Metabolic rate in fishes - Wiley Online Library

Journal of Fish Biology (2016) 88, 1–9 doi:10.1111/jfb.12873, available online at wileyonlinelibrary.com

EDITORIAL Metabolic rate in fishes: definitions, methods and significance for conservation physiology There is a growing interest in the potential for physiological tools and know-how to be applied in conservation research, the emerging field of conservation physiology. For fishes, there is a long-standing hypothesis whereby their bioenergetics and respiratory physiology should be of particular significance for their ecological performance (Fry, 1971). Water can be challenging as a respiratory medium, because it only contains a few milligrams of oxygen per litre (Schmidt-Nielsen, 1990). The physiology and oxygen demands of ectothermic fishes can be profoundly affected by environmental temperature. Oxygen can also easily become depleted in aquatic habitats, and such episodes of hypoxia are becoming more prevalent because of human effects (Diaz & Rosenberg, 1995; Breitburg et al., 2009). It has been argued, therefore, that the capacity of fishes to provide oxygen for vital processes and important activities, such as swimming or digesting and assimilating food, may define the habitats they can colonize successfully (Fry, 1971). As such, there is interest in how traits of metabolism and energetics may be applied in conservation physiology. Such applications can be direct, in experimental contexts, and also indirect, to parameterize models to project the potential effects of environmental changes, such as eutrophication, contamination and ongoing climate change. A core element of energetics is, of course, the metabolic rate that, in fishes, is typically measured as rate of oxygen uptake. There has been a surge in interest for measuring oxygen uptake in fishes but newcomers to the field may well find the terminology and definitions for major metabolic traits confusing. Further, methods and protocols are dispersed throughout the literature, without much integration and comparison. This special issue (SI) arose to respond to these issues, following conversations among fish ecophysiologists at conferences organized within the context of the European Cooperation in Science and Technology (COST) Action Conservation Physiology of Marine Fishes (COST FA1004). The first objective of this SI was to provide literature reviews for the major metabolic traits and recommend appropriate techniques to measure and calculate different metabolic rates. This includes instructions to construct appropriate experimental setups and software to analyse the data. That is, the aim was to make this SI the essential guide that ecophysiologists can consult before planning experiments to measure the metabolic rate of fishes. To this effect, the SI provides 13 papers that explain why the metabolic rate of fishes can be estimated by measuring oxygen uptake, and discuss the different levels of metabolic rate that are of interest, and terminology to deal with metabolic capacity and shortages of dissolved oxygen (hypoxia). These papers provide detailed instructions for proper design of equipment and protocols to measure these metabolic traits accurately and reliably in fishes in the laboratory, 1 © 2016 The Fisheries Society of the British Isles

2

EDITORIAL

including fish larvae and fishes with bimodal respiration (that can extract oxygen from both water and air). Techniques to estimate the metabolic rate of free-ranging fishes are also reviewed. A second objective was to provide perspectives on the ecological significance of metabolic rate in fishes and review some examples of how the respiratory physiology and metabolism of fishes can be used in a conservation context. This comprises five papers, starting with a review of why the metabolic rate of fishes is of ecological relevance, followed by a perspective arguing for the continuing relevance of the oxygen capacity limited thermal tolerance (OCLTT) hypothesis. There are reviews of aspects of how the respiratory physiology and performance of salmonids can be of significance for conservation research, and the section concludes with a discussion on integration of fish respiratory metabolism into models. The SI concludes with three research articles applying respiratory metabolism in studies with conservation objectives. Authors should note that the SI does not follow this Journal’s conventions for some abbreviations used. The SI Editors chose to use the most widely used terms, such as standard metabolic rate (SMR) or active metabolic rate (AMR) that, in Journal convention, would be Rs and Ra , respectively. This decision was made because many of the review papers dealt with changes in terminology over time and needed to track these using the original authors’ terms.

