The physiological mechanisms limiting and adjusting cold and heat tolerance have regained interest in the light of global warming and associated shifts in the geographical distribution of ectothermic animals. Recent comparative studies, largely carried out on marine ectotherms, indicate that the processes and limits of thermal tolerance are linked with the adjustment of aerobic scope and capacity of the whole animal as a crucial step in thermal adaptation on top of parallel adjustments at the molecular or membrane level. In accordance with Shelford's law of tolerance decreasing whole animal aerobic scope characterises the onset of thermal limitation at low and high pejus thresholds (pejus=getting worse). The drop in aerobic scope of an animal indicated by falling oxygen levels in the body fluids and or the progressively limited capacity of circulatory and ventilatory mechanisms. At high temperatures, excessive oxygen demand causes insufficient oxygen levels in the body fluids, whereas at low temperatures the aerobic capacity of mitochondria may become limiting for ventilation and circulation. Further cooling or warming beyond these limits leads to low or high critical threshold temperatures (T(c)) where aerobic scope disappears and transition to an anaerobic mode of mitochondrial metabolism and progressive insufficiency of cellular energy levels occurs. The adjustments of mitochondrial densities and their functional properties appear as a critical process in defining and shifting thermal tolerance windows. The finding of an oxygen limited thermal tolerance owing to loss of aerobic scope is in line with Taylor's and Weibel's concept of symmorphosis, which implies that excess capacity of any component of the oxygen delivery system is avoided. The present study suggests that the capacity of oxygen delivery is set to a level just sufficient to meet maximum oxygen demand between the average highs and lows of environmental temperatures. At more extreme temperatures only time limited passive survival is supported by anaerobic metabolism or the protection of molecular functions by heat shock proteins and antioxidative defence. As a corollary, the first line of thermal sensitivity is due to capacity limitations at a high level of organisational complexity, i.e. the integrated function of the oxygen delivery system, before individual, molecular or membrane functions become disturbed. These interpretations are in line with the more general consideration that, as a result of the high level of complexity of metazoan organisms compared with simple eukaryotes and then prokaryotes, thermal tolerance is reduced in metazoans. A similar sequence of sensitivities prevails within the metazoan organism, with the highest sensitivity at the organismic level and wider tolerance windows at lower levels of complexity. However, the situation is different in that loss in aerobic scope and progressive hypoxia at the organismic level define the onset of thermal limitation which then transfers to lower hierarchical levels and causes cellular and molecular disturbances. Oxygen limitation contributes to oxidative stress and, finally, denaturation or malfunction of molecular repair, e.g. during suspension of protein synthesis. The sequence of thermal tolerance limits turns into a hierarchy, ranging from systemic to cellular to molecular levels.

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Climate variations and the physiological basis of temperature dependent biogeography: systemic to molecular hierarchy of thermal tolerance in animals.

