Hypoxia Acclimation Research Articles

Evolved changes in the intracellular distribution and physiology of muscle mitochondria in high‐altitude native deer mice

Mitochondrial function changes over time at high altitudes, but the potential benefits of these changes for hypoxia resistance remains unclear. We used high-altitude-adapted populations of deer mice, which exhibit enhanced aerobic performance in hypoxia, to examine whether changes in mitochondrial physiology or intracellular distribution in the muscle contribute to hypoxia resistance. Permeabilized muscle fibres from the gastrocnemius muscle had higher respiratory capacities in high-altitude mice than in low-altitude mice. Highlanders also had higher mitochondrial volume densities, due entirely to an enriched abundance of subsarcolemmal mitochondria, such that more mitochondria were situated near the cell membrane and adjacent to capillaries. There were several effects of hypoxia acclimation on mitochondrial function, some of which were population specific, but they differed from the evolved changes in high-altitude natives, which probably provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes. High-altitude natives that have evolved to live in hypoxic environments provide a compelling system to understand how animals can overcome impairments in oxygen availability. We examined whether these include changes in mitochondrial physiology or intracellular distribution that contribute to hypoxia resistance in high-altitude deer mice (Peromyscus maniculatus). Mice from populations native to high and low altitudes were born and raised in captivity, and as adults were acclimated to normoxia or hypobaric hypoxia (equivalent to 4300m elevation). We found that highlanders had higher respiratory capacities in the gastrocnemius (but not soleus) muscle than lowlanders (assessed using permeabilized fibres with single or multiple inputs to the electron transport system), due in large part to higher mitochondrial volume densities in the gastrocnemius. The latter was attributed to an increased abundance of subsarcolemmal (but not intermyofibrillar) mitochondria, such that more mitochondria were situated near the cell membrane and adjacent to capillaries. Hypoxia acclimation had no significant effect on these population differences, but it did increase mitochondrial cristae surface densities of mitochondria in both populations. Hypoxia acclimation also altered the physiology of isolated mitochondria by affecting respiratory capacities and cytochrome c oxidase activities in population-specific manners. Chronic hypoxia decreased the release of reactive oxygen species by isolated mitochondria in both populations. There were subtle differences in O2 kinetics between populations, with highlanders exhibiting increased mitochondrial O2 affinity or catalytic efficiency in some conditions. Our results suggest that evolved changes in mitochondrial physiology in high-altitude natives are distinct from the effects of hypoxia acclimation, and probably provide a better indication of adaptive traits that improve performance and hypoxia resistance at high altitudes.

Ventilatory acclimatization to hypoxia in deer mice native to high altitudes

Hypoxia at high altitudes constrains O2 supply to support metabolism, thermoregulation in the cold, and the aerobic scope needed for exercise. Many highland taxa appear to have physiological specializations for overcoming these challenges, but relatively little is known about the importance of hypoxia acclimation responses in high‐altitude natives, such as ventilatory acclimatization to hypoxia (VAH) and erythropoiesis. Highland taxa often possess a high haemoglobin‐O2 binding affinity, which safeguards arterial O2 saturation (SaO2) and tissue O2 delivery in hypoxia, but could foreseeably affect hypoxia acclimation responses. We examined this issue by comparing captive populations of deer mice (Peromyscus maniculatus) from high and low altitudes. Mice from each population were acclimated to normoxia and moderate hypoxia (12 kPa O2, simulating the PO2 at the native altitude of highlanders at 4300m) for 6–8 weeks. Highland mice were also acclimated to more severe hypoxia (9 kPa, the PO2 at ~7000m), which induces the same SaO2 in highlanders that lowlanders experience at 12 kPa. In lowlanders, hypoxia acclimation augmented the hypoxic ventilatory response (HVR), increased SaO2 in hypoxia, blunted the hypoxic depression of body temperature, increased blood [haemoglobin], and induced right ventricle hypertrophy (a sign of maladaptive pulmonary hypertension). Hypoxia acclimation had little effect on the HVR in highlanders, such that the hypoxic control of breathing appeared to be fixed like that in hypoxia‐acclimated lowlanders. Right ventricle hypertrophy did not occur in either moderate or severe hypoxia in highlanders, suggesting that they have blunted hypoxic pulmonary vasoconstriction and hypertension. The increase in blood [haemoglobin] was lower in highlanders than in lowlanders at 12 kPa, but highlanders mounted a robust increase in severe hypoxia (9 kPa), suggesting that differences in SaO2 and not the erythropoietic process contribute to differences in blood [haemoglobin] between populations. Therefore, high‐altitude adaptation appears to have blunted several hypoxia acclimation responses in deer mice.Support or Funding InformationSupported by NSERC of Canada.

