Cold acclimation induces life stage-specific responses in the cardiac proteome of western painted turtles (Chrysemys picta bellii): implications for anoxia tolerance.

Western painted turtles (Chrysemys picta bellii) are the most anoxia-tolerant tetrapod. Survival time improves at low temperature and during ontogeny, such that adults acclimated to 3°C survive far longer without oxygen than either warm-acclimated adults or cold-acclimated hatchlings. As protein synthesis is rapidly suppressed to save energy at the onset of anoxia exposure, this study tested the hypothesis that cold acclimation would evoke preparatory changes in protein expression to support enhanced anoxia survival in adult but not hatchling turtles. To test this, adult and hatchling turtles were acclimated to either 20°C (warm) or 3°C (cold) for 5 weeks, and then the heart ventricles were collected for quantitative proteomic analysis. The relative abundance of 1316 identified proteins was compared between temperatures and developmental stages. The effect of cold acclimation on the cardiac proteome was only evident in the context of an interaction with life stage, suggesting that ontogenic differences in anoxia tolerance may be predicated on successful maturation of the heart. The main differences between the hatchling and adult cardiac proteomes reflect an increase in metabolic scope with age that included more myoglobin and increased investment in both aerobic and anaerobic energy pathways. Mitochondrial structure and function were key targets of the life stage- and temperature-induced changes to the cardiac proteome, including reduced Complex II proteins in cold-acclimated adults that may help down-regulate the electron transport system and avoid succinate accumulation during anoxia. Therefore, targeted cold-induced changes to the cardiac proteome may be a contributing mechanism for stage-specific anoxia tolerance in turtles.

Social stress increases plasma cortisol and reduces forebrain cell proliferation in subordinate male zebrafish (Danio rerio).

Many animals, including zebrafish (Danio rerio), form social hierarchies through competition for limited resources. Socially subordinate fish may experience chronic stress, leading to prolonged elevation of the glucocorticoid stress hormone cortisol. As elevated cortisol levels can impair neurogenesis, the present study tested the hypothesis that social stress suppresses cell proliferation in the telencephalon of subordinate zebrafish via a cortisol-mediated mechanism. Cell proliferation was assessed using incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). After 48 and 96 h of social interaction, subordinate male zebrafish exhibited elevated plasma cortisol concentrations and significantly lower numbers of BrdU+ cells in the dorsal but not ventral regions of the telencephalon compared with dominant or group-housed control male fish. After a 2 week recovery in a familiar group of conspecifics, the number of BrdU+ cells that co-labelled with a neuronal marker (NeuN) was modestly reduced in previously subordinate male fish, suggesting that the reduction of cell proliferation during social stress may result in fewer cells recruited into the neuronal population. In contrast to male social hierarchies, subordinate female zebrafish did not experience elevated plasma cortisol, and the number of BrdU+ cells in the dorsal telencephalic area was comparable among dominant, subordinate and group-housed control female fish. Treating male zebrafish with metyrapone, a cortisol synthesis inhibitor, blocked the cortisol response to social subordination and attenuated the suppression of brain cell proliferation in the dorsal telencephalic area of subordinate fish. Collectively, these data support a role for cortisol in regulating adult neurogenesis in the telencephalon of male zebrafish during social stress.

Corticotropin-releasing factor regulates caspase-3 and may protect developing zebrafish from stress-induced apoptosis.