DEFINITIONS AND METHODS Physiologists typically use oxygen uptake when they want to measure the metabolic rate of fishes, without a second thought, generally not bothering to convert their results into energy units. Nelson (2016) gives a thorough historical account of the study of metabolic rate, particularly in fishes. This is a must-read for anyone interested in measuring energy-use in animals in general. Aerobic energy production is so much more efficient than anaerobic production and usually makes up the bulk of energy production in animals, including fishes. Further, respirometry is much easier to perform than direct calorimetry, particularly in water, and almost all studies of metabolic rate in fishes used respirometry as a proxy for metabolic rate. Nelson (2016) then discusses the error when assuming that all of the energy used by fishes is produced by aerobic processes, and foresees gains in the understanding of the relative contribution of aerobic and anaerobic energy production, in different situations, as better equipment becomes available to perform direct calorimetry on fishes. Svendsen et al. (2016a) compare three methods used to measure oxygen uptake ̇ 2 ) in fishes and argue that intermittent-flow (or stop-flow) respirometry offers (MO the most advantages and should always be preferred. The rest of this review paper ̇ 2. makes recommendations for the design of respirometers and computation of MO The assumptions of the method are considered. The authors discuss the sources of noise and errors and common problems. In particular, the importance of mixing the water in the respirometer well and the need to control and correct for background respiration (including the methods to do so). The authors provide detailed instructions to build a low-cost but high-performance intermittent-flow respirometry system and describe free software, written by one of the authors, to run respirometry experiments ̇ 2 in real-time. and compute MO

© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, 88, 1–9

EDITORIAL

3

Two further papers deal with the design of intermittent-flow respirometers. First, Svendsen et al. (2016b) deal with the issues of respirometer size and of the relative contributions of experimental error v. biological variability to the variability observed ̇ 2 . Obviously, the decrease in dissolved oxygen in consecutive measurements of MO caused by a small fish inside a very large respirometer will be more difficult to measure accurately than that of the same small fish in a small respirometer. The authors compared different ratios of respirometer volume with fish volume and make recommendations for which ratios of respirometer volume to fish volume provide reasonable ̇ 2 measurements. In addition, Svendsen et al. (2016b) designed a sysprecision in MO ̇ 2 of a fish in a respirometer, without the need for a fish! This tem that replicates the MO ̇ 2 by the simuallowed them to control the rate of oxygen decrease (i.e. the rate of MO ̇ 2 . Their lated fish) and compare this with results from a real live fish with a similar MO ̇ 2 in fishes results suggest that most of the variability observed in the studies of MO is real biological variability with only a small proportion being technical experimental error. Poor methodology can, however, increase experimental error. Thus Rogers et al. (2016) demonstrate that insufficient mixing of water inside the respirometer is a ̇ 2 was underestimated in the absence of a mixing device, major source of problems. MO demonstrating that the presence of a fish in a respirometer is not always sufficient to prevent water stratification. It was not possible to measure background respiration of an empty chamber accurately without mixing. This paper confirms the need to use an efficient mixing device in intermittent-flow respirometry and to correct for background respiration. Chabot et al. (2016b) review the concept and terminology of minimum sustainable ̇ 2 in fishes, which is properly called the standard metabolic rate (SMR). level of MO SMR is central to fish energetics. It is an obligatory expense, on top of which all other costs are added. It is required to calculate the metabolic aerobic scope (AS), a measure of metabolic capacity that is useful to understand the effects of the environment on fish physiology, e.g. within the OCLTT paradigm (Farrell, 2016). The authors describe the different experimental conditions and approaches that can be used to measure SMR. One of the most frequently used methods, because of simplicity and low cost, is to place fishes in a respirometer, maintain conditions that should result in calm inactivity, but without any simultaneous measurement of locomotor activity. Such experiments result ̇ 2 and authors then typically use a relatively small number of the in variable levels of MO lowest values to estimate SMR. Chabot et al. (2016b) point out that with the very short ̇ 2 can be measured with modern instruments, and considering durations over which MO potential physiological variability within the animal, a proportion of measurements ̇ 2 will in fact be below SMR. The paper compares many different calculation of MO methods to estimate SMR with this type of data and makes recommendations as to which ones should be used or avoided. They provide a script in the R language that calculates all the methods discussed in the paper. Along with SMR, maximum metabolic rate (MMR) is the other variable required to assess the AS of fishes. Norin & Clark (2016) offer a critical historical review of the methods used to measure MMR in fishes. They demonstrate that the methods have to be adapted to the fishes’ propensity to swim for extended durations in swim flumes (tunnels) and to simultaneously digest food and swim. The importance of accurately measuring MMR is emphasized. The review summarizes the results from a large number of studies on many species of marine fishes. In addition, the authors address issues such as the effects on MMR of body size, temperature, oxygen availability and even


