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Investigating real-time stress responses during common aquaculture practices is necessary for improving the welfare of farmed fish. Recently, Star-Oddi DST milli-HRT bio-loggers capable of measuring heart rate (as an indicator for stress) in freely swimming fish have proved to be useful in this regard. However, despite the growing popularity of these devices, they have not yet been stringently evaluated and validated for use in fish. The present study is the first to do so by simultaneously recording heart rate of a commercially important salmonid species (rainbow trout, Oncorhynchus mykiss) using the abovementioned bio-loggers, as well as an established non-invasive wireless heart rate recording technique that has been demonstrated to provide more accurate recordings than those collected using traditional methods requiring a physical connection to recording equipment. The measurement error of the bio-loggers was 72 h. Following the successful validation of these bio-loggers, the effects of common aquaculture practices such as netting, grouping and enforced activity on the cardiac performance of freely swimming rainbow trout were investigated. Prior to the subsequent exposure to the abovementioned stressors, mean heart rates of trout were ~42 beats min−1. Following netting, trout increased heart rates by 57%, which recovered within 3 h. Grouping subsequently induced a more pronounced and prolonged stress response, as heart rates initially increased by 86% and then remained elevated throughout the 8 h recovery period. Consequently, trout were only able to further increase heart rates by 7% following enforced activity. This diminished cardiac response demonstrates a situation where the cumulative allostatic load induced by prior stressors is approaching a level where the animal struggles to perform normal physiological processes in the face of further challenges. These findings are important for the aquaculture industry as exposure to multiple stressors without sufficient recovery times has the potential to result in the collapse of cardiac scope, which combined with unfavourable environmental conditions could lead to increased mortality.

Farmed aquatic animals represent an increasingly important source of food for a growing human population. However, the aquaculture industry faces several challenges with regard to producing a profitable, ethical and environmentally sustainable product, which are exacerbated by the ongoing intensification of operations and increasingly extreme and unpredictable climate conditions. Fortunately, bio-sensors capable of measuring a range of environmental, behavioural and physiological variables (e.g. temperature, dissolved gases, depth, acceleration, ventilation, heart rate, blood flow, glucose and l -lactic acid) represent exciting and innovative tools for assessing the health and welfare of farmed animals in aquaculture. Here, we illustrate how these state-of-the-art technologies can provide unique insights into variables pertaining to the inner workings of the animal to elucidate animal–environment interactions throughout the production cycle, as well as to provide insights on how farmed animals perceive and respond to environmental and anthropogenic perturbations. Using examples based on current challenges (i.e. sub-optimal feeding strategies, sub-optimal animal welfare and environmental changes), we discuss how bio-sensors can contribute towards optimizing the growth, health and welfare of farmed animals under dynamically changing on-farm conditions. While bio-sensors currently represent tools that are primarily used for research, the continuing development and refinement of these technologies may eventually allow farmers to use real-time environmental and physiological data from their stock as ‘early warning systems' and/or for refining day-to-day operations to ethically and sustainably optimize production. This article is part of the theme issue ‘Measuring physiology in free-living animals (Part I)’.

Few studies have addressed how reduced water salinity affects cardiovascular and metabolic function in marine euryhaline fishes, despite its relevance for predicting impacts of natural salinity variations and ongoing climate change on marine fish populations. Here, shorthorn sculpin (Myoxocephalus scorpius) were subjected to different durations of reduced water salinity from 33 to 15 ppt. Routine metabolic rate decreased after short-term acclimation (4–9 days) to 15 ppt, which corresponded with similar reductions in cardiac output. Likewise, standard metabolic rate decreased after acute transition (3 h) from 33 to 15 ppt, suggesting a reduced energetic cost of osmoregulation at 15 ppt. Interestingly, gut blood flow remained unchanged across salinities, which contrasts with previous findings in freshwater euryhaline teleosts (e.g., rainbow trout) exposed to different salinities. Although plasma osmolality, [Na+], [Cl−] and [Ca2+] decreased in 15 ppt, there were no signs of cellular osmotic stress as plasma [K+], [hemoglobin] and hematocrit remained unchanged. Taken together, our data suggest that shorthorn sculpin are relatively weak plasma osmoregulators that apply a strategy whereby epithelial ion transport mechanisms are partially maintained across salinities, while plasma composition is allowed to fluctuate within certain ranges. This may have energetic benefits in environments where salinity naturally fluctuates, and could provide shorthorn sculpin with competitive advantages if salinity fluctuations intensify with climate change in the future.

