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Fish in the wild often contend with complex flows that are produced by natural and artificial structures. Research into fish interactions with turbulence often investigates metrics such as turbulent kinetic energy (TKE) or fish positional location, with less focus on the specific interactions between vortex organization and body swimming kinematics. Here, we compared the swimming kinematics of rainbow trout (Oncorhynchus mykiss) holding station in flows produced by two different 3×5 cylinder arrays. We systematically utilized computational fluid dynamics to identify one array that produced a Kármán vortex street with high vortex periodicity (KVS array) and another that produced low periodicity, similar to a parallel vortex street (PVS array), both validated with particle image velocimetry. The only difference in swimming kinematics between cylinder arrays was an increased tail beat amplitude in the KVS array. In both cylinder arrays, the tail beat frequency decreased and snout amplitude increased compared with the freestream. The center of mass amplitude was greater in the PVS array than in only the freestream, however, suggesting some buffeting of the body by the fluid. Notably, we did not observe Kármán gaiting in the KVS array as in previous studies. We hypothesize that this is because (1) vorticity was dissipated in the region where fish held station or (2) vortices were in-line rather than staggered. These results are the first to quantify the kinematics and behavior of fishes swimming in the wake of multiple cylinder arrays, which has important implications for biomechanics, fluid dynamics and fisheries management., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Increasing evidence shows that larger fish are more vulnerable to acute warming than smaller individuals of the same species. This size-dependency of thermal tolerance has been ascribed to differences in aerobic performance, largely owing to a decline in oxygen supply relative to demand. To shed light on these ideas, we examined metabolic allometry in 130 rainbow trout ranging from 12 to 358 g under control conditions (17°C) and in response to acute heating (to 25°C), with and without supplemental oxygen (100% versus 150% air saturation). Under normoxia, high temperature caused an average 17% reduction in aerobic scope compared with 17°C. Aerobic performance disproportionally deteriorated in bigger fish as the scaling exponent (b) for aerobic scope declined from b=0.87 at 17°C to b=0.74 at 25°C. Hyperoxia increased maximum metabolic rate and aerobic scope at both temperatures and disproportionally benefited larger fish at 25°C as the scaling exponent for aerobic scope was reestablished to the same level as at 17°C (b=0.86). This suggests that hyperoxia may provide metabolic refuge for larger individuals, allowing them to sustain aerobic activities when facing acute warming. Notably, the elevated aerobic capacity afforded by hyperoxia did not appear to improve thermal resilience, as mortality in 25°C hyperoxia (13.8%, n=4) was similar to that in normoxia (12.1%, n=4), although we caution that this topic warrants more targeted research. We highlight the need for mechanistic investigations of the oxygen transport system to determine the consequences of differential metabolic scaling across temperature in a climate warming context., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Complex hydrodynamics abound in natural streams, yet the selective pressures these impose upon different size classes of fish are not well understood. Attached vortices are produced by relatively large objects that block freestream flow, which fish routinely utilize for flow refuging. To test how flow refuging and the potential harvesting of energy (as seen in Kármán gaiting) vary across size classes in rainbow trout (Oncorhynchus mykiss; fingerling, 8 cm; parr, 14 cm; adult, 22 cm; n=4 per size class), we used a water flume (4100 l; freestream flow at 65 cm s-1) and created vortices using 45 deg wing dams of varying size (small, 15 cm; medium, 31 cm; large, 48 cm). We monitored microhabitat selection and swimming kinematics of individual trout and measured the flow field in the wake of wing dams using time-resolved particle image velocimetry (PIV). Trout of each size class preferentially swam in vortices rather than the freestream, but the capacity to flow refuge varied according to the ratio of vortex width to fish length (WV:LF). Consistent refuging behavior was exhibited when WV:LF≥1.5. All size classes exhibited increased wavelength and Strouhal number and decreased tailbeat frequency within vortices compared with freestream, suggesting that swimming in vortices requires less power output. In 17% of the trials, fish preferentially swam in a manner that suggests energy harvesting from the shear layer. Our results can inform efforts toward riparian restoration and fishway design to improve salmonid conservation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Pectoral fins play a crucial role in fish locomotion. Despite fishes living in complex fluid environments that exist in rivers and tidal flows, the role of the pectoral fins in navigating turbulent flows is not well understood. This study investigated the kinematics and muscle activity of pectoral fins in rainbow trout as they held station in the unsteady flows behind a D-section cylinder. We observed two distinct pectoral fin behaviors, one during braking and the other during Kármán gaiting. These behaviors were correlated to whole-body movements in response to the hydrodynamic conditions of specific regions in the cylinder wake. Sustained fin extensions during braking, where the fin was held out to maintain its position away from the body and against the flow, were associated with the cessation of forward body velocity, where the fish avoided the suction region directly downstream of the cylinder. Transient fin extensions and retractions during Kármán gaiting controlled body movements in the cross-stream direction. These two fin behaviors had different patterns of muscle activity. All braking events required recruitment from both the abductor and adductor musculature to actively extend a pectoral fin. In contrast, over 50% of fin extension movements during Kármán gaiting proceed in the absence of muscle activity. We reveal that in unsteady fluid environments, pectoral fin movements are the result of a complex combination of passive and active mechanisms that deviate substantially from canonical labriform locomotion, the implications of which await further work on the integration of sensory and motor systems., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Muscle shortening underpins most skeletal motion and ultimately animal performance. Most animal muscle generates its greatest mechanical output over a small, homogeneous range of shortening magnitudes and speeds. However, homogeneous muscle shortening is difficult to achieve for swimming fish because the whole body deforms like a bending beam: as the vertebral column flexes laterally, longitudinal muscle strain increases along a medio-lateral gradient. Similar dorsoventral strain gradients have been identified as the vertebral column flexes dorsally during feeding in at least one body location in one fish. If fish bodies also deform like beams during dorsoventral feeding motions, this would suggest the dorsal body (epaxial) muscles must homogenize both dorsoventral and mediolateral strain gradients. We tested this hypothesis by measuring curvature of the anterior vertebral column with XROMM and muscle shortening in 14 epaxial subregions with fluoromicrometry during feeding in rainbow trout (Oncorhynchus mykiss). We compared measured strain with the predicted strain based on beam theory's curvature-strain relationship. Trout flexed the vertebrae dorsally and laterally during feeding strikes, yet when flexion in both planes was included, the strain predicted by beam theory was strongly and significantly correlated with measured strain (P<0.01, R2=0.60). Beam theory accurately predicted strain (slope=1.15, compared with ideal slope=1) across most muscle subregions, confirming that epaxial muscles experience dorsoventral and mediolateral gradients in longitudinal strain. Establishing this deformation-curvature relationship is a crucial step to understanding how these muscles overcome orthogonal strain gradients to produce powerful feeding and swimming behaviours., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

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.)

Methionine is a key factor in modulating the cellular availability of the main biological methyl donor S -adenosylmethionine (SAM), which is required for all biological methylation reactions including DNA and histone methylation. As such, it represents a potential critical factor in nutritional programming. Here, we investigated whether early methionine restriction at first feeding could have long-term programmed metabolic consequences in rainbow trout. For this purpose, trout fry were fed with either a control diet (C) or a methionine-deficient diet (MD) for 2 weeks from the first exogenous feeding. Next, fish were subjected to a 5 month growth trial with a standard diet followed by a 2 week challenge (with the MD or C diet) to test the programming effect of the early methionine restriction. The results showed that, whatever the dietary treatment of fry, the 2 week challenge with the MD diet led to a general mitochondrial defect associated with an increase in endoplasmic reticulum stress, mitophagy and apoptosis, highlighting the existence of complex cross-talk between these different functions. Moreover, for the first time, we also observed that fish fed the MD diet at the first meal later exhibited an increase in several critical factors of mitophagy, hinting that the early nutritional stimulus with methionine deficiency resulted in long-term programming of this cell function. Together, these data extend our understanding of the role of dietary methionine and emphasize the potential for this amino acid in the application of new feeding strategies, such as nutritional programming, to optimize the nutrition and health of farmed fish., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

