Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity.

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

Impact of stress phenotype, elevated temperature, and bacterin exposure on male Atlantic salmon (Salmo salar) growth, stress, and immune biomarker gene expression.

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

Cardiorespiratory physiology and swimming capacity of Atlantic salmon (Salmo salar) at cold temperatures.

We investigated how acclimation to 8, 4 and 1°C, and acute cooling from 8  to 1°C, affected the Atlantic salmon's aerobic and anaerobic metabolism, and cardiac function, during a critical swim speed (Ucrit) test. This study revealed several interesting temperature-dependent effects. First, while differences in resting heart rate (fH) between groups were predictable based on previous research (range ∼28-65 beats  min-1), with values for 1°C-acclimated fish slightly higher than those of acutely exposed conspecifics, the resting cardiac output () of 1°C-acclimated fish was much lower and compensated for by a higher resting blood oxygen extraction (MO2/). In contrast, the acutely exposed fish had a ∼2-fold greater resting stroke volume (VS) compared with that of the other groups. Second, increases in fH (1.2- to 1.4-fold) contributed little to during the Ucrit test, and the contributions of (VS) versus MO2/ to aerobic scope (AS) were very different in the two groups tested at 1°C (1°C-acclimated and 8-1°C fish). Finally, Ucrit was 2.08 and 1.69 body lengths (BL) s-1 in the 8 and 4°C-acclimated groups, but only 1.27 and 1.44 BL s-1 in the 1°C-acclimated and 8-1°C fish, respectively - this lower value in 1°C versus 8-1°C fish despite higher values for maximum metabolic rate and AS. These data: support recent studies which suggest that the capacity to increase fH is constrained at low temperatures; show that cardiorespiratory function at cold temperatures, and its response to increased demands, depends on exposure duration; and suggest that AS does not constrain swimming capacity in salmon when chronically exposed to temperatures approaching their lower limit.

The upper temperature and hypoxia limits of Atlantic salmon (Salmo salar) depend greatly on the method utilized.

In this study, Atlantic salmon were: (i) implanted with heart rate (fH) data storage tags (DSTs), pharmacologically stimulated to maximum fH, and warmed at 10°C h-1 (i.e. tested using a 'rapid screening protocol'); (ii) fitted with Doppler® flow probes, recovered in respirometers and given a critical thermal maximum (CTmax) test at 2°C h-1; and (iii) implanted with fH DSTs, recovered in a tank with conspecifics for 4 weeks, and had their CTmax determined at 2°C h-1. Fish in respirometers and those free-swimming were also exposed to a stepwise decrease in water oxygen level (100% to 30% air saturation) to determine the oxygen level at which bradycardia occurred. Resting fH was much lower in free-swimming fish than in those in respirometers (∼49 versus 69 beats min-1) and this was reflected in their scope for fH (∼104 versus 71 beats min-1) and CTmax (27.7 versus 25.9°C). Further, the Arrhenius breakpoint temperature and temperature at peak fH for free-swimming fish were considerably greater than for those tested in the respirometers and given a rapid screening protocol (18.4, 18.1 and 14.6°C; and 26.5, 23.2 and 20.2°C, respectively). Finally, the oxygen level at which bradycardia occurred was significantly higher in free-swimming salmon than in those in respirometers (∼62% versus 53% air saturation). These results: highlight the limitations of some lab-based methods of determining fH parameters and thermal tolerance in fishes; and suggest that scope for fH may be a more reliable and predictive measure of a fish's upper thermal tolerance than their peak fH.

Expression of Interleukin-1ß protein in vitro,exvivo and in vivo salmonid models.