Adrenal chromaffin cell function in high‐altitude deer mice (Peromyscus maniculatus)

Hypoxia at high altitudes can activate the sympathetic nervous system, which could become maladaptive in high‐altitude natives if it is maintained over chronic periods. The deer mouse (Peromyscus maniculatus) has the largest altitudinal range of all North American mammals, with populations living above 4300m elevation. We hypothesized that high‐altitude deer mice might have altered the physiology of chromaffin cells in the adrenal gland to avoid chronic activation of the sympathetic response. We used captive breeding colonies derived from wild populations at high and low altitudes, and compared them in normoxia and after 6–8 weeks of acclimation to hypoxia (12kPa O2, simulating the PO2 at ~4300m). High‐altitude mice show lower rates of catecholamine secretion from adrenal slices (measured via carbon fibre amperometry) in response to stimulation by a nicotinic acetylcholine receptor agonist (nicotine). Here, we used ratiometric Ca2+ imaging of cultured chromaffin cells to assess whether the variation in catecholamine secretion resulted from variation in the Ca2+ signal for vesicular catecholamine release. The rise of intracellular Ca2+ in response to nicotine (1, 5, 25, 125μM) did not vary between populations and was not affected by hypoxia acclimation, suggesting that events downstream of Ca2+ influx likely contribute to population differences in catecholamine secretion. This appeared to include differences in catecholamine stores, which were lower in highlanders compared to lowlanders in cultured cells from normoxia‐acclimated mice. Our data therefore suggest that chronic sympathetic activation may be avoided in deer mice at high altitudes by a reduction in catecholamine storage and release from the adrenal gland.

Long-term HIF-1α transcriptional activation is essential for heat-acclimation-mediated cross tolerance: mitochondrial target genes.

An important adaptive feature of heat acclimation (HA) is the induction of cross tolerance against novel stressors (HACT) Reprogramming of gene expression leading to enhanced innate cytoprotective features by attenuating damage and/or enhancing the response of "help" signals plays a pivotal role. Hypoxia-inducible factor-1α (HIF-1α), constitutively upregulated by HA (1 mo, 34°C), is a crucial transcription factor in this program, although its specific role is as yet unknown. By using a rat HA model, we studied the impact of disrupting HIF-1α transcriptional activation [HIF-1α:HIF-1β dimerization blockade by intraperitoneal acriflavine (4 mg/kg)] on its mitochondrial gene targets [phosphoinositide-dependent kinase-1 (PDK1), LON, and cyclooxygenase 4 (COX4) isoforms] in the HA rat heart. Physiological measures of cardiac HACT were infarct size after ischemia-reperfusion and time to rigor contracture during hypoxia in cardiomyocytes. We show that HACT requires transcriptional activation of HIF-1α throughout the course of HA and that this activation is accompanied by two metabolic switches: 1) profound upregulation of PDK1, which reduces pyruvate entry into the mitochondria, consequently increasing glycolytic lactate production; 2) remodeling of the COX4 isoform ratio, inducing hypoxic-tolerant COX4.2 dominance, and optimizing electron transfer and possibly ATP production during the ischemic and hypoxic insults. LON and COX4.2 transcript upregulation accompanied this shift. Loss of HACT despite elevated expression of the cytoprotective protein heat shock protein-72 concomitantly with disrupted HIF-1α dimerization suggests that HIF-1α is essential for HACT. The role of a PDK1 metabolic switch is well known in hypoxia acclimation but not in the HA model and its ischemic setting. Remodeling of COX4 isoforms by environmental acclimation is a novel finding.

Could hypoxia acclimation cause morphological changes and protect against Mn-induced oxidative injuries in silver catfish (Rhamdia quelen) even after reoxygenation?

