findings suggest a role for STC1 and PTHrP related to strategic energy mechanisms that involve the production of glucose and safeguard of liver glycogen reserves for stressful situations.

Non-invasive quantification of cardiac stroke volume in the edible crab Cancer pagurus.

BACKGROUND: Brachyuran crabs can effectively modulate cardiac stroke volume independently of heart rate in response to abiotic drivers. Non-invasive techniques can help to improve the understanding of cardiac performance parameters of these animals. This study demonstrates the in vivo quantification of cardiac performance parameters through magnetic resonance imaging (MRI) on the edible crab Cancer pagurus. Furthermore, the suitability of signal integrals of infra-red photoplethysmographs as a qualitative tool is assessed under severe hypoxia. RESULTS: Multi-slice self-gated cardiac cinematic (CINE) MRI revealed the structure and motion of the ventricle to quantify heart rates, end-diastolic volume, end-systolic volume, stroke volume and ejection fraction. CINE MRI showed that stroke volumes increased under hypoxia because of a reduction of end-systolic volumes at constant end-diastolic volumes. Plethysmograph recordings allowed for automated heart rate measurements but determination of a qualitative stroke volume proxy strongly depended on the position of the sensor on the animal. Both techniques revealed a doubling in stroke volumes after 6 h under severe hypoxia (water PO2 = 15% air saturation). CONCLUSIONS: MRI has allowed for detailed descriptions of cardiac performance in intact animals under hypoxia. The temporal resolution of quantitative non-invasive CINE MRI is limited but should encourage further refining. The stroke volume proxy based on plethysmograph recordings is feasible to complement other cardiac measurements over time. The presented methods allow for non-destructive in vivo determinations of multiple cardiac performance parameters, with the possibility to study neuro-hormonal or environmental effects on decapod cardio physiology.

Elevated pCO2 Affects Feeding Behavior and Acute Physiological Response of the Brown Crab Cancer pagurus.

Anthropogenic climate change exposes marine organisms to CO2 induced ocean acidification (OA). Marine animals may make physiological and behavioral adaptations to cope with OA. Elevated pCO2 may affect metabolism, feeding, and energy partition of marine crabs, and thereby affect their predator-prey dynamics with mussels. Therefore, we examined the effects of simulated future elevated pCO2 on feeding behavior and energy metabolism of the brown crab Cancer pagurus. Following 54 days of pre-acclimation to control CO2 levels (360 µatm) at 11°C, crabs were exposed to consecutively increased oceanic CO2 levels (2 weeks for 1200 and 2300 µatm, respectively) and subsequently returned to control CO2 level (390 µatm) for 2 weeks in order to study their potential to acclimate elevated pCO2 and recovery performance. Standard metabolic rate (SMR), specific dynamic action (SDA) and feeding behavior of the crabs were investigated during each experimental period. Compared to the initial control CO2 conditions, the SMRs of CO2 exposed crabs were not significantly increased, but increased significantly when the crabs were returned to normal CO2 levels. Conversely, SDA was significantly reduced under high CO2 and did not return to control levels during recovery. Under high CO2, crabs fed on smaller sized mussels than under control CO2; food consumption rates were reduced; foraging parameters such as searching time, time to break the prey, eating time, and handling time were all significantly longer than under control CO2, and prey profitability was significantly lower than that under control conditions. Again, a two-week recovery period was not sufficient for feeding behavior to return to control values. PCA results revealed a positive relationship between feeding/SDA and pH, but negative relationships between the length of foraging periods and pH. In conclusion, elevated pCO2 caused crab metabolic rate to increase at the expense of SDA. Elevated pCO2 affected feeding performance negatively and prolonged foraging periods. These results are discussed in the context of how elevated pCO2 may impair the competitiveness of brown crabs in benthic communities.

Metabolic efficiency in yeast Saccharomyces cerevisiae in relation to temperature dependent growth and biomass yield.

