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To assess the relationship among various measures of thermal tolerance and performance suggested for use in fish, we determined the critical thermal maximum (CTmax), critical swimming speed (Ucrit), maximum thermal tolerance while swimming [CTSmax] and realistic aerobic scope (ASR) of juvenile schoolmaster snapper (Lutjanus apodus). Their CTSmax (37.5±0.1°C) was only slightly lower than their CTmax (38.9±0.1°C) and this is probably because their maximum metabolic rate (MMR) and ASR during the former test were ∼42 and 65% higher, respectively. Furthermore, we did not observe a transition to unsteady (i.e. anaerobically fueled) swimming in the CTSmax test as we did in the Ucrit protocol. These data strongly suggest that thermal tolerance tests on fishes whose lifestyle involves schooling or sustained activity should be performed at ecologically relevant swimming speeds. Our results do not support the hypothesis that failure during a CTSmax test is due to a fish's inability to meet its tissue oxygen demands., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Fish can experience hyperoxia in shallow environments due to photosynthetic activity and this has been suggested to provide them with a metabolic refuge during acute warming. However, this hypothesis has never been tested on a tropical marine species. Thus, we fitted 29°C-acclimated wild schoolmaster snapper (Lutjanus apodus; a species known to experience diel hyperoxia in mangrove creeks and coastal waters) with Transonic® flow probes and exposed them to an acute increase in temperature (at 1°C h-1) in respirometers under normoxia and hyperoxia (150% air saturation), until their critical thermal maximum (CTmax). The CTmax of both groups was ∼39°C, and no differences in maximum cardiac function were recorded as the fish were warmed. However, temperature-induced factorial aerobic scope was significantly greater in fish tested under hyperoxia. These data suggest that hyperoxia will not protect coastal tropical fish species during marine heat waves, despite its effects on metabolic scope/capacity., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Commercial overexploitation and climate change can alter the physiology and behavior of marine organisms, although intraspecific phenotypic responses to such changes can vary greatly depending on the environment, species, and severity of the stressor. Under the pace-of-life syndrome (POLS) hypothesis, behavior, physiology, and life-history traits are linked, and thus, affected by selection targeting any aspect of organismal biology. However, these links are understudied in tropical marine fishes, and further work is needed to better understand the impacts of fisheries and climate change on wild stocks. Moreover, tropical regions have a greater reliance on fisheries; thus investigations should focus on species with substantial socioeconomic value to ensure benefits at the local level. This study aimed to address this need by measuring the behavior (boldness and activity), metabolism, and swimming performance (using a critical swim speed [U crit ] test) of schoolmaster snapper Lutjanus apodus in Eleuthera, the Bahamas. We report a strong positive correlation between boldness and activity, high repeatability of these behavioral metrics, and two groupings that were consistent with "proactive" and "reactive" behavioral types. These behavioral types differed significantly in their swimming performance, with reactive individuals having a 13.1% higher mean U crit . In contrast, no significant differences were found in the measured metabolic parameters between behavioral types. This study is the first to investigate the intraspecific links between behavior and physiology in a snapper species, using the novel and ecologically relevant comparison of U crit with behavioral syndrome types. These data suggest that additional research is needed to better predict the success of proactive/reactive tropical fish if overexploited and as influenced by climate change., (© 2024 Fisheries Society of the British Isles.)

Large-scale mortality events have occurred during the winter in Atlantic salmon sea cages in Eastern Canada and Iceland. Thus, in salmon held at 3 °C that were apparently healthy (i.e., asymptomatic) and that had 'early' and 'advanced' symptoms of 'winter syndrome'/'winter disease' (WS/WD), we measured hepatic lipid classes and fatty acid levels, and the transcript expression of 34 molecular markers of fatty liver disease (FLD; a clinical sign of WS/WD). In addition, we correlated our results with previously reported characteristics associated with this disease's progression in these same individuals. Total lipid and triacylglycerol (TAG) levels increased by ~50%, and the expression of 32 of the 34 genes was dysregulated, in fish with symptoms of FLD. TAG was positively correlated with markers of inflammation ( 5loxa , saa5 ), hepatosomatic index (HSI), and plasma aspartate aminotransferase levels, but negatively correlated with genes related to lipid metabolism ( elovl5b , fabp3a , cd36c ), oxidative stress ( catc ), and growth ( igf1 ). Multivariate analyses clearly showed that the three groups of fish were different, and that saa5 was the largest contributor to differences. Our results provide a number of biomarkers for FLD in salmon, and very strong evidence that prolonged cold exposure can trigger FLD in this ecologically and economically important species.

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6-16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lumpfish (Cyclopterus lumpus) mortalities have been reported during the summer at some North Atlantic salmon cage-sites where they serve as "cleaner fish." To better understand this species' physiology and whether limitations in their metabolic capacity and thermal tolerance can explain this phenomenon, we compared the aerobic scope (AS) of 6°C-acclimated lumpfish (~50 g and 8.8 cm in length at the beginning of experiments) when all individuals (N = 12) were given a chase to exhaustion, a critical swim speed (U crit ) test, and a critical thermal maximum (CT Max ) test (rate of warming 2°C h -1 ). The U crit and CT Max of the lumpfish were 2.36 ± 0.08 body lengths per second and 20.6 ± 0.3°C. The AS of lumpfish was higher during the U crit test (206.4 ± 8.5 mg O 2 kg -1  h -1 ) versus that measured in either the CT Max test or after the chase to exhaustion (141.0 ± 15.0 and 124.7 ± 15.5 mg O 2 kg -1  h -1 , respectively). Maximum metabolic rate (MMR), AS, and "realistic" AS (AS R ) measured using the three different protocols were not significantly correlated, indicating that measurements of metabolic capacity using one of these methods cannot be used to estimate values that would be obtained using another method. Additional findings include that (1) the lumpfish's metabolic capacity is comparable to that of Atlantic cod, suggesting that they are not as "sluggish" as previously suggested in the literature, and (2) their CT Max (20.6°C when acclimated to 6°C), in combination with their recently determined IT Max (20.6°C when acclimated to 10°C), indicates that high sea-cage temperatures are unlikely to be the primary cause of lumpfish mortalities at salmon sea-cages during the summer., (© 2024 Fisheries Society of the British Isles.)

Surprisingly, the impacts of environmental changes on the physiology of tropical/subtropical marine fishes have received limited attention. Given that (i) temperature is considered to be a key factor controlling the biology of fishes; (ii) no published data are available on the swimming performance, metabolic capacity or cardiac function of any of the ~165 grouper species worldwide; and (iii) the Nassau grouper is an endangered species of great ecological and socioeconomic significance in The Bahamas, we investigated how current summer/early fall (30°C) and winter (22°C) temperatures in South Eleuthera affected the aerobic metabolism and heart function of wild Nassau grouper when swum to exhaustion (i.e. to their critical swimming speed, U crit ). The Nassau grouper had a very low U crit at 30°C (i.e. <1 body lengths s -1 ), and a 30% lower swimming performance during the winter (at 22°C), and this was that was indicative of a reduced absolute aerobic scope (~185 vs. 290 mg O 2 kg -1  h -1 ) and values of maximum heart rate ([Formula: see text] HMax ) and scope for [Formula: see text] H that were only one-half of that achieved at 30°C (~60 vs. 120 and 29 vs. 61 beats min -1 , respectively). Overall, these data reveal that the Nassau grouper's aerobic and swimming capacity are well below values reported for other tropical/subtropical fishes and suggest that, despite a compensatory (~30-40%) increase in stroke volume, constraints on [Formula: see text] H near this species' lower thermal limit negatively affect its cardiac output and swimming performance. These findings have considerable ecological implications as Bahamian grouper populations migrate over long distances to spawn during the winter months, and given the predicted increase in temperature variability with climate change., (© The Author(s) 2023. Published by Oxford University Press and the Society for Experimental Biology.)

In this study, postsmolt male Atlantic salmon, previously identified as low responders (LRs) or high responders (HRs) based on poststress cortisol levels, had their head kidney and liver sampled at 12°C and 20°C before injection ( time 0 ) and after injection (i.e., at 12- and 24-h postinjection, respectively) with either Forte Micro (a multivalent vaccine containing bacterin, to capture peak antibacterial responses) or an equal volume of PBS. Quantitative real-time PCR (qPCR) was then used to measure the expression of 15 biomarker genes in the head kidney and 12 genes in the liver at each temperature/sampling point. Target transcripts were chosen that were related to growth, stress, and innate antibacterial immune responses. Many temperature, phenotype, and injection effects were found for individual genes within these three broad categories, and multivariate statistical analyses (i.e., principal component analysis and permutational multivariate analysis of variance) were used to look for overall patterns in transcript expression. These analyses revealed that HR salmon at 20°C mounted a more robust response ( P < 0.05) for the 10 head kidney immune-related transcripts when injected with Forte Micro than LR salmon. In contrast, the seven liver stress-related transcripts displayed a greater response ( P = 0.057) in LR versus HR fish with Forte Micro at 12°C. Overall, although this research did find some differences between LR and HR fish, it does not provide strong (conclusive) evidence that the selection of a particular phenotype would have major implications for the health of salmon over the temperature range examined. NEW & NOTEWORTHY This is the first paper to describe the impact of both temperature and bacterial stimulation on head kidney and liver transcript expression in Atlantic salmon characterized as LRs versus HRs. Notably, we found that HR salmon at 20°C mounted a more robust innate antibacterial immune response than LR salmon. In addition, LR fish at 12°C may ( P = 0.057) exhibit higher expression of stress-related transcripts in response to vaccine injection relative to HR fish.