Exposure to hypoxia has shown beneficial adjustments in different species, including silver catfish (Rhamdia quelen), especially in situations of aquatic contamination with pollutants such as manganese (Mn). Considering that hypoxia is seasonal in the natural aquatic environment, we decided to assess whether these adaptive mechanisms could be maintained when reoxygenation is established. Silver catfish acclimated to moderate hypoxia (∼3 mg L−1, 41% O2 saturation) for 10 days and subsequently exposed to Mn (∼8.1 mg L−1) for additional 10 days displayed lower (47%) Mn accumulation in the gills, and it was maintained (62.6%) after reoxygenation, in comparison to normoxia. Oxidative status in the gills allowed us to observe increased reactive species (RS) generation and protein carbonyl (PC) level together with decreased mitochondrial viability induced by Mn under normoxia. Inversely, while hypoxia per se was beneficial on RS generation and PC level, this acclimation was able to minimize Mn toxicity, as observed by the minor increase of RS generation and the minor reduction of mitochondrial viability, together with decreased PC level. Interestingly, after reoxygenation, part of the protective influences observed during hypoxia against Mn toxicity were maintained, as observed through a lower level of PC and higher mitochondrial viability in relation to the group exposed to Mn under normoxia. Only groups exposed to Mn under hypoxia showed increased activity of both catalase (CAT) and Na+/K+-ATPase in the gills, but, while CAT activity remained increased after reoxygenation, Na+/K+-ATPase activity was decreased by Mn, regardless of the oxygen level. Based on these outcomes, it is possible to propose that environment events of moderate hypoxia are able to generate rearrangements in the gills of silver catfish exposed to Mn, whose influence persists after water reoxygenation. These responses may be related to the adaptive development, reducing Mn toxicity to silver catfish.Moderate hypoxia generates rearrangements in the gills of Silver catfish, exerting beneficial and persistent protection against Mn toxicity.

Acclimation to hypoxia increases carbohydrate use during exercise in high-altitude deer mice.

The low O2 experienced at high altitude is a significant challenge to effective aerobic locomotion, as it requires sustained tissue O2 delivery in addition to the appropriate allocation of metabolic substrates. Here, we tested whether high- and low-altitude deer mice (Peromyscus maniculatus) have evolved different acclimation responses to hypoxia with respect to muscle metabolism and fuel use during submaximal exercise. Using F1 generation high- and low-altitude deer mice that were born and raised in common conditions, we assessed 1) fuel use during exercise, 2) metabolic enzyme activities, and 3) gene expression for key transporters and enzymes in the gastrocnemius. After hypoxia acclimation, highland mice showed a significant increase in carbohydrate oxidation and higher relative reliance on this fuel during exercise at 75% maximal O2 consumption. Compared with lowland mice, highland mice had consistently higher activities of oxidative and fatty acid oxidation enzymes in the gastrocnemius. In contrast, only after hypoxia acclimation did activities of hexokinase increase significantly in the muscle of highland mice to levels greater than lowland mice. Highland mice also responded to acclimation with increases in muscle gene expression for hexokinase 1 and 2 genes, whereas both populations increased mRNA expression for glucose transporters. Changes in skeletal muscle with acclimation suggest that highland mice had an increased capacity for the uptake and oxidation of circulatory glucose. Our results demonstrate that highland mice have evolved a distinct mode of hypoxia acclimation that involves an increase in carbohydrate use during exercise.

Circulatory mechanisms underlying adaptive increases in thermogenic capacity in high-altitude deer mice.

We examined the circulatory mechanisms underlying adaptive increases in thermogenic capacity in deer mice (Peromyscus maniculatus) native to the cold hypoxic environment at high altitudes. Deer mice from high- and low-altitude populations were born and raised in captivity to adulthood, and then acclimated to normoxia or hypobaric hypoxia (simulating hypoxia at ∼4300 m). Thermogenic capacity [maximal O2 consumption (V̇O2,max), during cold exposure] was measured in hypoxia, along with arterial O2 saturation (SaO2 ) and heart rate (fH). Hypoxia acclimation increased V̇O2,max by a greater magnitude in highlanders than in lowlanders. Highlanders also had higher SaO2 and extracted more O2 from the blood per heartbeat (O2 pulse=V̇O2,max/fH). Hypoxia acclimation increased fH, O2 pulse and capillary density in the left ventricle of the heart. Our results suggest that adaptive increases in thermogenic capacity involve integrated functional changes across the O2 cascade that augment O2 circulation and extraction from the blood.

