The OptoReg system: a simple and inexpensive solution for regulating water oxygen.

This paper describes an optocoupler-based regulation apparatus for saturation manipulation of oxygen in water (OptoReg). This system enables control of solenoid valves for oxygen and nitrogen gases using a FireSting-O2 meter, an optocoupler box and an electronic switch box. The hardware components connect to a computer through Universal Serial Bus (USB) cables. The control software is free and has a graphical user interface, making it easy to use. With the OptoReg system, any lab with a computer running Microsoft Windows operating system and a 4-channel FireSting-O2 meter can easily and cheaply set up four independently controlled systems for regulating water oxygen levels. Here, we describe how to assemble and run the OptoReg system and present a data set demonstrating the high precision and stability of the OptoReg system during static acclimation experiments and dynamic warming trials.

Physiological Mechanisms of Acute Upper Thermal Tolerance in Fish.

This review is focused on the questions of why fish exhibit heat failure at thermal extremes and which physiological mechanisms determine the acute upper thermal tolerance. We propose that rapid direct thermal impacts on fish act through three fundamental molecular mechanisms reaction rates, protein structure, and membrane fluidity. During acute warming, these molecular effects then lead to loss of equilibrium and death through various cellular, organ, and physiological pathways. These pathways include mitochondrial dysfunction, oxygen limitation, and impacted excitability of excitable cells and eventually lead to neural and/or muscular failure. The pathways may also lead to loss of homeostasis and subsequent heat failure. There is strong evidence in some species for oxygen limitation in these processes and strong evidence against it in other species and contexts. The limiting mechanisms during acute warming therefore appear to differ between species, life stages, and recent thermal history. We conclude that a single mechanism underpinning the acute upper thermal tolerance across species and contexts will not be found. Therefore, we propose future avenues of research that can elucidate major patterns of physiological thermal limitations in fish.

Integrative Approaches to Understanding Organismal Responses to Aquatic Deoxygenation.

AbstractOxygen bioavailability is declining in aquatic systems worldwide as a result of climate change and other anthropogenic stressors. For aquatic organisms, the consequences are poorly known but are likely to reflect both direct effects of declining oxygen bioavailability and interactions between oxygen and other stressors, including two-warming and acidification-that have received substantial attention in recent decades and that typically accompany oxygen changes. Drawing on the collected papers in this symposium volume ("An Oxygen Perspective on Climate Change"), we outline the causes and consequences of declining oxygen bioavailability. First, we discuss the scope of natural and predicted anthropogenic changes in aquatic oxygen levels. Although modern organisms are the result of long evolutionary histories during which they were exposed to natural oxygen regimes, anthropogenic change is now exposing them to more extreme conditions and novel combinations of low oxygen with other stressors. Second, we identify behavioral and physiological mechanisms that underlie the interactive effects of oxygen with other stressors, and we assess the range of potential organismal responses to oxygen limitation that occur across levels of biological organization and over multiple timescales. We argue that metabolism and energetics provide a powerful and unifying framework for understanding organism-oxygen interactions. Third, we conclude by outlining a set of approaches for maximizing the effectiveness of future work, including focusing on long-term experiments using biologically realistic variation in experimental factors and taking truly cross-disciplinary and integrative approaches to understanding and predicting future effects.

Estimating Discard Mortality in Commercial Fisheries without Fish Dying: A 3R Challenge.

Globally, it is estimated that around 10% of the fish that are caught are discarded. This is considered to be a wasteful human marine activity since these fish are often dead or dying. To reduce the high discard rates of commercial fisheries, the European Union (E.U.) has enacted a landing obligation that includes the ability to exempt "species for which scientific evidence demonstrates high survival rates". Therefore, discard survival studies (henceforth DSSs) have become one of the most politically prioritized fisheries research areas in European fisheries. International expert groups have produced guidance reports to promote best practices and to harmonize the methodologies. Nevertheless, there has not been any focus on how to implement animal welfare (AW) regulations experimentally. Discard survival studies are "frontrunners" in fisheries science research areas that are embedded by animal research welfare requirements and are expected to be more restrictive in the future because of an increased public focus on fish welfare. This paper focuses on AW regulations in relation to conducting DSSs, but the outreach is much broader. We investigate experimental procedures by bringing in relevant examples, using output results, and relating this information to relevant AW guidelines and regulations by focusing on implementing 3R principles.

