Metabolic rate increases with acclimation temperature and is associated with mitochondrial function in some tissues of threespine stickleback.

The metabolic rate (MO2) of eurythermal fishes changes in response to temperature, yet it is unclear how changes in mitochondrial function contribute to changes in MO2. We hypothesized that MO2 would increase with acclimation temperature in the threespine stickleback (Gasterosteus aculeatus) in parallel with metabolic remodeling at the cellular level but that changes in metabolism in some tissues, such as liver, would contribute more to changes in MO2 than others. Threespine stickleback were acclimated to 5, 12 and 20°C for 7 to 21 weeks. At each temperature, standard and maximum metabolic rate (SMR and MMR, respectively), and absolute aerobic scope (AAS) were quantified, along with mitochondrial respiration rates in liver, oxidative skeletal and cardiac muscles, and the maximal activity of citrate synthase (CS) and lactate dehydrogenase (LDH) in liver, and oxidative and glycolytic skeletal muscles. SMR, MMR and AAS increased with acclimation temperature, along with rates of mitochondrial phosphorylating respiration in all tissues. Low SMR and MMR at 5°C were associated with low or undetectable rates of mitochondrial complex II activity and a greater reliance on complex I activity in liver, oxidative skeletal muscle and heart. SMR was positively correlated with cytochrome c oxidase (CCO) activity in liver and oxidative muscle, but not mitochondrial proton leak, whereas MMR was positively correlated with CCO activity in liver. Overall, the results suggest that changes in MO2 in response to temperature are driven by changes in some aspects of mitochondrial function in some, but not all, tissues of threespine stickleback.

Warm acclimation alters antioxidant defences but not metabolic capacities in the Antarctic fish, Notothenia coriiceps.

The Southern Ocean surrounding the Western Antarctic Peninsula region is rapidly warming. Survival of members of the dominant suborder of Antarctic fishes, the Notothenioidei, will likely require thermal plasticity and adaptive capacity in key traits delimiting thermal tolerance. Herein, we have assessed the thermal plasticity of several cellular and biochemical pathways, many of which are known to be associated with thermal tolerance in notothenioids, including mitochondrial function, activities of aerobic and anaerobic enzymes, antioxidant defences, protein ubiquitination and degradation in cardiac, oxidative skeletal muscles and gill of Notothenia coriiceps warm acclimated to 4°C for 22 days or 5°C for 42 days. Levels of triacylglycerol (TAG) were measured in liver and oxidative and glycolytic skeletal muscles, and glycogen in liver and glycolytic muscle to assess changes in energy stores. Metabolic pathways displayed minimal thermal plasticity, yet antioxidant defences were lower in heart and oxidative skeletal muscles of warm-acclimated animals compared with animals held at ambient temperature. Despite higher metabolic rates at elevated temperature, energy storage depots of TAG and glycogen increase in liver and remain unchanged in muscle with warm acclimation. Overall, our studies reveal that N. coriiceps displays thermal plasticity in some key traits that may contribute to their survival as the Southern Ocean continues to warm.

Aerobic scope is not maintained at low temperature and is associated with cardiac aerobic capacity in the three-spined stickleback Gasterosteus aculeatus.