In seawater, rainbow trout ( Oncorhynchus mykiss ) drink and absorb water through the gastrointestinal tract to compensate for water passively lost to the hyperosmotic environment. Concomitantly, they exhibit elevated cardiac output and a doubling of gastrointestinal blood flow to provide additional O 2 to the gut and increase convective flux of absorbed ions and water. Yet, it is unknown how warming waters, which elevate tissue O 2 demand and the rate of diffusion of ions and water across the gills (i.e. the osmo-respiratory compromise), affects these processes. We measured cardiovascular and blood variables of rainbow trout acclimated to freshwater and seawater during acute warming from 11 to 17°C. Relative to freshwater-acclimated trout, cardiac output was 34% and 55% higher in seawater-acclimated trout at 11 and 17°C, respectively, which allowed them to increase gastrointestinal blood flow significantly more during warming (increases of 75% in seawater vs. 31% in freshwater). These adjustments likely served to mitigate the impact of warming on osmotic balance, as changes in ionic and osmotic blood composition were minor. Furthermore, seawater-acclimated trout seemingly had a lower tissue O 2 extraction, explaining why trout acclimated to freshwater and seawater often exhibit similar metabolic rates, despite a higher cardiac output in seawater. Our results highlight a novel role of gastrointestinal blood perfusion in the osmo-respiratory compromise in fish, and improve our understanding of the physiological changes euryhaline fishes must undergo when faced with interacting environmental challenges such as transient warming events., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Many fish naturally encounter a daily cycle of hypoxia, but it is unclear whether this exposure hardens hypoxia-intolerant fish to future hypoxia or leads to accumulated stress and death. The rainbow trout ( Oncorhynchus mykiss ) is a putatively hypoxia-sensitive species found in rivers and estuaries that may routinely experience hypoxic events. Trout were exposed to one of four 135 h treatments in a swim-tunnel respirometer: (1) air-saturated control (20.7 kPa P O 2 ); (2) diel cycling O 2 (20.7-4.2 kPa P O 2  over 24 h); (3) acute hypoxia (130 h at 20.7 kPa P O 2  followed by 5 h at 4.2 kPa P O 2 ); and (4) the mean oxygen tension (12.4 kPa P O 2 ) experienced by the diel cycled fish. Some responses were similar in diel O 2 cycled and mean P O 2 -treated fish, but overall, exposure to ecologically representative diel hypoxia cycles improved hypoxia tolerance. Diel hypoxia-induced protective responses included increased inducible HSP70 concentration and mean corpuscular hemoglobin concentration, as well as reduced plasma cortisol. Acclimation to diel hypoxia allowed metabolic rates to decline during hypoxia, reduced oxygen debt following subsequent exposures, and allowed fish to return to an anabolic phenotype. The data demonstrate that acute diel cycling hypoxia improves hypoxia tolerance in previously intolerant fish through the activation of cellular protective mechanisms and a reduction in metabolic O 2 requirements., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

As fish approach fatigue at high water velocities in a critical swimming speed ( U crit ) test, their swimming mode and oxygen cascade typically move to an unsteady state because they adopt an unsteady, burst-and-glide swimming mode despite a constant, imposed workload. However, conventional rate of oxygen uptake ( Ṁ O 2 ) sampling intervals (5-20 min) tend to smooth any dynamic fluctuations in active Ṁ O 2  ( Ṁ O 2 active ) and thus likely underestimate the peak Ṁ O 2 active Here, we used rainbow trout ( Oncorhynchus mykiss ) to explore the dynamic nature of Ṁ O 2 active near U crit using various sampling windows and an iterative algorithm. Compared with a conventional interval regression analysis of Ṁ O 2 active over a 10-min period, our new analytical approach generated a 23% higher peak Ṁ O 2 active Therefore, we suggest that accounting for such dynamics in Ṁ O 2 active with this new analytical approach may lead to more accurate estimates of maximum Ṁ O 2  in fishes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Cortisol-induced epithelial tightening of a primary cultured rainbow trout gill epithelium model occurs in association with reduced paracellular permeability and increased abundance of select barrier-forming tight junction (TJ) proteins. Corticosteroid receptor (CR) pharmacological blocker studies have suggested that to produce this tightening effect, cortisol acts on the mineralocorticoid receptor (MR) as well as glucocorticoid receptors (GRs). This study considered how cortisol influences model gill epithelium permeability and TJ properties by transcriptional knockdown of the gene encoding the MR ( mr -KD) using double-stranded RNA. Following mr -KD, a significant reduction in MR protein abundance was observed in the epithelium. The mr -KD epithelium demonstrated reduced transepithelial resistance (TER) and an increase in the paracellular flux of [ 3 H]polyethylene glycol (MW 400 kDa, PEG-400). Concurrently, mRNA abundance of gr2 and 11βhsd increased, indicating a possible compensatory response to mr -KD. Transcript abundance of claudin ( cldn ) -6 , -8d , - 23a and -28b decreased while that of cldn-20a increased in mr -KD preparations. Cortisol-induced epithelial tightening was enhanced in mr -KD preparations, suggesting that alterations in CRs and TJ composition augmented model epithelium barrier function in response to lowered MR abundance. Cortisol treatment significantly increased the transcript and protein abundance of TJ proteins such as Cldn-8d and -28b. However, in mr -KD preparations, Cldn-28b protein abundance did not significantly alter in response to cortisol treatment, while Cldn-8d abundance was significantly elevated. Data suggest that mr -KD compromises normal barrier function of a primary cultured rainbow trout gill epithelium in both the presence and absence of cortisol and that Cldn-28b protein abundance may be modulated by cortisol via the MR only., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Acute warming in fish increases heart rate ( f H ) and cardiac output to peak values, after which performance plateaus or declines and arrhythmia may occur. This cardiac response can place a convective limitation on systemic oxygen delivery at high temperatures. To test the hypothesis that autonomic cardiac regulation protects cardiac performance in rainbow trout during acute warming, we investigated adrenergic and cholinergic regulation during the onset and progression of cardiac limitations. We explored the direct effects of adrenergic stimulation by acutely warming an in situ working perfused heart until arrhythmia occurred, cooling the heart to restore rhythmicity and rewarming with increasing adrenergic stimulation. Adrenergic stimulation produced a clear, dose-dependent increase in the temperature and peak f H achieved prior to the onset of arrhythmia. To examine how this adrenergic protection functions in conjunction with cholinergic vagal inhibition in vivo , rainbow trout fitted with ECG electrodes were acutely warmed in a respirometer until they lost equilibrium (CT max ) with and without muscarinic (atropine) and β-adrenergic (sotalol) antagonists. Trout exhibited roughly equal and opposing cholinergic and adrenergic tone on f H that persisted up to critical temperatures. β-Adrenergic blockade significantly lowered peak f H by 14-17%, while muscarinic blockade significantly lowered the temperature for peak f H by 2.0 ° C. Moreover, muscarinic and β-adrenergic blockers injected individually or together significantly reduced CT max by up to 3 ° C, indicating for the first time that cardiac adrenergic stimulation and cholinergic inhibition can enhance acute heat tolerance in rainbow trout at the level of the heart and the whole animal., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Trout caeca are vermiform structures projecting from the anterior intestine of the gastrointestinal tract. Despite their simple gross morphology, these appendages are physically distinct along the anterior-posterior axis, and ultrastructural evidence suggests zonation of function within the structures. Individual caeca from three sections (anterior, middle and posterior) were removed from the intestine of freshwater rainbow trout and investigated for ion transport and enzyme activity. Ca 2+ absorption appeared as a combination of active and passive movement, with Michaelis-Menten kinetics observable under symmetrical conditions, and was inhibited by several pharmacological agents (ouabain, La 3+ and a calmodulin antagonist). There was a decrease in ion transport function from adjacent to the intestine (proximal) to the distal tip of each caecum, along with decreasing transport from anterior to posterior for the proximal portion alone. Feeding increased the J Max and K M for Ca 2+ absorption within all sections, whereas ion-poor water (IPW) exposure further increased the J Max and K M for Ca 2+ transport in the anterior and middle sections. Increased Na + /K + -ATPase (NKA) and citrate synthase (CS) activity rates paralleled trends seen in Ca 2+ transport. Feeding in freshwater and IPW exposure increased the glycolytic capacity of the caeca via increased pyruvate kinase (PK) and decreased lactate dehydrogenase (LDH) activity, while amino acid metabolism increased with IPW exposure through increased glutamate dehydrogenase (GDH) activity. Overall, feeding and IPW exposure each altered ionoregulation within the caeca of freshwater rainbow trout in a zone-specific pattern, with the anterior and proximal portions of the caeca being most affected. Increased carbohydrate and protein metabolism fueled the increased ATP demand of NKA through CS., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The collagen content of the rainbow trout heart increases in response to cold acclimation and decreases with acclimation to warm temperatures. This ability to remodel the myocardial extracellular matrix (ECM) makes these fish useful models to study the cellular pathways involved in collagen regulation in the vertebrate heart. Remodelling of the ECM in the mammalian heart is regulated, in part, by myofibroblasts which arise from pre-existing fibroblasts in response to transforming growth factor-β1 (TGF-β1). We have previously demonstrated that treatment of cultured rainbow trout cardiac fibroblasts with human TGF-β1 causes an increase in collagen production. Here, we showed that repetitive treatment of rainbow trout cardiac fibroblasts with a physiologically relevant concentration of human recombinant TGF-β1 results in a ∼29-fold increase in phosphorylated small mothers against decapentaplegic 2 (pSmad2); a 2.9-fold increase in vinculin protein, a 1.2-fold increase in cellular size and a 3-fold increase in filamentous actin (F-actin). These are common markers of the transition of fibroblasts to myofibroblasts. Cells treated with TGF-β1 also had highly organized cytoskeletal α-smooth muscle actin, as well as increased transcript abundances of mmp-9 , timp-2 and col1a1 Furthermore, using gelatin zymography, we demonstrated that TGF-β1 treatment causes a 5.3-fold increase in gelatinase activity. Together, these results suggest that trout cardiac fibroblasts have the capacity to differentiate into myofibroblasts and that this cell type can increase extracellular collagen turnover via gelatinase activity. Cardiac myofibroblasts are, therefore, likely involved in the remodelling of the cardiac ECM in the trout heart during thermal acclimation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