Paths towards greater consensus building in experimental biology.

In a recent editorial, the Editors-in-Chief of Journal of Experimental Biology argued that consensus building, data sharing, and better integration across disciplines are needed to address the urgent scientific challenges posed by climate change. We agree and expand on the importance of cross-disciplinary integration and transparency to improve consensus building and advance climate change research in experimental biology. We investigated reproducible research practices in experimental biology through a review of open data and analysis code associated with empirical studies on three debated paradigms and for unrelated studies published in leading journals in comparative physiology and behavioural ecology over the last 10 years. Nineteen per cent of studies on the three paradigms had open data, and 3.2% had open code. Similarly, 12.1% of studies in the journals we examined had open data, and 3.1% had open code. Previous research indicates that only 50% of shared datasets are complete and re-usable, suggesting that fewer than 10% of studies in experimental biology have usable open data. Encouragingly, our results indicate that reproducible research practices are increasing over time, with data sharing rates in some journals reaching 75% in recent years. Rigorous empirical research in experimental biology is key to understanding the mechanisms by which climate change affects organisms, and ultimately promotes evidence-based conservation policy and practice. We argue that a greater adoption of open science practices, with a particular focus on FAIR (Findable, Accessible, Interoperable, Re-usable) data and code, represents a much-needed paradigm shift towards improved transparency, cross-disciplinary integration, and consensus building to maximize the contributions of experimental biologists in addressing the impacts of environmental change on living organisms.

Assessment of hypoxia avoidance behaviours in a eurythermal fish at two temperatures using a modified shuttlebox system.

Behavioural avoidance responses of red drum (Sciaenops ocellatus) to aquatic hypoxia were investigated at 22 and 30°C using a modified shuttlebox system. Fish movement between a control side maintained at normoxia and a hypoxic side with stepwise decreasing water oxygen tension was analysed for entries into the hypoxic side, residence time per entry into the hypoxic side and total time in the hypoxic side. Acclimation to 30°C increased the oxygen threshold for the onset of hypoxia avoidance behaviours for entries and total time, while residence time per entry was unchanged.

Oxygen-dependence of upper thermal limits in crustaceans from different thermal habitats.

The critical thermal maximum (CTMAX) is the temperature at which animals exhibit loss of motor response because of a temperature-induced collapse of vital physiological systems. A central mechanism hypothesised to underlie the CTMAX of water-breathing ectotherms is insufficient tissue oxygen supply for vital maintenance functions because of a temperature-induced collapse of the cardiorespiratory system. The CTMAX of species conforming to this hypothesis should decrease with declining water oxygen tension (PO2) because they have oxygen-dependent upper thermal limits. However, recent studies have identified a number of fishes and crustaceans with oxygen-independent upper thermal limits, their CTMAX unchanged in progressive aquatic hypoxia. The previous studies, which were performed separately on cold-water, temperate and tropical species, suggest the oxygen-dependence of upper thermal limits and the acute thermal sensitivity of the cardiorespiratory system increases with decreasing habitat temperature. Here we directly test this hypothesis by assessing the oxygen-dependence of CTMAX in the polar Antarctic krill (Euphausia superba), as well as the temperate Baltic prawn (Palaemon adspersus) and brown shrimp (Crangon crangon). We found that P. adspersus and C. crangon maintain CTMAX in progressive hypoxia down to 40 mmHg, and that only E. superba have oxygen-dependent upper thermal limits at normoxia. In E. superba, the observed decline in CTMAX with water PO2 is further supported by heart-rate measurements showing a plateauing, and subsequent decline and collapse of heart performance at CTMAX. Our results support the hypothesis that the oxygen-dependence of upper thermal limits in water-breathing ectotherms and the acute thermal sensitivity of their cardiorespiratory system increases with decreasing habitat temperature.