Interleukin-1ß (IL-1ß) is one of the first cytokines expressed during immune responses, and its levels are affected by many factors, including stress. To date, it has only been possible to measure IL-1ß transcript (mRNA) expression quantitatively in fish using qPCR. This is because previous studies that measured IL-1ß protein concentrations in these taxa used western blotting, which only provides qualitative data. To advance our knowledge of fish IL-1ß biology, and because post-translational processing plays a critical role in the activation of this molecule, we developed a quantitative enzyme-linked immunosorbent assay (ELISA) to accurately measure the concentration of IL-1ß protein in several cell cultures and in vivo in salmonids. We compared changes in IL-1ß protein levels to the expression of its mRNA. The developed ELISA was quite sensitive and has a detection limit of 12.5 pg/mL. The tools developed, and information generated through this research, will allow for a more accurate and complete understanding of IL-1ß's role in the immune response of salmonids.The assay described here has the potential to significantly advance our ability to assess fish health and immune status.

Production of a monoclonal antibody specific to sablefish (Anoplopoma fimbria) IgM and its application in ELISA, western blotting, and immunofluorescent staining.

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

Cold-induced metabolic depression in cunner (Tautogolabrus adspersus): A multifaceted cellular event.

Metabolic depression and dormancy (i.e., stopping/greatly reducing activity and feeding) are strategies used by many animals to survive winter conditions characterized by food shortages and cold temperatures. However, controversy exists on whether the reduced metabolism of some fishes at cold temperatures is due to dormancy alone, or also involves active metabolic depression. Thus, we acclimated winter-dormant cunner [Tautogolabrus adspersus, a north temperate wrasse which in Newfoundland is at the northern limit of its distribution] and winter-active Atlantic salmon (Salmo salar) to winter (0°C; 8h light: 16h dark) and summer (10°C; 16h light: 8 h dark) conditions, and measured the thermal sensitivity of ATP-producing and O2-consuming processes in isolated liver mitochondria and hepatocytes when exposed in vitro to temperatures from 20 to 0°C and 10 to 0°C, respectively. We found that: 1) liver mitochondrial State 3 respiration and hepatocyte O2 consumption in cunner were only ~ one-third and two-thirds of that measured in salmon, respectively, at all measurement temperatures; 2) cunner mitochondria also have proton conductance and leak respiration (State 4) values that are only approximately one-third of those in salmon; 3) the mitochondria of cunner show a dramatic reduction in respiratory control ratio (from ~ 8 to 3), and a much greater drop in State 3 respiration, between 10 and 5°C (Q10 values in 10- and 0°C-acclimated fish of 14.5 and 141.2, respectively), as compared with salmon (3.9 and 9.6, respectively); and 4) lowering temperature from 5 to 0°C resulted in ~ 40 and 30% reductions in hepatocyte O2 consumption due to non-mitochondrial respiration and Na+-K+-ATPase activity, respectively, in cunner, but not in salmon. Collectively, these results highlight the intrinsic capacity for metabolic depression in hepatocytes and mitochondria of cunner, and clearly suggest that several cellular processes play a role in the reduced metabolic rates exhibited by some fishes at cold temperatures.

Can temperature-dependent changes in myocardial contractility explain why fish only increase heart rate when exposed to acute warming?

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.

Influence of Supplemental Dietary Cholesterol on Growth Performance, Indices of Stress, Fillet Pigmentation, and Upper Thermal Tolerance of Female Triploid Atlantic Salmon (Salmo salar).