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 of 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 rate was 4.3±0.5 ml O 2  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 O 2  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 as a result of a ∼2-fold increase in oxygen extraction. The high metabolic thermal sensitivity of the gut could have important implications for the overall aerobic capacity and performance of fish in a warming world and warrants further investigation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Morphological streamlining is often associated with physiological advantages for steady swimming in fishes. Though most commonly studied in pelagic fishes, streamlining also occurs in fishes that occupy high-flow environments. Before the installation of dams and water diversions, bonytail (Cyprinidae, Gila elegans ), a fish endemic to the Colorado River (USA), regularly experienced massive, seasonal flooding events. Individuals of G. elegans display morphological characteristics that may facilitate swimming in high-flow conditions, including a narrow caudal peduncle and a high aspect ratio caudal fin. We tested the hypothesis that these features improve sustained swimming performance in bonytail by comparing locomotor performance in G. elegans with that of the closely related roundtail chub ( Gila robusta ) and two non-native species, rainbow trout ( Oncorhynchus mykiss ) and smallmouth bass ( Micropterus dolomieu ), using a Brett-style respirometer and locomotor step-tests. Gila elegans had the lowest estimated drag coefficient and the highest sustained swimming speeds relative to the other three species. There were no detectible differences in locomotor energetics during steady swimming among the four species. When challenged by high-velocity water flows, the second native species examined in this study, G. robusta , exploited the boundary effects in the flow tank by pitching forward and bracing the pelvic and pectoral fins against the acrylic tank bottom to 'hold station'. Because G. robusta can station hold to prevent being swept downstream during high flows and G. elegans can maintain swimming speeds greater than those of smallmouth bass and rainbow trout with comparable metabolic costs, we suggest that management agencies could use artificial flooding events to wash non-native competitors downstream and out of the Colorado River habitat., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Salmonid individuals show a relatively high variability in the time required to abandon the gravel nest where they hatch, the so-called 'emergence time'. Different behavioral and physiological traits have been shown to be associated with emergence time in wild salmonids. In general, early- and late-emerging fish have traits resembling those of proactive and reactive stress coping styles, respectively. Proactive fish are considered to be more resilient to stress and probably to disease, so it was hypothesized that fish with different emergence times have different abilities to resist repeated episodes of stress without suffering deleterious effects on their welfare or health status. In this study, rainbow trout eyed eggs were hatched and larvae were fractionated according to their emergence time (early fraction: first 20% of fish to emerge; intermediate fraction: mid 20%; late fraction: last 20%). When the fish were 4 months old, they were exposed to a daily repeated stress protocol for 15 days. The next day, both naïve and repeatedly stressed fish were exposed to an acute stress challenge. Different plasma (cortisol, glucose, lactate) as well as CNS (serotonergic activity) stress markers were assessed to evaluate the stress resilience of the different groups. Furthermore, an intraperitoneal infection challenge with Flavobacterium psychrophilum was carried out to assess disease resilience. Altogether, the results showed that fish from different fractions displayed differences in activation of the hypothalamus-pituitary-inter-renal axis, indicating a higher stress resilience in the fish with early emergence times. However, those differences were not reflected in the ability of the different fractions to grow and perform well in terms of growth, or in the ability to overcome infection with bacteria, which was similar for all the emergence fractions. This suggests that discriminating fish according to emergence time would probably have little effect in improving the performance and the welfare of farmed fish., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The mobilization of mammalian lipid reserves is strongly stimulated during exercise to reach a maximum at moderate intensities, but the effects of swimming speed on fish lipolysis have never been quantified. Continuous infusion of 2-[ 3 H]glycerol was used to measure the rate of appearance of glycerol or lipolytic rate ( R a glycerol) in rainbow trout kept at rest, or during graded exercise in a swim tunnel up to critical swimming speed ( U crit ). Results show that R a glycerol is 1.67±0.18 µmol kg -1  min -1 in control animals, and remains at a steady level of 1.24±0.10 µmol kg -1  min -1 in exercising fish at all swimming intensities. Baseline lipolytic rate provides more than enough fatty acids from lipid reserves to accommodate all the oxidative fuel requirements for swimming at up to 2 body lengths per second (BL s -1 ), and more than 50% of the energy needed at U crit (3.4±0.1 BL s -1 ). Such 'excess lipolysis' also means that trout sustain high rates of fatty acid reesterification. Maintaining steady lipolysis at rest and throughout graded swimming is strikingly different from mammals that stimulate R a glycerol by twofold to fivefold to support exercise. Instead, trout act like 'lipolytic machines' that do not modulate R a glycerol even when their metabolic rate triples - a strategy that eliminates the need to increase lipolytic rate during exercise. This study also supports the notion that maintaining a high rate of reesterification (or triacylglycerol/fatty acid cycling) may be a mechanism widely used by ectotherms to achieve rapid membrane remodelling in variable environments., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Environmental conditions experienced during early life play an important role in the long-term metabolic status of individuals. The present study investigated whether hypoxia exposure [for 24 h: 2.5 mg O 2  l -1 (20% dissolved O 2 )] during the embryonic stage alone (hypoxic history) or combined with a 5-day high-carbohydrate (60%) diet stimulus at first feeding (HC dietary history) can affect glucose metabolism later in life, i.e. in juvenile fish. After 19 weeks of growth, we observed a decrease in final body mass in fish with an HC dietary history. Feed efficiency was significantly affected by both hypoxic and HC dietary histories. After a short challenge test (5 days) performed with a 30% carbohydrate diet in juvenile trout, our results also showed that, in trout that experienced hypoxic history, mRNA levels of gluconeogenic genes in liver and glucose transport genes in both liver and muscle were significantly increased at the juvenile stage. Besides, mRNA levels of glycolytic genes were decreased in fish with an HC dietary history. Both hypoxic and dietary histories barely affected plasma metabolites or global epigenetic modifications in juvenile fish after the challenge test. In conclusion, our results demonstrated that an acute hypoxic stimulus during early development alone or combined with a hyperglucidic stimulus at first feeding can modify growth performance and glucose metabolism at the molecular level in juvenile trout., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