4

EDITORIAL

less often studied variables, such as salinity, reproduction and circadian and seasonal cycles. Additional topics include variability in MMR between individuals and species and the possible link between MMR and SMR. Finally, the authors relate MMR of fishes to lifestyle and ecology, anticipating progress in this area with the advent of accelerometer and heart rate telemetry (Metcalfe et al., 2016a). The postprandial increase in oxygen uptake, known as specific dynamic action (SDA), represents the cost of food assimilation. Measuring SDA makes it possible to compare the assimilation cost of different food items or different rations, in different environmental conditions (e.g. temperature and dissolved oxygen). SDA is relatively easy to measure in fishes that remain quiescent in respirometers. Many fishes, however, can be active at some point during the long experiments that are required to measure SDA. In the past, the best that could be done was to manipulate the experimental conditions with the aim of reducing such activity, but any activity ̇ 2 in a manner that was inextricable from the SDA. Chabot nonetheless increased MO et al. (2016a) report on a statistical technique, non-parametric quantile regression, ̇ 2 following the ingestion of a which makes it possible to track the increase in MO meal without considering high values that are caused by stress or activity. Removing the influence of activity not only provides a more accurate estimate of SDA but also reduces any variation among individuals that is caused by different levels and patterns of activity, thereby improving the sensitivity of comparisons between, for example, different treatment groups. The authors provide R scripts facilitating the use of this technique. As with older fishes, it is necessary to measure the metabolic rate of larval fishes to improve the understanding of the relationships between environmental factors and ̇ 2 of fish larvae their physiology. Peck & Moyano (2016) show that measuring the MO is fraught with specific difficulties, owing to their small size. The authors provide a historical review of the methods and results of 63 studies involving over 50 fish species, indicating which type of metabolic rate was measured (standard, routine or active), the effect of larval size (allometric relationships) and the effect of temperature (Q10 ). Methods have to be adapted, and respirometer design and materials are of the outmost importance. The authors make recommendations on methods and quality control that will facilitate inter-studies comparisons in the future. One of the more unusual aspects of fish respiratory physiology is the existence of many species with bimodal respiration. Lefevre et al. (2016a) review measurements of ̇ 2 by such fishes. Measuring MO ̇ 2 from aquatic and aerial phases, simultaneously, MO poses a number of challenges and, until recently, this involved direct intervention and manipulation by the experimenter. This is unsatisfactory for bimodal fishes, because disturbance inhibits spontaneous surfacing behaviours. The review describes two methods to perform automated intermittent-flow respirometry based around a straightforward design of bimodal respirometer. A simple method is described that requires little equipment but offers less control over potential errors, compared with a more technically refined method that requires some knowledge of electronics and digital control systems. The review also describes the challenges of measuring bimodal respiration by air-breathing fishes during aerobic exercise, with guidance for potential pitfalls. It concludes with perspectives on how advances in probe technology might lead to new research avenues in the study of this fascinating group of fishes.