Interleukin-1β (IL-1β) is one of the first cytokines expressed during immune responses, and its levels are affected by many factors, including stress. To date, it has only been possible to measure IL-1β transcript (mRNA) expression quantitatively in fish using qPCR. This is because previous studies that measured IL-1β protein concentrations in these taxa used western blotting, which only provides qualitative data. To advance our knowledge of fish IL-1β biology, and because post-translational processing plays a critical role in the activation of this molecule, we developed a quantitative enzyme-linked immunosorbent assay (ELISA) to accurately measure the concentration of IL-1β protein in several cell cultures and in vivo in salmonids. We compared changes in IL-1β protein levels to the expression of its mRNA. The developed ELISA was quite sensitive and has a detection limit of 12.5 pg/mL. The tools developed, and information generated through this research, will allow for a more accurate and complete understanding of IL-1β's role in the immune response of salmonids.The assay described here has the potential to significantly advance our ability to assess fish health and immune status., Competing Interests: Declaration of competing interest Dr. Brian Dixon is Co-Editor-in-Chief of the journal Developmental and Comparative Immunology. Thus, Dr. Dixon was not involved in any decisions with regards to this manuscript. This manuscript was subject to all of the journal's usual procedures, and peer review was handled by other members of the editorial team. Jack Iwanczyk is an employee of CedarLane labs, the company that produced the antibodies used in this research. Cedarlane labs hopes to market this assay in the future, but Jack Iwanczyk will derive no financial benefit from those sales., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In this study, Atlantic salmon were: (i) implanted with heart rate (fH) data storage tags (DSTs), pharmacologically stimulated to maximum fH, and warmed at 10°C h-1 (i.e. tested using a 'rapid screening protocol'); (ii) fitted with Doppler® flow probes, recovered in respirometers and given a critical thermal maximum (CTmax) test at 2°C h-1; and (iii) implanted with fH DSTs, recovered in a tank with conspecifics for 4 weeks, and had their CTmax determined at 2°C h-1. Fish in respirometers and those free-swimming were also exposed to a stepwise decrease in water oxygen level (100% to 30% air saturation) to determine the oxygen level at which bradycardia occurred. Resting fH was much lower in free-swimming fish than in those in respirometers (∼49 versus 69 beats min-1) and this was reflected in their scope for fH (∼104 versus 71 beats min-1) and CTmax (27.7 versus 25.9°C). Further, the Arrhenius breakpoint temperature and temperature at peak fH for free-swimming fish were considerably greater than for those tested in the respirometers and given a rapid screening protocol (18.4, 18.1 and 14.6°C; and 26.5, 23.2 and 20.2°C, respectively). Finally, the oxygen level at which bradycardia occurred was significantly higher in free-swimming salmon than in those in respirometers (∼62% versus 53% air saturation). These results: highlight the limitations of some lab-based methods of determining fH parameters and thermal tolerance in fishes; and suggest that scope for fH may be a more reliable and predictive measure of a fish's upper thermal tolerance than their peak fH., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

We investigated how acclimation to 8, 4 and 1°C, and acute cooling from 8  to 1°C, affected the Atlantic salmon's aerobic and anaerobic metabolism, and cardiac function, during a critical swim speed (Ucrit) test. This study revealed several interesting temperature-dependent effects. First, while differences in resting heart rate (fH) between groups were predictable based on previous research (range ∼28-65 beats  min-1), with values for 1°C-acclimated fish slightly higher than those of acutely exposed conspecifics, the resting cardiac output () of 1°C-acclimated fish was much lower and compensated for by a higher resting blood oxygen extraction (ṀO2/). In contrast, the acutely exposed fish had a ∼2-fold greater resting stroke volume (VS) compared with that of the other groups. Second, increases in fH (1.2- to 1.4-fold) contributed little to during the Ucrit test, and the contributions of (VS) versus ṀO2/ to aerobic scope (AS) were very different in the two groups tested at 1°C (1°C-acclimated and 8-1°C fish). Finally, Ucrit was 2.08 and 1.69 body lengths (BL) s-1 in the 8 and 4°C-acclimated groups, but only 1.27 and 1.44 BL s-1 in the 1°C-acclimated and 8-1°C fish, respectively - this lower value in 1°C versus 8-1°C fish despite higher values for maximum metabolic rate and AS. These data: support recent studies which suggest that the capacity to increase fH is constrained at low temperatures; show that cardiorespiratory function at cold temperatures, and its response to increased demands, depends on exposure duration; and suggest that AS does not constrain swimming capacity in salmon when chronically exposed to temperatures approaching their lower limit., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Ulcer diseases are a recalcitrant issue at Atlantic salmon ( Salmo salar ) aquaculture cage-sites across the North Atlantic region. Classical ulcerative outbreaks (also called winter ulcer disease) refer to a skin infection caused by Moritella viscosa . However, several bacterial species are frequently isolated from ulcer disease events, and it is unclear if other undescribed pathogens are implicated in ulcer disease in Atlantic salmon. Although different polyvalent vaccines are used against M. viscosa , ulcerative outbreaks are continuously reported in Atlantic salmon in Canada. This study analyzed the phenotypical and genomic characteristics of Vibrio sp. J383 isolated from internal organs of vaccinated farmed Atlantic salmon displaying clinical signs of ulcer disease. Infection assays conducted on vaccinated farmed Atlantic salmon and revealed that Vibrio sp. J383 causes a low level of mortalities when administered intracelomic at doses ranging from 10 7 -10 8 CFU/dose. Vibrio sp. J383 persisted in the blood of infected fish for at least 8 weeks at 10 and 12 °C. Clinical signs of this disease were greatest 12 °C, but no mortality and bacteremia were observed at 16 °C. The Vibrio sp. J383 genome (5,902,734 bp) has two chromosomes of 3,633,265 bp and 2,068,312 bp, respectively, and one large plasmid of 201,166 bp. Phylogenetic and comparative analyses indicated that Vibrio sp. J383 is related to V. splendidus , with 93% identity. Furthermore, the phenotypic analysis showed that there were significant differences between Vibrio sp. J383 and other Vibrio spp, suggesting J383 is a novel Vibrio species adapted to cold temperatures.

Sablefish (Anoplopoma fimbria) are an emerging aquaculture species native to the continental shelf of the northern Pacific Ocean. There is limited information on both innate and adaptive immunity for this species and new tools are needed to determine antibody response following vaccination or disease outbreaks. In this paper, a monoclonal antibody, UI-25A, specific to sablefish IgM was produced in mice. Western blotting confirmed UI-25A recognizes the heavy chain of IgM and does not cross react to proteins or carbohydrates in serum of four other teleost species. An ELISA was developed to measure Aeromonas salmonicida specific IgM in the plasma of sablefish from a previous experiment where fish were immunized with a proprietary A. salmonicida vaccine. UI-25A was used in Western blot analyses to identify immunogenic regions of A. salmonicida recognized by this specific IgM from vaccinated sablefish. Immunofluorescent staining also demonstrated the ability of UI-25A to recognize membrane-bound IgM and identify IgM + cells in the head kidney. These results demonstrate the usefulness of UI-25A as a tool to improve the understanding of antibody-mediated immunity in sablefish as well as to provide valuable information for vaccine development and expansion of aquaculture efforts for this fish species., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The salmon aquaculture industry must be proactive at developing mitigation tools/strategies to offset the potential negative impacts of climate change. Therefore, this study examined if additional dietary cholesterol could enhance salmon production at elevated temperatures. We hypothesized that supplemental cholesterol could aid in maintaining cell rigidity, reducing stress and the need to mobilize astaxanthin muscle stores, and improving salmon growth and survival at high rearing temperatures. Accordingly, postsmolt female triploid salmon were exposed to an incremental temperature challenge (+0.2°C day -1 ) to mimic conditions that they experience in sea cages in the summer, with temperature held at both 16 and 18°C for several weeks [i.e., 3 weeks at 16°C, followed by an increase at 0.2°C day -1 to 18°C (10 days), then 5 weeks at 18°C] to prolong their exposure to elevated temperatures. From 16°C onwards, the fish were fed either a control diet, or one of two nutritionally equivalent experimental diets containing supplemental cholesterol [+1.30%, experimental diet # 1 (ED1); or +1.76%, experimental diet # 2 (ED2)]. Adding cholesterol to the diet did not affect the salmon's incremental thermal maximum (IT Max ), growth, plasma cortisol, or liver stress-related transcript expression. However, ED2 appeared to have a small negative impact on survival, and both ED1 and ED2 reduced fillet "bleaching" above 18°C as measured using SalmoFan™ scores. Although the current results suggest that supplementing salmon diets with cholesterol would have few/minimal benefits for the industry, ≤ 5% of the female triploid Atlantic salmon used in this study irrespective of diet died before temperature reached 22°C. These latter data suggest that it is possible to produce all female populations of reproductively sterile salmon that can withstand summer temperatures in Atlantic Canada., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Eric H. Ignatz et al.)