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European whitefish (Coregonus lavaretus) is an aquaculture species with the potential for expanded cultivation in the fresh and brackish waters of Northern Europe. Yet, relatively little species-specific information is available regarding the stress responses and associated welfare implications for this species in captivity. We addressed this knowledge gap by using a combination of implantable heart rate bio-loggers and a range of traditional stress indicators (e.g. haematological parameters and plasma concentrations of cortisol, glucose and ions) to comprehensively evaluate the physiological responses of freely swimming whitefish in captivity, as well as when subjected to aquaculture practices and stressors that commonly occur prior to and during slaughter. Whitefish appeared to recover rapidly from surgery, as resting heart rate decreased within 36 h to stabilize at similar to 25 beats min(-1) for the next 18 days when fish were left relatively undisturbed (i.e. personnel were only present when feeding fish). In contrast with previous studies on farmed rainbow trout and Atlantic salmon, whitefish did not exhibit a clear circadian heart rate rhythm, which may be related to species-specific differences in diurnal locomotor activity. Whitefish also appear to have a well-developed capacity for thermal acclimation of heart rate, as daily resting heart rate did not change during the undisturbed period despite an increase in body temperature from similar to 6.8 to 11.2 degrees C. Following acute stressors such as crowding and transportation, the physiological response of whitefish typically involved transient elevations in heart rate, plasma cortisol and glucose, and red blood cell swelling, while plasma [K+] decreased. In contrast, the heart rate of whitefish plummeted following the combination of brailing (i.e. to haul in fish with a brail/net) and CO2 exposure prior to slaughter, while plasma cortisol, glucose and [Ca2+] significantly increased. An unforeseen finding concerns the substantial and long-lasting physiological stress response observed in whitefish when held in close proximity (i.e. within similar to 10 m) to a rainbow trout net pen, as the mean heart rate of whitefish increased from similar to 32 to 43 beats min(-1) (i.e. an increase of similar to 34%). This may represent an innate physiological response to the threat of predation, which consequently increases the allostatic load and energetic expenditure of whitefish when farmed alongside salmonids. To conclude, this study highlights the importance of performing long-term, species-specific evaluations of freely swimming fish in real aquaculture settings, and provides a platform for further research aiming to determine the welfare implications of simultaneously farming predatory and prey species in close proximity.

We would like to thank Hedrick and colleagues for their thoughtful comments and for the opportunity to further elaborate on the splenic blood-boosting strategy of Pagothenia borchgrevinki ([Brijs et al., 2020][1]). [Hedrick et al. (2020)][2] argue that the reduction in spleen mass observed in our

Bio-logging devices can provide unique insights on the life of freely moving animals. However, implanting these devices often requires invasive surgery that causes stress and physiological side-effects. While certain medications in connection to surgeries have therapeutic capacity, others may have aversive effects. Here, we hypothesized that the commonly prescribed prophylactic treatment with enrofloxacin would increase the physiological recovery rate and reduce the presence of systemic inflammation following the intraperitoneal implantation of a heart rate bio-logger in rainbow trout (Oncorhynchus mykiss). To assess post-surgical recovery, heart rate was recorded for 21 days in trout with or without enrofloxacin treatment. Contrary to our hypothesis, treated trout exhibited a prolonged recovery time and elevated resting heart rates during the first week of post-surgical recovery compared to untreated trout. In addition, an upregulated mRNA expression of TNFα in treated trout indicate a possible inflammatory response 21 days post-surgery. Interestingly, the experience level of the surgeon was observed to have a long-lasting impact on heart rate. In conclusion, our study showed no favorable effects of enrofloxacin treatment. Our findings highlight the importance of adequate post-surgical recovery times and surgical training with regards to improving the welfare of experimental animals and reliability of research outcomes.

Welfare of farmed fish has become of increasing concern for consumers, producers, interest groups and authorities. To improve fish welfare, it is necessary to find indicators that can identify stressful situations early enough so that an intervention can take place before detrimental effects occur. By using heart rate bio-loggers in freely swimming rainbow trout (Oncorhynchus mykiss), combined with plasma cortisol levels and a range of haematological and blood chemistry parameters, we assessed the severity of stress responses induced by a range of common aquaculture practices before and during harvest. Following surgery, transportation and reintroduction with conspecifics in the sea cage, it took ~4 days for heart rate to stabilize and for a clear circadian rhythm in heart rate to emerge (i.e. average circadian fluctuation in heart rate of ~25 to 27 beats min−1). The presence or absence of this circadian rhythm in heart rate could inform researchers in the aquaculture industry whether or not specific farming routines induce unnecessary and prolonged stress. The elevations in heart rate caused by common farming practises such as crowding and transportation (e.g. heart rate increased by ~8 and 9 beats min−1 above what would normally be expected for that time of day, respectively) corresponded well with increases in plasma cortisol levels. Stressful farming practises or events (indicated by elevated plasma cortisol levels) such as air exposure during brailing and aquatic hypoxia triggered a hypoxic bradycardia until fish were released back into oxygenated water whereupon heart rate significantly increased to repay the accumulated oxygen debt. Repeated stress induced by multiple farming practises (i.e. combined stressors of crowding, brailing and transportation) clearly had a cumulative and long-lasting effect as heart rate peaked at ~25 beats min−1 above what would normally be expected for that time of day. Heart rate also remained significantly elevated by ~9 beats min−1 the following morning, suggesting that if rainbow trout need to be subjected to multiple stressors during consequtive days, it is recommended that sufficient time for recovery is provided between stressors. This study demonstrates that heart rate monitoring can be useful to assess stress levels of freely swimming fish in sea cages. Moreover, the use of implantable bio-loggers opens up a broad range of possible applications that will allow researchers to investigate the effects of environmental and/or anthropogenic stressors on the welfare of fish under conditions more realistic to the aquaculture industry.