In mammals, ceramides are involved in the modulation of the orexigenic effects of ghrelin (GHRL). We previously demonstrated in rainbow trout that intracerebroventricular (ICV) treatment with ceramide (2.5 µg/100 g fish) resulted in an anorexigenic response, i.e. a response opposed to that described in mammals, where ceramide treatment is orexigenic. Therefore, we hypothesized that the putative interaction between GHRL and ceramide must be different in fish. Accordingly, in a first experiment, we observed that ceramide levels in the hypothalamus of rainbow trout did not change after ICV treatment with GHRL. In a second experiment, we assessed whether the effects of GHRL treatment on the regulation of food intake in rainbow trout changed in the presence of ceramide. Thus, we injected ICV GHRL and ceramide alone or in combination to evaluate in hypothalamus and hindbrain changes in parameters related to the metabolic control of food intake. The presence of ceramide generally counteracted the effects elicited by GHRL on fatty acid-sensing systems, the capacity of integrative sensors (AMPK, mTOR and SIRT-1), proteins involved in cellular signalling pathways (Akt and FoxO1) and neuropeptides involved in the regulation of food intake (AgRP, NPY, POMC and CART). The results are discussed in the context of regulation of food intake by metabolic and endocrine inputs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Stress and elevated cortisol levels are associated with pathological heart growth and cardiovascular disease in humans and other mammals. We recently established a link between heritable variation in post-stress cortisol production and cardiac growth in salmonid fish too. A conserved stimulatory effect of the otherwise catabolic steroid hormone cortisol is probably implied, but has to date not been established experimentally. Furthermore, whereas cardiac growth is associated with failure of the mammalian heart, pathological cardiac hypertrophy has not previously been described in fish. Here, we show that rainbow trout ( Oncorhynchus mykiss ) treated with cortisol in the diet for 45 days have enlarged hearts with lower maximum stroke volume and cardiac output. In accordance with impaired cardiac performance, overall circulatory oxygen-transporting capacity was diminished as indicated by reduced aerobic swimming performance. In contrast to the well-known adaptive/physiological heart growth observed in fish, cortisol-induced growth is maladaptive. Furthermore, the observed heart growth was associated with up-regulated signature genes of mammalian cardiac pathology, suggesting that signalling pathways mediating cortisol-induced cardiac remodelling in fish are conserved from fish to mammals. Altogether, we show that excessive cortisol can induce pathological cardiac remodelling. This is the first study to report and integrate the etiology, physiology and molecular biology of cortisol-induced pathological remodelling in fish., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

By filtering relevant sensory inputs and initiating stress responses, the brain is an essential organ in stress coping and adaptation. However, exposure to chronic or repeated stress can lead to allostatic overload, where neuroendocrinal and behavioral reactions to stress become maladaptive. This work examines forebrain mechanisms involved in allostatic processes in teleost fishes. Plasma cortisol, forebrain serotonergic (5-HTergic) neurochemistry, and mRNA levels of corticotropin-releasing factor (CRF), CRF-binding protein (CRF-BP), CRF receptors (CRFR1 and CRFR2), mineralocorticoid receptor (MR), glucocorticoid receptors (GR1 and GR2) and serotonin type 1A (5-HT 1A ) receptors (5-HT 1Aα and 5-HT 1Aβ ) were investigated at 1 h before and 0, 1 and 4 h after acute stress, in two groups of rainbow trout held in densities of 25 and 140 kg m -3 for 28 days. Generally, being held at 140 kg m -3 resulted in a less pronounced cortisol response. This effect was also reflected in lower forebrain 5-HTergic turnover, but not in mRNA levels in any of the investigated genes. This lends further support to reports that allostatic load causes fish to be incapable of mounting a proper cortisol response to an acute stressor, and suggests that changes in forebrain 5-HT metabolism are involved in allostatic processes in fish. Independent of rearing densities, mRNA levels of 5-HT 1Aα and MR were downregulated 4 h post-stress compared with values 1 h post-stress, suggesting that these receptors are under feedback control and take part in the downregulation of the hypothalamic-pituitary-interrenal (HPI) axis after exposure to an acute stressor., (© 2016. Published by The Company of Biologists Ltd.)

Fishes may exploit environmental vortices to save in the cost of locomotion. Previous work has investigated fish refuging behind a single cylinder in current, a behavior termed the Kármán gait. However, current-swept habitats often contain aggregations of physical objects, and it is unclear how the complex hydrodynamics shed from multiple structures affect refuging in fish. To begin to address this, we investigated how the flow fields produced by two D-shaped cylinders arranged in tandem affect the ability of rainbow trout (Oncorhynchus mykiss) to Kármán gait. We altered the spacing of the two cylinders from l/D of 0.7 to 2.7 (where l=downstream spacing of cylinders and D=cylinder diameter) and recorded the kinematics of trout swimming behind the cylinders with high-speed video at Re=10,000-55,000. Digital particle image velocimetry showed that increasing l/D decreased the strength of the vortex street by an average of 53% and decreased the frequency that vortices were shed by ∼20% for all speeds. Trout were able to Kármán gait behind all cylinder treatments despite these differences in the downstream wake; however, they Kármán gaited over twice as often behind closely spaced cylinders (l/D=0.7, 1.1, and 1.5). Computational fluid dynamics simulations show that when cylinders are widely spaced, the upstream cylinder generates a vortex street that interacts destructively with the downstream cylinder, producing weaker, more widely spaced and less-organized vortices that discourage Kármán gaiting. These findings are poised to help predict when fish may seek refuge in natural habitats based on the position and arrangement of stationary objects., (© 2016. Published by The Company of Biologists Ltd.)

Time course studies are critical for understanding regulatory mechanisms and temporal constraints in ectothermic animals acclimating to warmer temperatures. Therefore, we investigated the dynamics of heart rate and its neuro-humoral control in rainbow trout ( ITALIC! Onchorhynchus mykissL.) acclimating to 16°C for 39 days after being acutely warmed from 9°C. Resting heart rate was 39 beats min(-1)at 9°C, and increased significantly when fish were acutely warmed to 16°C ( ITALIC! Q10=1.9), but then declined during acclimation ( ITALIC! Q10=1.2 at day 39), mainly due to increased cholinergic inhibition while the intrinsic heart rate and adrenergic tone were little affected. Maximum heart rate also increased with warming, although a partial modest decrease occurred during the acclimation period. Consequently, heart rate scope exhibited a complex pattern with an initial increase with acute warming, followed by a steep decline and then a subsequent increase, which was primarily explained by cholinergic inhibition of resting heart rate., (© 2016. Published by The Company of Biologists Ltd.)

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.)

The boundary layers of rainbow trout, Oncorhynchus mykiss, swimming at 1.02±0.09 L s(-1) (mean±s.d., N=4), were measured by the particle image velocimetry (PIV) technique at a Reynolds number of 4×10(5). The boundary layer profile showed unsteadiness, oscillating above and beneath the classical logarithmic law of the wall with body motion. Across the entire surface regions that were measured, local Reynolds numbers based on momentum thickness, which is the distance that is perpendicular to the fish surface through which the boundary layer momentum flows at free-stream velocity, were greater than the critical value of 320 for the laminar-to-turbulent transition. The skin friction was dampened on the convex surface while the surface was moving towards a free-stream flow and increased on the concave surface while retreating. These observations contradict the result of a previous study using different species swimming by different methods. Boundary layer compression accompanied by an increase in local skin friction was not observed. Thus, the overall results may not support absolutely the Bone-Lighthill boundary layer thinning hypothesis that the undulatory motions of swimming fish cause a large increase in their friction drag because of the compression of the boundary layer. In some cases, marginal flow separation occurred on the convex surface in the relatively anterior surface region, but the separated flow reattached to the fish surface immediately downstream. Therefore, we believe that a severe impact due to induced drag components (i.e. pressure drag) on the swimming performance, an inevitable consequence of flow separation, was avoided., (© 2015. Published by The Company of Biologists Ltd.)