The salmon aquaculture industry must be proactive at developing mitigation tools/strategies to offset the potential negative impacts of climate change. Therefore, this study examined if additional dietary cholesterol could enhance salmon production at elevated temperatures. We hypothesized that supplemental cholesterol could aid in maintaining cell rigidity, reducing stress and the need to mobilize astaxanthin muscle stores, and improving salmon growth and survival at high rearing temperatures. Accordingly, postsmolt female triploid salmon were exposed to an incremental temperature challenge (+0.2°C day-1) to mimic conditions that they experience in sea cages in the summer, with temperature held at both 16 and 18°C for several weeks [i.e., 3 weeks at 16°C, followed by an increase at 0.2°C day-1 to 18°C (10 days), then 5 weeks at 18°C] to prolong their exposure to elevated temperatures. From 16°C onwards, the fish were fed either a control diet, or one of two nutritionally equivalent experimental diets containing supplemental cholesterol [+1.30%, experimental diet #1 (ED1); or +1.76%, experimental diet #2 (ED2)]. Adding cholesterol to the diet did not affect the salmon's incremental thermal maximum (ITMax), growth, plasma cortisol, or liver stress-related transcript expression. However, ED2 appeared to have a small negative impact on survival, and both ED1 and ED2 reduced fillet "bleaching" above 18°C as measured using SalmoFan™ scores. Although the current results suggest that supplementing salmon diets with cholesterol would have few/minimal benefits for the industry, ≤ 5% of the female triploid Atlantic salmon used in this study irrespective of diet died before temperature reached 22°C. These latter data suggest that it is possible to produce all female populations of reproductively sterile salmon that can withstand summer temperatures in Atlantic Canada.

Phenotypic stress response does not influence the upper thermal tolerance of male Atlantic salmon (Salmo salar).

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

Temperature effects on the contractile performance and efficiency of oxidative muscle from a eurythermal versus a stenothermal salmonid.

We compared the thermal sensitivity of oxidative muscle function between the eurythermal Atlantic salmon (Salmo salar) and the more stenothermal Arctic char (Salvelinus alpinus; which prefers cooler waters). Power output was measured in red skeletal muscle strips and myocardial trabeculae, and efficiency (net work/energy consumed) was measured for trabeculae, from cold (6°C) and warm (15°C) acclimated fish at temperatures from 2 to 26°C. The mass-specific net power produced by char red muscle was greater than in salmon, by 2-to 5-fold depending on test temperature. Net power first increased, then decreased, when the red muscle of 6°C-acclimated char was exposed to increasing temperature. Acclimation to 15°C significantly impaired mass-specific power in char (by ∼40-50%) from 2 to 15°C, but lessened its relative decrease between 15 and 26°C. In contrast, maximal net power increased, and then plateaued, with increasing temperature in salmon from both acclimation groups. Increasing test temperature resulted in a ∼3- to 5-fold increase in maximal net power produced by ventricular trabeculae in all groups, and this effect was not influenced by acclimation temperature. Nonetheless, lengthening power was higher in trabeculae from warm-acclimated char, and char trabeculae could not contract as fast as those from salmon. Finally, the efficiency of myocardial net work was approximately 2-fold greater in 15°C-acclimated salmon than char (∼15 versus 7%), and highest at 20°C in salmon. This study provides several mechanistic explanations as to their inter-specific difference in upper thermal tolerance, and potentially why southern char populations are being negatively impacted by climate change.

Does hydrostatic pressure influence lumpfish (Cyclopterus lumpus) heart rate and its response to environmental challenges?

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

The Atlantic salmon's stress- and immune-related transcriptional responses to moderate hypoxia, an incremental temperature increase, and these challenges combined.

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

The transcriptomic responses of Atlantic salmon (Salmo salar) to high temperature stress alone, and in combination with moderate hypoxia.

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

Research on sablefish (Anoplopoma fimbria) suggests that limited capacity to increase heart function leaves hypoxic fish susceptible to heat waves.

Studies of heart function and metabolism have been used to predict the impact of global warming on fish survival and distribution, and their susceptibility to acute and chronic temperature increases. Yet, despite the fact that hypoxia and high temperatures often co-occur, only one study has examined the effects of hypoxia on fish thermal tolerance, and the consequences of hypoxia for fish cardiac responses to acute warming have not been investigated. We report that sablefish (Anoplopoma fimbria) did not increase heart rate or cardiac output when warmed while hypoxic, and that this response was associated with reductions in maximum O2 consumption and thermal tolerance (CTmax) of 66% and approximately 3°C, respectively. Further, acclimation to hypoxia for four to six months did not substantially alter the sablefish's temperature-dependent physiological responses or improve its CTmax. These results provide novel, and compelling, evidence that hypoxia can impair the cardiac and metabolic response to increased temperatures in fish, and suggest that some coastal species may be more vulnerable to climate change-related heat waves than previously thought. Further, they support research showing that cross-tolerance and physiological plasticity in fish following hypoxia acclimation are limited.