Ventilatory acclimatization to hypoxia in mice: Methodological considerations

We examined ventilatory acclimatization to hypoxia (VAH) in CD1 mice, and contrasted results obtained using the barometric method on unrestrained mice with pneumotachography and pulse oximetry on restrained mice. Responses to progressive step reductions in O2 fraction (21%–8%) were assessed in mice acclimated to normoxia and hypobaric hypoxia (barometric pressure of 60kPa for 6–8 weeks). Hypoxia acclimation increased the hypoxic ventilatory response (primarily by increasing breathing frequency rather than tidal volume), arterial O2 saturation (SaO2) and heart rate in deep hypoxia, hypoxic chemosensitivity (ventilatory O2/CO2 equivalents versus SaO2), and respiratory water loss, and it blunted the hypoxic depression of metabolism and body temperature. Although some effects of hypoxia acclimation were qualitatively similar between methods, the effects were often greater in magnitude when assessed using pneumotachography. Furthermore, whereas hypoxia acclimation reduced ventilatory O2 equivalent and increased pulmonary O2 extraction in barometric experiments, it had the opposite effects in pneumotachography experiments. Our findings highlight the importance of considering the impact of how breathing is measured on the apparent responses to hypoxia.

Hypoxia acclimation and subsequent reoxygenation partially prevent Mn-induced damage in silver catfish

This study investigated if hypoxia acclimation modifies the hematological and oxidative profiles in tissues of Mn-exposed silver catfish (Rhamdia quelen), and if such modifications persist upon subsequent reoxygenation. Silver catfish acclimated to hypoxia (~3mgL−1) for 10days and subsequently exposed to Mn (~8.1mgL−1) for additional 10days exhibited lower Mn accumulation in plasma, liver and kidney, even after reoxygenation, as compared to normoxia-acclimated fish. Hypoxia acclimation increased per se red blood cells count and hematocrit, suggesting adaptations under hypoxia, while the reoxygenation process was also related to increased hematocrit and hemoglobin per se. Fish exposed to Mn under normoxia for 20days showed decreased red blood cells count and hematocrit, while reoxygenation subsequent to hypoxia increased red blood cells count. Hypoxia acclimation also prevented Mn-induced oxidative damage, observed by increased reactive species generation and higher protein carbonyl levels in both liver and kidney under normoxia. Mn-exposed fish under hypoxia and after reoxygenation showed decreased plasma transaminases in relation to the normoxia group. Moreover, acclimation to hypoxia increased reduced glutathione levels, catalase activity and Na+/K+-ATPase activity in liver and kidney during Mn exposure, remaining increased even after reoxygenation. These findings show that previous acclimation to hypoxia generates physiological adjustments, which drive coordinated responses that ameliorate the antioxidant status even after reoxygenation. Such responses represent a physiological regulation of this teleost fish against oxygen restriction and/or Mn toxicity in order to preserve the stability of a particular tissue or system.

Studies on the association of angiotensinogen polymorphisms and hypoxia acclimatization

To research the effects of gene polymorphisms of AGT at G-217A and T174M loci on incidence and the hypoxia acclimation to acute mountain sickness (AMS). Step 1:61 Han students origin low landers were exposed in acute hypoxia at 4 800 m altitude-equivalent for 6 hours, and took the supine bicycle exercise for 20 min at quantitative load of 60 r/min, 80 W after entering the cabin for 30 min, then the AMS were evaluated by the Lake Louise acute mountain sickness scoring system (LLS). The physical index of heart rate (HR), ambulatory blood pressure and oxygen saturation (SpO2) were measured during exercise. Step 2:The physical index was measured again in acute hypoxic exposure after 3 weeks increasing hypoxia exercise (At an increasing altitude of 2 500 m, 3 500 m, 4 800 m, time of 2 hours/day, 4 days/week, and in moderate intensity of exercise). PCR-RFLP was used to determine the genotypes and alleles frequencies of AGT at G-217A and T174M loci. In step 1 the hypoxic exposure, for the G-217A locus in AGT, there was no significant difference between GG and GA+AA gene tester. While in step 2 the hypoxic exposure, the SpO2 of GG gene was lower than GA+AA gene obviously (P<0.05). No significant differences of AMS incidence, VE, SpO2, HR and blood pressure were detected in different genotypes and alleles at T174M locusin both hypoxic exposures. The G-217A may be the genetics sign of hypoxia acclimation. There is no obvious rel-evancy between the T174M polymorphism and the occurrence of AMS/hypoxia acclimation.