EDITORIAL

5

Hypoxia is often treated as a presence or absence variable. Either dissolved oxygen is below a threshold and the habitat is hypoxic, or it is above and the habitat is normoxic. Although categorization can be useful in assessing the progression of hypoxia in time, it greatly underestimates the true effect of hypoxia on fishes, by ignoring differences in sensitivity among species and individuals, and also the many non-lethal effects of chronic hypoxia. Claireaux & Chabot (2016) use the AS concept of Fry (1971) to show that the metabolic capacity can be reduced even at mild levels of hypoxia, with progressively more debilitating effects as hypoxia progresses. This is illustrated by the limiting oxygen level (LOL) curve. This curve shows that when fishes are exposed to hypoxia when their oxygen uptake is relatively elevated, they will appear to be oxygen conformers, such that the difference between oxygen conformers and oxygen regulators may be an artefact of the method used, in particular, the success with which the experiments achieved calm, quiescent fishes before manipulating dissolved oxygen. The authors also present a commonly used measure of hypoxia tolerance in fishes, the critical oxygen level or O2crit , providing scripts in the R language to calculate and plot the results. Finally, the review considers how the concept of degree days, so common in plant ecology, can be used to understand how long fishes survive once oxygen levels drop below O2crit . Snyder et al. (2016) compare two methods commonly used to decrease dissolved oxygen, within a respirometer, during experiments to measure O2crit . Some studies simply keep the respirometer closed for the entire duration of the experiment, which obviously results in progressive hypoxia and hypercapnia. The alternative is to continue to flush the respirometer, but to control the oxygen content of the flushing water, reducing it in a stepwise manner. The authors provide an experimental comparison of both methods, with the shiner perch Cymatogaster aggregata Gibbons 1854. They recommend stepwise control of flush water, because C. aggregata were less hypoxia tolerant when they decreased the oxygen content by themselves, probably because of the more rapid rate of decrease of oxygen and of the inevitable accumulation of metabolites, mostly carbon dioxide, in the closed respirometers. Training early stage researchers to use adequate equipment and techniques to measure the oxygen uptake of fishes is of paramount importance. Rosewarne et al. (2016) provide step-by-step instructions to teach respirometry techniques tailored to the measurement of oxygen uptake and the impact of hypoxia in fishes. The authors provide a list of materials required to perform intermittent-flow respirometry, a detailed protocol to measure SMR, MMR and AS, and explain the calculations involved in transforming the changes in oxygen content in the respirometer into oxygen uptake, including how to correct for background respiration. Finally, they explain how the students can investigate the effect of declining oxygen levels on these metabolic variables. Altogether, the paper constitutes an excellent tutorial allowing readers to put together the techniques and recommendations from many of the papers in the SI. At present, it is a major challenge to estimate the metabolic rate of fishes in their natural habitat, especially marine fishes in their vast underwater realm. Metcalfe et al. (2016a) review current methods for estimating metabolic rate indirectly, by calibration against other physiological variables. They focus on electronic devices that record body acceleration in three dimensions (accelerometry). Correlations between dynamic body acceleration and rate of oxygen uptake can be developed, to then estimate activity-specific energy expenditure of fishes in semi-natural or natural habitats, over


6

EDITORIAL

extended periods. This technique can, therefore, be applied in conservation physiology studies aimed at understanding the behavioural and energetic decisions that wild fishes may make as a function of environmental conditions. This is an area of conservation physiology of fishes that is expected to expand rapidly.

METABOLIC RATE AND CONSERVATION PHYSIOLOGY Metcalfe et al. (2016b) review current knowledge of the extent and causes of individual variations in metabolic phenotype (SMR, MMR and AS) of fishes and the links between metabolic phenotype, performance and behaviour. For instance, SMR can be related to behavioural traits, such as dominance, risk taking and growth rate, and fishes with high SMR may be advantaged when food is abundant, but disadvantaged during periods of food shortage. Such trade-offs are probably involved in the maintenance of diversity in metabolic phenotypes through genetic diversity. Considering possible effects of MMR on the ability to catch prey and avoid predators and of AS with performance in hypoxic conditions, the authors emphasize the need for more research relating MMR and AS to behaviour and fitness of fishes. Farrell (2016) provides a perspectives paper in support of the Fry (1971) AS paradigm, and its derivative the OCLTT hypothesis, as universal frameworks to explain how temperature constrains physiological performance in fishes. The Fry and OCLTT paradigms are both based upon respiratory metabolism, and the notion that the capacity to provide oxygen for metabolic demands is constrained at thermal extremes. The paper argues that competition among activities, and the vascular beds that support these, makes cardiac performance capacity a key mechanism underlying thermal constraints on aerobic performance, which requires more careful study. The objective is that, through better understanding of its underlying mechanisms, the OCLTT hypothesis can support predictive ecological applications for fishes in a rapidly changing world. Eliason & Farrell (2016) provide an authoritative review of the research into the anadromous migratory life cycle of Pacific salmon Oncorhynchus spp., in the Fraser River catchment. This constitutes what is perhaps the world’s most emblematic example of research into the conservation physiology of fishes, which also happens to focus upon respiratory physiology and energy metabolism. Among the earliest studies of fish respiratory performance during aerobic exercise, and how this was affected by temperature, this research was initiated over 50 years ago on these species. The review discusses how traits of energy metabolism relate to a migratory lifestyle, in particular how the semelparous spawning strategy has put strong selection on adaptations for powerful and efficient upriver migration. Also, how homing to specific natal streams has led to strong local adaptation, of cardiorespiratory capacity and temperature tolerance of populations, in response to the specific challenges of their migration route. Ongoing climate change is raising serious conservation concerns and, despite the long history of study of these species, the review concludes by identifying critical knowledge gaps that remain. Enders & Boisclair (2016) take Atlantic salmon Salmo salar L 1758 parr as a case study to review evidence that variations in environmental conditions have significant effects on energy metabolism. They focus on water temperature, presence of shelter and variations in current speed. They argue therefore that the accuracy of models of