A mechanistic oxygen- and temperature-limited metabolic niche framework.

The abundance and distribution of fishes and other water-breathing ectotherms are partially shaped by the capacities of individuals to perform ecologically relevant functions, which collectively determine whole-organism performance. Aerobic scope (AS) quantifies the capacity of the cardiorespiratory system to supply tissues with oxygen for fuelling such functions. Aquatic hypoxia and water temperature are principal environmental factors affecting the AS of water-breathing ectotherms. Although it is intuitive that animal energetics will be of ecological significance, many studies argue against a hypothesized overarching link between AS, whole-organism performance, and shifts in the abundance and distribution of water-breathing ectotherms with environmental change. Consequently, relationships between AS and ecologically relevant performance traits must be established for individual species. This article proposes a mechanistic framework for integrating and correlating experimental traits for assessing the AS, anaerobic capacity (AC) and range boundaries of water-breathing ectotherms exposed to progressive aquatic hypoxia and rising water temperature. The framework also describes cardiorespiratory thermal tolerance and proposes an empirical definition of the mechanism underlying the critical thermal maximum in species with oxygen-dependent upper thermal limits. Incorporating performance traits, exemplified with preference and avoidance responses, may provide information about the role of metabolism in shaping whole-organism performance, and the potential applicability of AS and AC in species distribution models. This article is part of the theme issue 'Physiological diversity, biodiversity patterns and global climate change: testing key hypotheses involving temperature and oxygen'.

Effects of salinity and hypoxia-induced hyperventilation on oxygen consumption and cost of osmoregulation in the estuarine red drum (Sciaenops ocellatus).

Understanding the physiological responses of fishes to salinity changes and aquatic hypoxia is essential for the conservation of marine species. Salinity changes affect the osmotic gradient across the gill epithelium, while hypoxia increases gill ventilation and the flow of water over the gills. Both processes affect the diffusive movement of ions and water across the gill epithelium, and the rate of active ion transport required for maintaining osmotic homeostasis. Consequently, salinity and hypoxia may affect the energetic cost of osmoregulation, and consequently the energy available for other physiological functions such as migration, growth, and reproduction. Historically, studies have assessed the costs of osmoregulation and ventilation in fishes via standard metabolic rate (SMR); however, few studies have used a multi-stressor approach that fully accounts for the osmorespiratory compromise. Here, we determined the combined effects of salinity and hypoxia on SMR, routine metabolic rate (RMR), and plasma ion concentrations in red drum (Sciaenops ocellatus) acclimated to salinities ranging from freshwater to hypersalinity. Surprisingly, there was no significant change in any parameter as a consequence of salinity or hypoxia, including the relatively extreme scenario of combined hypersalinity and hypoxia exposure. We conclude that changes in the osmotic gradient across the gill epithelium and the flow of water over the gills have a negligible effect on the whole animal energy budget of S. ocellatus, suggesting that the cost of osmoregulation is a minor component of basal metabolism regardless of oxygenation status.

Acclimation to prolonged hypoxia alters hemoglobin isoform expression and increases hemoglobin oxygen affinity and aerobic performance in a marine fish.

Hemoglobin (Hb) multiplicity is common in fish, yet despite its ubiquitous nature, the functional significance is unclear. Here we explore the hypothesis that Hb multiplicity plays a role in hypoxia tolerance using the red drum (Sciaenops ocellatus). Red drum is an economically and ecologically important species native to coastal regions and estuaries of the Gulf of Mexico - habitats that routinely experience pronounced hypoxic events. Using a transcriptomic approach, we demonstrate that red drum red blood cells express 7 and 5 Hbα and Hbß isoforms, respectively. Phylogenetic analysis grouped these isoforms into distinct isoHb clades, and provided evidence of lineage specific expression of particular isoHbs. In normoxia, three isoHbs predominated (Hbα-3.1, -3.2, and Hbß-3.1). A three-week hypoxia acclimation (48 mmHg) resulted in significant up-regulation of Hbα-2, Hbα-3.2, and Hbß-3.1, effectively switching the predominantly expressed isoforms. Changes in subunit expression were correlated with a decrease in non-stripped hemolysate P50. Similarly, hypoxia acclimation resulted in a 20% reduction in whole animal critical oxygen threshold (Pcrit). Hypoxia acclimation was not associated with changes in gill morphology, hematocrit, or relative ventricular mass. Overall, these data provide support for the hypothesis that Hb isoform switching can provide a physiological benefit to counteract environmental stress in fishes.