Mitochondrial physiology and reactive oxygen species production are altered by hypoxia acclimation in killifish (Fundulus heteroclitus).

Many fish encounter hypoxia in their native environment, but the role of mitochondrial physiology in hypoxia acclimation and hypoxia tolerance is poorly understood. We investigated the effects of hypoxia acclimation on mitochondrial respiration, O2kinetics, emission of reactive oxygen species (ROS), and antioxidant capacity in the estuarine killifish ( ITALIC! Fundulus heteroclitus). Killifish were acclimated to normoxia, constant hypoxia (5 kPa O2) or intermittent diel cycles of nocturnal hypoxia (12 h:12 h normoxia:hypoxia) for 28-33 days and mitochondria were isolated from liver. Neither pattern of hypoxia acclimation affected the respiratory capacities for oxidative phosphorylation or electron transport, leak respiration, coupling control or phosphorylation efficiency. Hypoxia acclimation also had no effect on mitochondrial O2kinetics, but ITALIC! P50(the O2tension at which hypoxia inhibits respiration by 50%) was lower in the leak state than during maximal respiration, and killifish mitochondria endured anoxia-reoxygenation without any impact on mitochondrial respiration. However, both patterns of hypoxia acclimation reduced the rate of ROS emission from mitochondria when compared at a common O2tension. Hypoxia acclimation also increased the levels of protein carbonyls and the activities of superoxide dismutase and catalase in liver tissue (the latter only occurred in constant hypoxia). Our results suggest that hypoxia acclimation is associated with changes in mitochondrial physiology that decrease ROS production and may help improve hypoxia tolerance.

Simultaneous nitrification and denitrification via nitrite in a pilot-scale modified anaerobic-anoxic-oxic reactor

The performance of simultaneous nitrification and denitrification (SND) via nitrite including partial nitrification and SND was investigated in a pilot-scale-modified anaerobic–anoxic–oxic reactor. Since partial nitrification and SND need a similar requirement on dissolved oxygen (DO) level, a low DO was kept in the operation for achieving SND via nitrite. Results showed that SND via nitrite succeeded between 0.4 and 1.6 mg/L DO concentration. The average total nitrogen (TN) removal efficiency was 67.5%. Four forms of biodenitrification were quantified via the estimation of material balance and calculation of SND. SND via nitrite compared with the other three forms made 32.3% contribution to biodenitrification. The operation of a wastewater treatment plant was continuously studied as a comparison with the pilot reactor. With the same raw water, SND not only saved 82.6% of oxygen consumption as well as 30.4% of carbon source, but also owned a higher effluent quality on TN at 11.2 mg/L. Denaturing gradient gel electrophoresis results showed that after long-term hypoxia acclimation, the dominant denitrifier community changed and the abundance ratio of ammonia-oxidizing bacteria/nitrite-oxidizing bacteria was increased.

Transcriptional events co-regulated by hypoxia and cold stresses in Zebrafish larvae.

BackgroundHypoxia and temperature stress are two major adverse environmental conditions often encountered by fishes. The interaction between hypoxia and temperature stresses has been well documented and oxygen is considered to be the limiting factor for the thermal tolerance of fish. Although both high and low temperature stresses can impair the cardiovascular function and the cross-resistance between hypoxia and heat stress has been found, it is not clear whether hypoxia acclimation can protect fish from cold injury.ResultsPre-acclimation of 96-hpf zebrafish larvae to mild hypoxia (5% O2) significantly improved their resistance to lethal hypoxia (2.5% O2) and increased the survival rate of zebrafish larvae after lethal cold (10°C) exposure. However, pre-acclimation of 96-hpf larvae to cold (18°C) decreased their tolerance to lethal hypoxia although their ability to endure lethal cold increased. RNA-seq analysis identified 132 up-regulated and 41 down-regulated genes upon mild hypoxia exposure. Gene ontology enrichment analyses revealed that genes up-regulated by hypoxia are primarily involved in oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development and cellular iron ion homeostasis. Hypoxia-inhibited genes are enriched in inorganic anion transport, sodium ion transport, very long-chain fatty acid biosynthetic process and cytidine deamination. A comparison with the dataset of cold-regulated gene expression identified 23 genes co-induced by hypoxia and cold and these genes are mainly associated with oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis. The alleviation of lipid peroxidation damage by both cold- and hypoxia-acclimation upon lethal cold stress suggests the association of these genes with cold resistance. Furthermore, the alternative promoter of hmbsb gene specifically activated by hypoxia and cold was identified and confirmed.ConclusionsAcclimation responses to mild hypoxia and cold stress were found in zebrafish larvae and pre-acclimation to hypoxia significantly improved the tolerance of larvae to lethal cold stress. RNA-seq and bioinformatics analyses revealed the biological processes associated with hypoxia acclimation. Transcriptional events co-induced by hypoxia and cold may represent the molecular basis underlying the protection of hypoxia-acclimation against cold injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1560-y) contains supplementary material, which is available to authorized users.