Metabolic thermal plasticity is central to the survival of fishes in a changing environment. The eurythermal three-spined stickleback Gasterosteus aculeatus displays thermal plasticity at the cellular level with an increase in the activity of key metabolic enzymes in response to cold acclimation. Nonetheless, it is unknown if these changes are sufficient to completely compensate for the depressive effects of cold temperature on whole organismal metabolic rate (MO2 ). The authors hypothesized that as a cold-tolerant, eurythermal fish, absolute aerobic scope (AAS), the difference between the maximum metabolic rate (MMR) and standard metabolic rate (SMR), would be maintained in G. aculeatus following acclimation to a range of temperatures that span its habitat temperatures. To test this hypothesis, G. aculeatus were acclimated to 5, 12 and 20°C for 20-32 weeks, and SMR, MMR and aerobic scope (AS) were quantified at each acclimation temperature. The maximal activity of citrate synthase (CS), a marker enzyme of aerobic metabolism, was also quantified in heart ventricles to determine if cardiac aerobic capacity is associated with AS at these temperatures. SMR increased with acclimation temperature and was significantly different among all three temperature groups. MMR was similar between animals at 5 and 12°C and between animals at 12 and 20°C but was 2.6-fold lower in fish at 5°C compared with those at 20°C, resulting in a lower AAS in fish at 5°C compared with those at 12 and 20°C. Correlated with a higher AAS in animals acclimated to 12 and 20°C was a larger relative ventricular mass and higher CS activity per 100 g body mass compared with animals at 5°C. Together, the results indicate that despite their eurythermal nature, AS is not maintained at low temperature but is associated with cardiac aerobic metabolic capacity.

Resilience of cardiac performance in Antarctic notothenioid fishes in a warming climate.

Warming in the region of the Western Antarctic Peninsula is occurring at an unprecedented rate, which may threaten the survival of Antarctic notothenioid fishes. Herein, we review studies characterizing thermal tolerance and cardiac performance in notothenioids - a group that includes both red-blooded species and the white-blooded, haemoglobinless icefishes - as well as the relevant biochemistry associated with cardiac failure during an acute temperature ramp. Because icefishes do not feed in captivity, making long-term acclimation studies unfeasible, we focus only on the responses of red-blooded notothenioids to warm acclimation. With acute warming, hearts of the white-blooded icefish Chaenocephalus aceratus display persistent arrhythmia at a lower temperature (8°C) compared with those of the red-blooded Notothenia coriiceps (14°C). When compared with the icefish, the enhanced cardiac performance of N. coriiceps during warming is associated with greater aerobic capacity, higher ATP levels, less oxidative damage and enhanced membrane integrity. Cardiac performance can be improved in N. coriiceps with warm acclimation to 5°C for 6-9 weeks, accompanied by an increase in the temperature at which cardiac failure occurs. Also, both cardiac mitochondrial and microsomal membranes are remodelled in response to warm acclimation in N. coriiceps, displaying homeoviscous adaptation. Overall, cardiac performance in N. coriiceps is malleable and resilient to warming, yet thermal tolerance and plasticity vary among different species of notothenioid fishes; disruptions to the Antarctic ecosystem driven by climate warming and other anthropogenic activities endanger the survival of notothenioids, warranting greater protection afforded by an expansion of marine protected areas.

Homeoviscous adaptation occurs with thermal acclimation in biological membranes from heart and gill, but not the brain, in the Antarctic fish Notothenia coriiceps.

As temperatures continue to rise, adjustments to biological membranes will be key for maintenance of function. It is largely unknown to what extent Antarctic notothenioids possess the capacity to remodel their biological membranes in response to thermal change. In this study, physical and biochemical properties were examined in membranes prepared from gill epithelia (plasma membranes), cardiac ventricles (microsomes, mitochondria), and brains (synaptic membranes, myelin, mitochondria) from Notothenia coriiceps following acclimation to 5 °C (or held at ambient temperature, 0 °C) for a minimum of 6 weeks. Fluidity was measured between 0 and 30 °C in all membranes, and polar lipid compositions and cholesterol contents were analyzed in a subset of biological membranes from all tissues. Osmotic permeability was measured in gills at 0 and 4 °C. Gill plasma membranes, cardiac mitochondria, and cardiac microsomes displayed reduced fluidity following acclimation to 5 °C, indicating compensation for elevated temperature. In contrast, no fluidity changes with acclimation were observed in any of the membranes prepared from brain. In all membranes, adjustments to the relative abundances of major phospholipid classes, and to the extent of fatty acid unsaturation, were undetectable following thermal acclimation. However, alterations in cholesterol contents and acyl chain length, consistent with the changes in fluidity, were observed in membranes from gill and cardiac tissue. Water permeability was reduced with 5 °C acclimation in gills, indicating near-perfect homeostatic efficacy. Taken together, these results demonstrate a homeoviscous response in gill and cardiac membranes, and limited plasticity in membranes from the nervous system, in an Antarctic notothenioid.