Effects of hypoxia and ocean acidification on the upper thermal niche boundaries of coral reef fishes.

Rising ocean temperatures are predicted to cause a poleward shift in the distribution of marine fishes occupying the extent of latitudes tolerable within their thermal range boundaries. A prevailing theory suggests that the upper thermal limits of fishes are constrained by hypoxia and ocean acidification. However, some eurythermal fish species do not conform to this theory, and maintain their upper thermal limits in hypoxia. Here we determine if the same is true for stenothermal species. In three coral reef fish species we tested the effect of hypoxia on upper thermal limits, measured as critical thermal maximum (CTmax). In one of these species we also quantified the effect of hypoxia on oxygen supply capacity, measured as aerobic scope (AS). In this species we also tested the effect of elevated CO2 (simulated ocean acidification) on the hypoxia sensitivity of CTmax We found that CTmax was unaffected by progressive hypoxia down to approximately 35 mmHg, despite a substantial hypoxia-induced reduction in AS. Below approximately 35 mmHg, CTmax declined sharply with water oxygen tension (PwO2). Furthermore, the hypoxia sensitivity of CTmax was unaffected by elevated CO2 Our findings show that moderate hypoxia and ocean acidification do not constrain the upper thermal limits of these tropical, stenothermal fishes.

Oxygen dependence of upper thermal limits in fishes.

Temperature-induced limitations on the capacity of the cardiorespiratory system to transport oxygen from the environment to the tissues, manifested as a reduced aerobic scope (maximum minus standard metabolic rate), have been proposed as the principal determinant of the upper thermal limits of fishes and other water-breathing ectotherms. Consequently, the upper thermal niche boundaries of these animals are expected to be highly sensitive to aquatic hypoxia and other environmental stressors that constrain their cardiorespiratory performance. However, the generality of this dogma has recently been questioned, as some species have been shown to maintain aerobic scope at thermal extremes. Here, we experimentally tested whether reduced oxygen availability due to aquatic hypoxia would decrease the upper thermal limits (i.e. the critical thermal maximum, CTmax) of the estuarine red drum (Sciaenops ocellatus) and the marine lumpfish (Cyclopterus lumpus). In both species, CTmax was independent of oxygen availability over a wide range of oxygen levels despite substantial (>72%) reductions in aerobic scope. These data show that the upper thermal limits of water-breathing ectotherms are not always linked to the capacity for oxygen transport. Consequently, we propose a novel metric for classifying the oxygen dependence of thermal tolerance; the oxygen limit for thermal tolerance (PCTmax ), which is the water oxygen tension (PwO2 ) where an organism's CTmax starts to decline. We suggest that this metric can be used for assessing the oxygen sensitivity of upper thermal limits in water-breathing ectotherms, and the susceptibility of their upper thermal niche boundaries to environmental hypoxia.

Hyperventilation and blood acid-base balance in hypercapnia exposed red drum (Sciaenops ocellatus).