Control of Breathing and Adaptation to High‐altitude Hypoxia in Deer Mice (Peromyscus maniculatus)

High‐altitude environments pose multiple challenges, such as cold temperatures and low partial pressures of oxygen. This study compared the control of breathing and pulmonary gas exchange in populations of deer mice with highland and lowland ancestry to determine the physiological specializations important for living in high‐altitude environments. The hypoxic ventilatory response was measured using whole‐body plethysmography in response to step‐wise decreases in inspired oxygen fraction, from 21% to 8%, in mice acclimated to normoxia or hypobaric hypoxia (simulating the hypoxia at ~4300 m elevation). Total ventilation was similar between populations, but highland mice acclimated to normoxia had a more effective breathing pattern than lowland mice acclimated to normoxia, characterized by deeper less frequent breaths at a given total ventilation. Hypoxia acclimation had no effect on breathing pattern in highland mice, but it increased tidal volume and decreased breathing frequency in lowland mice. Highland ancestry and hypoxia acclimation also affected respiratory water loss and thermoregulatory responses to hypoxia. Therefore, both highland ancestry and hypoxia acclimation influence the control of breathing and pulmonary gas exchange in deer mice. Supported by NSERC of Canada.

High-altitude ancestry and hypoxia acclimation have distinct effects on exercise capacity and muscle phenotype in deer mice.

The hypoxic and cold environment at high altitudes requires that small mammals sustain high rates of O2 transport for exercise and thermogenesis while facing a diminished O2 availability. We used laboratory-born and -raised deer mice (Peromyscus maniculatus) from highland and lowland populations to determine the interactive effects of ancestry and hypoxia acclimation on exercise performance. Maximal O₂consumption (V̇o(2max)) during exercise in hypoxia increased after hypoxia acclimation (equivalent to the hypoxia at ∼4,300 m elevation for 6-8 wk) and was consistently greater in highlanders than in lowlanders. V̇o(2max) during exercise in normoxia was not affected by ancestry or acclimation. Highlanders also had consistently greater capillarity, oxidative fiber density, and maximal activities of oxidative enzymes (cytochrome c oxidase and citrate synthase) in the gastrocnemius muscle, lower lactate dehydrogenase activity in the gastrocnemius, and greater cytochrome c oxidase activity in the diaphragm. Hypoxia acclimation did not affect any of these muscle traits. The unique gastrocnemius phenotype of highlanders was associated with higher mRNA and protein abundances of peroxisome proliferator-activated receptor γ (PPARγ). Vascular endothelial growth factor (VEGFA) transcript abundance was lower in highlanders, and hypoxia acclimation reduced the expression of numerous genes that regulate angiogenesis and energy metabolism, in contrast to the observed population differences in muscle phenotype. Lowlanders exhibited greater increases in blood hemoglobin content, hematocrit, and wet lung mass (but not dry lung mass) than highlanders after hypoxia acclimation. Genotypic adaptation to high altitude, therefore, improves exercise performance in hypoxia by mechanisms that are at least partially distinct from those underlying hypoxia acclimation.

Warm acclimation and oxygen depletion induce species-specific responses in salmonids.