Effects of hypoxic acclimation, muscle strain, and contraction frequency on nitric oxide-mediated myocardial performance in steelhead trout (Oncorhynchus mykiss).

Whether hypoxic acclimation influences nitric oxide (NO)-mediated control of fish cardiac function is not known. Thus, we measured the function/performance of myocardial strips from normoxic- and hypoxic-acclimated (40% air saturation; ∼8 kPa O2) trout at several frequencies (20-80 contractions·min-1) and two muscle strain amplitudes (8% and 14%) when exposed to increasing concentrations of the NO donor sodium nitroprusside (SNP) (10-9 to 10-4 M). Further, we examined the influence of 1) nitric oxide synthase (NOS) produced NO [by blocking NOS with 10-4 M NG-monomethyl-l-arginine (l-NMMA)] and 2) soluble guanylyl cyclase mediated, NOS-independent, NO effects (i.e., after blockade with 10-4 M ODQ), on myocardial contractility. Hypoxic acclimation increased twitch duration by 8%-10% and decreased mass-specific net power by ∼35%. However, hypoxic acclimation only had minor impacts on the effects of SNP and the two blockers on myocardial function. The most surprising finding of the current study was the degree to which contraction frequency and strain amplitude influenced NO-mediated effects on myocardial power. For example, at 8% strain, 10-4 SNP resulted in a decrease in net power of ∼30% at 20 min-1 but an increase of ∼20% at 80 min-1, and this effect was magnified at 14% strain. This research suggests that hypoxic acclimation has only minor effects on NO-mediated myocardial contractility in salmonids, is the first to report the high frequency- and strain-dependent nature of NO effects on myocardial contractility in fishes, and supports previous work showing that NO effects on the heart (myocardium) are finely tuned spatiotemporally.

Antigen presentation genes in gadoid species (haddock: Melanogrammus aeglefinus and Atlantic cod: Gadus morhua) raise questions about cross-presentation pathways and glycosylated beta-2-microglobulin.

The Atlantic cod immune system deviates from antigen presentation processes seen in other vertebrates in that it lacks the necessary genes for exogenous antigen presentation (i.e., MHC-II and li) and a key MHC-II interacting molecule necessary for T-helper cell function (i.e., CD4), while possessing an expanded repertoire of MHC-I genes that facilitate endogenous antigen presentation. These observations, combined with the identification of putative endosomal sorting signals in MHC-I cytoplasmic tails, have led to speculation that cod rely on cross-presentation of exogenous antigens to elicit cytotoxic T-lymphocyte responses against extracellular threats. In light of this suggestion, we investigated MHC-I transcriptional profiles and endosomal sorting signals in a closely related gadoid species, the haddock. Analysis of transcripts from one individual identified 13 unique MHC-I molecules, including two non-classical molecules as determined by the level of conservation at their peptide anchoring sites. This suggests that like the cod, the haddock has an expanded MHC-I repertoire. Analysis of haddock MHC-I cytoplasmic tail sequences revealed that the dileucine- and tyrosine-based endosomal signaling motifs, that are suggested to facilitate cross-presentation in cod, were absent. Closer examination of the cod signaling motifs, including their relative position in the cytoplasmic tail region, indicates that these motifs might be non-functional, further supporting the need for functional studies to assess cross-presentation. Finally, in silico analysis and in vitro N-type de-glycosylation experiments demonstrate that haddock and cod beta-2-microglobulin (ß2M) are glycosylated at the same NQT sequon. Interestingly, whole genome tBLASTn searches also revealed that putative ß2 M glycosylation sites appear frequently within the Gadiformes lineage, as the predictive NQT and other N-X-S/T sequons were located in ß2M orthologues from 19 of the 25 additional gadoid genomes analyzed. Though the exact significance of ß2M glycosylation has yet to be elucidated, phylogenetic comparisons predict that the same NQT glycosylation sequence occurs in 13 additional species comprising four different orders of Actinopterygii (Gymnotiformes, Esociformes, Beryciformes and Perciformes). This suggests either that this feature has arisen independently in multiple lineages or that it comes from a common ancestor and has been lost or modified in many species. Together, the analysis of gadoid MHC-I genes and ß2M molecules highlights the challenges in generalizing immune system paradigms across the most diverse vertebrate lineage (i.e., fish) and between fish and more well-studied mammals.