The corticotropin-releasing factor (CRF) system is expressed in the earliest stages of zebrafish development, long before its canonical function in the endocrine stress response is realized, and yet its function during embryogenesis is unknown. We tested the hypothesis that CRF protects embryos from stress-induced apoptosis. Here we confirm that a 1 h heat shock applied at either 6 h post-fertilization (hpf) or 30 hpf elicits an increase in caspase-3 activity, a key effector of apoptosis. Temporal changes in the expression of crf and its binding protein (crf-bp) during recovery from heat shock indicate that the CRF system is responsive to stressors experienced as early as gastrulation. Next, we heat shocked embryos that were microinjected with crf mRNA, and showed that caspase-3 induction is significantly reduced in embryos that overexpress CRF relative to control embryos. In addition, incubating embryos in the presence of the CRF receptor type 1 (CRF-R1) antagonist, antalarmin, during recovery from heat shock significantly increased caspase-3 activity, suggesting that CRF regulates caspase-3 via a CRF-R1-dependent pathway. Finally, we show that most heat shock-induced mortality occurred during the first hour of recovery, long before a significant increase in caspase-3 activity was detected. Indeed, the delayed caspase-3 induction coincided with a mortality plateau, and neither CRF overexpression nor antalarmin treatment altered heat shock induced mortality, supporting previous in vitro evidence that CRF-mediated cytoprotection occurs through the slow and tightly controlled apoptotic pathway. This study provides novel in vivo evidence that CRF regulates stress-induced apoptosis in a vertebrate model species, and demonstrates for the first time a function for the CRF system in early development that precedes its role in the endocrine stress response.

Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that may enhance oxygen delivery to the myocardium.

Oxygen supply to the heart of most teleosts, including salmonids, relies in part or in whole on oxygen-depleted venous blood. Given that plasma-accessible carbonic anhydrase (CA) in red muscle of rainbow trout has recently been shown to facilitate oxygen unloading from arterial blood under certain physiological conditions, we tested the hypothesis that plasma-accessible CA is present in the lumen of coho salmon (Oncorhynchus kisutch) hearts, and may therefore assist in the luminal oxygen supply to the spongy myocardium, which has no coronary circulation. We demonstrate a widespread distribution of CA throughout the heart chambers, including lumen-facing cells in the atrium, and confirm that the membrane-bound isoform ca4 is expressed in the atrium and ventricle of the heart. Further, we confirm that CA catalytic activity is available to blood in the atrial lumen using a modified electrometric ΔpH assay in intact atria in combination with either a membrane-impermeable CA inhibitor or specific cleavage of the Ca4 membrane anchor. Combined, these results support our hypothesis of the presence of an enhanced oxygen delivery system in the lumen of a salmonid heart, which could help support oxygen delivery when the oxygen content of venous blood becomes greatly reduced, such as after burst exercise and during environmental hypoxia.

Effect of cold acclimation on troponin I isoform expression in striated muscle of rainbow trout.

In vertebrates each of the three striated muscle types (fast skeletal, slow skeletal, and cardiac) contain distinct isoforms of a number of different contractile proteins including troponin I (TnI). The functional characteristics of these proteins have a significant influence on muscle function and contractility. The purpose of this study was to characterize which TnI gene and protein isoforms are expressed in the different muscle types of rainbow trout (Oncorhynchus mykiss) and to determine whether isoform expression changes in response to cold acclimation (4°C). Semiquantitative real-time PCR was used to characterize the expression of seven different TnI genes. The sequence of these genes, cloned from Atlantic salmon (Salmo salar) and rainbow trout, were obtained from the National Center for Biotechnology Information databases. One-dimensional gel electrophoresis and tandem mass spectrometry were used to identify the TnI protein isoforms expressed in each muscle type. Interestingly, the results indicate that each muscle type expresses the gene transcripts of up to seven TnI isoforms. There are significant differences, however, in the expression pattern of these genes between muscle types. In addition, cold acclimation was found to increase the expression of specific gene transcripts in each muscle type. The proteomics analysis demonstrates that fast skeletal and cardiac muscle contain three TnI isoforms, whereas slow skeletal muscle contains four. No other vertebrate muscle to date has been found to express as many TnI protein isoforms. Overall this study underscores the complex molecular composition of teleost striated muscle and suggests there is an adaptive value to the unique TnI profiles of each muscle type.

Central and peripheral glucocorticoid receptors are involved in the plasma cortisol response to an acute stressor in rainbow trout.