Thermal profiles reveal stark contrasts in properties of biological membranes from heart among Antarctic notothenioid fishes which vary in expression of hemoglobin and myoglobin.

Antarctic notothenioids are noted for extreme stenothermy, yet underpinnings of their thermal limits are not fully understood. We hypothesized that properties of ventricular membranes could explain previously observed differences among notothenioids in temperature onset of cardiac arrhythmias and persistent asystole. Microsomes were prepared using ventricles from six species of notothenioids, including four species from the hemoglobin-less (Hb-) family Channichthyidae (icefishes), which also differentially express cardiac myoglobin (Mb), and two species from the (Hb+) Nototheniidae. We determined membrane fluidity and structural integrity by quantifying fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and leakage of 5(6)-carboxyfluorescein, respectively, over a temperature range from ambient (0 °C) to 20 °C. Compositions of membrane phospholipids and cholesterol contents were also quantified. Membranes from all four species of icefishes exhibited greater fluidity than membranes from the red-blooded species N. coriiceps. Thermal sensitivity of fluidity did not vary among species. The greatest thermal sensitivity to leakage occurred between 0 and 5 °C for all species, while membranes from the icefish, Chaenocephalus aceratus (Hb-/Mb-) displayed leakage that was nearly 1.5-fold greater than leakage in N. coriiceps (Hb+/Mb+). Contents of phosphatidylethanolamine (PE) were approximately 1.5-fold greater in icefishes than in red-blooded fishes, and phospholipids had a higher degree of unsaturation in icefishes than in Hb + notothenioids. Cholesterol contents were lowest in Champsocephalus gunnari (Hb-/Mb-) and highest in the two Hb+/Mb + species, G. gibberifrons and N. coriiceps. Our results reveal marked differences in membrane properties and indicate a breach in membrane fluidity and structural integrity at a lower temperature in icefishes than in red-blooded notothenioids.

Maximum cardiac performance of Antarctic fishes that lack haemoglobin and myoglobin: exploring the effect of warming on nature's natural knockouts.

Antarctic notothenioids, some of which lack myoglobin (Mb) and/or haemoglobin (Hb), are considered extremely stenothermal, which raises conservation concerns since Polar regions are warming at unprecedented rates. Without reliable estimates of maximum cardiac output ([Formula: see text]), it is impossible to assess their physiological scope in response to warming seas. Therefore, we compared cardiac performance of two icefish species, Chionodraco rastrospinosus (Hb-Mb+) and Chaenocephalus aceratus (Hb-Mb-), with a related notothenioid, Notothenia coriiceps (Hb+Mb+) using an in situ perfused heart preparation. The maximum [Formula: see text], heart rate (f H), maximum cardiac work (W C) and relative ventricular mass of N. coriiceps at 1°C were comparable to temperate-water teleosts, and acute warming to 4°C increased f H and W C, as expected. In contrast, icefish hearts accommodated a higher maximum stroke volume (V S) and maximum [Formula: see text] at 1°C, but their unusually large hearts had a lower f H and maximum afterload tolerance than N. coriiceps at 1°C. Furthermore, maximum V S, maximum [Formula: see text] and f H were all significantly higher for the Hb-Mb+ condition compared with the Hb-Mb- condition, a potential selective advantage when coping with environmental warming. Like N. coriiceps, both icefish species increased f H at 4°C. Acutely warming C. aceratus increased maximum [Formula: see text], while C. rastrospinosus (like N. coriiceps) held at 4°C for 1 week maintained maximum [Formula: see text] when tested at 4°C. These experiments involving short-term warming should be followed up with long-term acclimation studies, since the maximum cardiac performance of these three Antarctic species studied seem to be tolerant of temperatures in excess of predictions associated with global warming.

