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

Atlantic Salmon (Salmo salar) Cage-Site Distribution, Behavior, and Physiology During a Newfoundland Heat Wave

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

Recombinant interleukin-1β dilates steelhead trout coronary microvessels: effect of temperature and role of the endothelium, nitric oxide and prostaglandins

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

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 (fH) 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 fH response to chasing and to increasing temperature (to 22°C) at atmospheric pressure. Pressure-induced increases in fH (e.g. from 48 to 61 bpm at 12°C) were associated with hyperactivity. The magnitude of the rise in fH 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 fH when control fish were exposed to hypoxia. Exhaustive exercise and increasing temperature to 22°C resulted in a maximum fH of 77 and 81 bpm, respectively. Our research shows that pressure influences the fH response to environmental challenges and provides the first evidence that lumpfish have a limited capacity to increase fH

The Relationship between Myoglobin, Aerobic Capacity, Nitric Oxide Synthase Activity and Mitochondrial Function in Fish Hearts

The dynamic interactions between nitric oxide (NO) and myoglobin (Mb) in the cardiovascular system have received considerable attention. The loss of Mb, the principal O2 carrier and a NO scavenger/producer, in the heart of some red-blooded fishes provides a unique opportunity for assessing this globin’s role in NO homeostasis and mitochondrial function. We measured Mb content, activities of enzymes of NO and aerobic metabolism [NO Synthase (NOS) and citrate synthase, respectively] and mitochondrial parameters [Complex-I and -I+II respiration, coupling efficiency, reactive oxygen species production/release rates and mitochondrial sensitivity to inhibition by NO (i.e., NO IC50)] in the heart of three species of red-blooded fish. The expression of Mb correlated positively with NOS activity and NO IC50, with low NOS activity and a reduced NO IC50 in the Mb-lacking lumpfish (Cyclopterus lumpus) as compared to the Mb-expressing Atlantic salmon (Salmo salar) and short-horned sculpin (Myoxocephalus scorpius). Collectively, our data show that NO levels are fine-tuned so that NO homeostasis and mitochondrial function are preserved; indicate that compensatory mechanisms are in place to tightly regulate [NO] and mitochondrial function in a species without Mb; and strongly suggest that the NO IC50 for oxidative phosphorylation is closely related to a fish’s hypoxia tolerance

The Innate Immune Response of Atlantic Salmon (Salmo salar) Is Not Negatively Affected by High Temperature and Moderate Hypoxia

Climate change is predicted to increase water temperatures and decrease oxygen levels in freshwater and marine environments, however, there is conflicting information regarding the extent to which these conditions may impact the immune defenses of fish. In this study, Atlantic salmon were exposed to: (1) normoxia (100–110% air saturation) at 12°C; (2) an incremental temperature increase (1°C per week from 12 to 20°C), and then held at 20°C for an additional 4 weeks; and (3) “2” with the addition of moderate hypoxia (~65–75% air saturation). These conditions realistically reflect what farmed salmon in some locations are currently facing, and future conditions in Atlantic Canada and Europe, during the summer months. The salmon were sampled for the measurement of head kidney constitutive anti-bacterial and anti-viral transcript expression levels, and blood parameters of humoral immune function. Thereafter, they were injected with either the multi-valent vaccine Forte V II (contains both bacterial and viral antigens) or PBS (phosphate-buffer-saline), and the head kidney and blood of these fish were sampled at 6, 12, 24, and 48 h post-injection (HPI). Our results showed that: (1) neither high temperature, nor high temperature + moderate hypoxia, adversely affected respiratory burst, complement activity or lysozyme concentration; (2) the constitutive transcript expression levels of the anti-bacterial genes il1β, il8-a, cox2, hamp-a, stlr5-a, and irf7-b were up-regulated by high temperature; (3) while high temperature hastened the peak in transcript expression levels of most anti-bacterial genes by 6–12 h following V II injection, it did not affect the magnitude of changes in transcript expression; (4) anti-viral (viperin-b, mx-b, and isg15-a) transcript expression levels were either unaffected, or downregulated, by acclimation temperature or V II injection over the 48 HPI; and (5) hypoxia, in addition to high temperature, did not impact immune transcript expression. In conclusion, temperatures up to 20°C, and moderate hypoxia, do not impair the capacity of the Atlantic salmon's innate immune system to respond to bacterial antigens. These findings are surprising, and highlight the salmon's capacity to mount robust innate immune responses (i.e., similar to control fish under optimal conditions) under conditions approaching their upper thermal limit

Heat-shock responsive genes identified and validated in Atlantic cod (Gadus morhua) liver, head kidney and skeletal muscle using genomic techniques