Cortisol, the primary circulating corticosteroid in teleosts, is elevated during stress following activation of the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol exerts genomic effects on target tissues in part by activating glucocorticoid receptors (GR). Despite a well-established negative feedback loop involved in plasma cortisol regulation, the role of GR in the functioning of the HPI axis during stress in fish is still unclear. We used mifepristone (a GR antagonist) to suppress GR signaling in rainbow trout (Oncorhynchus mykiss) and assessed the resultant changes to HPI axis activity. We show for the first time that mifepristone caused a functional knockdown of GR by depleting protein expression 40-75%. The lower GR protein expression corresponded with a compensatory up-regulation of GR mRNA levels across tissues. Mifepristone treatment completely abolished the stressor-induced elevation in plasma cortisol and glucose levels seen in the control fish. A reduction in corticotropin-releasing factor (CRF) mRNA abundance in the hypothalamic preoptic area was also observed, suggesting that GR signaling is involved in maintaining basal CRF levels. We further characterized the effect of mifepristone treatment on the steroidogenic capacity of interrenal tissue in vitro. A marked reduction in cortisol production following adrenocorticotropic hormone stimulation of head kidney pieces was observed from mifepristone treated fish. This coincided with the suppression of steroidogenic acute regulatory protein, but not P450 side chain cleavage mRNA abundances. Overall, our results underscore a critical role for central and peripheral GR signaling in the regulation of plasma cortisol levels during stress in fish.

Diluted Bitumen Affects Multiple Physiological Systems in Sockeye Salmon (Oncorhynchus nerka) Embryo to Juvenile Life Stages.

An understanding of the risks associated with diluted bitumen (dilbit) transport through Pacific salmon habitat necessitates the identification and quantification of hazards posed to early life stages. Sockeye from the embryo to juvenile stage (8 months old) were exposed to four concentrations of the water-soluble fraction of Cold Lake dilbit (summer blend; concentrations of 0, 13.7, 34.7, and 124.5 µg/L total polycyclic aromatic compounds). Significant mortality (up to 18% over controls) only occurred in the embryo to swim-up fry stage. Impaired growth was seen in the alevin, swim-up, and juvenile stages (maximum reduction 15% in mass but not fork length). Reductions in both critical (maximum 24% reductions) and burst (maximum 47% reductions) swimming speed in swim-up fry and juveniles were seen. Alterations in energy substrate reserves (reductions in soluble protein and glycogen content, elevations in whole-body lipid and triglyceride levels) at all stages may underlie the effects seen in swimming and growth. Dilbit exposure induced a preexercise physiological stress response that affected the recovery of postexercise biochemistry (cortisol, glycogen, lactate, triglyceride concentrations). The transcript abundance of the cytochrome P450 1A gene (cyp1a) was quantified in alevin head regions (containing the heart) and in the hearts of swim-up fry and juveniles and showed a concentration-dependent increase in the expression of cyp1a at all life stages. Environ Toxicol Chem 2022;41:1937-1949. © 2022 SETAC.

Age matters: Comparing life-stage responses to diluted bitumen exposure in coho salmon (Oncorhynchus kisutch).

Millions of liters of diluted bitumen (dilbit), a crude oil product from Canada's oil sands region, is transported through critical Pacific salmon habitat each day. While the toxicity of the water-soluble fraction of dilbit (WSFd) to early life-stages of salmon is known, quantitative data on life-stage differences in sensitivity to WSFd is missing. To fill this knowledge gap, we exposed two juvenile life-stages of coho salmon (O. kisutch) in parallel to very low (parts per billion), environmentally-relevant concentrations of WSFd for acute (48 h) and sub-chronic (4 wk) durations. The relative sensitivities of the two life-stages (fry and parr) were assessed by comparing the timing and magnitude of biological responses using common organismal and molecular endpoints of crude oil exposure. A significant reduction in body condition occurred in both fry and parr after 4 wk exposure to WSFd. Both life-stages also experienced a concentration-dependent decrease in time-to-loss-of-equilibrium during a hypoxia challenge test at both 48 h and 4 wk of exposure. Although organismal responses were similar, molecular responses were distinct between life-stages. In general, unexposed fry had higher baseline values of hepatic phase I biotransformation indicators than unexposed parr, but induction of EROD activity and cyp1a mRNA expression in response to WSFd exposure was greater in parr than in fry. Neither gst nor hsp70 mRNA expression, markers of phase II biotransformation and cell stress, respectively, were reliably altered by WSFd exposure in either life-stage. Taken together, results of this study do not support differential sensitivities of coho fry and parr to WSFd. All the same, the potential for ontogenic differences in the expression and induction of phase I biotransformation need to be considered because age does matter for these endpoints if they are used as bioindicators of exposure in post-spill impact assessments.