Mitochondrial membranes in cardiac muscle from Antarctic notothenioid fishes vary in phospholipid composition and membrane fluidity.

Antarctic notothenioid fishes are highly stenothermal, yet their tolerance for warming is species-dependent. Because a body of literature points to the loss of cardiac function as underlying thermal limits in ectothermic animals, we investigated potential relationships among properties of ventricular mitochondrial membranes in notothenioids with known differences in both cardiac mitochondrial metabolism and organismal thermal tolerance. Fluidity of mitochondrial membranes was quantified by fluorescence depolarization for the white-blooded Chaenocephalus aceratus and the red-blooded Notothenia coriiceps. In these same membranes, lipid compositions and products of lipid peroxidation, the latter of which can disrupt membrane order, were analyzed in both species and in a second icefish, Pseudochaenichthys georgianus. Mitochondrial membranes from C. aceratus were significantly more fluid than those of the more thermotolerant species N. coriiceps (P < .0001). Consistent with this, ratios of total phosphatidylethanolamine (PE) to total phosphatidylcholine (PC) were lower in membranes from both species of icefishes, compared to those of N. coriiceps (P < .05). However, membranes of N. coriiceps displayed a greater unsaturation index (P < .0001). No differences among species were found in membrane products of lipid peroxidation. With rising temperatures, greater contents of PC in mitochondrial membranes from ventricles of icefishes are likely to promote membrane hyperfluidization at a lower temperature than for cardiac mitochondrial membranes from the red-blooded notothenioid. We propose that physical and chemical properties of the mitochondrial membranes may contribute to some of the observed differences in thermal sensitivity of physiological function among these species.

Physical, chemical, and functional properties of neuronal membranes vary between species of Antarctic notothenioids differing in thermal tolerance.

Disruption of neuronal function is likely to influence limits to thermal tolerance. We hypothesized that with acute warming the structure and function of neuronal membranes in the Antarctic notothenioid fish Chaenocephalus aceratus are more vulnerable to perturbation than membranes in the more thermotolerant notothenioid Notothenia coriiceps. Fluidity was quantified in synaptic membranes, mitochondrial membranes, and myelin from brains of both species of Antarctic fishes. Polar lipid compositions and cholesterol contents were analyzed in myelin; cholesterol was measured in synaptic membranes. Thermal profiles were determined for activities of two membrane-associated proteins, acetylcholinesterase (AChE) and Na+/K+-ATPase (NKA), from brains of animals maintained at ambient temperature or exposed to their critical thermal maxima (CTMAX). Synaptic membranes of C. aceratus were consistently more fluid than those of N. coriiceps (P < 0.0001). Although the fluidities of both myelin and mitochondrial membranes were similar among species, sensitivity of myelin fluidity to in vitro warming was greater in N. coriiceps than in C. aceratus (P < 0.001), which can be explained by lower cholesterol contents in myelin of N. coriiceps (P < 0.05). Activities of both enzymes, AChE and NKA, declined upon CTMAX exposure in C. aceratus, but not in N. coriiceps. We suggest that hyper-fluidization of synaptic membranes with warming in C. aceratus may explain the greater stenothermy in this species, and that thermal limits in notothenioids are more likely to be influenced by perturbations in synaptic membranes than in other membranes of the nervous system.

The effects of thermal acclimation on cardio-respiratory performance in an Antarctic fish (Notothenia coriiceps).

