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.

Specific dynamic action: the energy cost of digestion or growth?

The physiological processes underlying the post-prandial rise in metabolic rate, most commonly known as the 'specific dynamic action' (SDA), remain debated and controversial. This Commentary examines the SDA response from two opposing hypotheses: (i) the classic interpretation, where the SDA represents the energy cost of digestion, versus (ii) the alternative view that much of the SDA represents the energy cost of growth. The traditional viewpoint implies that individuals with a reduced SDA should grow faster given the same caloric intake, but experimental evidence for this effect remains scarce and inconclusive. Alternatively, we suggest that the SDA reflects an organism's efficacy in allocating the ingested food to growth, emphasising the role of post-absorptive processes, particularly protein synthesis. Although both viewpoints recognise the trade-offs in energy allocation and the dynamic nature of energy distribution among physiological processes, we argue that equating the SDA with 'the energy cost of digestion' oversimplifies the complexities of energy use in relation to the SDA and growth. In many instances, a reduced SDA may reflect diminished nutrient absorption (e.g. due to lower digestive efficiency) rather than increased 'free' energy available for somatic growth. Considering these perspectives, we summarise evidence both for and against the opposing hypotheses with a focus on ectothermic vertebrates. We conclude by presenting a number of future directions for experiments that may clarify what the SDA is, and what it is not.

Estimating maximum oxygen uptake of fishes during swimming and following exhaustive chase - different results, biological bases and applications.

The maximum rate at which animals take up oxygen from their environment (MO2,max) is a crucial aspect of their physiology and ecology. In fishes, MO2,max is commonly quantified by measuring oxygen uptake either during incremental swimming tests or during recovery from an exhaustive chase. In this Commentary, we compile recent studies that apply both techniques to the same fish and show that the two methods typically yield different mean estimates of MO2,max for a group of individuals. Furthermore, within a group of fish, estimates of MO2,max determined during swimming are poorly correlated with estimates determined during recovery from chasing (i.e. an individual's MO2,max is not repeatable across methods). One explanation for the lack of agreement is that these methods measure different physiological states, each with their own behavioural, anatomical and biochemical determinants. We propose that these methods are not directly interchangeable but, rather, each is suited to address different questions in fish biology. We suggest that researchers select the method that reflects the biological contexts of their study, and we advocate for the use of accurate terminology that acknowledges the technique used to elevate MO2 (e.g. peak MO2,swim or peak MO2,recovery). If the study's objective is to estimate the 'true' MO2,max of an individual or species, we recommend that pilot studies compare methods, preferably using repeated-measures designs. We hope that these recommendations contribute new insights into the causes and consequences of variation in MO2,max within and among fish species.

Supplemental oxygen does not improve growth but can enhance reproductive capacity of fish.

Fish tend to grow faster as the climate warms but attain a smaller adult body size following an earlier age at sexual maturation. Despite the apparent ubiquity of this phenomenon, termed the temperature-size rule (TSR), heated scientific debates have revealed a poor understanding of the underlying mechanisms. At the centre of these debates are prominent but marginally tested hypotheses which implicate some form of 'oxygen limitation' as the proximate cause. Here, we test the role of oxygen limitation in the TSR by rearing juvenile Galaxias maculatus for a full year in current-day (15°C) and forecasted (20°C) summer temperatures while providing half of each temperature group with supplemental oxygen (hyperoxia). True to the TSR, fish in the warm treatments grew faster and reached sexual maturation earlier than their cooler conspecifics. Yet, despite supplemental oxygen significantly increasing maximum oxygen uptake rate, our findings contradict leading hypotheses by showing that the average size at sexual maturation and the adult body size did not differ between normoxia and hyperoxia groups. We did, however, discover that hyperoxia extended the reproductive window, independent of fish size and temperature. We conclude that the intense resource investment in reproduction could expose a bottleneck where oxygen becomes a limiting factor.

Evidence for energy reallocation, not oxygen limitation, driving the deceleration in growth of adult fish.

