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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

How to quantify thermal acclimation capacity?

The capacity of organisms to acclimate will influence their ability to cope with ongoing global changes in thermal regimes. Here we highlight methodological issues associated with recent attempts to quantify variation in acclimation capacity among taxa and environments, and describe how these may introduce bias to conclusions. We then propose a measure of thermal acclimation capacity that more directly quantifies the process of acclimation. Future studies of variation in acclimation capacity should critically evaluate whether their chosen empirical metric accurately reflects the theoretical concept of acclimation.

Brain cooling marginally increases acute upper thermal tolerance in Atlantic cod.

Physiological mechanisms determining thermal limits in fishes are debated but remain elusive. It has been hypothesised that motor function loss, observed as loss of equilibrium during acute warming, is due to direct thermal effects on brain neuronal function. To test this, we mounted cooling plates on the heads of Atlantic cod (Gadus morhua) and quantified whether local brain cooling increased whole-organism acute upper thermal tolerance. Brain cooling reduced brain temperature by 2-6°C below ambient water temperature and increased thermal tolerance by 0.5 and 0.6°C on average relative to instrumented and uninstrumented controls, respectively, suggesting that direct thermal effects on brain neurons may contribute to setting upper thermal limits in fish. However, the improvement in thermal tolerance with brain cooling was small relative to the difference in brain temperature, demonstrating that other mechanisms (e.g. failure of spinal and peripheral neurons, or muscle) may also contribute to controlling acute thermal tolerance.

Density differences between water masses preclude laminar flow in two-current choice flumes.

Two-current choice flumes are used to measure preference and avoidance behaviour in response to chemical cues in aquatic animals. If used correctly, they produce two parallel, non-overlapping, laminar water currents in which the animal can move freely and choose between the two currents. As climate change is affecting water temperature, and altered precipitation patterns are changing water salinity, two-current choice flumes are increasingly being used to test the choice between water currents of different temperatures and salinities. This inevitably means that water currents of different densities are being used simultaneously in the flume. Here, we investigated the tolerance range for density differences due to temperature and salinity in five common flume designs. Through dye tests and stepwise modifications of temperatures and salinities we determined the limits for laminar and non-overlapping flows. We also developed an automated method for quantifying the overlap precisely and objectively. The tolerance for density differences between the water currents where laminar and non-overlapping flows were maintained was surprisingly low, withstanding ± 0.5 °C temperature differences, and ± 0.1 PSU salinity differences, i.e. a maximum density difference of 0.28 gL-1. Above these very narrow limits we found a range where the flumes showed partly overlapping, stratified water currents that preclude easy determination of cue preference. We conclude that two-current choice flumes are not suitable for testing the behavioural choices of aquatic animals using water currents of anything other than minor differences in temperature and/or salinity.

Exposure to elevated carbon dioxide does not impair short-term swimming behaviour or shelter-seeking in a predatory coral-reef fish.

Adult bluespotted rockcod Cephalopholis cyanostigma, a coral-reef grouper, were acclimated to either ambient (mean ± s.d. 406 ± 21 µatm; 1 atmos = 101325 Pa) or high pCO2 (945 ± 116 µatm) conditions in a laboratory for 8-9 days, then released at the water surface directly above a reef (depth c. 5 m) and followed on video camera (for 191 ± 21 s) by scuba divers until they sought cover in the reef. No differences were detected between groups in any of the six measured variables, which included the time fish spent immobile after release, tail beat frequency during swimming and the time required to locate and enter the protective shelter of the reef.

Responses of neurogenesis and neuroplasticity related genes to elevated CO2 levels in the brain of three teleost species.

The continuous increase of anthropogenic CO2 in the atmosphere resulting in ocean acidification has been reported to affect brain function in some fishes. During adulthood, cell proliferation is fundamental for fish brain growth and for it to adapt in response to external stimuli, such as environmental changes. Here we report the first expression study of genes regulating neurogenesis and neuroplasticity in brains of three-spined stickleback (Gasterosteus aculeatus), cinnamon anemonefish (Amphiprion melanopus) and spiny damselfish (Acanthochromis polyacanthus) exposed to elevated CO2 The mRNA expression levels of the neurogenic differentiation factor (NeuroD) and doublecortin (DCX) were upregulated in three-spined stickleback exposed to high-CO2 compared with controls, while no changes were detected in the other species. The mRNA expression levels of the proliferating cell nuclear antigen (PCNA) and the brain-derived neurotrophic factor (BDNF) remained unaffected in the high-CO2 exposed groups compared to the control in all three species. These results indicate a species-specific regulation of genes involved in neurogenesis in response to elevated ambient CO2 levels. The higher expression of NeuroD and DCX mRNA transcripts in the brain of high-CO2-exposed three-spined stickleback, together with the lack of effects on mRNA levels in cinnamon anemonefish and spiny damselfish, indicate differences in coping mechanisms among fish in response to the predicted-future CO2 level.

Cardiac oxygen limitation during an acute thermal challenge in the European perch: effects of chronic environmental warming and experimental hyperoxia.

Oxygen supply to the heart has been hypothesized to limit cardiac performance and whole animal acute thermal tolerance (CTmax) in fish. We tested these hypotheses by continuously measuring venous oxygen tension (Pvo2) and cardiovascular variables in vivo during acute warming in European perch (Perca fluviatilis) from a reference area during summer (18°C) and a chronically heated area (Biotest enclosure) that receives warm effluent water from a nuclear power plant and is normally 5-10°C above ambient (24°C at the time of experiments). While CTmax was 2.2°C higher in Biotest compared with reference perch, the peaks in cardiac output and heart rate prior to CTmax occurred at statistically similar Pvo2 values (2.3-4.0 kPa), suggesting that cardiac failure occurred at a common critical Pvo2 threshold. Environmental hyperoxia (200% air saturation) increased Pvo2 across temperatures in reference fish, but heart rate still declined at a similar temperature. CTmax of reference fish increased slightly (by 0.9°C) in hyperoxia, but remained significantly lower than in Biotest fish despite an improved cardiac output due to an elevated stroke volume. Thus, while cardiac oxygen supply appears critical to elevate stroke volume at high temperatures, oxygen limitation may not explain the bradycardia and arrhythmia that occur prior to CTmax Acute thermal tolerance and its thermal plasticity can, therefore, only be partially attributed to cardiac failure from myocardial oxygen limitations, and likely involves limiting factors on multiple organizational levels.