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.

A novel method for measuring acute thermal tolerance in fish embryos.

Aquatic ectotherms are vulnerable to thermal stress, with embryos predicted to be more sensitive than juveniles and adults. When examining the vulnerability of species and life stages to warming, comparable methodology must be used to obtain robust conclusions. Critical thermal methodology is commonly used to characterize acute thermal tolerances in fishes, with critical thermal maximum (CTmax) referring to the acute upper thermal tolerance limit. At this temperature, fish exhibit loss of controlled locomotion due to a temperature-induced collapse of vital physiological functions. While it is relatively easy to monitor behavioural responses and measure CTmax in larval and adult fish, this is more challenging in embryos, leading to a lack of data on this life stage, or that studies rely on potentially incomparable metrics. Here, we present a novel method for measuring CTmax in fish embryos, defined by the temperature at which embryos stop moving. Additionally, we compare this measurement with the temperature of the embryos' last heartbeat, which has previously been proposed as a method for measuring embryonic CTmax. We found that, like other life stages, late-stage embryos exhibited a period of increased activity, peaking approximately 2-3°C before CTmax. Measurements of CTmax based on last movement are more conservative and easier to record in later developmental stages than measurements based on last heartbeat, and they also work well with large and small embryos. Importantly, CTmax measurements based on last movement in embryos are similar to measurements from larvae and adults based on loss of locomotory control. Using last heartbeat as CTmax in embryos likely overestimates acute thermal tolerance, as the heart is still beating when loss of response/equilibrium is reached in larvae/adults. The last movement technique described here allows for comparisons of acute thermal tolerance of embryos between species and across life stages, and as a response variable to treatments.

The ecological relevance of critical thermal maxima methodology for fishes.

Critical thermal maxima methodology (CTM) has been used to infer acute upper thermal tolerance in fishes since the 1950s, yet its ecological relevance remains debated. In this study, the authors synthesize evidence to identify methodological concerns and common misconceptions that have limited the interpretation of critical thermal maximum (CTmax ; value for an individual fish during one trial) in ecological and evolutionary studies of fishes. They identified limitations of, and opportunities for, using CTmax as a metric in experiments, focusing on rates of thermal ramping, acclimation regimes, thermal safety margins, methodological endpoints, links to performance traits and repeatability. Care must be taken when interpreting CTM in ecological contexts, because the protocol was originally designed for ecotoxicological research with standardized methods to facilitate comparisons within study individuals, across species and contexts. CTM can, however, be used in ecological contexts to predict impacts of environmental warming, but only if parameters influencing thermal limits, such as acclimation temperature or rate of thermal ramping, are taken into account. Applications can include mitigating the effects of climate change, informing infrastructure planning or modelling species distribution, adaptation and/or performance in response to climate-related temperature change. The authors' synthesis points to several key directions for future research that will further aid the application and interpretation of CTM data in ecological contexts.

Extreme original data yield extreme decline effects.

Clements et al. respond to Munday's claim that his "reanalysis shows there is not an extreme decline effect in fish ocean acidification studies". They contend that extreme data reported in early studies authored by Dixson and Munday indeed result in an "extreme" decline effect in this field, and conclude that the decline effect is primarily driven by papers by particular authors.

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.

Brain dysfunction during warming is linked to oxygen limitation in larval zebrafish.

Understanding the physiological mechanisms that limit animal thermal tolerance is crucial in predicting how animals will respond to increasingly severe heat waves. Despite their importance for understanding climate change impacts, these mechanisms underlying the upper thermal tolerance limits of animals are largely unknown. It has been hypothesized that the upper thermal tolerance in fish is limited by the thermal tolerance of the brain and is ultimately caused by a global brain depolarization. In this study, we developed methods for measuring the upper thermal limit (CTmax) in larval zebrafish (Danio rerio) with simultaneous recordings of brain activity using GCaMP6s calcium imaging in both free-swimming and agar-embedded fish. We discovered that during warming, CTmax precedes, and is therefore not caused by, a global brain depolarization. Instead, the CTmax coincides with a decline in spontaneous neural activity and a loss of neural response to visual stimuli. By manipulating water oxygen levels both up and down, we found that oxygen availability during heating affects locomotor-related neural activity, the neural response to visual stimuli, and CTmax. Our results suggest that the mechanism limiting the upper thermal tolerance in zebrafish larvae is insufficient oxygen availability causing impaired brain function.

Reduced physiological plasticity in a fish adapted to stable temperatures.