The lifetime growth of almost all fishes follows a biphasic relationship - juvenile growth is rapid and adult growth subsequently decelerates. For a trend that is so ubiquitous, there is no general agreement as to the underlying mechanisms causing adult growth to decelerate. Ongoing theories argue that adult growth slows because either the gills fail to supply the body with surplus oxygen needed for continued somatic gain (i.e. oxygen limited), or sexual maturation induces a switch in energy allocation towards reproduction and away from growth (i.e. energy limited). Here, we empirically tested these notions by tracking the individual growth trajectories of ∼100 female Galaxias maculatus, ranging in size, during their first 3 months of adulthood. At a summer temperature of 20°C, we provided subsets of fish with additional energy (fed once versus twice a day), supplementary oxygen (normoxia versus hyperoxia), or a combination of the two, to assess whether we could change the trajectory of adult growth. We found that growth improved marginally with additional energy, yet remained unaffected by supplementary oxygen, thereby providing evidence for a role for energy reallocation in the deceleration of adult growth. Interestingly, additional dietary energy had a disproportionately larger effect on the growth of fish that matured at a greater size, revealing size-dependent variance in energy acquisition and/or allocation budgets at summer temperatures. Overall, these findings contribute towards understanding the mechanisms driving widespread declines in the body size of fish with climate warming.

Behavioural temperature regulation is a low priority in a coral reef fish (Plectropomus leopardus): insights from a novel behavioural thermoregulation system.

Current understanding of behavioural thermoregulation in aquatic ectotherms largely stems from systems such as 'shuttle boxes', which are generally limited in their capacity to test large-bodied species. Here, we introduce a controlled system that allows large aquatic ectotherms to roam freely in a tank at sub-optimal temperatures, using thermal refuges to increase body temperature to their thermal optimum as desired. Of the 10 coral grouper (Plectropomus leopardus; length ∼400 mm) implanted with thermal loggers, three fish maintained themselves at the ambient tank temperature of 17.5-20.5°C for the entire 2-4 days of the trial. Of the other seven fish, body temperature never exceeded ∼21.5°C, which was well below the temperature available in the thermal refuges (∼31°C) and below the species' optimal temperature of ∼27°C. This study adds to a growing literature documenting an unexpected lack of behavioural thermoregulation in aquatic ectotherms in controlled, heterothermal environments.

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.

Chronic experimental hyperoxia elevates aerobic scope: a valid method to test for physiological oxygen limitations in fish.

Experimental hyperoxia has been shown to enhance the maximum oxygen uptake capacity of fishes under acute conditions, potentially offering an avenue to test prominent physiological hypotheses attempting to explain impacts of climate warming on fish populations (e.g., gill-oxygen limitation driving declines in fish size). Such benefits of experimental hyperoxia must persist under chronic conditions if it is to provide a valid manipulation to test the relevant hypotheses, yet the long-term benefits of experimental hyperoxia to oxygen uptake capacity have not been examined. Here, the authors measured aerobic metabolic performance of Galaxias maculatus upon acute exposure to hyperoxia (150% air saturation) and after 5 months of acclimation, at both 15°C and 20°C. Acute hyperoxia elevated aerobic scope by 74%-94% relative to normoxic controls, and an elevation of 58%-73% persisted after 5 months of hyperoxia acclimation. When hyperoxia-acclimated fish were acutely transitioned back to normoxia, they maintained superior aerobic performance compared with normoxic controls, suggesting an acclimation of the underlying metabolic structures/processes. In demonstrating the long-term benefits of experimental hyperoxia on the aerobic performance of a fish, the authors encourage the use of such approaches to disentangle the role of oxygen in driving the responses of fish populations to climate warming.

Thermal performance curves for aerobic scope in a tropical fish (Lates calcarifer): flexible in amplitude but not breadth.

Aerobic metabolic scope is a popular metric to estimate the capacity for temperature-dependent performance in aquatic animals. Despite this popularity, little is known of the role of temperature acclimation and variability in shaping the breadth and amplitude of the thermal performance curve for aerobic scope. If daily thermal experience can modify the characteristics of the thermal performance curve, interpretations of aerobic scope data from the literature may be misguided. Here, tropical barramundi (Lates calcarifer) were acclimated for ∼4 months to cold (23°C), optimal (29°C) or warm (35°C) conditions, or to a daily temperature cycle between 23 and 35°C (with a mean of 29°C). Measurements of aerobic scope were conducted every 3-4 weeks at three temperatures (23, 29 and 35°C), and growth rates were monitored. Acclimation to constant temperatures caused some changes in aerobic scope at the three measurement temperatures via adjustments in standard and maximum metabolic rates, and growth rates were lower in the 23°C-acclimated group than in all other groups. The metabolic parameters and growth rates of the thermally variable group remained similar to those of the 29°C-acclimated group. Thus, acclimation to a variable temperature regime did not broaden the thermal performance curve for aerobic scope. We propose that thermal performance curves for aerobic scope are more plastic in amplitude than in breadth, and that the metabolic phenotype of at least some fish may be more dependent on the mean daily temperature than on the daily temperature range.