Hyperventilation is a common response in fish exposed to elevated water CO2. It is believed to lessen the respiratory acidosis associated with hypercapnia by lowering arterial PCO2, but the contribution of hyperventilation to blood acid-base compensation has yet to be quantified. Hyperventilation may also increase the flux of irons across the gill epithelium and the cost of osmoregulation, owing to the osmo-respiratory compromise. Therefore, hypercapnia exposed fish may increase standard metabolic rate (SMR) leaving less energy for physiological functions such as foraging, migration, growth and reproduction. Here we show that gill ventilation, blood PCO2 and total blood [CO2] increased in red drum (Sciaenops ocellatus) exposed to 1000 and 5000 µatm water CO2, and that blood PCO2 and total blood [CO2] decrease in fish during hypoxia induced hyperventilation. Based on these results we estimate the ventilatory contributions to total acid-base compensation in 1000 and 5000 µatm water CO2. We find that S. ocellatus only utilize a portion of its ventilatory capacity to reduce the acid-base disturbance in 1000 µatm water CO2. SMR was unaffected by both salinity and hypercapnia exposure indicating that the cost of osmoregulation is small relative to SMR, and that the lack of increased ventilation in 1000 µatm water CO2 despite the capacity to do so is not due to an energetic tradeoff between acid-base balance and osmoregulation. Therefore, while ocean acidification may impact ventilatory parameters, there will be little impact on the overall energy budget of S. ocellatus.

Respiratory plasticity is insufficient to alleviate blood acid-base disturbances after acclimation to ocean acidification in the estuarine red drum, Sciaenops ocellatus.

The changes in ocean chemistry stemming from anthropogenic CO2 release--termed ocean acidification (OA)--are predicted to have wide-ranging effects on fish and ultimately threaten global populations. The ability of fish to adapt to environmental change is currently unknown, but phenotypic plasticity has been highlighted as a crucial factor in determining species resilience. Here we show that red drum, a long-lived estuarine-dependent fish species native to the Gulf of Mexico, exhibit respiratory plasticity that increases CO2 excretion capacity when acclimated to OA conditions. Specifically, fish exposed to 14 days of 1000 µatm CO2 had a 32% reduction in branchial diffusion distance and increased expression of two putative CO2 channel proteins--rhag and rhcg1. No changes were observed in the erythrocyte CO2 transport pathways. Surprisingly, no significant changes in blood chemistry were observed between acclimated and acutely challenged animals; however, a non-significant 30 % drop in the magnitude of plasma C(CO2) elevation was observed. Reduced diffusion distance also comes with the cost of increased diffusive water loss, which would require greater osmoregulatory investment by the animal. OA exposure induced increased gill Na(+), K(+) ATPase activity and intestinal nkcc2 expression, supporting both the presumed osmotic stress and increased osmoregulatory investment. However, no differences in standard metabolic rate, maximum metabolic rate or aerobic scope were detected between control and OA acclimated individuals. Similarly, no differences in critical swim speed were detected between groups, suggesting the energetic cost related to respiratory plasticity is negligible against background metabolism. The current study demonstrated that red drum exhibit respiratory plasticity with only mild physiological trade-offs; however, this plasticity is insufficient to fully offset the OA-induced acid-base disturbance and as such is unlikely to impact species resilience.

Does oxygen limit thermal tolerance in arthropods? A critical review of current evidence.

