Implications of chronic hypoxia during development in red drum.

Respiratory plasticity is a beneficial response to chronic hypoxia in fish. Red drum, a teleost that commonly experiences hypoxia in the Gulf of Mexico, have shown respiratory plasticity following sublethal hypoxia exposure as juveniles, but implications of hypoxic exposure during development are unknown. We exposed red drum embryos to hypoxia (40% air saturation) or normoxia (100% air saturation) for 3 days post fertilization (dpf). This time frame encompasses hatch and exogenous feeding. At 3 dpf, there was no difference in survival and no change in size. After the 3-day hypoxia exposure, all larvae were moved and reared in common normoxic conditions. Fish were reared for ∼3 months and measured for implications of the developmental hypoxia exposure on swim performance and whole-animal aerobic metabolism. We used a cross design wherein fish from normoxia (N=24) were swam in Blazka swim tunnels in both hypoxia (40%, n=12) and normoxia (100%, n=12), and likewise for hypoxia-exposed fish (N=20, n=10 each group). Oxygen consumption, critical swim speed (Ucrit), critical oxygen threshold (Pcrit), and mitochondrial respiration were measured. Hypoxia-exposed fish had higher aerobic scope, maximum metabolic rate, and higher liver mitochondrial efficiency relative to control fish in normoxia. Interestingly, hypoxia-exposed fish showed increased hypoxia sensitivity (higher Pcrit), and recruit burst swimming at lower swim speeds relative to control fish. These data provide evidence that hypoxia exposure leads to a complex response in later life.

Acute toxicity testing of 6PPD-quinone on the estuarine-dependent sport fish, Sciaenops ocellatus.

Recently, large-scale fish kills in the Pacific Northwest were linked to tire wear particles (TWPs) left on roadways, with the lethality attributed to 6PPD-quinone. which has a median lethal concentration of <1 µg/L for selected salmonids. However, there remains a paucity of 6PPD-quinone toxicity values developed for estuarine fish species, which is particularly significant because estuaries receiving inflows from highly urbanized watersheds are especially vulnerable to TWP contamination. Therefore, the present study aimed to determine the toxicity of 6PPD-quinone to an economically and ecologically important estuarine-dependent fish-red drum (Sciaenops ocellatus). Here, we examined the relative sensitivities of three early life stages within red drum: embryonic, larval, and post-settlement for 24-72 hours, depending on the life stage. Exposure concentrations ranged from 10 µg/L to 500 µg/L. We also assessed the sub-lethal impacts of 6PPD-quinone exposure on development during embryonic and larval stages, including body and organ sizes. Our results indicate that red drum are not acutely sensitive to 6PPD-quinone at each early life stage tested. We also found that yolk-sac larvae did not exhibit sub-lethal morphological impacts in a dose-dependent manner, regardless of exposure during embryonic and larval stages. These data are the first to assess the impacts of 6PPD-quinone on estuarine-dependent non-model fishes.

Hypoxia acclimation improves mitochondrial efficiency in the aerobic swimming muscle of red drum (Sciaenops ocellatus).