The Southern Ocean has experienced stable, cold temperatures for over 10 million years, yet particular regions are currently undergoing rapid warming. To investigate the impacts of warming on cardiovascular oxygen transport, we compared the cardio-respiratory performance in an Antarctic notothenioid (Notothenia coriiceps) that was maintained at 0 or 5°C for 6.0-9.5 weeks. When compared at the fish's respective acclimation temperature, the oxygen consumption rate and cardiac output were significantly higher in 5°C-acclimated than 0°C-acclimated fish. The 2.7-fold elevation in cardiac output in 5°C-acclimated fish (17.4 vs. 6.5 ml min-1 kg-1) was predominantly due to a doubling of stroke volume, likely in response to increased cardiac preload, as measured by higher central venous pressure (0.15 vs. 0.08 kPa); tachycardia was minor (29.5 vs. 25.2 beats min-1). When fish were acutely warmed, oxygen consumption rate increased by similar amounts in 0°C- and 5°C-acclimated fish at equivalent test temperatures. In both acclimation groups, the increases in oxygen consumption rate during acute heating were supported by increased cardiac output achieved by elevating heart rate, while stroke volume changed relatively little. Cardiac output was similar between both acclimation groups until 12°C when cardiac output became significantly higher in 5°C-acclimated fish, driven largely by their higher stroke volume. Although cardiac arrhythmias developed at a similar temperature (~14.5°C) in both acclimation groups, the hearts of 5°C-acclimated fish continued to pump until significantly higher temperatures (CTmax for cardiac function 17.7 vs. 15.0°C for 0°C-acclimated fish). These results demonstrate that N. coriiceps is capable of increasing routine cardiac output during both acute and chronic warming, although the mechanisms are different (heart rate-dependent versus primarily stroke volume-dependent regulation, respectively). Cardiac performance was enhanced at higher temperatures following 5°C acclimation, suggesting cardiovascular function may not constrain the capacity of N. coriiceps to withstand a warming climate.

Exploring nature's natural knockouts: in vivo cardiorespiratory performance of Antarctic fishes during acute warming.

We tested the hypothesis that blackfin icefish (Chaenocephalus aceratus), one of the six species in the family Channichthyidae (the icefishes) that do not express haemoglobin and myoglobin, lack regulatory cardiovascular flexibility during acute warming and activity. The experimental protocols were designed to optimize the surgical protocol and minimize stress. First, minimally invasive heart rate (fH) measurements were made during a thermal ramp until cardiac failure in C. aceratus and compared with those from the closely related red-blooded black rockcod (Notothenia coriiceps). Then, integrative cardiovascular adjustments were more extensively studied using flow probes and intravascular catheters in C. aceratus during acute warming (from 0 to 8°C) at rest and after imposed activity. Chaenocephalus aceratus had a lower routine fH than N. coriiceps (9 beats min-1 versus 14 beats min-1) and a lower peak fH during acute warming (38 beats min-1 versus 55 beats min-1) with a similar cardiac breakpoint temperature (13 and 14°C, respectively). Routine cardiac output (Q̇) for C. aceratus at ∼0°C was much lower (26.6 ml min-1 kg-1) than previously reported, probably because fish in the present study had a low fH (12 beats min-1) indicative of a high routine vagal tone and low stress. Chaenocephalus aceratus increased oxygen consumption during acute warming and with activity. Correspondingly, Q̇ increased considerably (maximally 86.3 ml min-1 kg-1), as did vascular conductance (5-fold). Thus, unlike earlier suggestions, these data provide convincing evidence that icefish can mount a well-developed cardiovascular regulation of heart rate, cardiac output and vascular conductance, and this regulatory capacity provides flexibility during acute warming.

Cardiac mitochondrial metabolism may contribute to differences in thermal tolerance of red- and white-blooded Antarctic notothenioid fishes.