Anthropogenic activities are greatly altering the habitats of animals, whereby fish are already encountering several stressors simultaneously. The purpose of the current study was to investigate the capacity of fish to respond to two different environmental stressors (high temperature and overnight hypoxia) separately and together. We found that acclimation to increased temperature (from 7.7±0.02°C to 14.9±0.05°C) and overnight hypoxia (daily changes from normoxia to 63-67% oxygen saturation), simulating climate change and eutrophication, had both antagonistic and synergistic effects on the capacity of fish to tolerate these stressors. The thermal tolerance of Arctic char (Salvelinus alpinus) and landlocked salmon (Salmo salar m. sebago) increased with warm acclimation by 1.3 and 2.2°C, respectively, but decreased when warm temperature was combined with overnight hypoxia (by 0.2 and 0.4°C, respectively). In contrast, the combination of the stressors more than doubled hypoxia tolerance in salmon and also increased hypoxia tolerance in char by 22%. Salmon had 1.2°C higher thermal tolerance than char, but char tolerated much lower oxygen levels than salmon at a given temperature. The changes in hypoxia tolerance were connected to the responses of the oxygen supply and delivery system. The relative ventricle mass was higher in cold- than in warm-acclimated salmon but the thickness of the compact layer of the ventricle increased with the combination of warm and hypoxia acclimation in both species. Char had also significantly larger hearts and thicker compact layers than salmon. The results illustrate that while fish can have protective responses when encountering a single environmental stressor, the combination of stressors can have unexpected species-specific effects that will influence their survival capacity.

Hypoxia acclimation protects against oxidative damage and changes in prolactin and somatolactin expression in silver catfish (Rhamdia quelen) exposed to manganese

The aim of this study was to assess the Mn toxicity to silver catfish considering Mn accumulation and oxidative status in different tissues, as well as pituitary hormone expression after acclimation to hypoxia. Silver catfish acclimated to hypoxia for 10 days and successively exposed to Mn (9.8mgL−1) for an additional 10 days exhibited lower Mn accumulation in plasma, liver, kidneys and brain and prevented the hematocrit decrease observed in the normoxia group. Hypoxia acclimation also modified Mn-induced oxidative damage, which was observed by lower reactive species (RS) generation in gills and kidneys, decreased lipid peroxidation (LP) levels in gills, liver and kidneys and decreased protein carbonyl (PC) levels in liver, kidneys and brain. Manganese accumulation showed positive correlations with LP levels in gills and kidneys, as well as with PC levels in gills, liver and brain. In addition, hypoxia acclimation and Mn exposure increased catalase (CAT) activity in gills and kidneys and Na+/K+-ATPase activity in gills, liver and brain. Silver catfish that were acclimated under normoxia and exposed to Mn displayed increased pituitary prolactin (PRL) and decreased somatolactin (SL) expression. Interestingly, hypoxia acclimation prevented hormonal fluctuation of PRL and SL in fish exposed to Mn. These findings indicate that while the exposure of silver catfish to Mn under normoxia was related to metal accumulation and oxidative damage in tissues together with endocrine axis disruption, as represented by PRL and SL, hypoxia acclimation reduced waterborne Mn uptake, thereby minimizing oxidative damage and changes in hormonal profile. We hypothesized that moderate hypoxia is able to generate adaptive responses, which may be related to hormesis, thereby ameliorating Mn toxicity to silver catfish.

Hypoxia tolerance thresholds for post-smolt Atlantic salmon: Dependency of temperature and hypoxia acclimation

Atlantic salmon (Salmo salar L.) experience periodic drops in dissolved oxygen (O2) inside aquaculture sea cages. In order to evaluate whether fish function or welfare is compromised during such drops in O2 (termed hypoxia), it is necessary to establish the limit for acceptable O2 reductions; the hypoxia tolerance threshold. In the present study, effects of temperature (6, 12 and 18°C) and hypoxia acclimation (33days of hypoxic periods down to 50% O2 occurring every 6h at 16°C) on the routine oxygen consumption rate (ṀO2) and the limiting oxygen saturation (LOS, defined as the hypoxia tolerance threshold) were investigated in undisturbed, fed fish kept in groups with the aim of resembling commercial aquaculture conditions. ṀO2 was measured using open respirometry where a progressive decline in O2 was caused by fish O2 consumption during a period of low water turnover. LOS was defined as the O2 below which fish were no longer able to uphold routine ṀO2. Both ṀO2 and LOS were found to increase exponentially with temperature (Q10=2.7 for ṀO2 and 1.8 for LOS), but no effect of hypoxia acclimation was found. The mean (±SE) LOS at 6, 12, 16 and 18°C was 30±1, 39±1, 47±1 and 55±2% of air saturation respectively. The variation in LOS within and across temperatures was to a large extent explained by variation in ṀO2 (R2=0.93), suggesting that LOS increases with any factor that raises the metabolic rate of Atlantic salmon. This study provides aquaculturists and legislators with a lower limit for acceptable drops in O2 at temperatures relevant for the on-growing phase in Atlantic salmon aquaculture, and discusses the variation in LOS that can be expected at a given temperature.