Over the last decade, numerous studies have investigated the role of oxygen in setting thermal tolerance in aquatic animals, and there has been particular focus on arthropods. Arthropods comprise one of the most species-rich taxonomic groups on Earth, and display great diversity in the modes of ventilation, circulation, blood oxygen transport, with representatives living both in water (mainly crustaceans) and on land (mainly insects). The oxygen and capacity limitation of thermal tolerance (OCLTT) hypothesis proposes that the temperature dependent performance curve of animals is shaped by the capacity for oxygen delivery in relation to oxygen demand. If correct, oxygen limitation could provide a mechanistic framework to understand and predict both current and future impacts of rapidly changing climate. In arthropods, most studies testing the OCLTT hypothesis have considered tolerance to thermal extremes. These studies likely operate from the philosophical viewpoint that if the model can predict these critical thermal limits, then it is more likely to also explain loss of performance at less extreme, non-lethal temperatures, for which much less data is available. Nevertheless, the extent to which lethal temperatures are influenced by limitations in oxygen supply remains unresolved. Here we critically evaluate the support and universal applicability for oxygen limitation being involved in lethal temperatures in crustaceans and insects. The relatively few studies investigating the OCLTT hypothesis at low temperature do not support a universal role for oxygen in setting the lower thermal limits in arthropods. With respect to upper thermal limits, the evidence supporting OCLTT is stronger for species relying on underwater gas exchange, while the support for OCLTT in air-breathers is weak. Overall, strongest support was found for increased anaerobic metabolism close to thermal maxima. In contrast, there was only mixed support for the prediction that aerobic scope decreases near critical temperatures, a key feature of the OCLTT hypothesis. In air-breathers, only severe hypoxia (<2 kPa) affected heat tolerance. The discrepancies for heat tolerance between aquatic and terrestrial organisms can to some extent be reconciled by differences in the capacity to increase oxygen transport. As air-breathing arthropods are unlikely to become oxygen limited under normoxia (especially at rest), the oxygen limitation component in OCLTT does not seem to provide sufficient information to explain lethal temperatures. Nevertheless, many animals may simultaneously face hypoxia and thermal extremes and the combination of these potential stressors is particularly relevant for aquatic organisms where hypoxia (and hyperoxia) is more prevalent. In conclusion, whether taxa show oxygen limitation at thermal extremes may be contingent on their capacity to regulate oxygen uptake, which in turn is linked to their respiratory medium (air vs. water). Fruitful directions for future research include testing multiple predictions of OCLTT in the same species. Additionally, we call for greater research efforts towards studying the role of oxygen in thermal limitation of animal performance at less extreme, sub-lethal temperatures, necessitating studies over longer timescales and evaluating whether oxygen becomes limiting for animals to meet energetic demands associated with feeding, digestion and locomotion.

Some like it hot: Thermal tolerance and oxygen supply capacity in two eurythermal crustaceans.

Thermal sensitivity of the cardiorespiratory oxygen supply capacity has been proposed as the cardinal link underlying the upper boundary of the temperature niche in aquatic ectotherms. Here we examined the evidence for this link in two eurythermal decapods, the Giant tiger shrimp (Penaeus monodon) and the European crayfish (Astacus astacus). We found that both species have a temperature resistant cardiorespiratory system, capable of maintaining oxygen delivery up to their upper critical temperature (Tcrit). In neither species was Tcrit reduced in hypoxia (60% air saturation) and both species showed an exponential increase in heart and gill ventilation rates up to their Tcrit. Further, failure of action potential conduction in preparations of A. astacus motor neurons coincided with Tcrit, indicating that compromised nervous function may provide the underlying determinant for Tcrit rather than oxygen delivery. At high temperatures, absolute aerobic scope was maintained in P. monodon, but reduced in A. astacus. However, A. astacus also displayed reduced exercise intensity indicating that impaired muscle performance with resulting reduced tissue oxygen demand may explain the reduced scope rather than insufficient oxygen supply capacity. This interpretation agrees with early literature on aquatic ectotherms, correlating loss of nervous function with impaired locomotion as temperatures approach Tcrit.

Oxygen delivery does not limit thermal tolerance in a tropical eurythermal crustacean.

In aquatic environments, rising water temperatures reduce water oxygen content while increasing oxygen demand, leading several authors to propose cardiorespiratory oxygen transport capacity as the main determinant of aquatic animal fitness. It has also been argued that tropical species, compared with temperate species, live very close to their upper thermal limit and hence are vulnerable to even small elevations in temperature. Little, however, is known about physiological responses to high temperatures in tropical species. Here we report that the tropical giant freshwater shrimp (Macrobrachium rosenbergii) maintains normal growth when challenged by a temperature rise of 6°C above the present day average (from 27°C to 33°C). Further, by measuring heart rate, gill ventilation rate, resting and maximum oxygen uptake, and hemolymph lactate, we show that oxygen transport capacity is maintained up to the critical maximum temperature around 41°C. In M. rosenbergii heart rate and gill ventilation rate increases exponentially until immediately below critical temperatures and at 38°C animals still retained more than 76% of aerobic scope measured at 30°C, and there was no indication of anaerobic metabolism at the high temperatures. Our study shows that the oxygen transport capacity is maintained at high temperatures, and that other mechanisms, such as protein dysfunction, are responsible for the loss of ecological performance at elevated temperatures.