Studies in temperate fishes provide evidence that cardiac mitochondrial function and the capacity to fuel cardiac work contribute to thermal tolerance. Here, we tested the hypothesis that decreased cardiac aerobic metabolic capacity contributes to the lower thermal tolerance of the haemoglobinless Antarctic icefish, Chaenocephalus aceratus, compared with that of the red-blooded Antarctic species, Notothenia coriiceps. Maximal activities of citrate synthase (CS) and lactate dehydrogenase (LDH), respiration rates of isolated mitochondria, adenylate levels and changes in mitochondrial protein expression were quantified from hearts of animals held at ambient temperature or exposed to their critical thermal maximum (CTmax). Compared with C. aceratus, activity of CS, ATP concentration and energy charge were higher in hearts of N. coriiceps at ambient temperature and CTmax While state 3 mitochondrial respiration rates were not impaired by exposure to CTmax in either species, state 4 rates, indicative of proton leakage, increased following exposure to CTmax in C. aceratus but not N. coriiceps The interactive effect of temperature and species resulted in an increase in antioxidants and aerobic metabolic enzymes in N. coriiceps but not in C. aceratus Together, our results support the hypothesis that the lower aerobic metabolic capacity of C. aceratus hearts contributes to its low thermal tolerance.

The loss of hemoglobin and myoglobin does not minimize oxidative stress in Antarctic icefishes.

The unusual pattern of expression of hemoglobin (Hb) and myoglobin (Mb) among Antarctic notothenioid fishes provides an exceptional model system for assessing the impact of these proteins on oxidative stress. We tested the hypothesis that the lack of oxygen-binding proteins may reduce oxidative stress. Levels and activity of pro-oxidants and small-molecule and enzymatic antioxidants, and levels of oxidized lipids and proteins in the liver, oxidative skeletal muscle and heart ventricle were quantified in five species of notothenioid fishes differing in the expression of Hb and Mb. Levels of ubiquitinated proteins and rates of protein degradation by the 20S proteasome were also quantified. Although levels of oxidized proteins and lipids, ubiquitinated proteins, and antioxidants were higher in red-blooded fishes than in Hb-less icefishes in some tissues, this pattern did not persist across all tissues. Expression of Mb was not associated with oxidative damage in the heart ventricle, whereas the activity of citrate synthase and the contents of heme were positively correlated with oxidative damage in most tissues. Despite some tissue differences in levels of protein carbonyls among species, rates of degradation by the 20S proteasome were not markedly different, suggesting either alternative pathways for eliminating oxidized proteins or that redox tone varies among species. Together, our data indicate that the loss of Hb and Mb does not correspond with a clear pattern of either reduced oxidative defense or oxidative damage.

Molecular drivers of mitochondrial membrane proliferation in response to cold acclimation in threespine stickleback.

Little is known about how the synthesis of mitochondrial phospholipids is integrated into mitochondrial biogenesis in fish or mammals. Glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) catalyzes the addition of fatty acyl CoA to the sn-1 position of glycerol-3-phosphate, in what is considered the rate-limiting step in phospholipid biosynthesis. Previous studies have shown that mitochondrial volume density increases in oxidative skeletal muscle but not liver of Gasterosteus aculeatus (threespine stickleback) in response to cold acclimation. We hypothesized that maximal activity of GPAT would increase in oxidative skeletal muscle but not liver during cold acclimation, coinciding with mitochondrial biogenesis. GPAT activity was measured in liver and oxidative skeletal (pectoral adductor) muscle of threespine stickleback acclimated to 8°C or 20°C. In addition, mRNA levels of enzymes involved in phospholipid synthesis, including cytidine diphosphodiacylglycerol synthase-1 (CDS1), CDS2, GPAT1, GPAT2 and 1-acylglycerol 3-phosphate acyltransferase-2 (AGPAT2), were quantified in liver and pectoral muscle of stickleback harvested during cold acclimation. GPAT activity and transcript levels of AGPAT2 increased in response to cold acclimation in pectoral muscle but not liver. Transcript levels of GPAT1 increased in liver but not pectoral muscle. Overall our results suggest that the activity of GPAT, and possibly AGPAT as well, increase during cold acclimation and may contribute to mitochondrial phospholipid biosynthesis required for mitochondrial biogenesis.

Characterization of mitochondrial glycerol-3-phosphate acyltransferase in notothenioid fishes.