Environmental hypoxia (low dissolved oxygen) is a significant threat facing fishes. As fishes require oxygen to efficiently produce ATP, hypoxia can significantly limit aerobic capacity. However, some fishes show respiratory flexibility that rescues aerobic performance, including plasticity in mitochondrial performance. This plasticity may result in increased mitochondrial efficiency (e.g., less proton leak), increased oxygen storage capacity (increased myoglobin), and oxidative capacity (e.g., higher citrate synthase activity) under hypoxia. We acclimated a hypoxia-tolerant fish, red drum (Sciaenops ocellatus), to 8-days of constant hypoxia to induce a hypoxic phenotype. Fish were terminally sampled for cardiac and red muscle tissue to quantify oxidative phosphorylation, proton leak, and maximum respiration in tissue from both hypoxia-acclimated and control fish. Tissue was also collected to assess the plasticity of citrate synthase enzyme activity and mRNA expression for select oxygen storage and antioxidant pathway transcripts. We found that mitochondrial respiration rates were not affected by hypoxia exposure in cardiac tissue, though citrate synthase activity and myoglobin expression were higher following hypoxia acclimation. Interestingly, measures of mitochondrial efficiency in red muscle significantly improved in hypoxia-acclimated individuals. Hypoxia-acclimated fish had significantly higher OXPHOS Control Efficiency, OXPHOS Capacity and Coupling Control Ratios (i.e., LEAK/OXPHOS). There was no significant change to citrate synthase activity or myoglobin expression in red muscle. Overall, these results suggest that red muscle mitochondria of hypoxia-acclimated fish more efficiently utilize oxygen, which may explain previous reports in red drum of improved aerobic swimming performance in the absence of improved maximum metabolic rate following hypoxia acclimation.

The role of carbonic anhydrase-mediated tissue oxygen extraction in a marine teleost acclimated to hypoxia.

With the growing prevalence of hypoxia (O2 levels ≤2 mg l-1) in aquatic and marine ecosystems, there is increasing interest in the adaptive mechanisms fish may employ to better their performance in stressful environments. Here, we investigated the contribution of a proposed strategy for enhancing tissue O2 extraction - plasma-accessible carbonic anhydrase (CA-IV) - under hypoxia in a species of estuarine fish (red drum, Sciaenops ocellatus) that thrives in fluctuating habitats. We predicted that hypoxia-acclimated fish would increase the prevalence of CA-IV in aerobically demanding tissues to confer more efficient tissue O2 extraction. Furthermore, we predicted the phenotypic changes to tissue O2 extraction that occur with hypoxia acclimation may improve respiratory and swim performance under 100% O2 conditions (i.e. normoxia) when compared with performance in fish that have not been acclimated to hypoxia. Interestingly, there were no significant differences in relative CA-IV mRNA expression, protein abundance or enzyme activity between the two treatments, suggesting CA-IV function is maintained under hypoxia. Likewise, respiratory performance of hypoxia-acclimated fish was similar to that of control fish when tested in normoxia. Critical swim speed (Ucrit) was significantly higher in hypoxia-acclimated fish but translated to marginal ecological benefits with an increase of ∼0.3 body lengths per second. Instead, hypoxia-acclimated fish may have relied more heavily on anaerobic metabolism during their swim trials, utilizing burst swimming 1.5 times longer than control fish. While the maintenance of CA-IV may still be an important contributor for hypoxia tolerance, our evidence suggests hypoxia-acclimated red drum are using other mechanisms to cope in an O2-depleted environment.

Effects of hypoxia on swimming and sensing in a weakly electric fish.

Low dissolved oxygen (hypoxia) can severely limit fish performance, especially aerobically expensive behaviours including swimming and acquisition of sensory information. Fishes can reduce oxygen requirements by altering these behaviours under hypoxia, but the underlying mechanisms can be difficult to quantify. We used a weakly electric fish as a model system to explore potential effects of hypoxia on swim performance and sensory information acquisition, which enabled us to non-invasively record electric signalling activity used for active acquisition of sensory information during swimming. To quantify potential effects of hypoxia, we measured critical swim speed (Ucrit) and concurrent electric signalling activity under high- and low-dissolved oxygen concentrations in a hypoxia-tolerant African mormyrid fish, Marcusenius victoriae Fish were maintained under normoxia for 6 months prior to experimental treatments, and then acclimated for 8 weeks to normoxia or hypoxia and tested under both conditions (acute: 4 h exposure). Acute hypoxia exposure resulted in a significant reduction in both Ucrit and electric signalling activity in fish not acclimated to hypoxia. However, individuals acclimated to chronic hypoxia were characterized by a higher Ucrit under both hypoxia and normoxia than fish acclimated to normoxia. Following a 6 month re-introduction to normoxia, hypoxia-acclimated individuals still showed increased performance under acute hypoxic test conditions, but not under normoxia. Our results highlight the detrimental effects of hypoxia on aerobic swim performance and sensory information acquisition, and the ability of fish to heighten aerobic performance through acclimation processes that can still influence performance even months after initial exposure.

