Acute warming tolerance (CTmax) in zebrafish (Danio rerio) appears unaffected by changes in water salinity.

Tolerance against acute warming is an essential trait that can determine how organisms cope during heat waves, yet the mechanisms underlying it remain elusive. Water salinity has previously been suggested to modulate warming tolerance in fish and may therefore provide clues towards these limiting mechanisms. Here, using the critical thermal maximum (CTmax) test, we investigated whether short (2 hours) and long (10 days) term exposure to different water salinities (2 hours: 0-5 ppt, 10 days: 0-3 ppt) affected acute warming tolerance in zebrafish (N = 263). We found that water salinity did not affect the warming tolerance of zebrafish at either time point, indicating that salinity does not affect the mechanism limiting acute warming tolerance in zebrafish at these salinity ranges, and that natural fluctuations in salinity levels might not have a large impact on acute warming tolerance in wild zebrafish.

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.

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.

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.

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.