Best practices for non-lethal blood sampling of fish via the caudal vasculature.

Blood sampling through the caudal vasculature is a widely used technique in fish biology for investigating organismal health and physiology. In live fishes, it can provide a quick, easy and relatively non-invasive method for obtaining a blood sample (cf. cannulation and cardiac puncture). Here, a general set of recommendations are provided for optimizing the blood sampling protocol that reflects best practices in animal welfare and sample integrity. This includes selecting appropriate use of anaesthetics for blood sampling as well as restraint techniques for situations where sedation is not used. In addition, ideal sampling environments where the fish can freely ventilate and strategies for minimizing handling time are discussed. This study summarizes the techniques used for extracting blood from the caudal vasculature in live fishes, highlighting the phlebotomy itself, the timing of sampling events and acceptable blood sample volumes. This study further discuss considerations for selecting appropriate physiological metrics when sampling in the caudal region and the potential benefits that this technique provides with respect to long-term biological assessments. Although general guidelines for blood sampling are provided here, it should be recognized that contextual considerations (e.g., taxonomic diversity, legal matters, environmental constraints) may influence the approach to blood sampling. Overall, it can be concluded that when done properly, blood sampling live fishes through the caudal vasculature is quick, efficient and minimally invasive, thus promoting conditions where live release of focal animals is possible.

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.

Long-term acclimation to near-future ocean acidification has negligible effects on energetic attributes in a juvenile coral reef fish.

Increased levels of dissolved carbon dioxide (CO2) drive ocean acidification and have been predicted to increase the energy use of marine fishes via physiological and behavioural mechanisms. This notion is based on a theoretical framework suggesting that detrimental effects on energy use are caused by plasma acid-base disruption in response to hypercapnic acidosis, potentially in combination with a malfunction of the gamma aminobutyric acid type A (GABAA) receptors in the brain. However, the existing empirical evidence testing these effects primarily stems from studies that exposed fish to elevated CO2 for a few days and measured a small number of traits. We investigated a range of energetic traits in juvenile spiny chromis damselfish (Acanthochromis polyacanthus) over 3 months of acclimation to projected end-of-century CO2 levels (~ 1000 µatm). Somatic growth and otolith size and shape were unaffected by the CO2 treatment across 3 months of development in comparison with control fish (~ 420 µatm). Swimming activity during behavioural assays was initially higher in the elevated CO2 group, but this effect dissipated within ~ 25 min following handling. The transient higher activity of fish under elevated CO2 was not associated with a detectable difference in the rate of oxygen uptake nor was it mediated by GABAA neurotransmitter interference because treatment with a GABAA antagonist (gabazine) did not abolish the CO2 treatment effect. These findings contrast with several short-term studies by suggesting that end-of-century levels of CO2 may have negligible direct effects on the energetics of at least some species of fish.

Effects of an unprecedented summer heatwave on the growth performance, flesh colour and plasma biochemistry of marine cage-farmed Atlantic salmon (Salmo salar).