The aim of the current work was to elucidate if there is a connection between stress-induced decrease in coeliacomesenteric artery blood flow (i.e. gastrointestinal blood flow; GBF) and disruption of the intestinal primary barrier in rainbow trout Oncorhynchus mykiss. Upon initiation of a 15 min acute chasing stress, the GBF decreased instantly by c. 92%. The GBF then slowly increased and reached c. 28% of resting values at the end of the stress protocol. After the stress was ceased, the GBF slowly increased and returned to resting values within c. 45 min. Intestinal permeability assessment in an Ussing-chambers set-up revealed impaired intestinal barrier function 24 h after stress. When the stress-induced GBF reduction was mimicked by an experimental occlusion of the coeliacomesenteric artery for 15 min followed by 24 h recovery, no effect on intestinal barrier function was observed. These results suggest that no direct causal relationship can be found between the GBF reduction and development of intestinal barrier dysfunction following periods of acute stress in this species of fish.

Gill parasites on fish are likely to negatively influence their host by inhibiting respiration, oxygen transport capacity and overall fitness. The glochidia larvae of the endangered freshwater pearl mussel (FPM, Margaritifera margaritifera (Linnaeus, 1758)) are obligate parasites on the gills of juvenile salmonid fish. We investigated the effects of FPM glochidia encystment on the metabolism and haematology of brown trout (Salmo trutta Linnaeus, 1758). Specifically, we measured whole-animal oxygen uptake rates at rest and following an exhaustive exercise protocol using intermittent flow-through respirometry, as well as haematocrit, in infested and uninfested trout. Glochidia encystment significantly affected whole-animal metabolic rate, as infested trout exhibited higher standard and maximum metabolic rates. Furthermore, glochidia-infested trout also had elevated levels of haematocrit. The combination of an increased metabolism and haematocrit in infested fish indicates that glochidia encystment has a physiological effect on the trout, perhaps as a compensatory response to the potential respiratory stress caused by the glochidia. When relating glochidia load to metabolism and haematocrit, fish with low numbers of encysted glochidia were the ones with particularly elevated metabolism and haematocrit. Standard metabolic rate decreased with substantial glochidia loads towards levels similar to those of uninfested fish. This suggests that initial effects visible at low levels of encystment may be countered by additional physiological effects at high loads, e.g. potential changes in energy utilization, and also that high numbers of glochidia may restrict oxygen uptake by the gills.

Phenotypic scoring of wild animals under standardized laboratory conditions is important as it allows field ecologists and evolutionary biologists to understand the development and maintenance of interindividual differences in plastic traits (e.g. behaviour and physiology). However, captivity is associated with a shift from a natural familiar environment to an unfamiliar and artificial environment, which may affect estimates of plastic phenotypic traits. In this study, we tested how previous experience with laboratory environments and time spent in captivity affects behavioural (i.e. activity) and metabolic (i.e. standard and maximum metabolic rates) scoring of our model species, wild brown trout Salmo trutta. We found that individuals with previous experience of laboratory captivity (10.5 months earlier) showed higher activity in an open field test than individuals with no prior experience of laboratory captivity. Previous experience with captivity had no significant effect on metabolic rates. However, metabolic rates seemed to increase with increasing time spent in captivity prior to the collection of measurements. Although there are benefits of keeping wild animals in captivity prior to scoring, our results suggest that while allowing for sufficient acclimatization researchers should aim at minimizing time in captivity of wild animals to increase accuracy and ecological relevance of the scoring of plastic phenotypic traits.