Metabolic depression and dormancy (i.e., stopping/greatly reducing activity and feeding) are strategies used by many animals to survive winter conditions characterized by food shortages and cold temperatures. However, controversy exists on whether the reduced metabolism of some fishes at cold temperatures is due to dormancy alone, or also involves active metabolic depression. Thus, we acclimated winter-dormant cunner [Tautogolabrus adspersus, a north temperate wrasse which in Newfoundland is at the northern limit of its distribution] and winter-active Atlantic salmon (Salmo salar) to winter (0°C; 8h light: 16h dark) and summer (10°C; 16h light: 8 h dark) conditions, and measured the thermal sensitivity of ATP-producing and O2-consuming processes in isolated liver mitochondria and hepatocytes when exposed in vitro to temperatures from 20 to 0°C and 10 to 0°C, respectively. We found that: 1) liver mitochondrial State 3 respiration and hepatocyte O2 consumption in cunner were only ~ one-third and two-thirds of that measured in salmon, respectively, at all measurement temperatures; 2) cunner mitochondria also have proton conductance and leak respiration (State 4) values that are only approximately one-third of those in salmon; 3) the mitochondria of cunner show a dramatic reduction in respiratory control ratio (from ~ 8 to 3), and a much greater drop in State 3 respiration, between 10 and 5°C (Q10 values in 10- and 0°C-acclimated fish of 14.5 and 141.2, respectively), as compared with salmon (3.9 and 9.6, respectively); and 4) lowering temperature from 5 to 0°C resulted in ~ 40 and 30% reductions in hepatocyte O2 consumption due to non-mitochondrial respiration and Na+-K+-ATPase activity, respectively, in cunner, but not in salmon. Collectively, these results highlight the intrinsic capacity for metabolic depression in hepatocytes and mitochondria of cunner, and clearly suggest that several cellular processes play a role in the reduced metabolic rates exhibited by some fishes at cold temperatures., Competing Interests: The authors have declared that no competing interests exist.

Antibiotics are used to treat bacterial infections in fish aquaculture, and these drugs can interact with immune cells/the immune system and potentially leave fish vulnerable to viral, fungal, parasitic, or other bacterial infections. However, the effects of antibiotics on fish immunity have largely been overlooked by the aquaculture industry. We tested, at 12 and 20 °C, whether tetracycline and florfenicol (the most commonly used antibiotics in commercial aquaculture), affected the Atlantic salmon's capacity to respond to bacterial or viral stimulation. Atlantic salmon were acclimated to 12 or 20 °C and fed with tetracycline or florfenicol (100 and 10 mg kg of body weight -1 day -1 , respectively) medicated feed for 15 or 10 days, respectively. Thereafter, we evaluated their immune function prior to, and after, an intraperitoneal injection of Forte Micro (containing inactivated cultures of Aeromonas salmonicida, Vibrio anguillarum, Vibrio ordalii and Vibrio salmonicida) or the viral mimic polyriboinosinic polyribocytidylic acid (pIC). We measured the transcript expression levels of 8 anti-bacterial and 8 anti-viral putative biomarker genes, and the innate (leukocyte respiratory burst, plasma lysozyme activity and hemolytic activity of the alternative complement pathway) and cellular (relative number of erythrocytes, lymphocytes and thrombocytes, and granulocytes such as monocytes and neutrophils) responses to these challenges. Overall, we only found a few minor effects of either tetracycline or florfenicol on immune gene expression or function at either temperature. Although several studies have reported that antibiotics may negatively affect fish immune responses, our results show that industry-relevant dietary tetracycline and florfenicol treatments do not substantially impact the salmon's innate immune responses. Currently, this is the most comprehensive study on the effects of antibiotics administrated according to industry protocols on immune function in Atlantic salmon., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

No studies have examined the effects of cold temperatures (∼0-1 °C) on in vivo cardiac function and control, and metabolism, in salmonids. Thus, we examined: 1) how acclimation to 8 °C vs. acclimation (>3 weeks) or acute exposure (8-1 °C at 1 °C h -1 ) to 1 °C influenced cardiorespiratory parameters in resting Atlantic salmon; and 2) if/how the control of cardiac function was affected. Oxygen consumption ( M ˙ O 2 ) and cardiac function [i.e., heart rate ( f H ) and cardiac output ( Q ˙ ) ] were 50% lower in the acutely cooled and 1 o C-acclimated salmon as compared to 8 °C fish, whereas stroke volume (V S ) was unchanged. Intrinsic f H was not affected by whether the fish were acutely exposed or acclimated to 1 °C (values ∼51, 24 and 21 beats min -1 in 8 and 1 °C-acclimated fish, and 8-1 °C fish, respectively), and in all groups f H was primarily under adrenergic control/tone (cholinergic tone 13-18%; adrenergic tone 37-70%). However, β-adrenergic blockade resulted in a 50% increase in V S in the 1 o C-acclimated group, and this was surprising as circulating catecholamine levels were ∼1-3 nM in all groups. Overall, the data suggest that this species has a limited capacity to acclimate to temperatures approaching 0 °C. However, we cannot exclude the possibility that cardiac and metabolic responses are evoked when salmon are cooled to ∼ 0-1 °C, and that this prevented further declines in these parameters (i.e., they 'reset' quickly). Our data also provide further evidence that V S is temperature insensitive, and strongly suggest that changes in adrenoreceptor mediated control of venous pressure/capacitance occur when salmon are acclimated to 1 °C., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Gamperl reports financial support was provided by the 10.13039/501100000038Natural Sciences and Engineering Research Council of Canada., (© 2022 The Authors.)

Fish increase heart rate (fH), not stroke volume (VS), when acutely warmed as a way to increase cardiac output (Q). To assess whether aspects of myocardial function may have some basis in determining temperature-dependent cardiac performance, we measured work and power (shortening, lengthening and net) in isolated segments of steelhead trout (Oncorhynchus mykiss) ventricular muscle at the fish's acclimation temperature (14°C), and at 22°C, when subjected to increased rates of contraction (30-105 min-1, emulating increased fH) and strain amplitude (8-14%, mimicking increased VS). At 22°C, shortening power (indicative of Q) increased in proportion to fH, and the work required to re-lengthen (stretch) the myocardium (fill the heart) was largely independent of fH. In contrast, the increase in shortening power was less than proportional when strain was augmented, and lengthening work approximately doubled when strain was increased. Thus, the derived relationships between fH, strain and myocardial shortening power and lengthening work, suggest that increasing fH would be preferable as a mechanism to increase Q at high temperatures, or in fact may be an unavoidable response given constraints on muscle mechanics as temperatures rise. Interestingly, at 14°C, lengthening work increased substantially at higher fH, and the duration of lengthening (i.e. diastole) became severely constrained when fH was increased. These data suggest that myocardial contraction/twitch kinetics greatly constrain maximal fH at cool temperatures, and may underlie observations that fish elevate VS to an equal or greater extent than fH to meet demands for increased Q at lower temperatures., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. &nbsp; salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes , masoucida , smithia , and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55% ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. &nbsp; salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies' taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.