Rainbow trout (Oncorhynchus mykiss Walbaum) in southern Western Australia have undergone passive selection for over 19 generations to survive high water temperatures. Based on the conceptual model of 'oxygen- and capacity-limited thermal tolerance', we measured critical thermal maximum (CTmax), maximum heart rate (fH,max) and aerobic scope to test the hypothesis that these rainbow trout can maintain aerobic scope at high temperatures through a robust cardiac performance supporting oxygen delivery. Across five family groups CTmax averaged 29.0±0.02°C. Aerobic scope was maximized at 15.8±0.3°C (Topt), while the upper pejus temperature (Tpej, set at 90% of maximum aerobic scope) was 19.9±0.3°C. Although aerobic scope decreased at temperatures above Topt, the value at 25°C remained well over 40% of the maximum. Furthermore, pharmacologically stimulated fH,max increased with temperature, reaching a peak value between 23.5±0.4 and 24.0±0.4°C (Tmax) for three family groups. The Arrhenius breakpoint temperature (TAB) for fH,max was 20.3±0.3 to 20.7±0.4°C, while the average Q10 breakpoint temperature (TQB, when the incremental Q10<1.6) for fH,max was 21.6±0.2 to 22.0±0.4°C. Collectively, fH,max progressively became less temperature dependent beyond 20°C (TAB and TQB), which coincides with the upper Tpej for aerobic scope. Although upper thermal performance indices for both aerobic scope and fH,max were compared among family groups in this population, appreciable differences were not evident. Compared with other populations of rainbow trout, the present assessment is consistent with the prediction that this strain has undergone selection and shows the ability to tolerate higher water temperatures., (© 2015. Published by The Company of Biologists Ltd.)

Reducing temperature below the optimum of most vertebrate hearts impairs contractility and reduces organ function. However, a number of fish species, including the rainbow trout, can seasonally acclimate to low temperature. Such ability requires modification of physiological systems to compensate for the thermodynamic effects of temperature on biological processes. The current study tested the hypothesis that rainbow trout compensate for the direct effect of cold temperature by increasing cardiac contractility during cold acclimation. We examined cardiac contractility, following thermal acclimation (4, 11 and 17°C), by measuring the Ca(2+) sensitivity of force generation by chemically skinned cardiac trabeculae as well as ventricular pressure generation using a modified Langendorff preparation. We demonstrate, for the first time, that the Ca(2+) sensitivity of force generation was significantly higher in cardiac trabeculae from 4°C-acclimated trout compared with those acclimated to 11 or 17°C, and that this functional change occurred in parallel with a decrease in the level of cardiac troponin T phosphorylation. In addition, we show that the magnitude and rate of ventricular pressure generation was greater in hearts from trout acclimated to 4°C compared with those from animals acclimated to 11 or 17°C. Taken together, these results suggest that enhanced myofilament function, caused by modification of existing contractile proteins, is at least partially responsible for the observed increase in pressure generation after acclimation to 4°C. In addition, by examining the phenotypic plasticity of a comparative model we have identified a strategy, used in vivo, by which the force-generating capacity of cardiac muscle can be increased., (© 2014. Published by The Company of Biologists Ltd.)

The relationship between tail (or wing) beat frequency (f(tail)), amplitude (A) and forward velocity (U) in animals using oscillatory propulsion, when moving at a constant cruising speed, converges upon an optimum range of the Strouhal number (St = f(tail) · A/U). Previous work, based on observational data and supported by theory, shows St falling within the broad optimum range (0.2

Cortisol has been suggested to mediate the effect of stress on pineal melatonin synthesis in fish. Therefore, we aimed to determine how pineal melatonin synthesis is affected by exposing rainbow trout to different stressors, such as hypoxia, chasing and high stocking density. In addition, to test the hypothesis that cortisol is a mediator of such stress-induced effects, a set of animals were intraperitoneally implanted with coconut oil alone or containing cortisol (50 mg kg(-1) body mass) and sampled 5 or 48 h post-injection at midday and midnight. The specificity of such effect was also assessed in cultured pineal organs exposed to cortisol alone or with the general glucocorticoid receptor antagonist, mifepristone (RU486). Stress (in particular chasing and high stocking density) affected the patterns of plasma and pineal organ melatonin content during both day and night, with the greatest reduction occurring at night. The decrease in nocturnal melatonin levels in the pineal organ of stressed fish was accompanied by increased serotonin content and decreased AANAT2 enzymatic activity and mRNA abundance. Similar effects on pineal melatonin synthesis to those elicited by stress were observed in trout implanted with cortisol for either 5 or 48 h. These data indicate that stress negatively influences the synthesis of melatonin in the pineal organ, thus attenuating the day-night variations of circulating melatonin. The effect might be mediated by increased cortisol, which binds to trout pineal organ-specific glucocorticoid receptors to modulate melatonin rhythms. Our results in cultured pineal organs support this. Considering the role of melatonin in the synchronization of daily and annual rhythms, the results suggest that stress-induced alterations in melatonin synthesis could affect the availability of fish to integrate rhythmic environmental information.

A mechanistic understanding of how fishes swim in unsteady flows is challenging despite its prevalence in nature. Previous kinematic studies of fish Kármán gaiting in a vortex street behind a cylinder only report time-averaged measurements, precluding our ability to formally describe motions on a cycle-by-cycle basis. Here we present the first analytical model that describes the swimming kinematics of Kármán gaiting trout with 70-90% accuracy. We found that body bending kinematics can be modelled with a travelling wave equation, which has also been shown to accurately model free-stream swimming kinematics. However, free-stream swimming and Kármán gaiting are separated in the parameter space; the amplitude, wavelength and frequency values of the traveling wave equation are substantially different for each behavior. During Kármán gaiting, the wave is initiated at the body center, which is 0.2L (where L is total body length) further down the body compared with the initiation point in free-stream swimming. The wave travels with a constant speed, which is higher than the nominal flow speed just as in free-stream swimming. In addition to undulation, we observed that Kármán gaiting fish also exhibit substantial lateral translations and body rotations, which can constitute up to 75% of the behavior. These motions are periodic and their frequencies also match the vortex shedding frequency. There is an inverse correlation between head angle and body angle: when the body rotates in one direction, the head of the fish turns into the opposite direction. Our kinematic model mathematically describes how fish swim in vortical flows in real time and provides a platform to better understand the effects of flow variations as well as the contribution of muscle activity during corrective motions.

Changes in lactate kinetics as a function of exercise intensity have never been measured in an ectotherm. Continuous infusion of a tracer is necessary to quantify rates of lactate appearance (Ra) and disposal (Rd), but it requires double catheterization, which could interfere with swimming. Using rainbow trout, our goals were to: (1) determine the potential effects of catheters and blood sampling on metabolic rate (O2), total cost of transport (TCOT), net cost of transport (NCOT) and critical swimming speed (Ucrit), and (2) monitor changes in lactate fluxes during prolonged, steady-state swimming or graded swimming from rest to Ucrit. This athletic species maintains high baseline lactate fluxes of 24 μmol kg(-1) min(-1) that are only increased at intensities >2.4 body lengths (BL) s(-1) or 85% Ucrit. As the fish reaches Ucrit, Ra is more strongly stimulated (+67% to 40.4 μmol kg(-1) min(-1)) than Rd (+41% to 34.7 μmol kg(-1) min(-1)), causing a fourfold increase in blood lactate concentration. Without this stimulation of Rd during intense swimming, lactate accumulation would double. By contrast, steady-state exercise at 1.7 BL s(-1) increases lactate fluxes to ~30 μmol kg(-1) min(-1), with a trivial mismatch between Ra and Rd that only affects blood concentration minimally. Results also show that the catheterizations and blood sampling needed to measure metabolite kinetics in exercising fish have no significant impact on O2 or TCOT. However, these experimental procedures affect locomotion energetics by increasing NCOT at high speeds and by decreasing Ucrit.