Plasticity can allow organisms to maintain consistent performance across a wide range of environmental conditions. However, it remains largely unknown how costly plasticity is and whether a trade-off exists between plasticity and performance under optimal conditions. Biological rates generally increase with temperature, and to counter that effect, fish use physiological plasticity to adjust their biochemical and physiological functions. Zebrafish in the wild encounter large daily and seasonal temperature fluctuations, suggesting they should display high physiological plasticity. Conversely, laboratory zebrafish have been at optimal temperatures with low thermal fluctuations for over 150 generations. We treated this domestication as an evolution experiment and asked whether this has reduced the physiological plasticity of laboratory fish compared to their wild counterparts. We measured a diverse range of phenotypic traits, from gene expression through physiology to behavior, in wild and laboratory zebrafish acclimated to 15 temperatures from 10 °C to 38 °C. We show that adaptation to the laboratory environment has had major effects on all levels of biology. Laboratory fish show reduced plasticity and are thus less able to counter the direct effects of temperature on key traits like metabolic rates and thermal tolerance, and this difference is detectable down to gene expression level. Rapid selection for faster growth in stable laboratory environments appears to have carried with it a trade-off against physiological plasticity in captive zebrafish compared with their wild counterparts.

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.

Meta-analysis reveals an extreme "decline effect" in the impacts of ocean acidification on fish behavior.

Ocean acidification-decreasing oceanic pH resulting from the uptake of excess atmospheric CO2-has the potential to affect marine life in the future. Among the possible consequences, a series of studies on coral reef fish suggested that the direct effects of acidification on fish behavior may be extreme and have broad ecological ramifications. Recent studies documenting a lack of effect of experimental ocean acidification on fish behavior, however, call this prediction into question. Indeed, the phenomenon of decreasing effect sizes over time is not uncommon and is typically referred to as the "decline effect." Here, we explore the consistency and robustness of scientific evidence over the past decade regarding direct effects of ocean acidification on fish behavior. Using a systematic review and meta-analysis of 91 studies empirically testing effects of ocean acidification on fish behavior, we provide quantitative evidence that the research to date on this topic is characterized by a decline effect, where large effects in initial studies have all but disappeared in subsequent studies over a decade. The decline effect in this field cannot be explained by 3 likely biological explanations, including increasing proportions of studies examining (1) cold-water species; (2) nonolfactory-associated behaviors; and (3) nonlarval life stages. Furthermore, the vast majority of studies with large effect sizes in this field tend to be characterized by low sample sizes, yet are published in high-impact journals and have a disproportionate influence on the field in terms of citations. We contend that ocean acidification has a negligible direct impact on fish behavior, and we advocate for improved approaches to minimize the potential for a decline effect in future avenues of research.

Investigating the gill-oxygen limitation hypothesis in fishes: intraspecific scaling relationships of metabolic rate and gill surface area.

Many ectotherms have shown a reduction in maximum body size in the past decades in parallel with climate warming. Indeed, some models forecast a maximum body size decline of 14%-24% by 2050 for numerous fish species. The gill-oxygen limitation (GOL) hypothesis is perhaps the most prominent concept regarding the physiological mechanisms underlying the observed trends, implicating oxygen uptake limitations in driving the decline in fish body size with warming. Current scientific debates, however, demonstrate a clear need for a synthesis of existing empirical evidence to test the fundamental assumptions of the GOL hypothesis. Here, we perform a systematic literature review of the intraspecific allometry of gill surface area (GSA) and metabolic rate. Additionally, we introduce a new parameter, the ratio S, which provides a measure of GSA in relation to the metabolic requirements for maintenance (S SMR) and maximum activity (S AMR). Support for the GOL hypothesis would be evidenced by a universal decline in S with increasing body mass within each species, such that gills become less equipped to supply metabolic requirements as fish grow. In contrast to the predictions of the GOL hypothesis, we show that the scaling exponents for S SMR and S AMR are consistently close to zero, with only a few exceptions where S either increased or decreased. These findings suggest that the GSA of each species is sufficient to meet its oxygen requirements throughout life, and that growth is not universally restricted by oxygen uptake limitations across the gills. We identify the need to investigate hypotheses other than the GOL hypothesis to help explain the observed declines in maximum fish body sizes concurrent with climate warming, in order to facilitate accurate predictions of fish community structure and manage fisheries in the face of climate change.

Low potential for evolutionary rescue from climate change in a tropical fish.