Hearts of Antarctic icefishes (suborder Notothenioidei, family Channichthyidae) have higher densities of mitochondria, and mitochondria have higher densities of phospholipids, compared to red-blooded notothenioids. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting step in glycerolipid biosynthesis. There are four isoforms of GPAT in vertebrates; GPAT1 and GPAT2 are localized to the outer mitochondrial membrane, whereas GPAT3 and GPAT4 are localized to the endoplasmic reticulum membrane. We hypothesized that transcript levels of GPAT1 and/or GPAT2 would mirror densities of mitochondrial phospholipids and be higher in the icefish Chaenocephalus aceratus compared to the red-blooded species Notothenia coriiceps. Transcript levels of GPAT1 were quantified in heart ventricles and liver using qRT-PCR. Additionally, GPAT1 cDNA was sequenced in the Antarctic notothenioids, C. aceratus and N. coriiceps, and in the sub-Antarctic notothenioid, Eleginops maclovinus, to identify amino acid substitutions that may maintain GPAT1 function at cold temperature. Transcript levels of GPAT1 were higher in liver compared to heart ventricles but were not significantly different between the two species. In contrast, transcripts of GPAT2 were only detected in ventricle where they were 6.6-fold higher in C. aceratus compared to N. coriiceps. These data suggest GPAT1 may be more important for synthesizing triacylglycerol, whereas GPAT2 may regulate synthesis of phospholipids. GPAT1 amino acid sequences are highly conserved among the three notothenioids with 97.9-98.7% identity. Four amino acid substitutions within the cytosolic region of Antarctic notothenioid GPAT1 may maintain conformational changes necessary for binding and catalysis at cold temperature.

Expansion of capacities for iron transport and sequestration reflects plasma volumes and heart mass among white-blooded notothenioid fishes.

The family Channichthyidae or "icefishes" (suborder Notothenioidei) represents the only vertebrates lacking hemoglobin (Hb) as adults. Several icefish species also do not express cardiac myoglobin (Mb). We address how levels of proteins involved in iron (Fe) processing (transport, sequestration, and export) vary among white- and red-blooded notothenioids, and whether absence of Hb and/or Mb in channichthyids is accompanied by expansion of contents of Fe-binding proteins to protect against unchaperoned Fe. Levels of transferrin (Tf), ferritin (Ft), ceruloplasmin (Cp), and non-heme Fe were quantified in plasma, serum, and/or nonhematopoietic tissues (cardiac ventricle, skeletal muscle, and liver) from species of white-blooded (Chaenocephalus aceratus, Champsocephalus gunnari, Chionodraco rastrospinosus, Pseudochaenichthys georgianus) (the first two species not expressing Mb) and red-blooded (Notothenia coriiceps, Gobionotothen gibberifrons) notothenioids. We also measured levels of ascorbate (Asc), a mediator of Fe uptake. While plasma concentrations of Tf and tissue levels of Asc are similar among species, concentrations of plasma Asc are lower in white-blooded species. Concentrations of Ft and non-heme Fe and activities of Cp are also generally reduced in icefishes compared with red-blooded notothenioids. The presence of cardiac Mb in some icefish species does not appear to influence levels of proteins involved in Fe processing. To address further the question of Fe sequestration within a physiological context, we account for well-characterized differences in blood volume and heart mass among white- and red-blooded notothenioids. We report that total contents of plasma Tf are greater, while ventricle non-heme Fe is at least at parity in white- vs. red-blooded species.

New Lessons from an Old Fish: What Antarctic Icefishes May Reveal about the Functions of Oxygen-Binding Proteins.