Substantial increases in cardiac output (CO), stroke volume (SV), and gastrointestinal blood flow are essential for euryhaline rainbow trout ( Oncorhyncus mykiss) osmoregulation in seawater. However, the underlying hemodynamic mechanisms responsible for these changes are unknown. By examining a range of circulatory and cardiac morphological variables of seawater- and freshwater-acclimated rainbow trout, the present study revealed a significantly higher central venous pressure (CVP) in seawater-acclimated trout (~0.09 vs. −0.02 kPa). This serves to increase cardiac end-diastolic volume in seawater and explains the elevations in SV (~0.41 vs. 0.27 ml/kg) and CO (~21.5 vs. 14.2 ml·min−1·kg−1) when compared with trout in freshwater. Furthermore, these hemodynamic modifications coincided with a significant increase in the proportion of compact myocardium, which may be necessary to compensate for the increased wall tension associated with a larger stroke volume. Following a temperature increase from 10 to 16.5°C, both acclimation groups exhibited similar increases in heart rate (Q10of ~2), but SV tended to decrease in seawater-acclimated trout despite the fact that CVP was maintained in both groups. This resulted in CO of seawater- and freshwater-acclimated trout stabilizing at a similar level after warming (~26 ml·min−1·kg−1). The consistently higher CVP of seawater-acclimated trout suggests that factors other than compromised cardiac filling constrained the SV and CO of these individuals at high temperatures. The present study highlights, for the first time, the complex interacting effects of temperature and water salinity on cardiovascular responses in a euryhaline fish species.

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 CTmaxwas 2.2°C higher in Biotest compared with reference perch, the peaks in cardiac output and heart rate prior to CTmaxoccurred at statistically similar Pvo2values (2.3–4.0 kPa), suggesting that cardiac failure occurred at a common critical Pvo2threshold. Environmental hyperoxia (200% air saturation) increased Pvo2across temperatures in reference fish, but heart rate still declined at a similar temperature. CTmaxof 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.

Common slaughter procedures for African sharptooth catfish (Clarias gariepinus) include asphyxiation, ice chilling and exsanguination, which may all cause substantial suffering over prolonged periods of time before death. Therefore, comprehensive evaluations of potentially more humane slaughter procedures for this species are urgently needed. Here, we use a non-invasive electroencephalographic (EEG) method to assess the state of sensibility in African sharptooth catfish in response to various stunning methods (e.g. ice chilling, electrical stunning, electrical stunning followed by exsanguination, percussive stunning, and immersion in isoeugenol). Based on the abolition of visually evoked responses (VERs) on the EEG, ice slurry immersion induced insensibility between 2.6 and 7.6 min, during which catfish exhibited aversive behaviours. Once VERs were lost, they remained absent so long as catfish remained immersed in the ice slurry. Electrical stunning (i.e. exposure to ~1.7 A dm−2 at a water conductivity of ~997 μS cm−1) induced insensibility immediately but not irreversibly. Depending on the duration of the stun (i.e. from 1 to 10 s), catfish either regained VERs immediately or within 4.9 min after the completion of the electrical insult. However, when a 10 s electrical stun was immediately followed by exsanguination and immersion in an ice slurry, the duration of insensibility was sufficient to humanely kill catfish. When administered correctly, manual percussive stunning with a fish priest induced insensibility immediately and irreversibly. However, 36% of catfish regained VERs, which is likely explained by the difficulty associated with administering an accurate manual percussive stun of sufficient force on a live and struggling catfish. Catfish appeared to be sedated following immersion in isoeugenol (i.e. catfish were calm and easy to handle), yet VERs remained present at doses exceeding that recommended for euthanasia in salmonids, which indicates that this substance may not be suitable for stunning catfish. However, the potential for using isoeugenol as a pre-stunning sedative for improving handleability and reducing handling stress of this species warrants further investigation. In conclusion, this study clearly demonstrates that when singularly administered, none of the abovementioned stunning methods could reliably induce insensibility immediately and/or irreversibly without welfare implications. Yet, our findings indicate that these shortcomings can be resolved by using a combination of methods. This could include an electrical or percussive stun to immediately induce insensibility that should be immediately followed by exsanguination and immersion in an ice slurry to maintain insensibility until death.