Climate change is increasing global temperatures and intensifying the frequency and severity of extreme heat waves. How organisms will cope with these changes depends on their inherent thermal tolerance, acclimation capacity, and ability for evolutionary adaptation. Yet, the potential for adaptation of upper thermal tolerance in vertebrates is largely unknown. We artificially selected offspring from wild-caught zebrafish (Danio rerio) to increase (Up-selected) or decrease (Down-selected) upper thermal tolerance over six generations. Selection to increase upper thermal tolerance was also performed on warm-acclimated fish to test whether plasticity in the form of inducible warm tolerance also evolved. Upper thermal tolerance responded to selection in the predicted directions. However, compared to the control lines, the response was stronger in the Down-selected than in the Up-selected lines in which evolution toward higher upper thermal tolerance was slow (0.04 ± 0.008 °C per generation). Furthermore, the scope for plasticity resulting from warm acclimation decreased in the Up-selected lines. These results suggest the existence of a hard limit in upper thermal tolerance. Considering the rate at which global temperatures are increasing, the observed rates of adaptation and the possible hard limit in upper thermal tolerance suggest a low potential for evolutionary rescue in tropical fish living at the edge of their thermal limits.

Rapid-warming tolerance correlates with tolerance to slow warming but not growth at non-optimal temperatures in zebrafish.

Global warming is predicted to increase both acute and prolonged thermal challenges for aquatic ectotherms. Severe short- and medium-term thermal stress over hours to days may cause mortality, while longer sub-lethal thermal challenges may cause performance declines. The inter-relationship between the responses to short, medium and longer thermal challenges is unresolved. We asked if the same individuals are tolerant to both rapid and slow warming challenges, a question that has so far received little attention. Additionally, we investigated the possibility of a thermal syndrome where individuals in a population are distributed along a warm-type to cold-type axis. We tested whether different thermal traits correlate across individuals by acclimating 200 juvenile zebrafish (Danio rerio) to sub- or supra-optimal temperatures for growth (22 and 34°C) for 40 days and measuring growth and thermal tolerance at two different warming rates. We found that tolerance to rapid warming correlated with tolerance to slow warming in the 22°C treatment. However, individual tolerance to neither rapid nor slow warming correlated with growth at the supra-optimal temperature. We thus find some support for a syndrome-like organisation of thermal traits, but the lack of connection between tolerance and growth performance indicates a restricted generality of a thermal syndrome. The results suggest that tolerance to rapid warming may share underlying physiological mechanisms with tolerance to slower heating, and indicate that the relevance of acute critical thermal tolerance extends beyond the rapid ramping rates used to measure them.

Chronic Exposure to Oxazepam Pollution Produces Tolerance to Anxiolytic Effects in Zebrafish (Danio rerio).

Environmental concentrations of the anxiolytic drug oxazepam have been found to disrupt antipredator behaviors of wild fish. Most experiments exposed fish for a week, while evidence from mammals suggests that chronic exposure to therapeutic concentrations of benzodiazepines (such as oxazepam) results in the development of tolerance to the anxiolytic effects. If tolerance can also develop in response to the low concentrations found in the aquatic environment, it could mitigate the negative effects of oxazepam pollution. In the current study, we exposed wild-caught zebrafish to oxazepam (∼7 µg L-1) for 7 or 28 days and evaluated behavioral and physiological parameters at both time points. Females showed reduced diving responses to conspecific alarm pheromone after 7 days, but not after 28 days, indicating that they had developed tolerance to the anxiolytic effects of the drug. Zebrafish males were not affected by this oxazepam concentration, in line with earlier results. Serotonin turnover (ratio 5-HIAA/5-HT) was reduced in exposed females and males after 28 days, indicating that brain neurochemistry had not normalized. Post-confinement cortisol concentrations and gene expression of corticotropin-releasing hormone (CRH) were not affected by oxazepam. We did not find evidence that chronically exposed fish had altered relative expression of GABAA receptor subunits, suggesting that some other still unknown mechanism caused the developed tolerance.

Ocean acidification does not impair the behaviour of coral reef fishes.