Effects of hypoxic acclimation on contractile properties of the spongy and compact ventricular myocardium of steelhead trout (Oncorhynchus mykiss).

The trout ventricle has an outer compact layer supplied with well-oxygenated arterial blood from the coronary circulation, and an inner spongy myocardium supplied with oxygen poor venous blood. It was hypothesized that: (1) the spongy myocardium of steelhead trout (Oncorhynchus mykiss), given its routine exposure to low partial pressures of oxygen (PO2), would be better able to maintain contractile performance (work) when exposed to acute hypoxia (100 to 10% air saturation) relative to the compact myocardium, and would show little benefit from hypoxic acclimation; and (2) the compact myocardium from hypoxia-acclimated (40% air saturation) fish would be better able to maintain work during acute exposure to hypoxia relative to normoxia-acclimated individuals. Consistent with our expectations, when PO2 was acutely lowered, net work from the compact myocardium of normoxia-acclimated fish declined more (by ~ 73%) than the spongy myocardium (~ 50%), and more than the compact myocardium of hypoxia-acclimated fish (~ 55%), and hypoxic acclimation did not benefit the spongy myocardium in the face of reduced PO2. Further, while hypoxic acclimation resulted in a 25% (but not significant) decrease in net work of the spongy myocardium, the performance of the compact myocardium almost doubled. This research suggests that, in contrast to the spongy myocardium, performance of the compact myocardium is improved by hypoxic acclimation; and supports previous research suggesting that the decreased contractile performance of the myocardium upon exposure to lowered PO2 may be adaptive and mediated by mechanisms within the muscle itself.

Aeromonas salmonicida infection kinetics and protective immune response to vaccination in sablefish (Anoplopoma fimbria).

Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD50). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD50 was estimated as ~3 × 105 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6-8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs.

Thermal biology and swimming performance of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) are two commercially important marine fishes impacted by both overfishing and climate change. Increasing ocean temperatures are affecting the physiology of these species and causing changes in distribution, growth, and maturity. While the physiology of cod has been well investigated, that of haddock has received very little attention. Here, we measured the metabolic response to increasing temperatures, as well as the critical thermal maximum (CTmax), of cod acclimated to 8 and 12 °C and haddock acclimated to 12 °C. We also compared the swimming performance (critical swimming speed, U crit) of cod and haddock at 12 °C, as well as the U crit of 12 °C-acclimated cod acutely exposed to a higher-than-optimal temperature (16 °C). The CTmax for cod was 21.4 and 23.0 °C for 8- and 12 °C-acclimated fish, respectively, whereas that for the 12 °C-acclimated haddock was 23.9 °C. These values were all significantly different and show that haddock are more tolerant of high temperatures. The aerobic maximum metabolic rate (MMR) of swimming cod remained high at 16 °C, suggesting that maximum oxygen transport capacity was not limited at a temperature above optimal in this species. However, signs of impaired swimming (struggling) were becoming evident at 16 °C. Haddock were found to reach a higher U crit than cod at 12 °C (3.02 vs. 2.62 body lengths s-1, respectively), and at a lower MMR. Taken together, these results suggest that haddock perform better than cod in warmer conditions, and that haddock are the superior swimmer amongst the two species.