Thermal stressors during embryo incubation have limited ontogenic carryover effects in brook trout.

Winter climate is changing rapidly in northern latitudes, and these temperature events have effects on salmonid thermal biology. Stressors during winter egg incubation could reduce hatching success and physiological performance of fall-spawning fishes. Here we quantified the potential for ontogenic carryover effects from embryonic thermal stress in multiple wild and hatchery-origin populations of brook trout (Salvelinus fontinalis), a temperate ectotherm native to northeastern North America. Fertilized eggs from four populations were incubated over the winter in the laboratory in four differing thermal regimes: ambient stream-fed water, chronic warming (+2 °C), ambient with a mid-winter cold-shock, and short-term warming late during embryogenesis (to stimulate an early spring). We examined body size and upper thermal tolerance at the embryonic, fry (10 weeks post-hatch and 27-30 weeks post-hatch) and gravid adult (age 2+) life stages (overall N = 1482). In a separate experiment, we exposed developing embryos to acute seven-day heat stress events immediately following fertilization and at the eyed-egg stage, and then assessed upper thermal tolerance (CTmax) 37 weeks post-hatch. In all cases, fish were raised in common garden conditions after hatch (i.e., same temperatures). Our thermal treatments during incubation had effects that varied by life stage, with incubation temperature and life stage both affecting body size and thermal tolerance. Embryos incubated in warmer treatment groups had higher thermal tolerance; there was no effect of the mid-winter melt event on embryo CTmax. Ten weeks after hatch, fry from the ambient and cold-shock treatment groups had higher and less variable thermal tolerance than did the warmer treatment groups. At 27-30 post-hatch and beyond, differences in thermal tolerance among treatment groups were negligible. Collectively, our study suggests that brook trout only exhibit short-term carryover effects from thermal stressors during embryo incubation, with no lasting effects on phenotype beyond the first few months after hatch.

Validation of a new acoustic telemetry transmitter for the study of predation events in small fishes.

Acoustic telemetry has emerged as an important tool for studying the movement and behavior of aquatic animals. Predation-sensing acoustic transmitters combine the functions of typical acoustic transmitters with the added ability to identify the predation of tagged animals. The objective of this paper was to assess the performance of a newly miniaturized acid-based predation-sensing acoustic transmitter (Innovasea V3D; 0.33 g in air). We conducted staged predation events in the laboratory where acoustically tagged rainbow trout (Oncorhynchus mykiss) were fed to largemouth bass (Micropterus nigricans) at 3.3-7.0, 9.0-10.8, 16.0-20.0, and 22.0-25.8°C. We also conducted false-positive tests where tagged rainbow trout were held at 10.0 and 16.8°C without the risk of predation. Predation events were successfully identified in 92% of the staged predation trials. Signal lag (i.e., the time required for a predation tag to indicate that predation occurred) ranged from 0.11 to 6.29 days and decreased strongly with increasing water temperature and increased with increasing body mass of the tagged prey. Tag retention in the gut of the predator was much more variable than signal lag and was influenced by water temperature and individual predators but not by prey mass. No false positives were detected after 60 days at either temperature (n = 27 individuals). Although the relationships between water temperature, signal lag, and retention time are likely species-specific, the data reported here provide useful information for the use of these transmitters to study predation in wild fishes, especially for temperate, freshwater fish.

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.

CTmax in brook trout (Salvelinus fontinalis) embryos shows an acclimation response to developmental temperatures but is more variable than in later life stages.

Critical thermal maximum (CTmax ) is widely used to measure upper thermal tolerance in fish but is rarely examined in embryos. Upper thermal limits generally depend on an individual's thermal history, which molds plasticity. We examined how thermal acclimation affects thermal tolerance of brook trout (Salvelinus fontinalis) embryos using a novel method to assess CTmax in embryos incubated under three thermal regimes. Warm acclimation was associated with an increase in embryonic upper thermal tolerance. However, CTmax variability was markedly higher than is typical for juvenile or adult salmonids.

Physiological differences between wild and captive animals: a century-old dilemma.