Based on its generally accessible I/II redox couple and bioavailability, copper plays a wide variety of roles in nature that mostly involve electron transfer (ET), O2 binding, activation and reduction, NO2− and N2O reduction and substrate activation. Copper sites that perform ET are the mononuclear blue Cu site that has a highly covalent CuII-S(Cys) bond and the binuclear CuA site that has a Cu2S(Cys)2 core with a Cu-Cu bond that keeps the site delocalized (Cu(1.5)2) in its oxidized state. In contrast to inorganic Cu complexes, these metalloprotein sites transfer electrons rapidly often over long distances, as has been previously reviewed.1–4 Blue Cu and CuA sites will only be considered here in their relation to intramolecular ET in multi-center enzymes. The focus of this review is on the Cu enzymes (Figure 1). Many are involved in O2 activation and reduction, which has mostly been thought to involve at least two electrons to overcome spin forbiddenness and the low potential of the one electron reduction to superoxide (Figure 2).5,6 Since the Cu(III) redox state has not been observed in biology, this requires either more than one Cu center or one copper and an additional redox active organic cofactor. The latter is formed in a biogenesis reaction of a residue (Tyr) that is also Cu catalyzed in the first turnover of the protein. Recently, however, there have been a number of enzymes suggested to utilize one Cu to activate O2 by 1e− reduction to form a Cu(II)-O2•− intermediate (an innersphere redox process) and it is important to understand the active site requirements to drive this reaction. The oxidases that catalyze the 4e−reduction of O2 to H2O are unique in that they effectively perform this reaction in one step indicating that the free energy barrier for the second two-electron reduction of the peroxide product of the first two-electron step is very low. In nature this requires either a trinuclear Cu cluster (in the multicopper oxidases) or a Cu/Tyr/Heme Fe cluster (in the cytochrome oxidases). The former accomplishes this with almost no overpotential maximizing its ability to oxidize substrates and its utility in biofuel cells, while the latter class of enzymes uses the excess energy to pump protons for ATP synthesis. In bacterial denitrification, a mononuclear Cu center catalyzes the 1e- reduction of nitrite to NO while a unique µ4S2−Cu4 cluster catalyzes the reduction of N2O to N2 and H2O, a 2e− process yet requiring 4Cu’s. Finally there are now several classes of enzymes that utilize an oxidized Cu(II) center to activate a covalently bound substrate to react with O2.



Figure 1

Copper active sites in biology.





Figure 2

Latimer Diagram for Oxygen Reduction at pH = 7.0 Adapted from References 5 and 6.



This review presents in depth discussions of all these classes of Cu enzymes and the correlations within and among these classes. For each class we review our present understanding of the enzymology, kinetics, geometric structures, electronic structures and the reaction mechanisms these have elucidated. While the emphasis here is on the enzymology, model studies have significantly contributed to our understanding of O2 activation by a number of Cu enzymes and are included in appropriate subsections of this review. In general we will consider how the covalency of a Cu(II)–substrate bond can activate the substrate for its spin forbidden reaction with O2, how in binuclear Cu enzymes the exchange coupling between Cu’s overcomes the spin forbiddenness of O2 binding and controls electron transfer to O2 to direct catalysis either to perform two e− electrophilic aromatic substitution or 1e− H-atom abstraction, the type of oxygen intermediate that is required for H-atom abstraction from the strong C-H bond of methane (104 kcal/mol) and how the trinuclear Cu cluster and the Cu/Tyr/Heme Fe cluster achieve their very low barriers for the reductive cleavage of the O-O bond.

Much of the insight available into these mechanisms in Cu biochemistry has come from the application of a wide range of spectroscopies and the correlation of spectroscopic results to electronic structure calculations. Thus we start with a tutorial on the different spectroscopic methods utilized to study mononuclear and multinuclear Cu enzymes and their correlations to different levels of electronic structure calculations.

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Copper Active Sites in Biology

In this review I show that the '3/4-power scaling law' of metabolic rate is not universal, either within or among animal species. Significant variation in the scaling of metabolic rate with body mass is described mainly for animals, but also for unicells and plants. Much of this variation, which can be related to taxonomic, physiological, and/or environmental differences, is not adequately explained by existing theoretical models, which are also reviewed. As a result, synthetic explanatory schemes based on multiple boundary constraints and on the scaling of multiple energy-using processes are advocated. It is also stressed that a complete understanding of metabolic scaling will require the identification of both proximate (functional) and ultimate (evolutionary) causes. Four major types of intraspecific metabolic scaling with body mass are recognized [based on the power function R=aMb, where R is respiration (metabolic) rate, a is a constant, M is body mass, and b is the scaling exponent]: Type I: linear, negatively allometric (b 1) to one or two later phases of negative allometry (b<1). Ontogenetic changes in the metabolic intensity of four component processes (i.e. growth, reproduction, locomotion, and heat production) appear to be important in these different patterns of metabolic scaling. These changes may, in turn, be shaped by age (size)-specific patterns of mortality. In addition, major differences in interspecific metabolic scaling are described, especially with respect to mode of temperature regulation, body-size range, and activity level. A 'metabolic-level boundaries hypothesis' focusing on two major constraints (surface-area limits on resource/waste exchange processes and mass/volume limits on power production) can explain much, but not all of this variation. My analysis indicates that further empirical and theoretical work is needed to understand fully the physiological and ecological bases for the considerable variation in metabolic scaling that is observed both within and among species. Recommended approaches for doing this are discussed. I conclude that the scaling of metabolism is not the simple result of a physical law, but rather appears to be the more complex result of diverse adaptations evolved in the context of both physico-chemical and ecological constraints.