In rainbow trout development, a switch occurs from high-affinity embryonic hemoglobin (Hb) and round, embryonic erythrocytes to lower-affinity adult Hb and oval, adult erythrocytes. Our study investigated the early ontogeny of rainbow trout blood properties and the hypoxia response. We hypothesized that hypoxia exposure would delay the ontogenetic turnover of Hb and erythrocytes because retention of high-affinity embryonic Hb would facilitate oxygen loading. To test this hypothesis we developed a method of efficiently extracting blood from individual embryos and larvae and optimized several techniques for measuring hematological parameters on microliter (0.5-2.0 μl) blood samples. In chronic hypoxia (30% of oxygen saturation), stage-matched embryos and larvae possessed half the Hb concentration, erythrocyte counts and hematocrit observed in normoxia. Hypoxia-reared larvae also had threefold to sixfold higher mRNA expression of the embryonic Hb α-1, β-1 and β-2 subunits relative to stage-matched normoxia-reared larvae. Furthermore, in hypoxia, the round embryonic erythrocytic shape persisted into later developmental stages. Despite these differences, Hb-oxygen affinity (P50), cooperativity and the Root effect were unaltered in hypoxia-reared O. mykiss. The data support our hypothesis that chronic hypoxia delays the ontogenetic turnover of Hb and erythrocytes, but without the predicted functional consequences (i.e. higher than expected P50). These results also suggest that the Hb-oxygen affinity is protected during development in chronic hypoxia to favor oxygen unloading at the tissues. We conclude that in early trout development, the blood-oxygen transport system responds very differently to chronic hypoxia relative to adults, possibly because respiration depends relatively more on oxygen diffusion than convection.

The brain monoaminergic neurotransmitter systems are known to be involved in the integrated response to stress in vertebrates. However, present knowledge about the timing of their actions as well as their specific roles in the regulation of the endocrine axes that drive the stress response is incomplete. This is partly because of the complexity of the reciprocal interactions among the monoaminergic systems and other biochemical effectors of the stress response such as corticotropin-releasing factor (CRF), arginine vasotocin (AVT), adrenocorticotropic hormone (ACTH) and corticosteroids. In this study, we show for the first time in teleost fish (rainbow trout) the short- and mid-term time course of the response of the forebrain serotonergic and dopaminergic activities after exposure to an acute stressor. Other stress markers like the plasma levels of cortisol, glucose and lactate were also monitored, providing a context in which to precisely locate the monoaminergic activation within the fish acute stress response. Our results show that acute stress induced a rapid increase in forebrain serotonergic activity, which became elevated after only 15 s of chasing. Several hours after stress, serotonergic activity recovered its basal levels, in parallel with the recovery of other stress markers such as plasma catecholamines and cortisol. Dopaminergic activity was also increased after stress, but only in the telencephalon and only after 20 min. The increase in serotonergic activity happened before the elevation of plasma catecholamines, suggesting that this monoamine system could have a key role in triggering the initial steps of the activation of not only the hypothalamus-pituitary-inter-renal axis but also the brain-sympathetic-chromaffin axis in fish.

Ammonia has been documented as a respiratory gas that stimulates ventilation, and is sensed by peripheral neuroepithelial cells (NECs) in the gills in ammoniotelic rainbow trout. However, the hyperventilatory response is abolished in trout chronically exposed (1+ months) to high environmental ammonia [HEA; 250 μmol l(-1) (NH4)2SO4]. This study investigates whether the brain is involved in the acute sensitivity of ventilation to ammonia, and whether changes in brain metabolism are related to the loss of hyperventilatory responses in trout chronically exposed to HEA ('HEA trout'). Hyperventilation (via increased ventilatory amplitude rather than rate) and increased total ammonia concentration ([TAmm]) in brain tissue were induced in parallel by acute HEA exposure in control trout in a concentration-series experiment [500, 750 and 1000 μmol l(-1) (NH4)2SO4], but these inductions were abolished in HEA trout. Ventilation was correlated more closely to [TAmm] in brain rather than to [TAmm] in plasma or cerebrospinal fluid. The close correlation of hyperventilation and increased brain [TAmm] also occurred in control trout acutely exposed to HEA in a time-series analysis [500 μmol l(-1) (NH4)2SO4; 15, 30, 45 and 60 min], as well as in a methionine sulfoxamine (MSOX) pre-injection experiment [to inhibit glutamine synthetase (GSase)]. These correlations consistently suggest that brain [TAmm] is involved in the hyperventilatory responses to ammonia in trout. The MSOX treatments, together with measurements of GSase activity, TAmm, glutamine and glutamate concentrations in brain tissue, were conducted in both the control and HEA trout. These experiments revealed that GSase plays an important role in transferring ammonia to glutamate to make glutamine in trout brain, thereby attenuating the elevation of brain [TAmm] following HEA exposure, and that glutamate concentration is reduced in HEA trout. The mRNAs for the ammonia channel proteins Rhbg, Rhcg1 and Rhcg2 were expressed in trout brain, and the expression of Rhbg and Rhcg2 increased in HEA trout, potentially as a mechanism to facilitate the efflux of ammonia. In summary, the brain appears to be involved in the sensitivity of ventilation to ammonia, and brain ammonia levels are regulated metabolically in trout.

We examined whether exogenous glucose affects contractile performance of electrically paced ventricle strips from rainbow trout under conditions known to alter cardiomyocyte performance, ion regulation and energy demands. Physiological levels of d-glucose did not influence twitch force development for aerobic preparations (1) paced at 0.5 or 1.1 Hz, (2) at 15 or 23°C, (3) receiving adrenergic stimulation or (4) during reoxygenation with or without adrenaline after severe hypoxia. Contractile responses to ryanodine, an inhibitor of Ca(2+) release from the sarcoplasmic reticulum, were also not affected by exogenous glucose. However, glucose did attenuate the fall in twitch force during severe hypoxia. Glucose uptake was assayed in non-contracting ventricle strips using 2-[(3)H] deoxy-d-glucose (2-DG) under aerobic and hypoxic conditions, at different incubation temperatures and with different inhibitors. Based upon a lack of saturation of 2-DG uptake and incomplete inhibition of uptake by cytochalasin B and d-glucose, 2-DG uptake was mediated by a combination of facilitated transport and simple diffusion. Hypoxia stimulated lactate efflux sixfold to sevenfold with glucose present, but did not increase 2-DG uptake or reduce lactate efflux in the presence of cytochalasin B. Increasing temperature (14 to 24°C) also did not increase 2-DG uptake, but decreasing temperature (14 to 4°C) reduced 2-DG uptake by 45%. In conclusion, exogenous glucose improves mechanical performance under hypoxia but not under any of the aerobic conditions applied. The extracellular concentration of glucose and cold temperature appear to determine and limit cardiomyocyte glucose uptake, respectively, and together may help define a metabolic strategy that relies predominantly on intracellular energy stores.

We have little understanding of how fish hold station in unsteady flows. Here, we investigated the effect of flow speed and body size on the kinematics of rainbow trout Kármán gaiting behind a 5 cm diameter cylinder. We established a set of criteria revealing that not all fish positioned in a vortex street are Kármán gaiting. By far the highest probability of Kármán gaiting occurred at intermediate flow speeds between 30 and 70 cm s(-1). We show that trout Kármán gait in a region of the cylinder wake where the velocity deficit is about 40% of the nominal flow. We observed that the relationships between certain kinematic and flow variables are largely preserved across flow speeds. Tail-beat frequency matched the measured vortex shedding frequency, which increased linearly with flow speed. Body wave speed was about 25% faster than the nominal flow velocity. At speeds where fish have a high probability of Kármán gaiting, body wavelength was about 25% longer than the cylinder wake wavelength. Likewise, the lateral (i.e. cross-stream) amplitude of the tail tip was about 50% greater than the expected lateral spacing of the cylinder vortices, while the body center amplitude was about 70% less. Lateral body center acceleration increased quadratically with speed. Head angle decreased with flow speed. While these values are different from those found in fish swimming in uniform flow, the strategy for locomotion is the same; fish adjust to increasing flow by increasing their tail-beat frequency. Body size also played a role in Kármán gaiting kinematics. Tail-beat amplitudes of Kármán gaiting increased with body size, as in freestream swimming, but were almost three times larger in magnitude. Larger fish had a shorter body wavelength and slower body wave speed than smaller fish, which is a surprising result compared with freestream swimming, where body wavelength and wave speed increased with size. In contrast to freestream swimming, tail-beat frequency for Kármán gaiting fish did not depend on body size and was a function of the vortex shedding frequency.