The effects of diel-cycling hypoxia acclimation on the hypoxia tolerance, swimming capacity and growth performance of southern catfish (Silurus meridionalis)

To investigate the effects of diel-cycling hypoxia acclimation on the hypoxia tolerance, swimming and growth performance of juvenile southern catfish, we initially measured the critical oxygen tension (Pcrit), oxygen thresholds of aquatic surface respiration (ASR) and loss of equilibrium (LOE) of diel-cycling hypoxia-acclimated (15d, 7:00–21:00, dissolved oxygen level (DO)=7.0±0.2mgL−1; 21:00–7:00, DO=3.0±0.2mgL−1) and non-acclimated (15d, DO=7.0±0.2mgL−1) southern catfish at 25°C. We then measured the critical swimming speed (Ucrit) and metabolic rate (MR) of hypoxia-acclimated and non-acclimated fish (under both hypoxic and normoxic conditions). The feeding rate (FR), feeding efficiency (FE) and specific growth rate (SGR) of fish in hypoxia-acclimated and non-acclimated groups were also measured. The Pcrit, ASR and LOE of hypoxia-acclimated fish were significantly lower than those of non-acclimated fish. Hypoxia acclimation resulted in a significantly higher Ucrit when the individuals swam in hypoxia. The Ucrit, maximum metabolic rate (MMR) and metabolic scope (MS) of both the hypoxia-acclimated and non-acclimated fish all decreased with the decrease of DO. However, the Ucrit, MMR and MS decreased by 31, 43 and 54%, respectively, in non-acclimated fish, whereas these values decreased by 15, 28 and 29%, respectively, in hypoxia-acclimated fish, which suggests that hypoxia-acclimated fish were less sensitive to the DO decrease. The FR, FE and SGR all decreased by 21, 20 and 45%, respectively, in the hypoxia-acclimated group compared to the non-acclimated group. This result suggests that diel-cycling hypoxia acclimation improved the hypoxia tolerance and aerobic swimming performance of southern catfish, whereas impaired the growth performance. The high hypoxia tolerance and physiological plasticity to hypoxia-acclimated southern catfish may be related to its lower maintenance energy expenditure, sit-and-wait lifestyle and bottom-dwelling living environment condition (usually facing oxygen fluctuation). The growth performance of so-called ‘visceral type’ fish species, such as southern catfish, are more sensitive to hypoxia compared to other fish species because of their high peak post-prandial metabolic rate, which may be restrained by the limited aerobic metabolic scope in hypoxia.

Monitoring angiogenesis using a human compatible calibration for broadband near-infrared spectroscopy

Angiogenesis is a hallmark of many conditions, including cancer, stroke, vascular disease, diabetes, and high-altitude exposure. We have previously shown that one can study angiogenesis in animal models by using total hemoglobin (tHb) as a marker of cerebral blood volume (CBV), measured using broadband near-infrared spectroscopy (bNIRS). However, the method was not suitable for patients as global anoxia was used for the calibration. Here we determine if angiogenesis could be detected using a calibration method that could be applied to patients. CBV, as a marker of angiogenesis, is quantified in a rat cortex before and after hypoxia acclimation. Rats are acclimated at 370-mmHg pressure for three weeks, while rats in the control group are housed under the same conditions, but under normal pressure. CBV increased in each animal in the acclimation group. The mean CBV (%volume/volume) is 3.49%± 0.43% (mean ± SD) before acclimation for the experimental group, and 4.76%± 0.29% after acclimation. The CBV for the control group is 3.28%± 0.75%, and 3.09%± 0.48% for the two measurements. This demonstrates that angiogenesis can be monitored noninvasively over time using a bNIRS system with a calibration method that is compatible with human use and less stressful for studies using animals.