Background: Daily and seasonal changes in temperature are challenges that fish within aquaculture settings cannot completely avoid, and are known to elicit complex organismal and cellular stress responses. We conducted a large-scale gene discovery and transcript expression study in order to better understand the genes that are potentially involved in the physiological and cellular aspects of stress caused by heat-shock. We used suppression subtractive hybridization (SSH) cDNA library construction and characterization to identify transcripts that were dysregulated by heat-shock in liver, skeletal muscle and head kidney of Atlantic cod. These tissues were selected due to their roles in metabolic regulation, locomotion and growth, and immune function, respectively. Fish were exposed for 3 hours to an 8°C elevation in temperature, and then allowed to recover for 24 hours at the original temperature (i.e. 10°C). Tissue samples obtained before heat-shock (BHS), at the cessation of heat-shock (CS), and 3, 12, and 24 hours after the cessation of heat-shock (ACS), were used for reciprocal SSH library construction and quantitative reverse transcription - polymerase chain reaction (QPCR) analysis of gene expression using samples from a group that was transferred but not heat-shocked (CT) as controls.Results: We sequenced and characterized 4394 ESTs (1524 from liver, 1451 from head kidney and 1419 from skeletal muscle) from three "forward subtracted" libraries (enriched for genes up-regulated by heat-shock) and 1586 from the liver "reverse subtracted" library (enriched for genes down-regulated by heat-shock), for a total of 5980 ESTs. Several cDNAs encoding putative chaperones belonging to the heat-shock protein (HSP) family were found in these libraries, and "protein folding" was among the gene ontology (GO) terms with the highest proportion in the libraries. QPCR analysis of HSP90α and HSP70-1 (synonym: HSPA1A) mRNA expression showed significant up-regulation in all three tissues studied. These transcripts were more than 100-fold up-regulated in liver following heat-shock. We also identified HSP47, GRP78 and GRP94-like transcripts, which were significantly up-regulated in all 3 tissues studied. Toll-like receptor 22 (TLR22) transcript, found in the liver reverse SSH library, was shown by QPCR to be significantly down-regulated in the head kidney after heat-shock.Conclusion: Chaperones are an important part of the cellular response to stress, and genes identified in this work may play important roles in resistance to thermal-stress. Moreover, the transcript for one key immune response gene (TLR22) was down-regulated by heat-shock, and this down-regulation may be a component of heat-induced immunosuppression

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

Responses to hypoxia and recovery: Repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid, Astronotus ocellatus

Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by 3H-labelled phenylalanine incorporation rates, were determined in Astronotus ocellatus to investigate the cellular mechanisms behind hypoxia-induced metabolic depression and recovery. RoMR was significantly depressed, by approximately 50%, when dissolved oxygen levels reached 10% saturation (0.67±0.01 mg l-1 at 28±1°C). This depression in RoMR was accompanied by a 50-60% decrease in liver, heart and gill protein synthesis, but only a 30% decrease in brain protein synthesis. During recovery from hypoxia, an overshoot in RoMR to 270% of the normoxic rate was observed, indicating the accumulation of an oxygen debt during hypoxia. This conclusion was consistent with significant increase in plasma lactate levels during the hypoxic exposure, and the fact that lactate levels rapidly returned to pre-hypoxic levels. In contrast, a hyperactivation of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is attributed to an increase in cellular processes other than protein synthesis

Atlantic cod (Gadus morhua) hemoglobin genes: multiplicity and polymorphism

Background: Hemoglobin (Hb) polymorphism, assessed by protein gel electrophoresis, has been used almost exclusively to characterize the genetic structure of Atlantic cod (Gadus morhua) populations and to establish correlations with phenotypic traits such as Hb oxygen binding capacity, temperature tolerance and growth characteristics. The genetic system used to explain the results of gel electrophoresis entails the presence of one polymorphic locus with two major alleles (HbI-1; HbI-2). However, vertebrates have more than one gene encoding Hbs and recent studies have reported that more than one Hb gene is present in Atlantic cod. These observations prompted us to re-evaluate the number of Hb genes expressed in Atlantic cod, and to perform an in depth search for polymorphisms that might produce relevant phenotypes for breeding programs. Results: Analysis of Expressed Sequence Tags (ESTs) led to the identification of nine distinct Hb transcripts; four corresponding to the α Hb gene family and five to the β Hb gene family. To gain insights about the Hb genes encoding these transcripts, genomic sequence data was generated from heterozygous (HbI-1/2) parents and fifteen progeny; five of each HbI type, i.e., HbI-1/1, HbI-1/2 and HbI-2/2. β Hb genes displayed more polymorphism than α Hb genes. Two major allele types (β1A and β1B) that differ by two linked non-synonymous substitutions (Met55Val and Lys62Ala) were found in the β1 Hb gene, and the distribution of these β1A and β1B alleles among individuals was congruent with that of the HbI-1 and HbI-2 alleles determined by protein gel electrophoresis. RT-PCR and Q-PCR analysis of the nine Hb genes indicates that all genes are expressed in adult fish, but their level of expression varies greatly; higher expression of almost all Hb genes was found in individuals displaying the HbI-2/2 electrophoretic type. Conclusion: This study indicates that more Hb genes are present and expressed in adult Atlantic cod than previously documented. Our finding that nine Hb genes are expressed simultaneously in adult fish suggests that Atlantic cod, similarly to fish such as rainbow trout, carp, and goldfish, might be able to respond to environmental challenges such as chronic hypoxia or long-term changes in temperature by altering the level of expression of these genes. In this context, the role of the non-conservative substitution Lys62Ala found in the β1 Hb gene, which appears to explain the occurrence of the HbI-1 and HbI-2 alleles described by gel electrophoresis, and which was found to be present in other fish such as eel, emerald rockcod, rainbow trout and moray, requires further investigation