Laboratory-based research dominates the fields of comparative physiology and biomechanics. The power of lab work has long been recognized by experimental biologists. For example, in 1932, Georgy Gause published an influential paper in Journal of Experimental Biology describing a series of clever lab experiments that provided the first empirical test of competitive exclusion theory, laying the foundation for a field that remains active today. At the time, Gause wrestled with the dilemma of conducting experiments in the lab or the field, ultimately deciding that progress could be best achieved by taking advantage of the high level of control offered by lab experiments. However, physiological experiments often yield different, and even contradictory, results when conducted in lab versus field settings. This is especially concerning in the Anthropocene, as standard laboratory techniques are increasingly relied upon to predict how wild animals will respond to environmental disturbances to inform decisions in conservation and management. In this Commentary, we discuss several hypothesized mechanisms that could explain disparities between experimental biology in the lab and in the field. We propose strategies for understanding why these differences occur and how we can use these results to improve our understanding of the physiology of wild animals. Nearly a century beyond Gause's work, we still know remarkably little about what makes captive animals different from wild ones. Discovering these mechanisms should be an important goal for experimental biologists in the future.

Short-term acclimation dynamics in a coldwater fish.

Critical thermal maximum (CTmax) is widely used for measuring thermal tolerance but the strong effect of acclimation on CTmax is a likely source of variation within and among studies/species that makes comparisons more difficult. There have been surprisingly few studies focused on quantifying how quickly acclimation occurs or that combine temperature and duration effects. We studied the effects of absolute temperature difference and duration of acclimation on CTmax of brook trout (Salvelinus fontinalis), a well-studied species in the thermal biology literature, under laboratory conditions to determine how each of the two factors and their combined effects influence critical thermal maximum. Using an ecologically-relevant range of temperatures and testing CTmax multiple times between one and 30 days, we found that both temperature and duration of acclimation had strong effects on CTmax. As predicted, fish that were exposed to warmer temperatures longer had increased CTmax, but full acclimation (i.e., a plateau in CTmax) did not occur by day 30. Therefore, our study provides useful context for thermal biologists by demonstrating that the CTmax of fish can continue to acclimate to a new temperature for at least 30 days. We recommend that this be considered in future studies measuring thermal tolerance that intend to have their organisms fully acclimated to a given temperature. Our results also support using detailed thermal acclimation information to reduce uncertainty caused by local or seasonal acclimation effects and to improve the use of CTmax data for fundamental research and conservation planning.

The movement ecology of fishes.

Movement of fishes in the aquatic realm is fundamental to their ecology and survival. Movement can be driven by a variety of biological, physiological and environmental factors occurring across all spatial and temporal scales. The intrinsic capacity of movement to impact fish individually (e.g., foraging) with potential knock-on effects throughout the ecosystem (e.g., food web dynamics) has garnered considerable interest in the field of movement ecology. The advancement of technology in recent decades, in combination with ever-growing threats to freshwater and marine systems, has further spurred empirical research and theoretical considerations. Given the rapid expansion within the field of movement ecology and its significant role in informing management and conservation efforts, a contemporary and multidisciplinary review about the various components influencing movement is outstanding. Using an established conceptual framework for movement ecology as a guide (i.e., Nathan et al., 2008: 19052), we synthesized the environmental and individual factors that affect the movement of fishes. Specifically, internal (e.g., energy acquisition, endocrinology, and homeostasis) and external (biotic and abiotic) environmental elements are discussed, as well as the different processes that influence individual-level (or population) decisions, such as navigation cues, motion capacity, propagation characteristics and group behaviours. In addition to environmental drivers and individual movement factors, we also explored how associated strategies help survival by optimizing physiological and other biological states. Next, we identified how movement ecology is increasingly being incorporated into management and conservation by highlighting the inherent benefits that spatio-temporal fish behaviour imbues into policy, regulatory, and remediation planning. Finally, we considered the future of movement ecology by evaluating ongoing technological innovations and both the challenges and opportunities that these advancements create for scientists and managers. As aquatic ecosystems continue to face alarming climate (and other human-driven) issues that impact animal movements, the comprehensive and multidisciplinary assessment of movement ecology will be instrumental in developing plans to guide research and promote sustainability measures for aquatic resources.

Cardiac activity in walleye (Sander vitreus) during exposure to and recovery from chemical anaesthesia, electroanaesthesia and electrostunning.

Handling and conducting invasive procedures are necessary for aspects of fisheries science, invariably inducing a stress response and imposing energetic demands on fish. Anaesthesia or immobilisation techniques are often used in an attempt to mitigate stress and improve welfare, yet these also come with their own impacts on post-release recovery. Here, the authors investigated whether changes in cardiac activity (heart rates over time, heart rate maxima, and scopes) differed in adult walleye (Sander vitreus) anaesthetised with AQUI-S® 20E (eugenol), electroanaesthetised with a transcutaneous electrical nerve stimulation (TENS) unit or electrostunned with a commercially developed stunning unit. This experiment was divided into two trials. In the first trial, fish were implanted with heart rate loggers and left to recover for c. 4 days. In the second trial, fish were implanted with heart rate loggers, given 3 days to recover and re-exposed to their initial treatments (excluding surgery). Post-treatment cardiac activity was quantified for both trials. Although highly variable across individuals, the authors found no significant differences in heart rate changes over time or recovery times among treatments. Maximum heart rates were consistent among treatment groups, yet significant differences in heart rate scope provided further evidence of strong interindividual variation in the second trial. Based on these results, the authors did not identify any welfare-relevant differences or concerns associated with one treatment over another. Further investigations of the relationships between measures of cardiac function and other physiological stress markers would be beneficial towards identifying best practices for fish handling in fisheries science.