Fish can be identified as either low responders (LR) or high responders (HR) based on post-stress cortisol levels and whether they exhibit a proactive or reactive stress coping style, respectively. In this study, male Atlantic salmon (Salmo salar) from 17 families reared at 9 °C were repeatedly exposed to an acute handling stress over a period of four months, with plasma cortisol levels measured at 1 h post-stress. Fish were identified as either LR or HR if the total Z-score calculated from their cortisol responses fell into the lower or upper quartile ranges, respectively; with intermediate responders (IR) classified as the remainder. Salmon characterized as LR, IR or HR were then subjected to an incremental thermal challenge, where temperature was raised at 0.2 °C day -1 from their acclimation temperature (12 °C) to mimic natural sea-cage farming conditions during the summer in Newfoundland. Interestingly, feed intake remained high up to 22 °C, while previous studies have shown a decrease in salmon appetite after ∼16-18 °C. After the first three mortalities were recorded at elevated temperature, a subset of LR and HR salmon were exposed to another acute handling stress event at 23.6 °C. Basal and post-stress measurements of plasma cortisol, glucose and lactate did not differ between stress response phenotypes at this temperature. In the end, the average incremental thermal maximum (IT Max ) of LR and HR fish was not different (25.1 °C). In comparison, the critical thermal maximum (CT Max ; temperature increased at 2 °C h -1 ) of the remaining IR fish that had been held at 12 °C was 28.5 °C. Collectively, these results: 1) show that this population of Atlantic salmon is very thermally tolerant, and further question the relevance of CT Max in assessing responses to real-world temperature changes; and 2) indicate that characterization of stress phenotype at 9 °C is not predictive of their stress response or survival at high temperatures. Therefore, selection of fish based on phenotypic stress response at low temperatures may not be beneficial to incorporate into Atlantic salmon breeding programs, especially if the goal is to improve growth performance and survival at high temperatures in sea-cages., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Climate change is leading to increased water temperatures and reduced oxygen levels at sea-cage sites, and this is a challenge that the Atlantic salmon aquaculture industry must adapt to it if it needs to grow sustainably. However, to do this, the industry must better understand how sea-cage conditions influence the physiology and behavior of the fish. Method: We fitted ~2.5 kg Atlantic salmon on the south coast of Newfoundland with Star-Oddi milli-HRT ACT and Milli-TD data loggers (data storage tags, DSTs) in the summer of 2019 that allowed us to simultaneously record the fish's 3D acceleration (i.e., activity/behavior), electrocardiograms (and thus, heart rate and heart rate variability), depth, and temperature from early July to mid-October. Results: Over the course of the summer/fall, surface water temperatures went from ~10-12 to 18-19.5°C, and then fell to 8°C. The data provide valuable information on how cage-site conditions affected the salmon and their determining factors. For example, although the fish typically selected a temperature of 14-18°C when available (i.e., this is their preferred temperature in culture), and thus were found deeper in the cage as surface water temperatures peaked, they continued to use the full range of depths available during the warmest part of the summer. The depth occupied by the fish and heart rate were greater during the day, but the latter effect was not temperature-related. Finally, while the fish generally swam at 0.4-1.0 body lengths per second (25-60 cm s -1 ), their activity and the proportion of time spent using non-steady swimming (i.e., burst-and-coast swimming) increased when feeding was stopped at high temperatures. Conclusion: Data storage tags that record multiple parameters are an effective tool to understand how cage-site conditions and management influence salmon (fish) behavior, physiology, and welfare in culture, and can even be used to provide fine-scale mapping of environmental conditions. The data collected here, and that in recent publications, strongly suggest that pathogen (biotic) challenges in combination with high temperatures, not high temperatures + moderate hypoxia (~70% air saturation) by themselves, are the biggest climate-related challenge facing the salmon aquaculture industry outside of Tasmania., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gamperl, Zrini and Sandrelli.)

We compared the thermal sensitivity of oxidative muscle function between the eurythermal Atlantic salmon (Salmo salar) and the more stenothermal Arctic char (Salvelinus alpinus; which prefers cooler waters). Power output was measured in red skeletal muscle strips and myocardial trabeculae, and efficiency (net work/energy consumed) was measured for trabeculae, from cold (6°C) and warm (15°C) acclimated fish at temperatures from 2 to 26°C. The mass-specific net power produced by char red muscle was greater than in salmon, by 2-to 5-fold depending on test temperature. Net power first increased, then decreased, when the red muscle of 6°C-acclimated char was exposed to increasing temperature. Acclimation to 15°C significantly impaired mass-specific power in char (by ∼40-50%) from 2 to 15°C, but lessened its relative decrease between 15 and 26°C. In contrast, maximal net power increased, and then plateaued, with increasing temperature in salmon from both acclimation groups. Increasing test temperature resulted in a ∼3- to 5-fold increase in maximal net power produced by ventricular trabeculae in all groups, and this effect was not influenced by acclimation temperature. Nonetheless, lengthening power was higher in trabeculae from warm-acclimated char, and char trabeculae could not contract as fast as those from salmon. Finally, the efficiency of myocardial net work was approximately 2-fold greater in 15°C-acclimated salmon than char (∼15 versus 7%), and highest at 20°C in salmon. This study provides several mechanistic explanations as to their inter-specific difference in upper thermal tolerance, and potentially why southern char populations are being negatively impacted by climate change., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Studies on the effects of environmental changes with increasing depth (e.g. temperature and oxygen level) on fish physiology rarely consider how hydrostatic pressure might influence the observed responses. In this study, lumpfish ( Cyclopterus lumpus , 200-400 g), which can exhibit vertical migrations of over 100 m daily and can be found at depths of 500 m or more, were implanted with Star-Oddi micro-HRT loggers. Then, their heart rate ( f H ) was measured in a pressure chamber when exposed to the following: (i) increasing pressure (up to 80 bar; 800 m in depth) at 10°C or (ii) increasing temperature (12-20°C), decreasing temperature (12 to 4°C) or decreasing oxygen levels (101-55% air saturation at 12°C) in the absence or presence of 80 bar of pressure. Additionally, we determined their f H response to chasing and to increasing temperature (to 22°C) at atmospheric pressure. Pressure-induced increases in f H (e.g. from 48 to 61 bpm at 12°C) were associated with hyperactivity. The magnitude of the rise in f H with temperature was greater in pressure-exposed vs. control fish (i.e. by ~30 bpm vs. 45 bpm between 5°C and 20°C). However, the relative increase (i.e. slope of the relationship) was not different between groups. In contrast, 80 bar of pressure eliminated the small (5 bpm) increase in f H when control fish were exposed to hypoxia. Exhaustive exercise and increasing temperature to 22°C resulted in a maximum f H of 77 and 81 bpm, respectively. Our research shows that pressure influences the f H response to environmental challenges and provides the first evidence that lumpfish have a limited capacity to increase f H ., (© The Author(s) 2021. Published by Oxford University Press and the Society for Experimental Biology.)

The marine environment is predicted to become warmer, and more hypoxic, and these conditions may negatively impact the health and survival of coastal fish species, including wild and farmed Atlantic salmon (Salmo salar). Thus, we examined how: (1) moderate hypoxia (∼70% air saturation) at 12°C for 3 weeks; (2) an incremental temperature increase from 12°C to 20°C (at 1°C week-1) followed by 4 weeks at 20°C; and (3) treatment "2" combined with moderate hypoxia affected transcript expression in the liver of post-smolts as compared to control conditions (normoxia, 12°C). Specifically, we assessed the expression of 45 genes related to the heat shock response, oxidative stress, apoptosis, metabolism and immunity using a high-throughput qPCR approach (Fluidigm Biomark™ HD). The expression profiles of 27 "stress"-related genes indicated that: (i) moderate hypoxia affected the expression of several stress genes at 12°C; (ii) their expression was impacted by 16°C under normoxic conditions, and this effect increased until 20°C; (iii) the effects of moderate hypoxia were not additive to those at temperatures above 16°C; and (iv) long-term (4 weeks) exposure to 20°C, with or without hypoxia, resulted in a limited acclimatory response. In contrast, the expression of 15 immune-related genes was not greatly affected until temperatures reached 20°C, and this effect was particularly evident in fish exposed to the added challenge of hypoxia. These results provide valuable information on how these two important environmental factors affect the "stress" physiology and immunology of Atlantic salmon, and we identify genes that may be useful as hypoxia and/or temperature biomarkers in salmonids and other fishes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

The dynamic interactions between nitric oxide (NO) and myoglobin (Mb) in the cardiovascular system have received considerable attention. The loss of Mb, the principal O 2 carrier and a NO scavenger/producer, in the heart of some red-blooded fishes provides a unique opportunity for assessing this globin's role in NO homeostasis and mitochondrial function. We measured Mb content, activities of enzymes of NO and aerobic metabolism [NO Synthase (NOS) and citrate synthase, respectively] and mitochondrial parameters [Complex-I and -I+II respiration, coupling efficiency, reactive oxygen species production/release rates and mitochondrial sensitivity to inhibition by NO (i.e., NO IC 50 )] in the heart of three species of red-blooded fish. The expression of Mb correlated positively with NOS activity and NO IC 50 , with low NOS activity and a reduced NO IC 50 in the Mb-lacking lumpfish ( Cyclopterus lumpus ) as compared to the Mb-expressing Atlantic salmon (Salmo salar) and short-horned sculpin (Myoxocephalus scorpius ). Collectively, our data show that NO levels are fine-tuned so that NO homeostasis and mitochondrial function are preserved; indicate that compensatory mechanisms are in place to tightly regulate [NO] and mitochondrial function in a species without Mb; and strongly suggest that the NO IC 50 for oxidative phosphorylation is closely related to a fish's hypoxia tolerance.