The partial pressure of CO2 in the oceans has increased rapidly over the past century, driving ocean acidification and raising concern for the stability of marine ecosystems1-3. Coral reef fishes are predicted to be especially susceptible to end-of-century ocean acidification on the basis of several high-profile papers4,5 that have reported profound behavioural and sensory impairments-for example, complete attraction to the chemical cues of predators under conditions of ocean acidification. Here, we comprehensively and transparently show that-in contrast to previous studies-end-of-century ocean acidification levels have negligible effects on important behaviours of coral reef fishes, such as the avoidance of chemical cues from predators, fish activity levels and behavioural lateralization (left-right turning preference). Using data simulations, we additionally show that the large effect sizes and small within-group variances that have been reported in several previous studies are highly improbable. Together, our findings indicate that the reported effects of ocean acidification on the behaviour of coral reef fishes are not reproducible, suggesting that behavioural perturbations will not be a major consequence for coral reef fishes in high CO2 oceans.

Low concentrations of the benzodiazepine drug oxazepam induce anxiolytic effects in wild-caught but not in laboratory zebrafish.

Pollution by psychoactive pharmaceuticals has been found to disrupt anti-predator behaviors of wild fish. The challenge is now to identify which of the many psychoactive drugs pose the greatest threat. One strategy is to screen for behavioral effects of selected pharmaceuticals using a single, widely available fish species such as zebrafish. Here, we show that although such high-throughput behavioral screening might facilitate comparisons between pharmaceuticals, the choice of strain is essential. While wild-caught zebrafish exposed to concentrations of the anxiolytic drug oxazepam as low as 0.57 µg L-1 showed a reduction in the response to conspecific alarm pheromone, laboratory strain AB did not respond to the alarm cue, and consequently, the anxiolytic effect of oxazepam could not be measured. Adaptation to the laboratory environment may have rendered laboratory strains unfit for use in some ecotoxicological and pharmacological studies, since the results might not translate to wild fish populations.

Extreme blood-boosting capacity of an Antarctic fish represents an adaptation to life in a sub-zero environment.

Blood doping, the practice of boosting the oxygen carrying capacity of blood, is an illegal strategy used by human athletes to enhance aerobic capacity and athletic performance. Interestingly, the practice of boosting blood oxygen carrying capacity is also naturally prevalent in the animal kingdom via the splenic release of stored erythrocytes. Here, we demonstrate that an Antarctic notothenioid fish, the bald notothen (Pagothenia borchgrevinki), is a master of this practice. Because of the sub-zero environment these fish inhabit, they sequester a large proportion of erythrocytes in the spleen during times of inactivity to reduce the energetic and physiological costs associated with continuously pumping highly viscous blood around the body. However, in response to metabolically demanding situations (i.e. exercise and feeding), these fish contract the spleen to eject stored erythrocytes into circulation, which boosts blood oxygen carrying capacity by up to 207% (cf. exercise-induced increases of ∼40-60% in a range of other vertebrates and ∼5-25% in blood-doping athletes). By evaluating cardiorespiratory differences between splenectomized (unable to release erythrocytes from the spleen) and sham-operated individuals, we demonstrate the metabolic benefits (i.e. aerobic scope increase of 103%) and the cardiovascular trade-offs (i.e. ventral aortic blood pressure and cardiac workload increase of 12% and 30%, respectively) associated with the splenic blood-boosting strategy. In conclusion, this strategy provides bald notothens with an extraordinary facultative aerobic scope that enables an active lifestyle in the extreme Antarctic marine environment, while minimizing the energetic and physiological costs of transporting highly viscous blood during times of reduced energetic demand.

On the Observation of Wild Zebrafish (Danio rerio) in India.

Zebrafish is one of the world's most widely used laboratory species, and it is utilized to answer important research questions in disparate fields such as biomedicine, genetics, developmental biology, pharmacology, toxicology, physiology, and evolution. Despite their popularity, very little is known about the biology of zebrafish in their natural habitat. This may, in part, be due to the difficulties associated with undertaking field trips to the remote areas of northern India, Nepal, and Bangladesh, which is the natural distribution range of zebrafish. Here, we present a field report describing a recent trip where we, together with local collaborators, visited several rivers in West Bengal, India, to observe wild zebrafish and their habitat. We present an overview of our observations on the biology of wild zebrafish, and the great variability of the different environments where they were found. We also include data collected on water chemistry parameters at 12 zebrafish sites, and weight data and photos of fish from these sites. We present extensive underwater videos of wild zebrafish and photographs of the sites, including video footage of courtship behavior. We show that the breeding period of wild zebrafish can be extended from the previous record of April-August to April-October. In addition, we provide practical advice for future zebrafish expeditions to this rural and inaccessible area. The goals of this article are to shed some light on the ecology of wild zebrafish, and to facilitate scientists in their future research trips. We hope that by observing zebrafish in the wild, we can increase our understanding of the natural ecology of this important model organism.