An energetics-performance framework for wild fishes.

There is growing evidence that bioenergetics can explain relationships between environmental conditions and fish behaviour, distribution and fitness. Fish energetic needs increase predictably with water temperature, but metabolic performance (i.e., aerobic scope) exhibits varied relationships, and there is debate about its role in shaping fish ecology. Here we present an energetics-performance framework, which posits that ecological context determines whether energy expenditure or metabolic performance influence fish behaviour and fitness. From this framework, we present testable predictions about how temperature-driven variability in energetic demands and metabolic performance interact with ecological conditions to influence fish behaviour, distribution and fitness. Specifically, factors such as prey availability and the spatial distributions of prey and predators may alter fish temperature selection relative to metabolic and energetic optima. Furthermore, metabolic flexibility is a key determinant of how fish will respond to changing conditions, such as those predicted with climate change. With few exceptions, these predictions have rarely been tested in the wild due partly to difficulties in remotely measuring aspects of fish energetics. However, with recent advances in technology and measurement techniques, we now have a better capacity to measure bioenergetics parameters in the wild. Testing these predictions will provide a more mechanistic understanding of how ecological factors affect fish fitness and population dynamics, advancing our knowledge of how species and ecosystems will respond to rapidly changing 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.

Exploring relationships between oxygen consumption and biologger-derived estimates of heart rate in two warmwater piscivores.

Estimating metabolic rate in wild, free-swimming fish is inherently challenging. Here, we explored using surgically implanted heart rate biologgers to estimate metabolic rate in two warmwater piscivores, bowfin Amia calva (Linneaus 1766) and largemouth bass Micropterus salmoides (Lacepède 1802). Fish were surgically implanted with heart rate loggers, allowed to recover for 24 h, exposed to a netting and air exposure challenge, and then placed into respirometry chambers so that oxygen consumption rate (MO2 ) could be measured in parallel to heart rate (fH ) for a minimum of 20 h (ca. 20 estimates of MO2 ). Heart rate across the duration of the experiment (at 19°C) was significantly higher in largemouth bass (mean ± s.d., 45 ± 14 beats min-1 , range 18-86) than in bowfin (27 ± 9 bpm, range 16-98). Standard metabolic rate was also higher in largemouth bass (1.06 ± 0.19 mg O2  kg-1  min-1 , range 0.46-1.36) than in bowfin (0.89 ± 0.17 mg O2  kg-1  min-1 , range 0.61-1.28). There were weak relationships between fH and MO2 , with heart rate predicting 28% of the variation in oxygen consumption in bowfin and 23% in largemouth bass. The shape of the relationship differed somewhat between the two species, which is perhaps unsurprising given their profound differences in physiology and life history, illustrating the need to carry out species-specific validations. Both species showed some potential for a role of fH in efforts to estimate field metabolic rates, although further validation experiments with a wider range of conditions (e.g., digestive states, swimming activity) would likely help improve the strength of the MO2 -fH relationship for use in field applications.

One hundred research questions in conservation physiology for generating actionable evidence to inform conservation policy and practice.

Environmental change and biodiversity loss are but two of the complex challenges facing conservation practitioners and policy makers. Relevant and robust scientific knowledge is critical for providing decision-makers with the actionable evidence needed to inform conservation decisions. In the Anthropocene, science that leads to meaningful improvements in biodiversity conservation, restoration and management is desperately needed. Conservation Physiology has emerged as a discipline that is well-positioned to identify the mechanisms underpinning population declines, predict responses to environmental change and test different in situ and ex situ conservation interventions for diverse taxa and ecosystems. Here we present a consensus list of 10 priority research themes. Within each theme we identify specific research questions (100 in total), answers to which will address conservation problems and should improve the management of biological resources. The themes frame a set of research questions related to the following: (i) adaptation and phenotypic plasticity; (ii) human-induced environmental change; (iii) human-wildlife interactions; (iv) invasive species; (v) methods, biomarkers and monitoring; (vi) policy, engagement and communication; (vii) pollution; (viii) restoration actions; (ix) threatened species; and (x) urban systems. The themes and questions will hopefully guide and inspire researchers while also helping to demonstrate to practitioners and policy makers the many ways in which physiology can help to support their decisions.

