The environmental tolerances and metabolic physiology of sablefish (Anoplopoma fimbria).

Given the potential impacts of global warming, such as increases in temperature and the frequency/severity of hypoxia in marine ecosystems, it is important to study the impacts of these environmental challenges on sea-cage reared aquaculture species. This study focuses on the sablefish (Anoplopoma fimbria), an emerging aquaculture species that has a unique ecology in the wild. For instance, adults inhabit oxygen minimum zones and cool waters at depths up to 1500 m. Using Atlantic salmon (Salmo salar) (~1132 g adults) as a comparative species, we used intermittent-flow respirometry to characterize the tolerance and metabolic response of sablefish (~10 g juveniles and ~675 g adults) to acute increases in temperature (2 °C h-1) and decreases in oxygen level (~10% air saturation h-1). Adult sablefish were much more hypoxia tolerant than adult salmon [O2 level at loss of equilibrium ~5.4% vs. ~24.2% air saturation, respectively]. In addition, sablefish could withstand upper temperatures only slightly lower than salmon [critical thermal maximum (CTmax) ~24.9 °C vs. ~26.2 °C, respectively]. Sablefish juveniles were both less hypoxia and thermally tolerant than adults [critical O2 tension ~18.9% vs. ~15.8% air saturation; CTmax ~22.7 vs. ~24.9 °C, respectively]. Interestingly, many of these differences in environmental tolerance could not be explained by differences in metabolic parameters (aerobic scope or routine metabolic rate). Our findings show that sablefish are tolerant of high temperatures, and very tolerant of hypoxia, traits that are advantageous for an aquaculture species in the era of climate change.

The biodegradation of crude oil in the deep ocean.

Oil biodegradation at a simulated depth of 1500m was studied in a high-pressure apparatus at 5°C, using natural seawater with its indigenous microbes, and 3ppm of an oil with dispersant added at a dispersant:oil ratio of 1:15. Biodegradation of the detectable hydrocarbons was prompt and extensive (>70% in 35days), although slower by about a third than under otherwise identical conditions equivalent to the surface. The apparent half-life of biodegradation of the total detectable hydrocarbons at 15MPa was 16days (compared to 13days at atmospheric pressure), although some compounds, such as the four-ring aromatic chrysene, were degraded rather more slowly.

Transcriptome profiling reveals that feeding wild zooplankton to larval Atlantic cod (Gadus morhua) influences suites of genes involved in oxidation-reduction, mitosis, and selenium homeostasis.

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

Characterization and expression analyses of five interferon regulatory factor transcripts (Irf4a, Irf4b, Irf7, Irf8, Irf10) in Atlantic cod (Gadus morhua).

The interferon regulatory factor (IRF) family of genes encodes a group of transcription factors that have important roles not only in regulating the expression of Type I interferons (IFNs) and other genes in the IFN pathway, but also in growth, development and the regulation of oncogenesis. In this study, several IRF family members (Irf4a, Irf4b, Irf7, Irf8, Irf10) in Atlantic cod (Gadus morhua) were characterized at the cDNA and putative amino acid levels, allowing for phylogenetic analysis of these proteins in teleost fish, as well as the development of gene-specific primers used in RT-PCR and quantitative PCR (QPCR) analyses. Two Atlantic cod Irf10 splice variants were identified and their presence confirmed by sequencing of the Irf10 genomic region. RT-PCR showed that Irf7, Irf8 and both Irf10 transcripts were expressed in all 15 cod tissues tested, while Irf4a and Irf4b were absent in some tissues. QPCR analysis of spleen expression expanded upon this, and upon previous work. All IRF transcripts in the study were responsive to stimulation by the viral mimic poly(I:C), and all except Irf4a were responsive to exposure to formalin-killed Aeromonas salmonicida (ASAL). These IRF genes, thus, are likely important in the cod immune response to both viral and bacterial infections. Increased temperature (10 °C to 16 °C) was also observed to modulate the antibacterial responses of all IRF transcripts, and the antiviral responses of Irf4b and Irf10-v2. This research supports earlier studies which reported that elevated temperature modulates the expression of many immune genes in Atlantic cod.

Metabolic depression in cunner (Tautogolabrus adspersus) is influenced by ontogeny, and enhances thermal tolerance.