Cardiovascular disease may pose a major threat to the health and welfare of farmed fish. By investigating a range of established cardiovascular disease indicators, we aimed to determine the prevalence, severity and consequences of this affliction in farmed rainbow trout (Oncorhynchus mykiss) from an open cage farm in the Baltic Sea, an open cage farm in a freshwater lake, and a land-based recirculating aquaculture system. We also aimed to identify environmental, anthropogenic and physiological factors contributing towards the development of the disease. The majority of trout possessed enlarged hearts with rounded ventricles (mean height:width ratios of 1.0-1.1 c.f. similar to 1.3 in wild fish) and a high degree of vessel misalignment (mean angles between the longitudinal ventricular axis and the axis of the bulbus arteriosus of 28-31 degrees c.f. similar to 23 degrees in wild fish). The prevalence and severity of coronary arteriosclerosis was also high, as 92-100% of fish from the different aquaculture facilities exhibited coronary lesions. Mean lesion incidence and severity indices were 67-95% and 3.1-3.9, respectively, which resulted in mean coronary arterial blockages of 19-32%. To evaluate the functional significance of these findings, we modelled the effects of arterial blockages on coronary blood flow and experimentally tested the effects of coronary occlusion in a sub-sample of fish. The observed coronary blockages were estimated to reduce coronary blood flow by 34-54% while experimental coronary occlusion adversely affected the electrocardiogram of trout. Across a range of environmental (water current, predation), anthropogenic (boat traffic intensity, hatchery of origin, brand of feed pellets) and physiological factors (condition factor, haematological and plasma indices), the hatchery of origin was the main factor contributing towards the observed variation in the development of cardiovascular disease. Therefore, further research on the effects of selective breeding programs and rearing strategies on the development of cardiovascular disease is needed to improve the welfare and health of farmed fish.

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. Due to 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% (c.f. 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 increased 103%) and the cardiovascular trade-offs (i.e. ventral aortic blood pressure and cardiac workload increased 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.

Climate change may exacerbate salinity variations in aquatic ecosystems, potentially affecting fish performance. We show that long-term seawater exposure in rainbow trout increases cardiac output and reduces blood pressure and vascular resistance through altered α-adrenergic control. Possible impacts of salinity fluctuations on fish aerobic swimming and digestive capacity are discussed., Greater salinity variations resulting from ongoing climate change requires consideration in conservation management as this may impact on the performance of aquatic organisms. Euryhaline fish exhibit osmoregulatory flexibility and can exploit a wide range of salinities. In seawater (SW), they drink and absorb water in the intestine, which is associated with increased gastrointestinal blood flow. Yet, detailed information on other cardiovascular changes and their control across salinities is scant. Such knowledge is fundamental to understand how fish are affected during migrations between environments with different salinities, as well as by increased future salinity variability. We used rainbow trout (Oncorhynchus mykiss) as a euryhaline model species and determined dorsal aortic blood pressure, cardiac output and systemic vascular resistance in vivo after chronic freshwater—or SW-acclimation. We also assessed α-adrenergic control of blood pressure using pharmacological tools. Dorsal aortic blood pressure and systemic vascular resistance were reduced, whereas cardiac output increased in SW. α-Adrenergic stimulation with phenylephrine caused similar dose-dependent increases in resistance and pressure across salinities, indicating unaltered α-adrenoceptor sensitivity. α-Adrenergic blockade with prazosin decreased resistance and pressure across salinities, but the absolute reduction in resistance was smaller in SW. Yet, both pressure and resistance after prazosin remained consistently lower in SW. This shows that SW-acclimation lowers systemic resistance through reduced vascular α-adrenergic tone, along with other unknown vasodilating factors. The marked changes in adrenergic regulation of the vasculature across salinities discovered here may have implications for cardiovascular and aerobic performance of fishes, with possible impacts on fitness-related traits like digestion and exercise capacity. Moreover, the evolution of more complex circulatory control systems in teleost fishes compared with elasmobranchs and cyclostomes may have been an important factor in the evolution of euryhalinity, and may provide euryhaline teleosts with competitive advantages in more variable salinity environments of the future.

The fish gut is responsible for numerous potentially energetically costly processes, yet, little is known about its metabolism. Here, we provide the first in vivo measurements for aerobic metabolism of the gut in a teleost fish by measuring gut blood flow, as well as arterial and portal venous oxygen content. At 10°C, gut oxygen uptake rates were 4.3±0.5 ml O2 h−1 kg−1 (∼11% of whole animal oxygen uptake). Following acute warming to 15°C, gut blood flow increased ∼3.4-fold and gut oxygen uptake rate increased ∼3.7-fold (16.0±3.3 ml O2 h−1 kg−1), now representing ∼25% of whole animal oxygen uptake. Although gut blood flow decreased following an acute stress event at 15°C, gut oxygen uptake remained unchanged due to a ∼2-fold increase in oxygen extraction. The high metabolic thermal sensitivity of the gut discovered here could have important implications on the overall aerobic capacity and performance of fish and warrants further investigations.