Ontogeny of the corticotropin-releasing factor system in rainbow trout and differential effects of hypoxia on the endocrine and cellular stress responses during development.

To further our understanding of the development of the stress axis and the responsiveness of embryonic and larval fish to environmental stressors, this study examined the ontogeny of whole-body cortisol levels and of the corticotropin-releasing factor (CRF) system in rainbow trout, as well as the endocrine and cellular stress responses to hypoxia. After depletion of a maternal deposit, de novo synthesis of cortisol increased exponentially between the 'eyed' stage and first feeding. Whole body CRF mRNA levels dominated over those of the related peptide urotensin I (UI) from hatch through complete yolk sac absorption. The mRNA levels of CRF-binding protein (CRF-BP) closely paralleled those of CRF and UI throughout ontogeny except at first feeding when an increase in CRF gene expression was not matched by change in CRF-BP transcript abundance. In the hypoxia challenge, fish were exposed to 15% O(2) saturation for either 90 min or 24h at three key developmental stages: hatch, swim up and first feeding. While the embryos were unaffected, chronic hypoxia elicited a transient 2-fold increase in whole-body cortisol levels in the larval stages. The hypoxia challenge also generally suppressed the mRNA levels of CRF and CRF-BP, had no effect on the expression of UI, but had a marked stimulatory effect on heat shock protein 70 (Hsp70) gene expression. Taken together, these results suggest a role for the CRF system in the ontogenic regulation of corticosteroidogenesis and show that hypoxia has developmental stage-specific effects on the endocrine and cellular stress responses in rainbow trout.

Growing up gator: a proteomic perspective on cardiac maturation in an oviparous reptile, the American alligator (Alligator mississippiensis).

We recently described lasting changes in the cardiac proteome of American alligators (Alligator mississippiensis) reared under hypoxic conditions, that resemble what embryos encounter in natural nests. While these changes were consistent with functional differences in cardiac performance induced by developmental hypoxia, the magnitude of this response was dwarfed by a much greater effect of development alone (76% of the total differentially abundant proteins). This means that substantial differences in relative steady-state protein expression occur in the hearts of alligators as they mature from egg-bound embryos to 2-year-old juveniles, and this developmental program is largely resistant to variation in nest conditions. We therefore performed functional enrichment analysis of the 412 DA proteins that were altered by development but not hypoxia, to gain insight into the mechanisms of cardiac maturation in this ectotherm. We found that the cardiac proteome of alligators at 90% of embryonic development retained a considerable capacity for transcription and translation, suggesting the heart was still primarily invested in growth even as the animal approached hatching. By contrast, the cardiac proteome of 2-year-old juveniles was weighted towards structural and energetic processes typical of a working heart. We discuss our results in the context of differences in cardiac development between ectothermic and endothermic oviparous vertebrates, and argue that the robust developmental program of the alligator heart reflects a slow-paced ontogeny, unburdened by the requirement to support the elevated peripheral oxygen demand typical of endothermic animals from a young age.

Effects of diluted bitumen exposure on Atlantic salmon smolts: Molecular and metabolic responses in relation to swimming performance.