Whether hypoxic acclimation influences nitric oxide (NO)-mediated control of fish cardiac function is not known. Thus, we measured the function/performance of myocardial strips from normoxic- and hypoxic-acclimated (40% air saturation; ∼8 kPa O 2 ) trout at several frequencies (20-80 contractions·min -1 ) and two muscle strain amplitudes (8% and 14%) when exposed to increasing concentrations of the NO donor sodium nitroprusside (SNP) (10 -9 to 10 -4 M). Further, we examined the influence of 1 ) nitric oxide synthase (NOS) produced NO [by blocking NOS with 10 -4 M N G -monomethyl-l-arginine (l-NMMA)] and 2 ) soluble guanylyl cyclase mediated, NOS-independent, NO effects (i.e., after blockade with 10 -4 M ODQ), on myocardial contractility. Hypoxic acclimation increased twitch duration by 8%-10% and decreased mass-specific net power by ∼35%. However, hypoxic acclimation only had minor impacts on the effects of SNP and the two blockers on myocardial function. The most surprising finding of the current study was the degree to which contraction frequency and strain amplitude influenced NO-mediated effects on myocardial power. For example, at 8% strain, 10 -4 SNP resulted in a decrease in net power of ∼30% at 20 min -1 but an increase of ∼20% at 80 min -1 , and this effect was magnified at 14% strain. This research suggests that hypoxic acclimation has only minor effects on NO-mediated myocardial contractility in salmonids, is the first to report the high frequency- and strain-dependent nature of NO effects on myocardial contractility in fishes, and supports previous work showing that NO effects on the heart (myocardium) are finely tuned spatiotemporally.

Background: Increases in ocean temperatures and in the frequency and severity of hypoxic events are expected with climate change, and may become a challenge for cultured Atlantic salmon and negatively affect their growth, immunology and welfare. Thus, we examined how an incremental temperature increase alone (Warm & Normoxic-WN: 12 → 20 °C; 1 °C week - 1 ), and in combination with moderate hypoxia (Warm & Hypoxic-WH: ~ 70% air saturation), impacted the salmon's hepatic transcriptome expr\ession compared to control fish (CT: 12 °C, normoxic) using 44 K microarrays and qPCR., Results: Overall, we identified 2894 differentially expressed probes (DEPs, FDR < 5%), that included 1111 shared DEPs, while 789 and 994 DEPs were specific to WN and WH fish, respectively. Pathway analysis indicated that the cellular mechanisms affected by the two experimental conditions were quite similar, with up-regulated genes functionally associated with the heat shock response, ER-stress, apoptosis and immune defence, while genes connected with general metabolic processes, proteolysis and oxidation-reduction were largely suppressed. The qPCR assessment of 41 microarray-identified genes validated that the heat shock response (hsp90aa1, serpinh1), apoptosis (casp8, jund, jak2) and immune responses (apod, c1ql2, epx) were up-regulated in WN and WH fish, while oxidative stress and hypoxia sensitive genes were down-regulated (cirbp, cyp1a1, egln2, gstt1, hif1α, prdx6, rraga, ucp2). However, the additional challenge of hypoxia resulted in more pronounced effects on heat shock and immune-related processes, including a stronger influence on the expression of 14 immune-related genes. Finally, robust correlations between the transcription of 19 genes and several phenotypic traits in WH fish suggest that changes in gene expression were related to impaired physiological and growth performance., Conclusion: Increasing temperature to 20 °C alone, and in combination with hypoxia, resulted in the differential expression of genes involved in similar pathways in Atlantic salmon. However, the expression responses of heat shock and immune-relevant genes in fish exposed to 20 °C and hypoxia were more affected, and strongly related to phenotypic characteristics (e.g., growth). This study provides valuable information on how these two environmental challenges affect the expression of stress-, metabolic- and immune-related genes and pathways, and identifies potential biomarker genes for improving our understanding of fish health and welfare.

Studies of heart function and metabolism have been used to predict the impact of global warming on fish survival and distribution, and their susceptibility to acute and chronic temperature increases. Yet, despite the fact that hypoxia and high temperatures often co-occur, only one study has examined the effects of hypoxia on fish thermal tolerance, and the consequences of hypoxia for fish cardiac responses to acute warming have not been investigated. We report that sablefish ( Anoplopoma fimbria ) did not increase heart rate or cardiac output when warmed while hypoxic, and that this response was associated with reductions in maximum O 2 consumption and thermal tolerance (CT max ) of 66% and approximately 3°C, respectively. Further, acclimation to hypoxia for four to six months did not substantially alter the sablefish's temperature-dependent physiological responses or improve its CT max . These results provide novel, and compelling, evidence that hypoxia can impair the cardiac and metabolic response to increased temperatures in fish, and suggest that some coastal species may be more vulnerable to climate change-related heat waves than previously thought. Further, they support research showing that cross-tolerance and physiological plasticity in fish following hypoxia acclimation are limited.

In fish, the capacity of thermal acclimation to preserve cardiac mitochondrial function under future warming scenarios is important to understand given the central roles that cardiac energy metabolism and performance play in this taxa's thermal tolerance. We acclimated Atlantic salmon to 12 and 20°C (for >2 months), and investigated the effects of acute and chronic warming on cardiac mitochondrial respiration and reactive oxygen species (ROS) production (release rate) using high-resolution fluorespirometry. Further, we compared the sensitivity of mitochondrial respiration to nitric oxide (i.e. the NO IC 50 ), and assessed the mitochondrial response to anoxia-reoxygenation (AR). Acute exposure to 20°C increased maximal mitochondrial respiration by ∼55%; however, the mitochondria's complex I respiratory control ratio was 17% lower and ROS production was increased by ≥60%. Acclimation to 20°C: (1) preserved mitochondrial coupling and aerobic capacity; (2) decreased the mitochondria's ROS production by ∼30%; (3) increased the mitochondria's NO IC 50 by ∼23%; and (4) improved mitochondrial membrane integrity at 20°C. AR did not affect mitochondrial function at 12°C, but acute exposure to 20°C and AR depressed maximal mitochondrial respiration (by ∼9%) and coupling (by ∼16%) without impacting ROS production. Finally, warm acclimation did not improve the capacity of mitochondria to recover from AR, indicating that there was no 'cross-tolerance' between these challenges. Our findings provide compelling evidence that thermal plasticity of cardiac mitochondrial function contributes to the Atlantic salmon's capability to survive at ≥20°C for prolonged periods, but call into question whether this plasticity may allow them to withstand high temperatures when combined with other stressors., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The trout ventricle has an outer compact layer supplied with well-oxygenated arterial blood from the coronary circulation, and an inner spongy myocardium supplied with oxygen poor venous blood. It was hypothesized that: (1) the spongy myocardium of steelhead trout (Oncorhynchus mykiss), given its routine exposure to low partial pressures of oxygen (PO 2 ), would be better able to maintain contractile performance (work) when exposed to acute hypoxia (100 to 10% air saturation) relative to the compact myocardium, and would show little benefit from hypoxic acclimation; and (2) the compact myocardium from hypoxia-acclimated (40% air saturation) fish would be better able to maintain work during acute exposure to hypoxia relative to normoxia-acclimated individuals. Consistent with our expectations, when PO 2 was acutely lowered, net work from the compact myocardium of normoxia-acclimated fish declined more (by ~ 73%) than the spongy myocardium (~ 50%), and more than the compact myocardium of hypoxia-acclimated fish (~ 55%), and hypoxic acclimation did not benefit the spongy myocardium in the face of reduced PO 2 . Further, while hypoxic acclimation resulted in a 25% (but not significant) decrease in net work of the spongy myocardium, the performance of the compact myocardium almost doubled. This research suggests that, in contrast to the spongy myocardium, performance of the compact myocardium is improved by hypoxic acclimation; and supports previous research suggesting that the decreased contractile performance of the myocardium upon exposure to lowered PO 2 may be adaptive and mediated by mechanisms within the muscle itself.