The Commentary by Pörtner, Bock and Mark (Pörtner et al., 2017)elaborates on the oxygen- and capacity-limited thermal tolerance(OCLTT) hypothesis. Journal of Experimental BiologyCommentaries allow for personal and controversial views, yetthe journal also mandates that ‘opinion and fact must beclearly distinguishable’ (http://jeb.biologists.org/content/articletypes#comms). We contend that Pörtner et al. (2017) do not meetthis requirement, and that they present a biased account of theOCLTT hypothesis. We raise two main points: (1) Pörtner et al.(2017) do not do justice to the growing number of empirical studiesthat failed to support the OCLTT hypothesis when specificallytesting its predictions, and (2) in response to these studies, andwithout new empirical evidence to support OCLTT, Pörtner andcolleagues have gradually redefined the core assumptions of thehypothesis so that it is increasingly difficult to test and has lostpredictive power.

Anadromy is a distinctive life-history strategy in fishes that has evolved independently many times. In an evolutionary context, the benefits of anadromy for a species or population must outweigh the costs and risks associated with the habitat switch. The migration of fish across the freshwater-ocean boundary coincides with potentially energetically costly osmoregulatory modifications occurring at numerous levels of biological organization. By integrating whole animal and sub-cellular metabolic measurements, this study presents significant findings demonstrating how an anadromous salmonid (i.e. rainbow trout, Oncorhynchus mykiss) is able to transform from a hyper- to hypo-osmoregulatory state without incurring significant increases in whole animal oxygen consumption rate. Instead, underlying metabolic mechanisms that fuel the osmoregulatory machinery at the organ level (i.e. intestine) are modulated, as mitochondrial coupling and anaerobic metabolism are increased to satisfy the elevated energetic demands. This may have positive implications for the relative fitness of the migrating individual, as aerobic capacity may be maintained for locomotion (i.e. foraging and predator avoidance) and growth. Furthermore, the ability to modulate mitochondrial metabolism in order to maintain osmotic balance suggests that mitochondria of anadromous fish may have been a key target for natural selection, driving species adaptations to different aquatic environments.

Upon exposure to seawater, euryhaline teleosts need to imbibe and desalinate seawater to allow for intestinal ion and water absorption, as this is essential for maintaining osmotic homeostasis. Despite the potential benefits of increased mixing and transport of imbibed water for increasing the efficiency of absorptive processes, the effect of water salinity on intestinal motility in teleosts remains unexplored. By qualitatively and quantitatively describing in vivo intestinal motility of euryhaline rainbow trout (Oncorhynchus mykiss), this study demonstrates that, in freshwater, the most common motility pattern consisted of clusters of rhythmic, posteriorly propagating contractions that lasted ∼1–2 min followed by a period of quiescence lasting ∼4–5 min. This pattern closely resembles mammalian migrating motor complexes (MMCs). Following a transition to seawater, imbibed seawater resulted in a significant distension of the intestine and the frequency of MMCs increased twofold to threefold with a concomitant reduction in the periods of quiescence. The increased frequency of MMCs was also accompanied by ripple-type contractions occurring every 12–60 s. These findings demonstrate that intestinal contractile activity of euryhaline teleosts is dramatically increased upon exposure to seawater, which is likely part of the overall response for maintaining osmotic homeostasis as increased drinking and mechanical perturbation of fluids is necessary to optimise intestinal ion and water absorption. Finally, the temporal response of intestinal motility in rainbow trout transitioning from freshwater to seawater coincides with previously documented physiological modifications associated with osmoregulation and may provide further insight into the underlying reasons shaping the migration patterns of salmonids.

Thermal tolerance in fish may be related to an oxygen limitation of cardiac function. While the hearts of some fish species receive oxygenated blood via a coronary circulation, the influence of this oxygen supply on thermal tolerance and cardiac performance during warming remain unexplored. Here, we analyzed the effect in vivo of acute warming on coronary blood flow in adult sexually mature rainbow trout ( Onchorhynchus mykiss) and the consequences of chronic coronary ligation on cardiac function and thermal tolerance in juvenile trout. Coronary blood flow at 10°C was higher in females than males (0.56 ± 0.08 vs. 0.30 ± 0.08 ml·min−1·g ventricle−1), and averaged 0.47 ± 0.07 ml·min−1·g ventricle−1across sexes. Warming increased coronary flow in both sexes until 14°C, at which it peaked and plateaued at 0.78 ± 0.1 and 0.61 ± 0.1 ml·min−1·g ventricle−1in females and males, respectively. Thus, the scope for increasing coronary flow was 101% in males, but only 39% in females. Coronary-ligated juvenile trout exhibited elevated heart rate across temperatures, reduced Arrhenius breakpoint temperature for heart rate (23.0 vs. 24.6°C), and reduced upper critical thermal maximum (25.3 vs. 26.3°C). To further analyze the effects of coronary flow restriction on cardiac rhythmicity, electrocardiogram characteristics were determined before and after coronary occlusion in anesthetized trout. Occlusion resulted in reduced R-wave amplitude and an elevated S-T segment, indicating myocardial ischemia, while heart rate was unaffected. This suggests that the tachycardia in ligated trout across temperatures in vivo was mainly to compensate for reduced cardiac contractility to maintain cardiac output. Moreover, our findings show that coronary flow increases with warming in a sex-specific manner. This may improve whole animal thermal tolerance, presumably by sustaining cardiac oxygenation and contractility at high temperatures.