Beyond the '3/4-power law': variation in the intra- and interspecific scaling of metabolic rate in animals.

CO2 currently accumulating in the atmosphere permeates into ocean surface layers, where it may impact on marine animals in addition to effects caused by global warming. At the same time, several countries are developing scenarios for the disposal of anthropogenic CO2 in the worlds' oceans, especially the deep sea. Elevated CO2 partial pressures (hypercapnia) will affect the physiology of water breathing animals, a phenomenon also considered in recent discussions of a role for CO2 in mass extinction events in earth history. Our current knowledge of CO2 effects ranges from effects of hypercapnia on acid-base regulation, calcification and growth to influences on respiration, energy turnover and mode of metabolism. The present paper attempts to evaluate critical processes and the thresholds beyond which these effects may become detrimental. CO2 elicits acidosis not only in the water, but also in tissues and body fluids. Despite compensatory accumulation of bicarbonate, acid-base parameters (pH, bicarbonate and CO2 levels) and ion levels reach new steady-state values, with specific, long-term effects on metabolic functions. Even though such processes may not be detrimental, they are expected to affect long-term growth and reproduction and may thus be harmful at population and species levels. Sensitivity is maximal in ommastrephid squid, which are characterized by a high metabolic rate and extremely pH-sensitive blood oxygen transport. Acute sensitivity is interpreted to be less in fish with intracellular blood pigments and higher capacities to compensate for CO2 induced acid-base disturbances than invertebrates. Virtually nothing is known about the degree to which deep-sea fishes are affected by short or long term hypercapnia. Sensitivity to CO2 is hypothesized to be related to the organizational level of an animal, its energy requirements and mode of life. Long-term effects expected at population and species levels are in line with recent considerations of a detrimental role of CO2 during mass extinctions in the earth's history. Future research is needed in this area to evaluate critical effects of the various CO2 disposal scenarios.

/pdf/biological-impact-of-elevated-ocean-co2-concentrations-3pqkmtp446.pdf

Biological Impact of Elevated Ocean CO2 Concentrations: Lessons from Animal Physiology and Earth History

Measurements of aerobic scope [the difference between minimum and maximum oxygen consumption rate (![Graphic][1] and ![Graphic][2] , respectively)] are increasing in prevalence as a tool to address questions relating to fish ecology and the effects of climate change. However, there are underlying issues regarding the array of methods used to measure aerobic scope across studies and species. In an attempt to enhance quality control before the diversity of issues becomes too great to remedy, this paper outlines common techniques and pitfalls associated with measurements of ![Graphic][3] , ![Graphic][4] and aerobic scope across species and under different experimental conditions. Additionally, we provide a brief critique of the oxygen- and capacity-limited thermal tolerance (OCLTT) hypothesis, a concept that is intricately dependent on aerobic scope measurements and is spreading wildly throughout the literature despite little evidence for its general applicability. It is the intention of this paper to encourage transparency and accuracy in future studies that measure the aerobic metabolism of fishes, and to highlight the fundamental issues with assuming broad relevance of the OCLTT hypothesis.

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Aerobic scope measurements of fishes in an era of climate change: respirometry, relevance and recommendations

The convergent evolution of cephalopods and fish has often been discussed on an anatomical basis, but recent advances in the knowledge of cephalopod life cycles, physiology, and biochemistry suggest that there are constraints on cephalopods that prevent them from competing directly with fish. These advances are reviewed against the background of detailed information on fish from the perspective that the basic inefficiency of the jet-propulsion system has required bioenergetic, physiological, and biochemical adaptations in squid which maximize their metabolic rates. Such "high-energy" adaptations are suggested to have resulted in the short life history and semelparous reproductive patterns that seem to characterize these coleoid cephalopods. Conversely, the physiology and biochemistry offish give them distinct advantages for long lives and iteroparity.