The Atlantic cod immune system deviates from antigen presentation processes seen in other vertebrates in that it lacks the necessary genes for exogenous antigen presentation (i.e., MHC-II and li) and a key MHC-II interacting molecule necessary for T-helper cell function (i.e., CD4), while possessing an expanded repertoire of MHC-I genes that facilitate endogenous antigen presentation. These observations, combined with the identification of putative endosomal sorting signals in MHC-I cytoplasmic tails, have led to speculation that cod rely on cross-presentation of exogenous antigens to elicit cytotoxic T-lymphocyte responses against extracellular threats. In light of this suggestion, we investigated MHC-I transcriptional profiles and endosomal sorting signals in a closely related gadoid species, the haddock. Analysis of transcripts from one individual identified 13 unique MHC-I molecules, including two non-classical molecules as determined by the level of conservation at their peptide anchoring sites. This suggests that like the cod, the haddock has an expanded MHC-I repertoire. Analysis of haddock MHC-I cytoplasmic tail sequences revealed that the dileucine- and tyrosine-based endosomal signaling motifs, that are suggested to facilitate cross-presentation in cod, were absent. Closer examination of the cod signaling motifs, including their relative position in the cytoplasmic tail region, indicates that these motifs might be non-functional, further supporting the need for functional studies to assess cross-presentation. Finally, in silico analysis and in vitro N-type de-glycosylation experiments demonstrate that haddock and cod beta-2-microglobulin (β2M) are glycosylated at the same NQT sequon. Interestingly, whole genome tBLASTn searches also revealed that putative β2 M glycosylation sites appear frequently within the Gadiformes lineage, as the predictive NQT and other N-X-S/T sequons were located in β2M orthologues from 19 of the 25 additional gadoid genomes analyzed. Though the exact significance of β2M glycosylation has yet to be elucidated, phylogenetic comparisons predict that the same NQT glycosylation sequence occurs in 13 additional species comprising four different orders of Actinopterygii (Gymnotiformes, Esociformes, Beryciformes and Perciformes). This suggests either that this feature has arisen independently in multiple lineages or that it comes from a common ancestor and has been lost or modified in many species. Together, the analysis of gadoid MHC-I genes and β2M molecules highlights the challenges in generalizing immune system paradigms across the most diverse vertebrate lineage (i.e., fish) and between fish and more well-studied mammals., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mitochondrial function can provide key insights into how fish will respond to climate change, due to its important role in heart performance, energy metabolism and oxidative stress. However, whether warm acclimation can maintain or improve the energetic status of the fish heart when exposed to short-term heat stress is not well understood. We acclimated Atlantic salmon, a highly aerobic eurythermal species, to 12 and 20 °C, then measured cardiac mitochondrial functionality and integrity at 20 °C and at 24, 26 and 28 °C (this species' critical thermal maximum ± 2 °C). Acclimation to 20 °C vs. 12 °C enhanced many aspects of mitochondrial respiratory capacity and efficiency up to 24 °C, and preserved outer mitochondrial membrane integrity up to 26 °C. Further, reactive oxygen species (ROS) production was dramatically decreased at all temperatures. These data suggest that salmon acclimated to 'normal' maximum summer temperatures are capable of surviving all but the most extreme ocean heat waves, and that there is no 'tradeoff' in heart mitochondrial function when Atlantic salmon are acclimated to high temperatures (i.e., increased oxidative phosphorylation does not result in heightened ROS production). This study suggests that fish species may show quite different acclimatory responses when exposed to prolonged high temperatures, and thus, susceptibility to climate warming.

Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD 50 ). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD 50 was estimated as ~3 × 10 5 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD 50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6-8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Climate change is predicted to increase water temperatures and decrease oxygen levels in freshwater and marine environments, however, there is conflicting information regarding the extent to which these conditions may impact the immune defenses of fish. In this study, Atlantic salmon were exposed to: (1) normoxia (100-110% air saturation) at 12°C; (2) an incremental temperature increase (1°C per week from 12 to 20°C), and then held at 20°C for an additional 4 weeks; and (3) "2" with the addition of moderate hypoxia (~65-75% air saturation). These conditions realistically reflect what farmed salmon in some locations are currently facing, and future conditions in Atlantic Canada and Europe, during the summer months. The salmon were sampled for the measurement of head kidney constitutive anti-bacterial and anti-viral transcript expression levels, and blood parameters of humoral immune function. Thereafter, they were injected with either the multi-valent vaccine Forte V II (contains both bacterial and viral antigens) or PBS (phosphate-buffer-saline), and the head kidney and blood of these fish were sampled at 6, 12, 24, and 48 h post-injection (HPI). Our results showed that: (1) neither high temperature, nor high temperature + moderate hypoxia, adversely affected respiratory burst, complement activity or lysozyme concentration; (2) the constitutive transcript expression levels of the anti-bacterial genes il1 β, il8-a, cox2, hamp-a, stlr5-a , and irf7-b were up-regulated by high temperature; (3) while high temperature hastened the peak in transcript expression levels of most anti-bacterial genes by 6-12 h following V II injection, it did not affect the magnitude of changes in transcript expression; (4) anti-viral ( viperin-b, mx-b , and isg15-a ) transcript expression levels were either unaffected, or downregulated, by acclimation temperature or V II injection over the 48 HPI; and (5) hypoxia, in addition to high temperature, did not impact immune transcript expression. In conclusion, temperatures up to 20°C, and moderate hypoxia, do not impair the capacity of the Atlantic salmon's innate immune system to respond to bacterial antigens. These findings are surprising, and highlight the salmon's capacity to mount robust innate immune responses (i.e., similar to control fish under optimal conditions) under conditions approaching their upper thermal limit., (Copyright © 2020 Zanuzzo, Beemelmanns, Hall, Rise and Gamperl.)

Cardiac stroke volume (S V ) is compromised in Atlantic cod and rainbow trout following acclimation to hypoxia (i.e., 40% air saturation; ~8 kPa O 2 ) at 10-12°C, and this is not due to changes in heart morphometrics or maximum achievable in vitro end-diastolic volume. To examine if this diminished S V may be related to compromised myocardial contractility, we used the work-loop method to measure work and power in spongy myocardial strips from normoxic- and hypoxic-acclimated steelhead trout when exposed to decreasing Po 2 levels (21 to 1.5 kPa) at several frequencies (30-90 contractions/min) at 14°C (their acclimation temperature). Work required to lengthen the muscle, as during filling of the heart, was strongly frequency dependent (i.e., increased with contraction rate) but was not affected by hypoxic acclimation or test Po 2 . In contrast, although shortening work was less frequency dependent, this parameter and network (and power) 1 ) were consistently lower (by ~30-50 and ~15%, respectively) in strips from hypoxic-acclimated fish and 2 ) fell by ~40-50% in both groups from 20 to 1.5 kPa Po 2 , despite the already-reduced myocardial performance in the hypoxic-acclimated group. In addition, strips from hypoxic-acclimated trout showed a poorer recovery of net power (by ~15%) when returned to normoxia. These results strongly suggest that hypoxic acclimation reduces myocardial contractility, and in turn, may limit S V (possibly by increasing end-systolic volume), but that this diminished performance does not improve the capacity to maintain myocardial performance under oxygen limiting conditions.

In fishes, the effect of O 2 limitation on cardiac mitochondrial function remains largely unexplored. The sablefish ( Anoplopoma fimbria ) encounters considerable variations in environmental oxygen availability, and is an interesting model for studying the effects of hypoxia on fish cardiorespiratory function. We investigated how in vivo hypoxia acclimation (6 months at 40% then 3 weeks at 20% air saturation) and in vitro anoxia-reoxygenation affected sablefish cardiac mitochondrial respiration and reactive oxygen species (ROS) release rates using high-resolution fluorespirometry. Further, we investigated how hypoxia acclimation affected the sensitivity of mitochondrial respiration to nitric oxide (NO), and compared mitochondrial lipid and fatty acid (FA) composition between groups. Hypoxia acclimation did not alter mitochondrial coupled or uncoupled respiration, or respiratory control ratio, ROS release rates, P 50 or superoxide dismutase activity. However, it increased citrate synthase activity (by ∼20%), increased the sensitivity of mitochondrial respiration to NO inhibition (i.e., the NO IC 50 was 25% lower), and enhanced the recovery of respiration (by 21%) and reduced ROS release rates (by 25-30%) post-anoxia. In addition, hypoxia acclimation altered mitochondrial FA composition [increasing arachidonic acid (20:4ω6) and eicosapentaenoic acid (20:5ω3) proportions by 11 and 14%, respectively], and SIMPER analysis revealed that the phospholipid:sterol ratio was the largest contributor (24%) to the dissimilarity between treatments. Overall, these results suggest that hypoxia acclimation may protect sablefish cardiac bioenergetic function during or after periods of O 2 limitation, and that this may be related to alterations in mitochondrial sensitivity to NO and to adaptive changes in membrane composition (fluidity)., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Atlantic cod ( Gadus morhua ) and haddock ( Melanogrammus aeglefinus ) are two commercially important marine fishes impacted by both overfishing and climate change. Increasing ocean temperatures are affecting the physiology of these species and causing changes in distribution, growth, and maturity. While the physiology of cod has been well investigated, that of haddock has received very little attention. Here, we measured the metabolic response to increasing temperatures, as well as the critical thermal maximum (CT max ), of cod acclimated to 8 and 12 °C and haddock acclimated to 12 °C. We also compared the swimming performance (critical swimming speed, U crit ) of cod and haddock at 12 °C, as well as the U crit of 12 °C-acclimated cod acutely exposed to a higher-than-optimal temperature (16 °C). The CT max for cod was 21.4 and 23.0 °C for 8- and 12 °C-acclimated fish, respectively, whereas that for the 12 °C-acclimated haddock was 23.9 °C. These values were all significantly different and show that haddock are more tolerant of high temperatures. The aerobic maximum metabolic rate (MMR) of swimming cod remained high at 16 °C, suggesting that maximum oxygen transport capacity was not limited at a temperature above optimal in this species. However, signs of impaired swimming (struggling) were becoming evident at 16 °C. Haddock were found to reach a higher U crit than cod at 12 °C (3.02 vs. 2.62 body lengths s -1 , respectively), and at a lower MMR. Taken together, these results suggest that haddock perform better than cod in warmer conditions, and that haddock are the superior swimmer amongst the two species., Competing Interests: Jason A. Bailey is employed by Vattenbrukscentrum Ost, East Region Aquaculture Centre (ERAC). ERAC is a non-profit, government-funded, organisation with the goal of aiding and giving advice to interested people in the Swedish aquaculture industry. Neither Jason A. Bailey nor ERAC will gain any financial or material benefits from the publication of these results. Any work on the manuscript was performed prior to Jason A. Bailey’s employment with ERAC or during his personal time., (© 2019 Norin et al.)