Canada's oil sands industry continues to expand and the volume of diluted bitumen (dilbit) transported across North America is increasing, adding to spill risk and environmental contamination. Dilbit exposure is known to cause adverse effects in fish, but linking molecular and cellular changes with ecologically-relevant individual performance metrics is needed to better understand the potential consequences of a dilbit spill into the aquatic environment. Therefore, this study examined the effects of dilbit exposure on subcellular responses in cardiac and skeletal muscle in relation to swimming performance in a migratory fish species at risk of exposure, Atlantic salmon. Smolts were exposed subchronically to environmentally relevant concentrations of the water-soluble fraction of dilbit (WSFd) for 24 d, and then a subset of exposed fish underwent a depuration period of 7 or 14 d, for a total of 3 experimental time points. At each time point, repeat swimming performance was assessed using sequential critical swimming speed tests (Ucrit) separated by a 24 h rest period, and then several tissues were collected to determine biotransformation enzyme activation, energetic responses, and gene expression changes. Ucrit was unaffected in fish exposed to 67.9 µg/L total initial polycyclic aromatic compounds (PAC), but fish showed a decreased reliance on lipid metabolism for adenosine triphosphate (ATP) in the heart that was maintained through 7 d depuration. In contrast, Ucrit increased in fish exposed to 9.65 µg/L PAC, corresponding to an increased reliance on anaerobic metabolic pathways in cardiac and red skeletal muscle, with partial recovery after 7 d depuration. As expected, at both concentrations WSFd hepatic cyp 1A-mediated biotransformation reactions increased, as measured by EROD activity, which remained elevated for 7 d but not after 14 d depuration. Transcript abundance of cyp1a was also increased in muscle tissue and recovered by 14 d depuration. The expression of other stress-related genes increased in white muscle of dilbit-exposed fish, but were largely unchanged in cardiac and red muscle. The transcriptional profile of cardiac tissue was compared to that of sockeye salmon similarly exposed to WSFd in a previous experiment, and is provided in supplemental text. Combined, these results demonstrate that dilbit exposure alters gene expression and enzyme activities related to xenobiotic exposure, cellular stress, and muscle energetics in juvenile Atlantic salmon without impairing swimming performance, and that most of these changes are recoverable within 14 d depuration.

Effects of diluted bitumen exposure and recovery on the seawater acclimation response of Atlantic salmon smolts.

Petrogenic chemicals are common and widespread contaminants in the aquatic environment. In Canada, increased extraction of bitumen from the oil sands and transport of the major crude oil export product, diluted bitumen (dilbit), amplifies the risk of a spill and contamination of Canadian waterways. Fish exposed to sublethal concentrations of crude oil can experience a variety of adverse physiological effects including osmoregulatory dysfunction. As regulation of water and ion balance is crucial during the seawater transition of anadromous fish, the hypothesis that dilbit impairs seawater acclimation in Atlantic salmon smolts (a fish at risk of exposure in Canada) was tested. Smolts were exposed for 24 d to the water-soluble fraction of dilbit in freshwater, and then transferred directly to seawater or allowed a 1 wk depuration period in uncontaminated freshwater prior to seawater transfer. The seawater acclimation response was quantified at 1 and 7 d post-transfer using established hematological, tissue, and molecular endpoints including gill Na+/K+-ATPase gene expression (nka). All smolts, irrespective of dilbit exposure, increased serum Na+ concentrations and osmolality within 1 d of seawater transfer. The recovery of these parameters to freshwater values by 7 d post-transfer was likely driven by the increased expression and activity of Na+/K+-ATPase in the gill. Histopathological changes in the gill were not observed; however, CYP1A-like immunoreactivity was detected in the pillar cells of gill lamellae of fish exposed to 67.9 µg/L PAC. Concentration-specific changes in kidney expression of a transmembrane water channel, aquaporin 3, occurred during seawater acclimation, but were resolved with 1 wk of depuration and were not associated with histopathological changes. In conclusion, apart from a robust CYP response in the gill, dilbit exposure did not greatly impact common measures of seawater acclimation, suggesting that significant osmoregulatory dysfunction is unlikely to occur if Atlantic salmon smolts are exposed sub-chronically to dilbit.