EDITORIAL

7

fish metabolism will be improved if such environmental conditions are considered. This will increase the accuracy of energy budget calculations, which is valuable for conservation actions and for predicting effects of future climate change. The integration of physiological information into models is a major objective in conservation physiology. The review by Jørgensen et al. (2016) describes how models may put fish bioenergetics in a broader context, with the objective of making fish ecophysiological research more useful for science and society, in an era of climate change. Bioenergetics is the mechanistic foundation of many models. The paper argues that the predictive capacity of physiologically based models will be improved if they complement bioenergetics with consideration of important potential trade-offs that might influence behaviour and performance of the animal, particularly trade-offs linked to survival.

RESEARCH ARTICLES The research article by Lea et al. (2016) investigates how water temperature affects the mechanical efficiency of swimming and aerobic performance of brown trout Salmo trutta L. 1758. The results indicate that S. trutta modulated the relationship between tailbeat frequency and amplitude to optimize swimming efficiency as they were acutely warmed from 11 to 24∘ C. Despite this, there was evidence that AS for activity declined at temperatures above 18∘ C, which may indicate energetic trade-offs when temperatures rise above thermal optima. Very little is known about the ability of air-breathing fishes to tolerate future climate changes. Lefevre et al. (2016b) studied the significance of air breathing for sensitivity to acute warming in a South-east Asian tropical species, the Asian swamp eel Monopterus albus (Zuiew 1793). The species relied on air breathing to meet the metabolic challenges of acute warming. Acclimation to a temperature above current seasonal averages improved upper temperature tolerance, but this was not linked to increased reliance on air breathing or improved cardiac performance. The authors conclude that in this species, air breathing will not provide particular advantages in tolerating extreme warming events. The study adds to the evidence that oxygen delivery is not necessarily the only factor limiting temperature tolerance in ectotherms. The brief communication by Lucas et al. (2016) reports that contamination by pyrolytic polycyclic aromatic hydrocarbons has no negative effects on AS and swimming performance of adult zebrafish Danio rerio (Hamilton 1822). Ecotoxicology is a facet of conservation physiology where techniques of fish respiratory metabolism have already been applied extensively. In particular, to investigate how contaminants might constrain fish performance by increasing maintenance costs or by reducing scope for activity. Reporting negative results, in carefully performed ecotoxicology studies, is important to avoid bias in information transfer regarding toxicological effects of aquatic pollution. It is hoped that this SI will prove to be a useful guide for researchers who want to measure fish metabolic rate. The SI editors are grateful to all authors for their support of this SI, and to the Editorial Office at JFB for their forbearance with delays with compiling the various contributions. They are also very grateful to the referees for the articles, in particular those that reviewed more than one contribution, often doing so rapidly when these contributions were submitted late. The COST Action FA1004,


8

EDITORIAL

Conservation Physiology of Marine Fishes, supported the conferences and networking that led to this SI. D. Chabot Guest Editor D. J. McKenzie Guest Associate Editor J. F. Craig Editor-in-Chief