Cellular and mitochondrial metabolic capacity of the heart has been suggested to limit performance of fish at warm temperatures. We investigated this hypothesis by studying the effects of acute temperature increases (16, 23, 30, 32.5 and 36°C) on the thermal sensitivity of 10 key enzymes governing cardiac oxidative and glycolytic metabolism in two populations of European perch (Perca fluviatilis) field-acclimated to 15.5 and 22.5°C, as well as the effects of acclimation on cardiac lipid composition. In both populations of perch, the activity of glycolytic (pyruvate kinase and lactate dehydrogenase) and tricarboxylic acid cycle (pyruvate dehydrogenase and citrate synthase) enzymes increased with acute warming. However, at temperatures exceeding 30°C, a drastic thermally-induced decline in citrate synthase activity was observed in the cold- and warm-acclimated populations respectively, indicating a bottleneck for producing the reducing equivalents required for oxidative phosphorylation. Yet, the increase in aspartate aminotransferase and malate dehydrogenase activities occurring in both populations at temperatures exceeding 30°C, suggests that the malate-aspartate shuttle may aid to maintain cardiac oxidative capacities at high temperatures. Warm acclimation resulted in a reorganization of the lipid profile, a general depression of enzymatic activity and an increased fatty acid metabolism and oxidative capacity. While these compensatory mechanisms may contribute to maintain cardiac energy production at high temperatures, the activity of the electron transport system enzymes, such as complexes I and IV, declined at 36°C in both populations indicating a thermal limit of oxidative phosphorylation capacity in the heart of European perch.

Rhythmic contractions of the mammalian gastrointestinal tract can occur in the absence of neuronal or hormonal stimulation due to the generation of spontaneous electrical activity by interstitial cells of Cajal (ICC) that are electrically coupled to smooth muscle cells. The myogenically-driven component of gastrointestinal motility patterns in fish likely also involves ICC, however, little is known of their presence, distribution and function in any fish species. In the present study, we combined immunohistochemistry and in vivo recordings of intestinal motility to investigate the involvement of ICC in the motility of the proximal intestine in adult shorthorn sculpin (Myoxocephalus scorpius). Antibodies against anoctamin 1 (Ano1, a Ca2+-activated Cl− channel), revealed a dense network of multipolar, repeatedly branching cells in the myenteric region of the proximal intestine, similar in many regards to the mammalian ICC-MY network. The addition of benzbromarone, a potent blocker of Ano1, altered the motility patterns seen in vivo after neural blockade with TTX. The results indicate that ICC are integral for the generation and propagation of the majority of rhythmic contractile patterns in fish, although their frequency and amplitude can be modulated via neural activity.

Cardiac oxygenation is achieved via both coronary arterial and luminal venous oxygen supply routes in many fish species. However, the relative importance of these supplies for cardiac and aerobic metabolic performance is not fully understood. Here, we investigated how coronary artery ligation in rainbow trout (Oncorhynchus mykiss), implanted with heart rate loggers, affected cardiorespiratory performancein vivo. While coronary ligation significantly elevated resting heart rate, the standard metabolic rate was unchanged compared to sham-treated controls. However, coronary ligation reduced the maximum metabolic rate while heart rate remained unchanged following enforced exercise. Thus, coronary ligation reduced metabolic and heart rate scopes by 29% and 74%, respectively. Our findings highlight the importance of coronary oxygen supply for overall cardiorespiratory performance in salmonid fish, and suggest that pathological conditions that impair coronary flow (e.g. coronary arteriosclerosis) constrain the ability of fish to cope with metabolically demanding challenges such as spawning migrations and environmental warming.