Warming-induced "plastic floors" improve hypoxia vulnerability, not aerobic scope, in red drum (Sciaenops ocellatus).

Ocean warming is a prevailing threat to marine ectotherms. Recently the "plastic floors, concrete ceilings" hypothesis was proposed, which suggests that a warmed fish will acclimate to higher temperatures by reducing standard metabolic rate (SMR) while keeping maximum metabolic rate (MMR) stable, therefore improving aerobic scope (AS). Here we evaluated this hypothesis on red drum (Sciaenops ocellatus) while incorporating measures of hypoxia vulnerability (critical oxygen threshold; Pcrit) and mitochondrial performance. Fish were subjected to a 12-week acclimation to 20 °C or 28 °C. Respirometry was performed every 4 weeks to obtain metabolic rate and Pcrit; mitochondrial respirometry was performed on liver and heart samples at the end of the acclimation. 28 °C fish had a significantly higher SMR, MMR, and Pcrit than 20 °C controls at time 0, but SMR declined by 36.2 % over the 12-week acclimation. No change in SMR was observed in the control treatment. Contrary to expectations, SMR suppression did not improve AS relative to time 0 owing to a progressive decline in MMR over acclimation time. Pcrit decreased by 27.2 % in the warm-acclimated fishes, which resulted in temperature treatments having statistically similar values by 12-weeks. No differences in mitochondrial traits were observed in the heart - despite a Δ8 °C assay temperature - while liver respiratory and coupling control ratios were significantly improved, suggesting that mitochondrial plasticity may contribute to the reduced SMR with warming. Overall, this work suggests that warming induced metabolic suppression offsets the deleterious consequences of high oxygen demand on hypoxia vulnerability, and in so doing greatly expands the theoretical range of metabolically available habitats for red drum.

Pyrene drives reduced brain size during early life exposure in an estuarine fish, the red drum (Sciaenops ocellatus).

Crude oil and the constituent polycyclic aromatic hydrocarbons (PAHs) induce a consistent suite of sub-lethal effects in early life stage fishes. It has been suggested that 3-ring PAHs drive cardiotoxicity and that all other impacts are downstream consequences of these cardiac effects. However, recent studies have documented behavioral alterations that may not be linked to cardiotoxicity. This raises the question of whether the 3-ring PAHs that drive cardiotoxicity are also responsible for the observed neurological impairments. To explore this question, we exposed embryonic red drum (Sciaenops ocellatus) - a species that exhibits greater sensitivity to craniofacial malformations than cardiotoxicity - to individual 2-ring, 3-ring, and 4-ring PAHs for 48 h after which they were assessed for sub-lethal developmental malformations. No effects were observed following exposure to naphthalene, anthracene, dibenzothiophene, phenanthrene and fluorene at doses equivalent to the ΣPAH50 effective concentration 50 for craniofacial malformation in red drum. Conversely, pyrene caused complete lethality at the original dose, and a 5× diluted dose resulted in significantly reduced brain size and spine length. Similar sub-lethal effects were also observed in chrysene at the 1× dose. These results indicate that 4-ring PAHs are driving malformations in developing red drum and suggest oil induced impairments in this species are not a downstream consequence of 3-ring PAH induced cardiac malformations.

Respiratory plasticity improves aerobic performance in hypoxia in a marine teleost.