Ontogeny of the corticotropin-releasing factor system in zebrafish.

The corticotropin-releasing factor (CRF) system in fish functions to maintain homeostasis during stress in part by regulating cortisol production via the hypothalamus-pituitary-interrenal (HPI) axis. Towards understanding the role of the CRF system in vertebrate development, we describe the ontogeny of the CRF system, cortisol, and the stress response in the zebrafish, Danio rerio. Early embryonic expression of mRNA encoding CRF, urotensin I (UI), CRF-binding protein (CRF-BP), and two CRF receptors (CRF-R1 and CRF-R2) suggest a function in the early organization of the developing embryo. The expression patterns of CRF, UI, and CRF-BP in the larval brain are consistent with the adult distribution patterns for these genes and support HPI-axis independent functions. The relative amounts of CRF and UI mRNA in the heads and tails of developing and adult zebrafish suggest that CRF functions primarily in the brain while UI also plays an important role in the caudal neurosecretory system. The amount of cortisol in developing zebrafish is low and relatively constant through the first 6 days of development. The commencement of feeding after 4 dpf, however, significantly increases basal cortisol production. Finally, we show that zebrafish larvae are able to respond to an osmotic stressor as early as 3 dpf. Overall, results from this study establish the zebrafish as a model species for research on stress during ontogeny and offer new insights into an HPI-axis independent function for the CRF system during embryogenesis.

Hypoxia-induced reprogramming of the cardiac phenotype in American alligators (Alligator mississippiensis) revealed by quantitative proteomics.

Hypoxic exposure during development can have a profound influence on offspring physiology, including cardiac dysfunction, yet many reptile embryos naturally experience periods of hypoxia in buried nests. American alligators experimentally exposed to developmental hypoxia demonstrate morphological and functional changes to the heart that persist into later life stages; however, the molecular bases of these changes remain unknown. We tested if targeted and persistent changes in steady-state protein expression underlie this hypoxic heart phenotype, using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics. Alligator eggs were reared under normoxia or 10% hypoxia, then either sampled (embryo) or returned to normoxia for 2 years (juvenile). Three salient findings emerge from the integrated analysis of the 145 differentially expressed proteins in hypoxia-reared animals: (1) significant protein-protein interaction networks were identified only in up-regulated proteins, indicating that the effects of developmental hypoxia are stimulatory and directed; (2) the up-regulated proteins substantially enriched processes related to protein turnover, cellular organization, and metabolic pathways, supporting increased resource allocation towards building and maintaining a higher functioning heart; and (3) the juvenile cardiac proteome retained many of the signature changes observed in embryonic hearts, supporting long-term reprogramming of cardiac myocytes induced by hypoxia during critical periods of development.

Developmental and latent effects of diluted bitumen exposure on early life stages of sockeye salmon (Oncorhynchus nerka).

The early life stages of Pacific salmon are at risk of environmental exposure to diluted bitumen (dilbit) as Canada's oil sands industry continues to expand. The toxicity and latent effects of dilbit exposure were assessed in sockeye salmon (Oncorhynchus nerka) exposed to water-soluble fractions of dilbit (WSFd) from fertilization to the swim-up stage, and then reared in clean water for 8 months. Mortality was significantly higher in WSFd-exposed embryos, with cumulative mortality up to 4.6-fold higher in exposed relative to unexposed embryos. The sublethal effects of WSFd exposure included transcriptional up-regulation of cyp1a, a concentration-dependent delay in the onset and progression of hatching, as well as increased prevalence of developmental deformities at total polycyclic aromatic hydrocarbon (TPAH) concentrations ≥35 µg L-1. Growth and body composition were negatively affected by WSFd exposure, including a concentration-specific decrease in soluble protein concentration and increases in total body lipid and triglyceride concentrations. Mortality continued during the first 2 months after transferring fish to clean water, reaching 53% in fish exposed to 100 µg L-1 TPAH; but there was no latent impact on swimming performance, heart mass, or heart morphology in surviving fish after 8 months. A latent effect of WSFd exposure on brain morphology was observed, with fish exposed to 4 µg L-1 TPAH having significantly larger brains compared to other treatment groups after 8 months in clean water. This study provides comprehensive data on the acute, sub-chronic, and latent impacts of dilbit exposure in early life stage sockeye, information that is critical for a proper risk analysis of the impact of a dilbit spill on this socioeconomically important fish species.