To examine the effect of ontogeny on metabolic depression in the cunner (Tautogolabrus adspersus), and to understand how ontogeny and the ability to metabolically depress influence this species' upper thermal tolerance: 1) the metabolic rate of 9°C-acclimated cunner of three size classes [0.2-0.5 g, young of the year (YOY); 3-6 g, small; and 80-120 g, large (adult)] was measured during a 2°C per day decrease in temperature; and 2) the metabolic response of the same three size classes of cunner to an acute thermal challenge [2°C h(-1) from 10°C until Critical Thermal Maximum, CTMax] was examined, and compared to that of the Atlantic cod (Gadus morhua). The onset-temperature for metabolic depression in cunner increased with body size, i.e. from 5°C in YOY cunner to 7°C in adults. In contrast, the extent of metabolic depression was ∼80% (Q10 = ∼15) for YOY fish, ∼65% (Q10 = ∼8) for small fish and ∼55% (Q10 = ∼5) for adults, and this resulted in the metabolic scaling exponent (b) gradually increasing from 0.84 to 0.92 between 9°C to 1°C. All size classes of cunner had significantly (approximately 60%) lower routine metabolic rates at 10°C than Atlantic cod. However, there was no species' difference in the temperature-induced maximum metabolic rate, and this resulted in factorial metabolic scope values that were more than two-fold greater for cunner, and CTMax values that were 6-9°C higher (∼21 vs. 28°C). These results: 1) show that ontogeny influences the temperature of initiation and the extent of metabolic depression in cunner, but not O2 consumption when in a hypometabolic state; and 2) suggest that the evolution of cold-induced metabolic depression in this northern wrasse species has not resulted in a trade-off with upper thermal tolerance, but instead, an enhancement of this species' metabolic plasticity.

Temperature and sex dependent effects on cardiac mitochondrial metabolism in Atlantic cod (Gadus morhua L.).

To test the hypothesis that impaired mitochondrial respiration limits cardiac performance at warm temperatures, and examine if any effect(s) are sex-related, the consequences of high temperature on cardiac mitochondrial oxidative function were examined in 10°C acclimated, sexually immature, male and female Atlantic cod. Active (State 3) and uncoupled (States 2 and 4) respiration were measured in isolated ventricular mitochondria at 10, 16, 20, and 24°C using saturating concentrations of malate and pyruvate, but at a submaximal (physiological) level of ADP (200µM). In addition, citrate synthase (CS) activity was measured at these temperatures, and mitochondrial respiration and the efficiency of oxidative phosphorylation (P:O ratio) were determined at [ADP] ranging from 25-200µM at 10 and 20°C. Cardiac morphometrics and mitochondrial respiration at 10°C, and the thermal sensitivity of CS activity (Q10=1.51), were all similar between the sexes. State 3 respiration at 200µM ADP increased gradually in mitochondria from females between 10 and 24°C (Q10=1.48), but plateaued in males above 16°C, and this resulted in lower values in males vs. females at 20 and 24°C. At 10°C, State 4 was ~10% of State 3 values in both sexes [i.e. a respiratory control ratio (RCR) of ~10] and P:O ratios were approximately 1.5. Between 20 and 24°C, State 4 increased more than State 3 (by ~70 vs. 14%, respectively), and this decreased RCR to ~7.5. The P:O ratio was not affected by temperature at 200µM ADP. However, (1) the sensitivity of State 3 respiration to increasing [ADP] (from 25 to 200µM) was reduced at 20 vs. 10°C in both sexes (Km values 105±7 vs. 68±10µM, respectively); and (2) mitochondria from females had lower P:O values at 25 vs. 100µM ADP at 20°C, whereas males showed a similar effect at 10°C but a much more pronounced effect at 20°C (P:O 1.05 at 25µM ADP vs. 1.78 at 100µMADP). In summary, our results demonstrate several sex-related differences in ventricular mitochondrial function in Atlantic cod, and suggest that myocardial oxidative function and possibly phosphorylation efficiency may be limited at temperatures of 20°C or above, particularly in males. These observations could partially explain why cardiac function in Atlantic cod plateaus just below this species׳ critical thermal maximum (~22°C) and may contribute to yet unidentified sex differences in thermal tolerance and swimming performance.

Variation in embryonic mortality and maternal transcript expression among Atlantic cod (Gadus morhua) broodstock: a functional genomics study.