The loss of expression of the oxygen-binding protein hemoglobin (Hb) in the family Channichthyidae (suborder Notothenioidei) of Antarctic fishes is considered a disaptation that has persisted because of the unusual conditions prevailing in the Southern Ocean during the evolution of the family. The loss of expression of the intracellular oxygen-binding protein myoglobin (Mb) in heart ventricles is more of a conundrum because it occurred at four points during the radiation of the family, suggesting weakened selective pressure maintaining expression of the protein. Yet, studies have shown that when present, Mb enhances function. Here, I discuss potential reasons for weakened selective pressure maintaining Mb expression in light of the multiple functions proposed for Mb. Additionally, I discuss results from recent studies exploring the possibility that the loss of Hb and Mb may be advantageous because it reduces the production of reactive oxygen species, levels of oxidized proteins, and the energetic costs associated with replacing oxidatively damaged proteins.

Hyperoxia Does Not Extend Critical Thermal Maxima (CTmax) in White- or Red-Blooded Antarctic Notothenioid Fishes.

Understanding what limits the capacity of organisms to tolerate increasing temperatures is a critical objective in comparative biology. Using an experimental system of Antarctic notothenioid fishes, we sought to determine whether a mismatch between oxygen demand and oxygen supply was responsible for setting thermal tolerance limits. Previous studies have shown that Antarctic icefishes (family Channichthyidae), which lack hemoglobin, have lower critical thermal maxima (CTmax) than red-blooded notothenioids collected from the same region of the Antarctic (Western Antarctic Peninsula). In addition, within the notothenioid fishes there exists a positive correlation between CTmax and hematocrit. We tested the hypothesis that the lower CTmax of icefishes is associated with reduced oxygen supply. We employed an experimental heat ramp (4°C h(-1)) to determine CTmax under both normoxic and hyperoxic conditions and quantified correlates of oxygen limitation (lactate levels and expression of hypoxia-inducible factor-1α) in white-blooded Chaenocephalus aceratus and red-blooded Notothenia coriiceps. Hyperoxia, corresponding to a three- to fourfold increase in seawater Po2, did not extend CTmax in either species despite an overall mitigation in the rise of plasma and muscle lactate compared with the normoxic treatment. Our results also indicate that cardiac HIF-1α mRNA levels were insensitive to changes in both temperature and oxygen treatments. The absence of a change in CTmax with hyperoxia is likely to represent the contribution of factors beyond oxygen supply to physiological failure at elevated temperatures.

Cold acclimation increases levels of some heat shock protein and sirtuin isoforms in threespine stickleback.

Molecular chaperones [heat shock proteins (HSPs)] increase in response to rapid changes in temperatures, but long-term acclimation to cold temperature may also warrant elevations in HSPs. In fishes, cold acclimation increases mitochondrial density and oxidative stress in some tissues, which may increase demand for HSPs. We hypothesized that levels of HSPs, as well as sirtuins (SIRTs), NAD-dependent deacetylases that mediate changes in metabolism and responses to oxidative stress (including increases in HSPs), would increase during cold acclimation of threespine stickleback (Gasterosteus aculeatus). Transcript levels of hsp70, hsc70, hsp60 and hsp90-α, sirts1-4, as well as protein levels of HSP60, HSP90 and HSC70 were quantified in liver and pectoral adductor muscle of stickleback during cold acclimation from 20 °C to 8 °C. In liver, cold acclimation stimulated a transient increase in mRNA levels of hsp60 and hsc70. Transcript levels of sirt1 and sirt2 also increased in response to cold acclimation and remained elevated. In pectoral muscle, mRNA levels of hsp60, hsp90-α, hsc70 and sirt1 all transiently increased in response to cold acclimation, while levels of sirts2-4 remained constant or declined. Similar to transcript levels, protein levels of HSC70 increased in both liver and pectoral muscle. Levels of HSP90 also increased in liver after 4 weeks at 8 °C. HSP60 remained unchanged in both tissues, as did HSP90 in pectoral muscle. Our results indicate that while both HSPs and SIRTs increase in response to cold acclimation in stickleback, the response is tissue and isoform specific, likely reflecting differences in metabolism and oxidative stress.