Calcitonin gene-related peptide (CGRP) and its receptors are widely distributed in the tissues of teleost fish, including the brain, but little is known about the ventilatory and cardiovascular effects of the peptide in these vertebrates. The present study was undertaken to compare the central and peripheral actions of graded doses (5-50 pmol) of trout CGRP on ventilatory and cardiovascular variables in unanesthetized rainbow trout. Compared with vehicle, intracerebroventricular injection of CGRP significantly elevated the ventilation frequency (f(V)) and the ventilation amplitude (V(AMP)) and, consequently, the total ventilation (V(TOT)). The maximum hyperventilatory effect of CGRP (V(TOT): +300%), observed at a dose of 50 pmol, was mostly due to its stimulatory action on V(AMP) (+200%) rather than f(V) (+30%). In addition, CGRP produced a significant and dose-dependent increase in mean dorsal aortic blood pressure (P(DA)) (50 pmol: +40%) but the increase in heart rate (f(H)) was not significant. Intra-arterial injections of CGRP were without effect on the ventilatory variables but significantly and dose-dependently elevated P(DA) (50 pmol: +36%) without changing f(H). At the highest dose tested, this hypertensive phase was preceded by a rapid and transient hypotensive response. In conclusion, our study suggests that endogenous CGRP within the brain of the trout may act as a potent neurotransmitter and/or neuromodulator in the regulation of cardio-ventilatory functions. In the periphery, endogenous CGRP may act as a local and/or circulating hormone preferentially involved in vasoregulatory mechanisms.

Teleost fish lack independent olfactory organs for odorant and pheromone detection. Instead, they have a single sensory epithelium with two populations of receptor neurons, ciliated and microvillous, that are conserved among vertebrates, and a unique receptor cell type named the olfactory crypt cell. Crypt cells were shown to be chemosensory neurons that project to specific areas in the olfactory bulb, but their odorant tuning and overall function remain unclear. Reproduction in fish is generally synchronized by sex pheromonal signaling between males and females, but the sensors responsible for pheromone detection remain unknown. In crucian carp, a seasonal variation in the population of olfactory crypt cells and their brain projections pathways, involved in reproduction, led to the hypothesis of a role as sex pheromone detectors. In the present study, morphology and localization of olfactory crypt cells were compared between juvenile and mature rainbow trout of both sexes, and calcium imaging was used to visualize responses of crypt cells from the three groups to common social and food-related odorants, sex hormones and conspecific tissue extracts. Crypt cells from mature trout were found to be larger than those of juvenile specimens, and preferentially localized to the apical surface of the olfactory epithelium. Although a fraction of crypt cells of all groups responded to common odorants such as amino acids and bile salts, cells from mature trout showed a characteristic preference for gonadal extracts and hormones from the opposite sex. These results support an involvement of olfactory crypt cells in reproduction-related olfactory signaling in fishes.

Trigeminally innervated, mechanically sensitive chemoreceptors (M) were previously identified in rainbow trout, Oncorhynchus mykiss, but it is not known whether these receptors are responsive only to noxious, chemical irritants or have a general chemosensory function. This study aimed to characterise the stimulus-response properties of these receptors in comparison with polymodal nociceptors (P). Both P and M gave similar response profiles to acetic acid concentrations. The electrophysiological properties were similar between the two different afferent types. To determine whether the receptors have a nociceptive function, a range of chemical stimulants was applied to these receptors, including non-noxious stimuli such as ammonium chloride, bile, sodium bicarbonate and alarm pheromone, and potentially noxious chemical irritants such as acetic acid, carbon dioxide, low pH, citric acid, citric acid phosphate buffer and sodium chloride. Only irritant stimuli evoked a response, confirming their nociceptive function. All receptor afferents tested responded to carbon dioxide (CO(2)) in the form of mineral water or soda water. The majority responded to 1% acetic acid, 2% citric acid, citric acid phosphate buffer (pH 3) and 5.0 mol l(-1) NaCl. CO(2) receptors have been characterised in the orobranchial cavity and gill arches in fish; however, this is the first time that external CO(2) receptors have been identified on the head of a fish. Because the fish skin is in constant contact with the aqueous environment, contaminants with a low pH or hypercapnia may stimulate the nociceptive system in fish.

Hydrogen sulfide (H(2)S) has been shown to affect gastrointestinal (GI) motility and signaling in mammals and O(2)-dependent H(2)S metabolism has been proposed to serve as an O(2) 'sensor' that couples hypoxic stimuli to effector responses in a variety of other O(2)-sensing tissues. The low P(O2) values and high H(2)S concentrations routinely encountered in the GI tract suggest that H(2)S might also be involved in hypoxic responses in these tissues. In the present study we examined the effect of H(2)S on stomach, esophagus, gallbladder and intestinal motility in the rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) and we evaluated the potential for H(2)S in oxygen sensing by examining GI responses to hypoxia in the presence of known inhibitors of H(2)S biosynthesis and by adding the sulfide donor cysteine (Cys). We also measured H(2)S production by intestinal tissue in real time and in the presence and absence of oxygen. In tissues exhibiting spontaneous contractions, H(2)S inhibited contraction magnitude (area under the curve and amplitude) and frequency, and in all tissues it reduced baseline tension in a concentration-dependent relationship. Longitudinal intestinal smooth muscle was significantly more sensitive to H(2)S than other tissues, exhibiting significant inhibitory responses at 1-10 μmol l(-1) H(2)S. The effects of hypoxia were essentially identical to those of H(2)S in longitudinal and circular intestinal smooth muscle; of special note was a unique transient stimulatory effect upon application of both hypoxia and H(2)S. Inhibitors of enzymes implicated in H(2)S biosynthesis (cystathionine β-synthase and cystathionine γ-lyase) partially inhibited the effects of hypoxia whereas the hypoxic effects were augmented by the sulfide donor Cys. Furthermore, tissue production of H(2)S was inversely related to O(2); addition of Cys to intestinal tissue homogenate stimulated H(2)S production when the tissue was gassed with 100% nitrogen (~0% O(2)), whereas addition of oxygen (~10% O(2)) reversed this to net H(2)S consumption. This study shows that the inhibitory effects of H(2)S on the GI tract of a non-mammalian vertebrate are identical to those reported in mammals and they provide further evidence that H(2)S is a key mediator of the hypoxic response in a variety of O(2)-sensitive tissues.

Steelhead rainbow trout acclimated to either freshwater (FW) or seawater (SW) were exposed to high environmental ammonia (HEA, 1000 μmol l(-1) NH(4)HCO(3), pH 7.8-8.0) for 24 h. SW trout restored ammonia excretion more rapidly (3-6 h versus 9-12 h in FW), despite higher production rates and lower plasma pH. Plasma total ammonia levels stabilized at comparable levels below the external HEA concentration, and blood acid-base disturbances were small at both salinities. The electrochemical gradients for NH(4)(+) entry (F(NH(4))(+)) were the same in the two salinities, but only because FW trout allowed their transepithelial potential to rise by ∼15 mV during HEA exposure. Elevation of plasma [cortisol] during HEA exposure was more prolonged in SW fish. Plasma [glucose] increased in SW, but decreased in FW trout. Plasma [urea-N] also decreased in FW, in concert with elevated urea transporter (UT) mRNA expression in the gills. Of 13 branchial transporters, baseline mRNA expression levels were higher for Rhcg1, NHE2, NKCC1a and UT, and lower for NBC1 and NKA-α1a in SW trout, whereas NKA-α1b, NHE3, CA2, H(+)-ATPase, Rhag, Rhbg and Rhcg2 did not differ. Of the Rh glycoprotein mRNAs responding to HEA, Rhcg2 was greatly upregulated in both FW and SW, Rhag decreased only in SW and Rhcg1 decreased only in FW. H(+)-ATPase mRNA increased in FW whereas NHE2 mRNA increased in SW; NHE3 did not respond, and V-type H(+)-ATPase activity declined in SW during HEA exposure. Branchial Na(+),K(+)-ATPase activity was much higher in SW gills, but could not be activated by NH(4)(+). Overall, the more effective response of SW trout was explained by differences in physical chemistry between SW and FW, which greatly reduced the plasma NH(3) tension gradient for NH(3) entry, as well as by the higher [Na(+)] in SW, which favoured Na(+)-coupled excretion mechanisms. At a molecular level, responses in SW trout showed subtle differences from those in FW trout, but were very different than in the SW pufferfish. Upregulation of Rhcg2 appears to play a key role in the response to HEA in both FW and SW trout, and NH(4)(+) does not appear to move through Na(+),K(+)-ATPase.