Canonized view on temperature effects on growth rate of microorganisms is based on assumption of protein denaturation, which is not confirmed experimentally so far. We develop an alternative concept, which is based on view that limits of thermal tolerance are based on imbalance of cellular energy allocation. Therefore, we investigated growth suppression of yeast Saccharomyces cerevisiae in the supraoptimal temperature range (30-40°C), i.e. above optimal temperature (Topt). The maximal specific growth rate (µmax) of biomass, its concentration and yield on glucose (Yx/glc) were measured across the whole thermal window (5-40°C) of the yeast in batch anaerobic growth on glucose. Specific rate of glucose consumption, specific rate of glucose consumption for maintenance (mglc), true biomass yield on glucose (Yx/glc(true)), fractional conservation of substrate carbon in product and ATP yield on glucose (Yatp/glc) were estimated from the experimental data. There was a negative linear relationship between ATP, ADP and AMP concentrations and specific growth rate at any growth conditions, whilst the energy charge was always high (~0.83). There were two temperature regions where mglc differed 12-fold, which points to the existence of a 'low' (within 5-31°C) and a 'high' (within 33-40°C) metabolic mode regarding maintenance requirements. The rise from the low to high mode occurred at 31-32°C in step-wise manner and it was accompanied with onset of suppression of µmax. High mglc at supraoptimal temperatures indicates a significant reduction of scope for growth, due to high maintenance cost. Analysis of temperature dependencies of product formation efficiency and Yatp/glc revealed that the efficiency of energy metabolism approaches its lower limit at 26-31°C. This limit is reflected in the predetermined combination of Yx/glc(true), elemental biomass composition and degree of reduction of the growth substrate. Approaching the limit implies a reduction of the safety margin of metabolic efficiency. We hypothesize that a temperature increase above Topt (e.g. >31°C) triggers both an increment in mglc and suppression of µmax, which together contribute to an upshift of Yatp/glc from the lower limit and thus compensate for the loss of the safety margin. This trade-off allows adding 10 more degrees to Topt and extends the thermal window up to 40°C, sustaining survival and reproduction in supraoptimal temperatures. Deeper understanding of the limits of thermal tolerance can be practically exploited in biotechnological applications.

Temperature, metabolic power and the evolution of endothermy.

Endothermy has evolved at least twice, in the precursors to modern mammals and birds. The most widely accepted explanation for the evolution of endothermy has been selection for enhanced aerobic capacity. We review this hypothesis in the light of advances in our understanding of ATP generation by mitochondria and muscle performance. Together with the development of isotope-based techniques for the measurement of metabolic rate in free-ranging vertebrates these have confirmed the importance of aerobic scope in the evolution of endothermy: absolute aerobic scope, ATP generation by mitochondria and muscle power output are all strongly temperature-dependent, indicating that there would have been significant improvement in whole-organism locomotor ability with a warmer body. New data on mitochondrial ATP generation and proton leak suggest that the thermal physiology of mitochondria may differ between organisms of contrasting ecology and thermal flexibility. Together with recent biophysical modelling, this strengthens the long-held view that endothermy originated in smaller, active eurythermal ectotherms living in a cool but variable thermal environment. We propose that rather than being a secondary consequence of the evolution of an enhanced aerobic scope, a warmer body was the means by which that enhanced aerobic scope was achieved. This modified hypothesis requires that the rise in metabolic rate and the insulation necessary to retain metabolic heat arose early in the lineages leading to birds and mammals. Large dinosaurs were warm, but were not endotherms, and the metabolic status of pterosaurs remains unresolved.

Geographical variation in thermal tolerance within Southern Ocean marine ectotherms.

Latitudinal comparisons of the Southern Ocean limpet, Nacella concinna, and clam, Laternula elliptica, acclimated to 0.0 degrees C, were used to assess differences in thermal response to two regimes, 0.0, 5.1 to 10.0 degrees C and 2.5, 7.5 to 12.5 degrees C, raised at 5.0 degrees C per week. At each temperature, tissue energy status was measured through a combination of O(2) consumption, intracellular pH, cCO(2), citrate synthase (CS) activity, organic acids (succinate, acetate, propionate), adenylates (ATP, ADP, AMP, ITP, PLA (phospho-L-arginine)) and heart rate. L. elliptica from Signy (60 degrees S) and Rothera (67 degrees S), which experience a similar thermal regime (-2 to +1 degrees C) had the same lethal (7.5-10.0 degrees C), critical (5.1-7.5 degrees C) and pejus (<5.1 degrees C;=getting worse) limits with only small differences in biochemical response. N. concinna, which experiences a wider thermal regime (-2 to +15.8 degrees C), had higher lethal limits (10.0-12.5 degrees C). However, at their Northern geographic limit N. concinna, which live in a warmer environment (South Georgia, 54 degrees S), had a lower critical limit (5.1-10.0 degrees C; O(2), PLA and organic acids) than Rothera and Signy N. concinna (10.0-12.5 degrees C). This lower limit indicates that South Georgia N. concinna have different biochemical responses to temperatures close to their thermal limit, which may make them more vulnerable to future warming trends.

Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia.