Identification of the actc1c cardiac actin gene in zebrafish.

Zebrafish is rapidly becoming a key model organism for studying a variety of biological processes from molecules to organisms. Interactions involving actin, a contractile protein and part of the cytoskeleton, are regulated by actin binding proteins in the majority of physiological processes in eukaryotic cells. To understand the contribution of actin proteins to the physiological processes of zebrafish, it is important to identify the diverse isoforms of actin encoded by its genome; however, significant sequence identity complicates isoform assignments. Through a combination of human-directed sequence and functional analysis, we have assigned and performed localization of actc1c, a previously undesignated cardiac actin gene, and propose an updated assignment of α-actin protein isoform identities in zebrafish.

Localization of corticotropin-releasing factor, urotensin I, and CRF-binding protein gene expression in the brain of the zebrafish, Danio rerio.

Our current understanding of the corticotropin-releasing factor (CRF) system distribution in the teleost brain is restricted by limited immunohistochemical studies and a lack of complete transcriptional distribution maps. The present study used in situ hybridization to localize and compare CRF, urotensin I (UI), and CRF-binding protein (CRF-BP) expression in the brain of adult zebrafish (Danio rerio). All three peptides were localized in the preoptic area, periventricular hypothalamic and tectal regions, and dorsal part of the trigeminal motor nucleus. CRF and UI were both expressed in several nuclei of the dorsal telencephalon, whereas CRF and CRF-BP were both expressed in the ventral nucleus of the ventral telencephalon. Sole expression of CRF and CRF-BP was apparent in the olfactory bulbs and superior raphe nucleus, respectively, whereas only UI was observed in the corpus mamillare, nucleus of the medial longitudinal fascicle, dorsal tegmental nucleus, nucleus lateralis valvulae, and nucleus interpeduncularis. A major finding of this study was the general regional overlapping of CRF-BP with its ligands and a tendency to be expressed in tandem with CRF rather than UI. Overall, the mRNA expression patterns outlined in this study support the stress-related neuroendocrine, autonomic, and behavioral functions generally ascribed to the vertebrate CRF system and suggest some unique functional roles for CRF and UI in the teleost brain.

Novel insights into cardiac remodelling revealed by proteomic analysis of the trout heart during exercise training.

The changes in the cardiac proteome of rainbow trout (Oncorhynchus mykiss) were quantified during the early phases (4, 7, and 14d) of a typical exercise-training regime to provide a comprehensive overview of the cellular changes responsible for developing a trained heart phenotype. Enhanced somatic growth during the 14d experiment was paralleled by cardiac growth to maintain relative ventricular mass. This was reflected in the cardiac proteome by the increased abundance of contractile proteins and cellular integrity proteins as early as Day 4, including a pronounced and sustained increase in blood vessel epicardial substance - an intercellular adhesion protein expressed in the vertebrate heart. An unexpected finding was that proteins involved in energy pathways, including glycolysis, ß-oxidation, the TCA cycle, and the electron transport chain, were generally present at lower levels relative to Day 0 levels, suggesting a reduced investment in the maintenance of energy production pathways. However, as the fish demonstrated somatic and cardiac growth during the exercise-training program, this change did not appear to influence cardiac function. The in-depth analysis of temporal changes in the cardiac proteome of trout during the early stages of exercise training reveals novel insights into cardiac remodelling in an important model species. BIOLOGICAL SIGNIFICANCE: Rainbow trout hearts have a remarkable ability for molecular, structural, and functional plasticity, and the inherent athleticism of these fish makes them ideal models for studies in comparative exercise physiology. Indeed, several decades of research using exercise-trained trout has shown both conserved and unique aspects of cardiac plasticity induced by a sustained increase in the workload of the heart. Despite a strong appreciation for the outcome of exercise training, however, the temporal events that generate this phenotype are not known. This study interrogates the early stages of exercise training using in-depth proteomic analysis to understand the molecular pathways of cardiac remodelling. Two major and novel findings emerge: (1) structural remodelling is initiated very early in training, as evidenced by a general increase in proteins associated with muscle contraction and integrity at Day 4, and (2) the abundance of proteins directly involved in energy production are decreased during 14d of exercise training, which contrasts the general acceptance of an exercise-induced increase in aerobic capacity of muscle, and suggests that regulation of energy pathways occurs at a different biological level than protein abundance.