Early life stage mortality is an important issue for Atlantic cod aquaculture, yet the impact of the cod maternal (egg) transcriptome on egg quality and mortality during embryonic development is poorly understood. In the present work, we studied embryonic mortality and maternal transcript expression using eggs from 15 females. Total mortality at 7days post-fertilization (7 dpf, segmentation stage) was used as an indice of egg quality. A 20,000 probe (20K) microarray experiment compared the 7hours post-fertilization (7 hpf, ~2-cell stage) egg transcriptome of the two lowest quality females (>90% mortality at 7 dpf) to that of the highest quality female (~16% mortality at 7 dpf). Forty-three microarray probes were consistently differentially expressed in both low versus high quality egg comparisons (25 higher expressed in low quality eggs, and 18 higher expressed in high quality eggs). The microarray experiment also identified many immune-relevant genes [e.g. interferon (IFN) pathway genes ifngr1 and ifrd1)] that were highly expressed in eggs of all 3 females regardless of quality. Twelve of the 43 candidate egg quality-associated genes, and ifngr1, ifrd1 and irf7, were included in a qPCR study with 7 hpf eggs from all 15 females. Then, the genes that were confirmed by qPCR to be greater than 2-fold differentially expressed between 7 hpf eggs from the lowest and highest quality females (dcbld1, ddc, and acy3 more highly expressed in the 2 lowest quality females; kpna7 and hacd1 more highly expressed in the highest quality female), and the 3 IFN pathway genes, were included in a second qPCR study with unfertilized eggs. While some maternal transcripts included in these qPCR studies were associated with extremes in egg quality, there was little correlation between egg quality and gene expression when all females were considered. Both dcbld1 and ddc showed greater than 100-fold differences in transcript expression between females and were potentially influenced by family. The Atlantic cod ddc (dopa decarboxylase) complete cDNA was characterized, and has a 1461bp open reading frame encoding a 486 amino acid protein that contains all eight residues of the conserved pyridoxal 5'-phosphate binding site including the catalytic lysine. This study provides valuable new information and resources related to the Atlantic cod egg transcriptome. Some of these microarray-identified, qPCR-confirmed, Atlantic cod egg transcripts (e.g. ddc, kpna7) play important roles during embryonic development of other vertebrate species, and may have similar functions in Atlantic cod.

Increased ventricular stiffness and decreased cardiac function in Atlantic cod (Gadus morhua) at high temperatures.

We employed the work loop method to study the ability of ventricular and atrial trabeculae from Atlantic cod to sustain power production during repeated contractions at acclimation temperatures (10°C) and when acutely warmed (20°C). Oxygen tension (Po2) was lowered from 450 to 34% air saturation to augment the thermal stress. Preparations worked under conditions simulating either a large stroke volume (35 contractions/min rate, 8-12% muscle strain) or a high heart rate (70 contractions/min, 2-4% strain), with power initially equal under both conditions. The effect of declining Po2 on power was similar under both conditions but was temperature and tissue dependent. In ventricular trabeculae at 10°C (and atria at 20°C), shortening power declined across the full range of Po2 studied, whereas the power required to lengthen the muscle was unaffected. Conversely, in ventricular trabeculae at 20°C, there was no decline in shortening power but an increase in lengthening power when Po2 fell below 100% air saturation. Finally, when ventricular trabeculae were paced at rates of up to 115 contractions/min at 20°C (vs. the maximum of 70 contractions/min in vivo), they showed marked increases in both shortening and lengthening power. Our results suggest that although elevated heart rates may not impair ventricular power as they commonly do isometric force, limited atrial power and the increased work required to expand the ventricle during diastole may compromise ventricular filling and hence, stroke volume in Atlantic cod at warm temperatures. Neither large strains nor high contraction rates convey an apparent advantage in circumventing this.

A moderate increase in ambient temperature modulates the Atlantic cod (Gadus morhua) spleen transcriptome response to intraperitoneal viral mimic injection.

BACKGROUND: Atlantic cod (Gadus morhua) reared in sea-cages can experience large variations in temperature, and these have been shown to affect their immune function. We used the new 20K Atlantic cod microarray to investigate how a water temperature change which, simulates that seen in Newfoundland during the spring-summer (i.e. from 10°C to 16°C, 1°C increase every 5 days) impacted the cod spleen transcriptome response to the intraperitoneal injection of a viral mimic (polyriboinosinic polyribocytidylic acid, pIC). RESULTS: The temperature regime alone did not cause any significant increases in plasma cortisol levels and only minor changes in spleen gene transcription. However, it had a considerable impact on the fish spleen transcriptome response to pIC [290 and 339 significantly differentially expressed genes between 16°C and 10°C at 6 and 24 hours post-injection (HPI), respectively]. Seventeen microarray-identified transcripts were selected for QPCR validation based on immune-relevant functional annotations. Fifteen of these transcripts (i.e. 88%), including DHX58, STAT1, IRF7, ISG15, RSAD2 and IκBα, were shown by QPCR to be significantly induced by pIC. CONCLUSIONS: The temperature increase appeared to accelerate the spleen immune transcriptome response to pIC. We found 41 and 999 genes differentially expressed between fish injected with PBS vs. pIC at 10°C and sampled at 6HPI and 24HPI, respectively. In contrast, there were 656 and 246 genes differentially expressed between fish injected with PBS vs. pIC at 16°C and sampled at 6HPI and 24HPI, respectively. Our results indicate that the modulation of mRNA expression of genes belonging to the NF-κB and type I interferon signal transduction pathways may play a role in controlling temperature-induced changes in the spleen's transcript expression response to pIC. Moreover, interferon effector genes such as ISG15 and RSAD2 were differentially expressed between fish injected with pIC at 10°C vs. 16°C at 6HPI. These results substantially increase our understanding of the genes and molecular pathways involved in the negative impacts of elevated ambient temperature on fish health, and may also be valuable to our understanding of how accelerated global climate change could impact cold-water marine finfish species.