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.

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.

Post-exercise respirometry underestimates maximum metabolic rate in juvenile salmon.

Experimental biologists now routinely quantify maximum metabolic rate (MMR) in fishes using respirometry, often with the goal of calculating aerobic scope and answering important ecological and evolutionary questions. Methods used for estimating MMR vary considerably, with the two most common methods being (i) the 'chase method', where fish are manually chased to exhaustion and immediately sealed into a respirometer for post-exercise measurement of oxygen consumption rate (M O2), and (ii) the 'swim tunnel method', whereby M O2 is measured while the fish swims at high speed in a swim tunnel respirometer. In this study, we compared estimates for MMR made using a 3-min exhaustive chase (followed by measurement of M O2 in a static respirometer) versus those made via maximal swimming in a swim tunnel respirometer. We made a total of 134 estimates of MMR using the two methods with juveniles of two salmonids (Atlantic salmon Salmo salar and Chinook salmon Oncorhynchus tshawytscha) across a 6°C temperature range. We found that the chase method underestimated 'true' MMR (based on the swim tunnel method) by ca. 20% in these species. The gap in MMR estimates between the two methods was not significantly affected by temperature (range of ca. 15-21°C) nor was it affected by body mass (overall range of 53.5-236 g). Our data support some previous studies that have suggested the use of a swim tunnel respirometer generates markedly higher estimates of MMR than does the chase method, at least for species in which a swim tunnel respirometer is viable (e.g. 'athletic' ram ventilating fishes). We recommend that the chase method could be used as a 'proxy' (i.e. with a correction factor) for MMR in future studies if supported by a species-specific calibration with a relevant range of temperatures, body sizes or other covariates of interest.

Effects of intracoelomic transmitter implantation on metabolic rate, swimming performance, growth and survival in juveniles of two salmonids.

In this study, we investigated the effects of acoustic tag implantation on standard and routine metabolic rate (SMR and RMR, estimated via oxygen consumption), critical swimming speed (Ucrit ), survival and growth in juveniles of rainbow trout Oncorhynchus mykiss and lake trout Salvelinus namaycush. Tag burdens ranged from 1.8% to 7.5% across the two species. Growth rates in acoustic-tagged fish were equal to or higher than those in other treatments. Acoustic-tagged S. namaycush had a marginally lower Ucrit than controls but that effect was not replicated in the O. mykiss experiment. Tagging did not have clear effects on metabolic rate but there was an interaction whereby SMR and RMR tended to increase with time since surgery in tagged O. mykiss but not in other treatments (the same trend did not occur in S. namaycush). Survival was high across treatments (mean 98% survival among O. mykiss, 97.5% among S. namaycush). There were no statistically significant effects of tag burden (percentage of body mass) except for a weak negative relationship with growth rate (across species) and a weak positive relationship with Ucrit but only in the O. mykiss. Collectively, our findings suggest there were minor, context-dependent effects of acoustic tagging in juvenile S. namaycush and O. mykiss during an eight-week laboratory experiment. Further research will be required to assess whether tagging can cause meaningful behavioural effects in these species in captivity or in the wild and whether there is a tag burden threshold above which deleterious effects consistently occur.

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.

Dermal injuries caused by purse seine capture result in lasting physiological disturbances in coho salmon.

Fish vitality can be measured by classifying reflex impairments (i.e., a visual impression of the ability to respond to induced stimuli) and visible injuries. These metrics can predict survival probability following release from fisheries, and monitoring physiological disturbances following capture can help understand mechanisms of mortality. To test the hypothesis that severity of injury and reflex impairment influences the time course of physiological recovery, coho salmon (Oncorhynchus kisutch) were held for up to 84-h following capture by purse seine. We classified reflex impairments and visible dermal injuries, and through repeated blood sampling, assessed metrics indicative of stress, exhaustion, and osmoregulatory disturbances. Reflex-impairments and blood lactate levels suggested fish were exhausted upon capture but recovered after 48 h. Conversely, fish with dermal injuries showed disruptions to ion homeostasis that were greater in more severely injured fish and increased over time. While reflex impairments may predict short term post-release mortality, the prolonged physiological disturbances caused by dermal injuries are likely to be responsible for delayed mortality; our results suggest that disruptions to ion homeostasis is a possible mechanism of post-release mortality.

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.