Ammonia, the third respiratory gas in teleost fish, acts as an acute stimulant to ventilation in ammoniotelic rainbow trout. We investigated whether this sensitivity is maintained in trout chronically exposed (1+ months) to high environmental ammonia [HEA, 250 μmol l(-1) (NH(4))(2)SO(4)] in the water, and whether gill neuroepithelial cells (NECs) are involved in ammonia sensing. Hyperventilation was induced both by acute external (NH(4))(2)SO(4) exposure [250 or 500 μmol l(-1) (NH(4))(2)SO(4)] and by intra-arterial (NH(4))(2)SO(4) injection (580 μmol kg(-1) of ammonia) in control trout, but these responses were abolished in chronic HEA animals. Hyperventilation in response to acute ammonia exposure persisted after bilateral removal of each of the four gill arch pairs separately or after combined removal of arches III and IV, but was delayed by removal of gill arch I, and eliminated by combined removal of arches I and II. NECs, identified by immunolabeling against 5-HT, were mainly organized in two lines along the filament epithelium in all four gill arches. In control trout, NECs were slightly smaller but more abundant on arches I and II than on arches III and IV. Chronic HEA exposure reduced the density of the NECs on all four arches, and their size on arches I and II only. Fura-2 fluorescence imaging was used to measure intracellular free calcium ion concentration ([Ca(2+)](i)) responses in single NECs in short-term (24-48 h) culture in vitro. [Ca(2+)](i) was elevated to a comparable extent by perfusion of 30 mmol l(-1) KCl and 1 mmol l(-1) NH(4)Cl, and these [Ca(2+)](i) responses presented in two different forms, suggesting that ammonia may be sensed by multiple mechanisms. The [Ca(2+)](i) responses to high ammonia were attenuated in NECs isolated from trout chronically exposed to HEA, especially in ones from gill arch I, but responses to high K(+) were unchanged. We conclude that the hyperventilatory response to ammonia is lost after chronic waterborne HEA exposure, and that NECs, especially the ones located in gill arches I and II, are probably ammonia chemoreceptors that participate in ventilatory modulation in trout.

The intestine of marine teleosts produces carbonate precipitates from ingested calcium as part of their osmoregulatory strategy in seawater. The potential for estrogens to control the production of intestinal calcium carbonate and so influence osmoregulation was investigated in seawater-acclimated rainbow trout following intraperitoneal implantation of 17β-estradiol (E2) at two doses (0.1 and 10 μg E2 g(-1)). Levels of plasma vitellogenin provided an indicator of estrogenic effect, increasing significantly by three and four orders of magnitude at the low and high doses, respectively. Plasma osmolality and muscle water content were unaffected, whereas E2-treated fish maintained lower plasma [Na(+)] and [Cl(-)]. Plasma [Ca(2+)] and [Mg(2+)] and muscle [Ca(2+)] increased with vitellogenin induction, whereas the intestinal excretion of calcium carbonate was reduced. This suggests that elevated levels of circulating E2 may enhance Ca(2+) uptake via the gut and simultaneously reduce CaCO(3) formation, which normally limits intestinal availability of Ca(2+). Increasing E2 caused an elevation of [Na(+)] and [Cl(-)] and a reduction of [HCO(3(-))] in intestinal fluid. We speculate that E2 may influence a number of intestinal ion transport processes that ultimately may influence water absorption: (1) reduced NaCl cotransport, (2) reduced Cl(-) uptake via Cl(-)/HCO(3(-)) exchange and (3) reduced precipitation of Ca(2+) and Mg(2+) carbonates. Despite these effects on intestinal ion and water transport, overall osmoregulatory status was not compromised in E2-treated fish, suggesting the possibility of compensation by other organs.

β-Adrenergic stimulation of the mammalian heart increases heart rate, the strength of contraction as well as the kinetics of force generation and relaxation. These effects are due to the phosphorylation of select membrane and thin filament proteins by cAMP-activated protein kinase (PKA). At the level of the sarcomere, it is typically the phosphorylation of cardiac myosin binding protein C (cMyBP-C) and cardiac troponin I (cTnI) that is responsible for the change in the kinetics of contraction and relaxation. Trout cTnI (ScTnI) lacks two critical PKA targets within the N-terminus of the protein that, when phosphorylated in mammalian cTnI, cause a reduction in myofilament Ca(2+) affinity. To determine what role the contractile element plays in the response of the trout heart to β-adrenergic stimulation, we characterized the influence of PKA treatment on the Ca(2+) activation of skinned preparations dissected from ventricular trabeculae. In these experiments, isometric force generation and the rate of force development were measured over a range of Ca(2+) concentrations. The results demonstrate that PKA treatment does not influence the Ca(2+) sensitivity of force generation but it decreases maximum force generation by 25% and the rate of force re-development at maximal activation by 46%. Analysis of the trabeculae preparations for phosphoproteins revealed that PKA treatment phosphorylated myosin light chain 2 but not cTnI or cMyBP-C. These results indicate that the function of the trout cardiac contractile element is altered by PKA phosphorylation but in a manner different from that in mammalian heart.

Salmonid embryos develop in cool waters over relatively long periods of time. Interestingly, hypoxic conditions have been found to be relatively common in some nesting sites (redds). The goals of this study were to determine the ontogeny of cardiac regulation in rainbow trout early life stages and how this is influenced by chronic hypoxia. The heart rate response to cholinergic and adrenergic receptor stimulation or inhibition was measured in individuals reared in normoxic (100% O(2) saturation) or hypoxic (30% O(2) saturation) conditions from fertilization to embryonic stages 22, 26 and 29, and larval stages 30 and 32. In normoxia, heart rate increased in response to β-adrenergic receptor stimulation (isoproterenol) as early as embryonic stage 22, and decreased with the antagonist propranolol after this stage. Cholinergic stimulation (acetylcholine) was ineffective at all stages, but atropine (acetylcholine antagonist) increased heart rate at larval stage 32. This demonstrates that cardiac β-adrenergic receptors are functional at early life stages, while cholinergic receptors are not responsive until after hatching. Collectively, embryos had cardio-acceleration control mechanisms in place just after the heartbeat stage, while cardio-inhibitory control was not functional until after hatching. Chronic hypoxia exposure triggered bradycardia, increased the response to adrenergic stimulation in embryos and larvae, and delayed the onset of cholinergic control in larvae. In non-motile stages, therefore, survival in chronic low oxygen may depend on the ability to alter the cardiac ontogenic program to meet the physiological requirements of the developing fish.

When faced with limited resources, juvenile salmonid fish form dominance hierarchies that result in social stress for socially subordinate individuals. Social stress, in turn, can have consequences for the ability of the fish to respond to additional stressors such as pathogens or exposure to pollutants. In the present study, the possibility that social stress affects the ability of rainbow trout (Oncorhynchus mykiss) to tolerate acute increases in water temperature was investigated. To this end, we first evaluated physiological and cellular stress responses following a 1 h heat shock in juvenile fish in dominance hierarchies. We measured stress hormone (cortisol and catecholamines) concentrations and blood, brain and liver tissue levels of three heat shock proteins (HSPs), the stress inducible HSP70, the constitutive HSC70 and HSP90, in dominant and subordinate trout. No effects of social status on the hormonal response to the heat stress were detected, but the cellular heat shock response in the brain and liver of dominant and subordinate individuals was inhibited. We then assessed thermal tolerance in dominant and subordinate fish through critical thermal maximum temperature (CT(max)) trials and measured HSPs following the heat shock. Subordinate fish were less thermally tolerant than their dominant counterparts. We conclude that social stress impacts the ability of fish to respond, on a cellular scale and in a tissue-specific manner, to increases in water temperature, with likely consequences for overall fitness.