The preservation of ion balance and pH despite environmental fluctuations is essential for the maintenance of vital cellular functions. While several ion transporters contribute to acid-base regulation in fish, the involvement and expression of key transporters under hypercapnia remain to be established. Here, two members of the HCO(3)(-) transporter family (Na(+)/HCO(3)(-) cotransporter NBC1 and Cl(-)/HCO(3)(-) exchanger AE1) were described for the first time in gills of marine fish. Benthic eelpout Zoarces viviparus were acclimated to 10,000 ppm CO(2). Hypercapnia did not affect whole animal oxygen consumption over a period of 4 days. During a time series of 6 wk NBC1 mRNA levels first decreased by about 40% (8 to 24 h) but finally increased about threefold over control. mRNA expression of AE1 decreased transiently by 50% at day 4 but recovered to control levels only. Reduced mRNA levels were also found for two Na(+)/H(+) exchangers (NHE1A, NHE1B) during the first days (by 50-60% at days 1 and 2), followed by restoration of control levels. This pattern was mirrored in a slight decrease of NHE1 protein contents and its subsequent recovery. In contrast, Na(+)-K(+)-ATPase mRNA and protein contents, as well as maximum activity, rose steadily from the onset of hypercapnia, and reached up to twofold control levels at the end. These results indicate shifting acclimation patterns between short- and long-term CO(2) exposures. Overall, ion gradient-dependent transporter mRNA levels were transiently downregulated in the beginning of the disturbance. Upregulation of NBC1 on long timescales stresses the importance of this transporter in the hypercapnia response of marine teleosts. Long-term rearrangements include Na(+)-K(+)-ATPase at higher densities and capacities, indicating a shift to elevated rates of ion and acid-base regulation under environmental hypercapnia.

The integrative and evolutionary biology of gas-binding copper proteins: an introduction.

This article summarizes the contributions given at the symposium "The Benefits of Gas-binding Proteins. Integrative and Evolutionary Physiology of Copper Proteins: Molecules to Organisms and their Environment," presented at the First International Congress of Respiratory Biology, August 14-16, at Bad Honnef/Bonn, Germany.

Chronological and physiological ageing in a polar and a temperate mud clam.

We investigated chronological and physiological ageing of two mud clams with regard to the "rate of living theory" (Pearl, 1928) and the "free radical theory of ageing" (Harman, 1956). The Antarctic Laternula elliptica (Pholadomyoida) and the temperate Mya arenaria (Myoida) represent the same ecotype (benthic infaunal filter feeders), but differ in maximum life span, 36 and 13 years, respectively. L. elliptica has a two-fold lower standard metabolic rate than M. arenaria, but its life long energy turnover at maximal age is three times higher. When comparing the two species within the lifetime window of M. arenaria, antioxidant capacities (glutathione, catalase) are higher and tissue oxidation (ratio of oxidised to reduced glutathione, lipofuscin accumulation) is lower in the polar L. elliptica than in the temperate mud clam. Tissue redox state in L. elliptica remained stable throughout all ages, whereas it increased dramatically in aged M. arenaria. Our results indicate that metabolic rates and maintenance of tissue redox state are major factors determining maximum lifespan in the investigated mud clams.

High-energy phosphate metabolism during exercise and recovery in temperate and Antarctic scallops: an in vivo 31P-NMR study.

In vivo (31)P-nuclear magnetic resonance (NMR) spectroscopy was used to measure the levels of ATP, phospho-l-arginine (PLA), and inorganic phosphate in the adductor muscle of the Antarctic scallop Adamussium colbecki and two temperate species, Aequipecten opercularis and Pecten maximus. Graded exercise regimes from light (one to two contractions) to exhausting (failing to respond to further stimulation) were imposed on animals of each species at its habitat temperature (0 degrees vs. 12 degrees C, respectively). NMR spectroscopy allowed noninvasive measurement of metabolite levels and intracellular pH at high time resolution (30-120-s intervals) during exercise and throughout the recovery period. Significant differences were shown between the magnitude and form of the metabolic response with increasing levels of exercise in each species. After exhaustion, short-term (first 15 min) muscle alkalosis was followed by acidosis of up to 0.2 pH units during the recovery process. Aequipecten opercularis had similar resting muscle PLA levels compared with either P. maximus or A. colbecki but used a fivefold greater proportion of this store per contraction and was able to perform only half as many claps (maximum of 24) as the other species before exhaustion. All species regenerated their PLA store at a similar rate despite different environmental temperatures. These findings argue for some cold compensation of muscular performance and recovery capacities in the Antarctic scallop, albeit at levels of performance similar to scallops with low activity lifestyles from temperate latitudes.