Doppler and B-mode ultrasonography and electrocardiography (ECG) were used to determine cardiac hemodynamics and electrical characteristics in 12°C-acclimated and metomidate-anesthetized northern pike, rainbow trout and white sturgeon (7-9 per species) at 12°C and 20°C, and at a comparable heart rate (f H , ~60 beats/min). Despite similar relative ventricle masses and cardiac output (Q), interspecific differences were observed at 12°C in f H , ventricular filling and ejection, stroke volume, the duration ECG intervals, and cardiac valve cross-sectional areas. Vis-a-fronte filling of the atrium due to ventricular contraction was observed in all species. However, biphasic ventricular filling (i.e., due to central venous pressure and then atrial contraction) was only observed in rainbow trout and white sturgeon. Changes in atrial and ventricular performance varied between the species as temperature increased from 12°C to 20°C. Rainbow trout had the highest thermal sensitivity for f H (Q 10  = 3.73), which doubled Q, and the largest increase in transvalvular blood velocity during ventricular filling. Conversely, northern pike had the lowest Q 10 for f H (1.58) and did not increase Q. At ~60 beats/min, the rainbow trout heart had the shortest period of electrical activity, which also resulted in the longest recovery period (TP interval) between successive beats. The QT interval at ~60 beats/min was also longer in the white sturgeon versus the other species. These results suggest that interspecific differences in fish cardiac hemodynamics may be related to cardiac morphology, the duration of electrical impulses through the heart, cardiac thermal sensitivity, and valve dimensions., (© 2019 Wiley Periodicals, Inc.)

Given climate change projections, the limited ability of fish reared in sea-cages to behaviourally thermoregulate, and that thermal tolerance may be heritable, studies that examine family-related differences in upper thermal tolerance are quite relevant to the aquaculture industry. Thus, we investigated the upper thermal tolerance of 15 Atlantic cod (Gadus morhua L.) families by challenging them with acute (2 °C h -1 ) and incremental (1 °C every 4 days) temperature increases (CT max and IT max tests, respectively) under normoxia (~ 100% air saturation) and mild hypoxia (~ 75% air sat.). The cod's CT max was 22.5 ± 0.1 °C (mean ± S.E.) during normoxia and 21.8 ± 0.1 °C during hypoxia (P < 0.001); and these two CT max values were significantly correlated across families. In both the normoxic and hypoxic IT max tests, feed intake fell by ~50% between 17 and 18 °C, and stopped entirely by 21 °C. No mortalities were observed under 20 °C in the normoxic and hypoxic IT max tests, and the IT max value was ~21.7 °C in both groups. Differences in the upper thermal tolerance between families were only observed in the CT max experiment. No correlation was found between the specific growth rate and the CT max of the families. Further, no correlation existed between CT max and IT max . This study is the first to compare the thermal tolerance of fish families to both CT max and IT max challenges, and the data: 1) suggest that the Atlantic cod is quite tolerant of acute (i.e., hours) or short-term (i.e., weeks) exposure to high water temperatures (i.e., up to 20 °C); 2) indicate that it might be difficult to select fish with higher IT max values; and 3) question the relevance of CT max for selecting fish that are destined for sea-cages where temperatures slowly warm over the summer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

A successful spawning migration in salmon depends on their athletic ability, and thus on efficient cardiovascular oxygen (O 2 ) transport. Most teleost fishes have highly pH-sensitive haemoglobins (Hb) that can release large amounts of O 2 when the blood is acidified at the tissues. We hypothesized that plasma-accessible carbonic anhydrase (paCA; the enzyme that catalyses proton production from CO 2 ) is required to acidify the blood at the tissues and promote tissue O 2 extraction. Previous studies have reported an elevated tissue O 2 extraction in hypoxia-acclimated teleosts that may also be facilitated by paCA. Thus, to create experimental contrasts in tissue O 2 extraction, Atlantic salmon were acclimated to normoxia or hypoxia (40% air saturation for more than six weeks), and the role of paCA in enhancing tissue O 2 extraction was tested by inhibiting paCA at rest and during submaximal exercise. Our results show that: (i) in both acclimation groups, the inhibition of paCA increased cardiac output by one-third, indicating a role of paCA in promoting tissue O 2 extraction during exercise, recovery and at rest; (ii) the recruitment of paCA was plastic and increased following hypoxic acclimation; and (iii) maximal exercise performance in salmon, and thus a successful spawning migration, may not be possible without paCA.

Given the potential impacts of global warming, such as increases in temperature and the frequency/severity of hypoxia in marine ecosystems, it is important to study the impacts of these environmental challenges on sea-cage reared aquaculture species. This study focuses on the sablefish (Anoplopoma fimbria), an emerging aquaculture species that has a unique ecology in the wild. For instance, adults inhabit oxygen minimum zones and cool waters at depths up to 1500 m. Using Atlantic salmon (Salmo salar) (~1132 g adults) as a comparative species, we used intermittent-flow respirometry to characterize the tolerance and metabolic response of sablefish (~10 g juveniles and ~675 g adults) to acute increases in temperature (2 °C h -1 ) and decreases in oxygen level (~10% air saturation h -1 ). Adult sablefish were much more hypoxia tolerant than adult salmon [O 2 level at loss of equilibrium ~5.4% vs. ~24.2% air saturation, respectively]. In addition, sablefish could withstand upper temperatures only slightly lower than salmon [critical thermal maximum (CT max ) ~24.9 °C vs. ~26.2 °C, respectively]. Sablefish juveniles were both less hypoxia and thermally tolerant than adults [critical O 2 tension ~18.9% vs. ~15.8% air saturation; CT max ~22.7 vs. ~24.9 °C, respectively]. Interestingly, many of these differences in environmental tolerance could not be explained by differences in metabolic parameters (aerobic scope or routine metabolic rate). Our findings show that sablefish are tolerant of high temperatures, and very tolerant of hypoxia, traits that are advantageous for an aquaculture species in the era of climate change., (Copyright © 2019 Elsevier Inc. All rights reserved.)