References Breitburg, D. L., Hondorp, D. W., Davias, L. A. & Diaz, R. J. (2009). Hypoxia, nitrogen, and fisheries: integrating effects across local and global landscapes. Annual Review of Marine Science 1, 329–349. Chabot, D., Koenker, R. & Farrell, A. P. (2016a). The measurement of specific dynamic action in fishes. Journal of Fish Biology 88, 152–172. Chabot, D., Steffensen, J. F. & Farrell, A. P. (2016b). The determination of standard metabolic rate in fishes. Journal of Fish Biology 88, 81–121. Claireaux, G. & Chabot, D. (2016). Responses by fishes to environmental hypoxia: integration through Fry’s concept of aerobic metabolic scope. Journal of Fish Biology 88, 232–251. Diaz, R. J. & Rosenberg, R. (1995). Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanography and Marine Biology: An Annual Review 33, 245–303. Eliason, E. J. & Farrell, A. P. (2016). Oxygen uptake in Pacific salmon Oncorhynchus spp.: when ecology and physiology meet. Journal of Fish Biology 88, 359–388. Enders, E. C. & Boisclair, D. (2016). Effects of environmental fluctuations on fish metabolism: Atlantic salmon Salmo salar as a case study. Journal of Fish Biology 88, 344–358. Farrell, A. P. (2016). Pragmatic perspective on aerobic scope: peaking, plummeting, pejus and apportioning. Journal of Fish Biology 88, 322–343. Fry, F. E. J. (1971). The effects of environmental factors on the physiology of fish. In Fish Physiology (Hoar, W. S. & Randall, D. J., eds), Vol. 6, pp. 1–98. New York, NY: Academic Press. Jørgensen, C., Enberg, K. & Mangel, M. (2016). Modelling and interpreting fish bioenergetics – a role for behaviour, life history traits, and survival trade-offs. Journal of Fish Biology 88, 389–402. Lea, J. M. D., Keen, A. N., Nudds, R. L. & Shiels, H. A. (2016). Kinematics and energetics of swimming performance during acute warming in brown trout Salmo trutta. Journal of Fish Biology 88, 403–417. Lefevre, S., Bayley, M. & McKenzie, D. J. (2016a). Measuring oxygen uptake in fishes with bimodal respiration. Journal of Fish Biology 88, 206–231. Lefevre, S., Findorf, I., Bayley, M., Huong, D. T. T. & Wang, T. (2016b). Increased temperature tolerance of the air-breathing Asian swamp eel Monopterus albus after high-temperature acclimation is not explained by improved cardio-respiratory performance. Journal of Fish Biology 88, 418–432. Lucas, J., Bonnieux, A., Lyphout, L., Cousin, X., Miramand, P. & Lefrançois, C. (2016). Trophic contamination by pyrolytic polycyclic aromatic hydrocarbons does not affect aerobic metabolic scope in zebrafish Danio rerio. Journal of Fish Biology 88, 433–442. Metcalfe, J. D., Wright, S., Tudorache, C. & Wilson, R. (2016a). Recent advances in telemetry for estimating the energy metabolism of wild fishes. Journal of Fish Biology 88, 284–297. Metcalfe, N., Van Leeuwen, T. E. & Killen, S. S. (2016b). Does individual variation in metabolic phenotype predict fish behaviour and performance? Journal of Fish Biology 88, 298–321.


EDITORIAL

9

Nelson, J. (2016). Oxygen consumption rate v. rate of energy utilization of fishes: a comparison and brief history of the two measurements. Journal of Fish Biology 88, 10–25. Norin, T. & Clark, T. D. (2016). Measurement and relevance of maximum metabolic rate in fishes. Journal of Fish Biology 88, 122–151. Peck, M. A. & Moyano, M. (2016). Measuring respiration rates in marine fish larvae: challenges and advances. Journal of Fish Biology 88, 173–205. Rogers, G. G., Tenzing, P. & Clark, T. D. (2016). Experimental methods in aquatic respirometry: the importance of mixing devices and accounting for background respiration. Journal of Fish Biology 88, 65–80. Rosewarne, P. J., Wilson, J. M. & Svendsen, J. C. (2016). Measuring maximum and standard metabolic rates using intermittent flow respirometry: a student laboratory investigation of aerobic metabolic scope and environmental hypoxia in aquatic breathers. Journal of Fish Biology 88, 265–283. Schmidt-Nielsen, K. (1990). Animal Physiology: Adaptation and Environment. Cambridge: Cambridge University Press. Snyder, S., Nadler, L. E., Domenici, P., Bayley, J. S., Johansen, J., Svendsen, M. B. S. & Steffensen, J. F. (2016). Effect of closed versus intermittent-flow respirometry on hypoxia tolerance in aquatic breathers. Journal of Fish Biology 88, 252–264. Svendsen, M. B., Bushnell, P. & Steffensen, J. F. (2016a). Design and setup of an intermittent-flow respirometry system for aquatic organisms. Journal of Fish Biology 88, 26–50. Svendsen, M. B., Bushnell, P., Christensen, E. A. F. & Steffensen, J. F. (2016b). Sources of variation in oxygen consumption of aquatic animals demonstrated by simulated constant oxygen consumption and respirometers of different size. Journal of Fish Biology 88, 51–64.