Proteomic analysis of sockeye salmon serum as a tool for biomarker discovery and new insight into the sublethal toxicity of diluted bitumen.

Pipelines carrying diluted bitumen (dilbit) from Canada's oil sands traverse North America, including the freshwater habitat of Pacific salmon, posing a risk of environmental release and aquatic exposure. Swimming performance is impacted in juvenile sockeye (Oncorhynchus nerka) exposed to dilbit; therefore biomarkers of dilbit exposure will be valuable for monitoring at-risk salmon stocks. This study characterized changes in the serum proteome of sockeye exposed to a sub-lethal and environmentally relevant concentration of dilbit using isobaric tags for relative and absolute quantitation (iTRAQ), and included a range of experimental conditions to permit identification of biomarkers that are robust across time (1 and 4wk) and exercise level (at rest and following a swim test). Over 500 proteins were identified and quantified in sockeye serum, with dilbit exposure significantly altering the abundance of 24 proteins irrespective of time and exercise, including proteins associated with immune and inflammatory responses, coagulation, and iron homeostasis. An increase in creatine kinase (CK) activity in serum of dilbit-exposed salmon confirmed the higher CK protein abundance measured using iTRAQ. The combination of 4wk dilbit exposure and a swim test had a greater effect on the serum proteome than either treatment alone, including a marked increase in tissue leakage proteins, suggesting that aerobic exercise exacerbates the serum proteome response to dilbit, and the increased cellular damage could impede exercise recovery. This study provides a foundation for the development of bio-monitoring tools for salmon stock assessments, and offers new insights into the sub-lethal toxicity of crude oil exposure in fish.

Effects of diluted bitumen exposure on juvenile sockeye salmon: From cells to performance.

Diluted bitumen (dilbit; the product of oil sands extraction) is transported through freshwater ecosystems critical to Pacific salmon. This is concerning, because crude oil disrupts cardiac development, morphology, and function in embryonic fish, and cardiac impairment in salmon can have major consequences on migratory success and fitness. The sensitivity of early life-stage salmon to dilbit and its specific cardiotoxic effects are unknown. Sockeye salmon parr were exposed to environmentally relevant concentrations of the water-soluble fraction (WSF) of dilbit for 1 wk and 4 wk, followed by an examination of molecular, morphological, and organismal endpoints related to cardiotoxicity. We show that parr are sensitive to WSF of dilbit, with total polycyclic aromatic hydrocarbon (PAH) concentrations of 3.5 µg/L sufficient to induce a liver biomarker of PAH exposure, and total PAH of 16.4 µg/L and 66.7 µg/L inducing PAH biomarkers in the heart. Furthermore, WSF of dilbit induces concentration-dependent cardiac remodeling coincident with performance effects: fish exposed to 66.7 µg/L total PAH have relatively fewer myocytes and more collagen in the compact myocardium and impaired swimming performance at 4 wk, whereas the opposite changes occur in fish exposed to 3.5 µg/L total PAH. The results demonstrate cardiac sensitivity to dilbit exposure that could directly impact sockeye migratory success. Environ Toxicol Chem 2017;36:354-360. © 2016 SETAC.