It has been suggested that exposure to high temperature or hypoxia may confer tolerance to the other oxygen-limited stressor (i.e., 'cross-tolerance'). Thus, we investigated if chronic hypoxia-acclimation (>3 months at 40% air saturation) improved the steelhead trout's critical thermal maximum (CT Max ), or affected key physiological variables that could impact upper thermal tolerance. Neither CT Max (24.7 vs. 25.3°C) itself, nor oxygen consumption ( [Formula: see text] ), haematocrit, blood haemoglobin concentration, or heart rate differed between hypoxia- and normoxia-acclimated trout when acutely warmed. However, the cardiac output (Q̇) of hypoxia-acclimated fish plateaued earlier compared to normoxia-acclimated fish due to an inability to maintain stroke volume (S V ), and this resulted in a ~50% lower maximum Q̇. Despite this reduced maximum cardiac function, hypoxia-acclimated trout were able to consume more O 2 per volume of blood pumped as evidenced by the equivalent [Formula: see text] . These results provide additional evidence that long-term hypoxia improves tissue oxygen utilization, and that this compensates for diminished cardiac pumping capacity. The limited S V in hypoxia-acclimated trout in vivo was not associated with changes in cardiac morphology or in vitro maximum S V , but the affinity and density of myocardial ß-adrenoreceptors were lower and higher, respectively, than in normoxia-acclimated fish. These data suggest that alterations in ventricular filling dynamics or myocardial contractility constrain cardiac function in hypoxia-acclimated fish at high temperatures. Our results do not support (1) 'cross-tolerance' between high temperature and hypoxia when hypoxia is chronic, or (2) that cardiac function is always the determinant of temperature-induced changes in fish [Formula: see text] , and thus thermal tolerance, as suggested by the oxygen- and capacity-limited thermal tolerance (OCLTT) theory., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Temperature-induced limitations on the capacity of the cardiorespiratory system to transport oxygen from the environment to the tissues, manifested as a reduced aerobic scope (maximum minus standard metabolic rate), have been proposed as the principal determinant of the upper thermal limits of fishes and other water-breathing ectotherms. Consequently, the upper thermal niche boundaries of these animals are expected to be highly sensitive to aquatic hypoxia and other environmental stressors that constrain their cardiorespiratory performance. However, the generality of this dogma has recently been questioned, as some species have been shown to maintain aerobic scope at thermal extremes. Here, we experimentally tested whether reduced oxygen availability due to aquatic hypoxia would decrease the upper thermal limits (i.e. the critical thermal maximum, CT max ) of the estuarine red drum (Sciaenops ocellatus) and the marine lumpfish (Cyclopterus lumpus). In both species, CT max was independent of oxygen availability over a wide range of oxygen levels despite substantial (>72%) reductions in aerobic scope. These data show that the upper thermal limits of water-breathing ectotherms are not always linked to the capacity for oxygen transport. Consequently, we propose a novel metric for classifying the oxygen dependence of thermal tolerance; the oxygen limit for thermal tolerance (P CT max ), which is the water oxygen tension (Pw O 2 ) where an organism's CT max starts to decline. We suggest that this metric can be used for assessing the oxygen sensitivity of upper thermal limits in water-breathing ectotherms, and the susceptibility of their upper thermal niche boundaries to environmental hypoxia., (© 2016. Published by The Company of Biologists Ltd.)

The microsporidian Loma morhua infects Atlantic cod (Gadus morhua) in the wild and in culture and results in the formation of xenomas within the gill filaments, heart and spleen. Given the importance of the two former organs to metabolic capacity and thermal tolerance, the cardiorespiratory performance of cod with a naturally acquired infection of Loma was measured during an acute temperature increase (2 °C h(-1)) from 10 °C to the fish's critical thermal maximum (CT(Max)). In addition, oxygen consumption and swimming performance were measured during two successive critical swimming speed (U(crit)) tests at 10 °C. While Loma infection had a negative impact on cod cardiac function at warm temperatures, and on metabolic capacity in both the CT(Max) and U(crit) tests (i.e. a reduction of 30-40%), it appears that the Atlantic cod can largely compensate for these Loma-induced cardiorespiratory limitations. For example, (i) CT(Max) (21.0 ± 0.3 °C) and U(crit) (~1.75 BL s(-1)) were very comparable to those reported in previous studies using uninfected fish from the same founder population; and (ii) our data suggest that tissue oxygen extraction, and potentially the capacity for anaerobic metabolism, is enhanced in fish infected with this microsporidian., (© 2015 The Authors Journal of Fish Diseases Published by John Wiley & Sons Ltd.)

The cunner (Tautogolabrus adspersus) is a fish with a wide latitudinal distribution that is capable of going into metabolic depression during the winter months, and thus, represents a unique model to investigate the impacts of cold temperatures on the stress response. In this study, we measured resting (pre-stress) plasma cortisol levels in 10 °C and 0 °C acclimated cunner from Newfoundland, and both catecholamine and cortisol levels after they were given a standardized handling stress (i.e. 1 min air exposure). In addition, we cloned and characterized cDNAs for several key genes of the cortisol-axis [cytochrome P450scc, steroidogenic acute regulatory protein (StAR) and a glucocorticoid receptor (GR) most likely to be an ortholog of the teleost GR2], determined the tissue distribution of their transcripts, and measured their constitutive (i.e. pre-stress) transcript levels in individuals acclimated to both temperatures. In cunner acclimated to 0 °C, post-stress epinephrine and norepinephrine levels were much lower (by approximately 9- and 5-fold, respectively) compared to 10 °C acclimated fish, and these fish had relatively low resting cortisol levels (~15 ngml(-1)) and showed a typical post-stress response. In contrast, those acclimated to 10 °C had quite high resting cortisol levels (~75 ngml(-1)) that actually decreased (to ~20 ngml(-1)) post-stress before returning to pre-stress levels. Finally, fish acclimated to 10 °C had higher P450scc transcript levels in the head kidney and lower levels of GR transcript in both the head kidney and liver. Taken together, these results suggest that: (1) temperature has a profound effect on the stress response of this species; and (2) although the ancestors of this species inhabited warm waters (i.e. they are members of the family Labridae), populations of cunner from colder regions may show signs of stress at temperatures as low as 10 °C., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Larval nutrition and growth are key issues for wild and cultured cod. While it was shown previously that larval cod fed wild zooplankton grow faster than those fed only rotifers, the mechanisms involved in this enhanced growth are not completely understood. We used microarrays to identify larval cod transcripts that respond to feeding with small amounts of wild zooplankton (5-10 % of live prey items). The larval transcriptome was compared between 3 treatment groups [fed rotifers (RA), rotifers with protein hydrolysate (RA-PH), or rotifers with zooplankton (RA-Zoo)] at 9-10 mm length [26-30 days post-hatch (dph)] to identify a robust suite of zooplankton-responsive genes (i.e. differentially expressed between RA-Zoo and both other groups)., Results: The microarray experiment identified 147 significantly up-regulated and 156 significantly down-regulated features in RA-Zoo compared with both RA and RA-PH. Gene ontology terms overrepresented in the RA-Zoo responsive gene set included "response to selenium ion" and several related to cell division and oxidation-reduction. Ten selenoprotein-encoding genes, and 2 genes involved in thyroid hormone generation, were up-regulated in RA-Zoo compared with both other groups. Hierarchical clustering of RA-Zoo responsive genes involved in oxidation-reduction and selenium homeostasis demonstrated that only the zooplankton treatment had a considerable and consistent impact on the expression of these genes. Fourteen microarray-identified genes were selected for QPCR involving 9-13 mm larvae, and 13 of these were validated as differentially expressed between RA-Zoo and both other groups at ~9 mm. In contrast, in age-matched (34-35 dph; ~11 mm RA and RA-PH, ~13 mm RA-Zoo) and size-matched (~13 mm) older larvae, only 2 and 3 genes, respectively, showed the same direction of RA-Zoo-responsive change as in ~9 mm larvae., Conclusions: The modulation of genes involved in selenium binding, redox homeostasis, and thyroid hormone generation in ~9 mm RA-Zoo larvae in this study may be in response to the relatively high levels of selenium, iodine, and LC-PUFA (potentially causing oxidative stress) in zooplankton. Nonetheless, only a subset of zooplankton-responsive genes in ~9 mm larvae remained so in older larvae, suggesting that the observed transcriptome changes are largely involved in initiating the period of growth enhancement.

Interleukin (IL)-1β is associated with hypotension and cardiovascular collapse in mammals during heat stroke, and the mRNA expression of this pro-inflammatory cytokine increases dramatically in the blood of Atlantic cod (Gadus morhua) at high temperatures. These data suggest that release of IL-1β at high temperatures negatively impacts fish cardiovascular function and could be a primary determinant of upper thermal tolerance in this taxa. Thus, we measured the concentration-dependent response of isolated steelhead trout (Oncorhynchus mykiss) coronary microvessels (<150 μm in diameter) to recombinant (r) IL-1β at two temperatures (10 and 20°C). Recombinant IL-1β induced a concentration-dependent vasodilation with vessel diameter increasing by approximately 8 and 30% at 10(-8) and 10(-7) mol l(-1), respectively. However, this effect was not temperature dependent. Both vessel denudation and cyclooxygenase blockade (by indomethacin), but not the nitric oxide (NO) antagonist L-NIO, inhibited the vasodilator effect of rIL-1β. In contrast, the concentration-dependent dilation caused by the endothelium-dependent calcium ionophore A23187 was completely abolished by L-NIO and indomethacin, suggesting that both NO and prostaglandin signaling mechanisms exist in the trout coronary microvasculature. These data: (1) are the first to demonstrate a functional link between the immune and cardiovascular systems in fishes; (2) suggest that IL-1β release at high temperatures may reduce systemic vascular resistance, and thus, the capacity of fish to maintain blood pressure; and (3) provide evidence that both NO and prostaglandins play a role in regulating coronary vascular tone, and thus, blood flow., (© 2015. Published by The Company of Biologists Ltd.)