Ocean deoxygenation is a pressing concern in the face of climate change. In response to prolonged hypoxia, fishes have demonstrated the ability to dynamically regulate hemoglobin (Hb) expression to enhance oxygen (O2) uptake. Here, we examined hypoxia-inducible Hb expression in red drum (Sciaenops ocellatus) and the subsequent implications on Hb-O2 binding affinity and aerobic scope. Fish were acclimated to 30 % air saturation for 1 d, 4 d, 8 d, 2 w, or 6 w, and red blood cells were collected for gene expression and biochemical profiling. Hypoxia acclimation induced significant up-regulation of one Hb subunit isoform (hbα 2) relative to control by 4 d with consistent upregulation thereafter. Hematocrit increased in hypoxia, with no changes in the allosteric modulator [NTP] at any time point. Changes in Hb expression co-occurred with a reduced Root effect (~26 % in normoxia, ~14 % in hypoxia) at a physiologically relevant pH while increasing O2 binding affinity (i.e., lower P50). These changes correlated with increased maximum metabolic rate and aerobic scope relative to controls when fish were tested in hypoxia. These results demonstrate an important role for Hb multiplicity in improving O2 affinity and maximizing respiratory performance in hypoxia.

The effects of temperature on oil-induced respiratory impairment in red drum (Sciaenops ocellatus).

The 2010 Deepwater Horizon (DWH) crude oil spill, among the largest environmental disasters in U.S. history, affected numerous economically important fishes. Exposure to crude oil can lead to reduced cardiac function, limiting oxygen transport, ATP production, and aerobic performance. However, crude oil exposure is not the only stressor that affects aerobic performance, and increasing environmental temperatures are known to significantly increase metabolic demands in fishes. As the DWH spill was active during warm summer months in the Gulf of Mexico, it is important to understand the combined effects of oil and temperature on a suite of metabolic parameters. Therefore, we investigated the effects of 24h crude oil exposure on the aerobic metabolism and hypoxia tolerance of red drum (Sciaenops ocellatus) following 3 week chronic exposure to four ecologically relevant temperatures (18 °C, 22 °C, 25 °C, 28 °C). Our results show that individuals acclimated to higher temperatures had significantly higher standard metabolic rate than individuals at lower temperatures, which resulted in significantly decreased critical oxygen threshold and reduced recovery from exercise. As predicted, crude oil exposure resulted in lower maximum metabolic rates (MMR) across the temperature range, and a significantly reduced ability to recover from exercise. The lowest temperature acclimation showed the smallest effect of oil on MMR, while the highest temperature showed the smallest effect on exercise recovery. Reduced respiratory performance and hypoxia tolerance are likely to have meaningful impacts on the fitness of red drum, especially with climate-induced temperature increases and continued oil exploration in the Gulf of Mexico.

The additive effects of oil exposure and hypoxia on aerobic performance in red drum (Sciaenops ocellatus).

Aerobic scope, the difference between standard metabolic requirements and maximum metabolic capacity, is considered a particularly important metric influencing ecological success in fishes. Crude oil exposure can impair cardiorespiratory function in fishes, which reduces maximum metabolic rate, aerobic scope, and may impair ecological performance. Oil exposure is not the only environmental stressor that can affect aerobic scope, especially in areas affected by crude oil spills. Hypoxia (low dissolved oxygen) is also known to constrain maximum metabolic rate, yet there has been little effort to explore how hypoxia may influence the magnitude of metabolic injury following oil exposure. Therefore, our goal was to investigate the effects of acute oil exposure and hypoxia on the metabolic performance of red drum (Sciaenops ocellatus), an economically important fish common in the Gulf of Mexico. Here, sub-adult red drum were exposed to crude oil for 24 h before being exposed to hypoxic conditions following exhaustive exercise. Our results show that hypoxia exposure combined with crude oil exposure results in significantly reduced aerobic scope, which was additive compared to the reductions caused by each stressor alone. We also quantified hypoxia tolerance among treatments following exposure, and our results showed no changes to hypoxia tolerance among individuals, regardless of exposure to hypoxia or oil. These data offer insight into the metabolic constraints facing fishes exposed to oil while concurrently subjected to hypoxia, a notable climate change stressor.