Global seawater temperatures are increasing and becoming more variable, with consequences for all marine animals including those in food production systems. In several countries around the world,arming of Atlantic salmon (Salmo salar) occurs towards the upper end of the thermal tolerance window for this species, and marked effects on salmon production during summers have been experienced but never empirically investigated. This project tracked the effects of an extreme summer heatwave on two different cohorts of fish stocked into farm cages either during early winter (EW) or late winter (LW). The farm site experienced an unprecedented high water temperature event, with a peak water temperature of 22.9 °C and 117 days above 18 °C. Fish in both EW and LW cohorts experienced a temperature-induced cessation of voluntary feed intake as well as inefficient osmoregulatory, liver and renal function during high temperature periods. Flesh colour declined primarily in the dorsal and ventral regions of the fillet and secondarily along the midline, with over 20% of fish demonstrated a complete loss of flesh colour during the months of March and April. A return to feeding in autumn occurred faster in some fish and caused a marked bimodal size distribution to appear within both the EW and LW cohorts as autumn progressed. However, the LW cohort returned to feeding at seawater temperatures of 20.2 °C, compared with 18.6 °C for the EW cohort. There was a strong positive relationship between fillet colour recovery and residual condition index (RCI). These findings identified alkaline phosphatase as a potential marker to non-destructively track individual fish for signs of recovery after a thermal stress event, and shed light on the physiological consequences of marine heatwaves on fishes. This study also identified that supporting feed intake or promoting a return to feeding may help mitigate the negative impacts of climate warming on cultured Atlantic salmon.

Negligible differences in metabolism and thermal tolerance between diploid and triploid Atlantic salmon (Salmo salar).

The mechanisms that underlie thermal tolerance in aquatic ectotherms remain unresolved. Triploid fish have been reported to exhibit lower thermal tolerance than diploids, offering a potential model organism to better understand the physiological drivers of thermal tolerance. Here, we compared triploid and diploid juvenile Atlantic salmon (Salmo salar) in freshwater to investigate the proposed link between aerobic capacity and thermal tolerance. We measured specific growth rates (SGR) and resting (aerobic) metabolic rates (RMR) in freshwater at 3, 7 and 9 weeks of acclimation to 10, 14 and 18°C. Additionally, maximum metabolic rates (MMR) were measured at 3 and 7 weeks of acclimation, and critical thermal maxima (CTmax) were measured at 9 weeks. Mass, SGR and RMR differed between ploidies across all temperatures at the beginning of the acclimation period, but all three metrics were similar across ploidies by week 7. Aerobic scope (MMR-RMR) remained consistent across ploidies, acclimation temperatures and time. At 9 weeks, CTmax was independent of ploidy, but correlated positively with acclimation temperature despite the similar aerobic scope between acclimation groups. Our findings suggest that acute thermal tolerance is not modulated by aerobic scope, and the altered genome of triploid Atlantic salmon does not translate to reduced thermal tolerance of juvenile fish in freshwater.

Baseline and stress-induced blood properties of male and female Darwin's small ground finch (Geospiza fuliginosa) of the Galapagos Islands.

Birds are renowned for exhibiting marked sex-specific differences in activity levels and reproductive investment during the breeding season, potentially impacting circulating blood parameters associated with stress and energetics. Males of many passerines often do not incubate, but they experience direct exposure to intruder threat and exhibit aggressive behaviour during the nesting phase in order to defend territories against competing males and predators. Nesting females often have long bouts of inactivity during incubation, but they must remain vigilant of the risks posed by predators and conspecific intruders approaching the nest. Here, we use 33 free-living male (n = 16) and female (n = 17) Darwin's small ground finches (Geospiza fuliginosa) on Floreana Island (Galapagos Archipelago) to better understand how sex-specific roles during the reproductive period impact baseline and stress-induced levels of plasma corticosterone (CORT), blood glucose and haematocrit. Specifically, we hypothesise that males are characterised by higher baseline values given their direct and relatively frequent exposure to intruder threat, but that a standardised stress event (capture and holding) overrides any sex-specific differences. In contrast with expectations, baseline levels of all blood parameters were similar between sexes (13.4 ±â€¯1.9 ng ml-1 for CORT, 13.7 ±â€¯0.4 mmol l-1 for glucose, 58.3 ±â€¯0.8% for haematocrit). Interestingly, females with higher body condition had lower baseline haematocrit. All blood parameters changed with time since capture (range 1.2-41.3 min) in both sexes, whereby CORT increased linearly, haematocrit decreased linearly, and glucose increased to a peak at ∼20 min post-capture and declined to baseline levels thereafter. Our results do not support the hypothesis that sex-specific roles during the reproductive period translate to differences in blood parameters associated with stress and energetics, but we found some evidence that blood oxygen transport capacity may decline as finches increase in body condition.

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.