Thermal plasticity of cardiorespiratory function allows ectotherms like fish to cope with seasonal temperature changes and is critical for resilience to climate change. Yet, the chronic thermal effects on cardiovascular homeostatic reflexes in fish are little understood although this may have important implications for physiological performance and overall resilience to climate warming. We compared cardiac autonomic control and baroreflex regulation of heart rate in perch (Perca fluviatilis L.) from a reference area in the Baltic Sea at 18–19°C with conspecifics from the ‘Biotest enclosure’, a chronically heated ecosystem receiving warmed effluent water (24–25°C) from a nuclear power plant. Resting heart rates of Biotest fish displayed clear thermal compensation and were 58.3±2.3 beats min−1 compared with 52.4±2.6 beats min−1 in reference fish at their respective environmental temperatures (Q10: 1.2). The thermally-compensated heart rate of Biotest fish was a combined effect of elevated inhibitory cholinergic tone (105% in Biotest fish versus 70% in reference fish) and reduced intrinsic cardiac pacemaker rate. A barostatic response was evident in both groups, as pharmacologically-induced increases and decreases in blood pressure resulted in atropine-sensitive bradycardia and tachycardia, respectively. Yet, the tachycardia in Biotest fish was significantly greater, presumably due to the larger scope for vagal release. Acclimation of Biotest fish to 18°C for 3 weeks abolished differences in intrinsic heart rate and autonomic tones, suggesting considerable short-term thermal plasticity of cardiovascular control in this species. The heightened hypotensive tachycardia in Biotest perch may represent an important mechanism of ectothermic vertebrates that safeguards tissue perfusion pressure when tissue oxygen demand is elevated by environmental warming.

The large-scale migrations of anadromous fish species from freshwater to seawater have long been considered particularly enigmatic, as this life history necessitates potentially energetically costly changes in behaviour and physiology. A significant knowledge gap concerns the integral role of cardiovascular responses, which directly link many of the well-documented adaptations (i.e. through oxygen delivery, water and ion transport) allowing fish to maintain osmotic homeostasis in the sea. Using long-term recordings of cardiorespiratory variables and a novel method for examining drinking dynamics, we show that euryhaline rainbow trout (Oncorhynchus mykiss) initiate drinking long before the surrounding environment reaches full seawater salinity (30–33 ppt), suggesting the presence of an external osmo-sensing mechanism. Onset of drinking was followed by a delayed, yet substantial increase in gastrointestinal blood flow through increased pulse volume exclusively, as heart rate remained unchanged. While seawater entry did not affect whole animal energy expenditure, enhanced gastrointestinal perfusion represents a mechanism crucial for ion and water absorption, as well as possibly increasing local gastrointestinal oxygen supply. Collectively, these modifications are essential for anadromous fish to maintain homeostasis at sea, whilst conserving cardiac and metabolic scope for activities directly contributing to fitness and reproductive success.

A progressive inability of the cardiorespiratory system to maintain systemic oxygen supply at elevated temperatures has been suggested to reduce aerobic scope and the upper thermal limit of aquatic ectotherms. However, few studies have directly investigated the dependence of thermal limits on oxygen transport capacity. By manipulating oxygen availability (via environmental hyperoxia) and blood oxygen carrying capacity (via experimentally-induced anemia) in European perch (Perca fluviatilis, Linneaus), we investigated the effects of oxygen transport capacity on aerobic scope and the critical thermal maximum (CTmax). Hyperoxia resulted in a two-fold increase in aerobic scope at the control temperature of 23°C, but this did not translate to an elevated CTmax in comparison with control fish (34.6±0.1°C vs. 34.0±0.5°C, respectively). Anemia (∼43% reduction in haemoglobin concentration) did not cause a reduction in aerobic scope nor CTmax (33.8±0.3°C) compared with control fish. Additionally, oxygen consumption rates of anemic perch during thermal ramping increased in a similar exponential manner as in control fish, highlighting that perch have an impressive capacity to compensate for a substantial reduction in blood oxygen carrying capacity. Taken together, these results indicate that oxygen limitation is not a universal mechanism determining the CTmax of fishes.

This is the first study to catalogue the diverse array of in vivo motility patterns in a teleost fish and how they are affected by feeding. Video recordings of exteriorised proximal intestine from fasted and fed shorthorn sculpin (Myoxocephalus scorpius) were used to generate spatio-temporal maps to portray and quantify motility patterns. Propagating and non-propagating contractions were observed to occur at different frequencies and durations. The most apparent difference between the feeding states was that bands of relatively high amplitude contractions propagating slowly in the anal direction were observed in all fasted fish (n=10) but only 35% of the fed fish (n=11). Additionally, fed fish displayed a reduced frequency (0.21±0.03 vs. 0.32±0.06 contractions per minute) and rhythmicity of these contractions compared to fasted fish. Although the underlying mechanisms of these slow anally-propagating contractions differ from mammalian MMCs, we believe that they may play a similar role in shorthorn sculpin during the interdigestive period, to potentially remove food remnants and prevent the establishment of pathogens. "Ripples" were the most prevalent contraction type in shorthorn sculpin and may be important during mixing and absorption. Persistence of shallow ripples and pendular movements of longitudinal muscle after TTX (1 µM) treatment suggests these contractions were myogenic in origin. The present study highlights both similarities and differences in motility patterns between shorthorn sculpin and other vertebrates, as well as providing a platform to examine other aspects of gastrointestinal functions in fish, including the impacts of environmental changes.