The constraints on cephalopods: why squid aren't fish

Influence of water temperature and oxygenation on the aerobic metabolic scope of Atlantic cod (Gadus morhua)

Testing a new acoustic telemetry technique to quantify long-term, fine-scale movements of aquatic animals

Acoustic telemetry has emerged as a leading approach to infer diel, tidal and lunar rhythmicity in the movements of aquatic organisms in a range of taxa. Typically, studies examine the relative frequency of detections from individuals tagged with acoustic transmitters, and then infer patterns in the species’ behaviour, but studies to date have not controlled for factors that may influence tag detection patterns in the absence of animal behaviour. We compared patterns in acoustic detections from tagged cuttlefish Sepia apama and several fixed-location control tags, and used these data to highlight the danger of misinterpreting patterns in the absence of adequate controls. Cuttlefish and control tags displayed similar detection patterns, and correcting cuttlefish-detection data for the influence of environmental factors resulted in the opposite pattern of cuttlefish activity displayed
prior to correction. This study highlights the danger of using acoustic data to infer animal behaviour in the absence of adequate controls.

/pdf/interpreting-diel-activity-patterns-from-acoustic-telemetry-1w6prw5e3u.pdf

Interpreting diel activity patterns from acoustic telemetry: the need for controls

Atlantic cod (Gadus morhua L.), acclimated to 5 dC, were equipped with ultrasonic transmitters which allowed the continuous monitoring of their vertical movements and heart rate. Fish were then placed in a 125 m3 tower tank in which the various thermal conditions they encounter in their natural environment were reproduced. Physiological and behavioural responses of cod were followed in parallel to the induced environmental changes. The experimental conditions studied in the tower tank were also reproduced in a swimming respirometer, where oxygen consumption and heart rate could be monitored within the activity range of a free-swimming animal. In a homogeneous water column, a rise in temperature induced marked increases in fish swimming activity, heart rate and heart beat-to-beat variability. In a thermally stratified environment, voluntary activity also increased when the thermal structure of the water column was altered, though no temperature-dependent changes in heart rate were observed. In this case, fish avoided the new temperature conditions, exhibiting distinct thermoregulatory behaviour. Stratification of the water column also prompted daily cyclic changes in fish distribution, animals tending to be in deeper and colder water layers during the day and in shallower and warmer layers at night. Respirometry experiments revealed that the thermoregulatory behaviour observed in free-ranging fish was probably driven by the energetic expedient of maintaining the physiological status quo s i.e. avoiding bioenergically costly reacclimation processes. Indeed, acute temperature increases or decreases of 2.5 dC led to marked differences in oxygen consumption, with metabolic rate changes of 15 and 30 %, respectively. The persistent linear relationship between heart rate and oxygen consumption allowed us to estimate, from the heart rate recorded in free-swimming fish, the entire range of metabolic responses that cod underwent voluntarily while experiencing a thermally stratified water column. The most profound metabolic effect, however, was observed with feeding, when oxygen consumption increased by as much as 80 %, resulting in an estimated 90 % reduction in their subsequent scope for activity.

/pdf/physiology-and-behaviour-of-free-swimming-atlantic-cod-gadus-3fxvbdtxau.pdf

Dale M. Webber

Papers

The constraints on cephalopods: why squid aren't fish

Influence of water temperature and oxygenation on the aerobic metabolic scope of Atlantic cod (Gadus morhua)

Testing a new acoustic telemetry technique to quantify long-term, fine-scale movements of aquatic animals

Interpreting diel activity patterns from acoustic telemetry: the need for controls

Physiology and behaviour of free-swimming Atlantic cod (Gadus morhua) facing fluctuating temperature conditions