Intermittent normobaric hypoxia alters substrate partitioning and muscle oxygenation in individuals with obesity: implications for fat burning.

This single-blind, crossover study aimed to measure and evaluate the short-term metabolic responses to continuous and intermittent hypoxic patterns in individuals with obesity. Indirect calorimetry was used to quantify changes in resting metabolic rate (RMR), carbohydrate (CHOox, %CHO), and fat oxidation (FATox, %FAT) in nine individuals with obesity pre and post: 1) breathing normoxic air [normoxic sham control (NS-control)], 2) breathing continuous hypoxia (CH), or 3) breathing intermittent hypoxia (IH). A mean peripheral oxygen saturation ([Formula: see text]) of 80-85% was achieved over a total of 45 min of hypoxia. Throughout each intervention, pulmonary gas exchanges, oxygen consumption (V̇o2) carbon dioxide production (V̇co2), and deoxyhemoglobin concentration (Δ[HHb]) in the vastus lateralis were measured. Both RMR and CHOox measured pre- and postinterventions were unchanged following each treatment: NS-control, CH, or IH (all P > 0.05). Conversely, a significant increase in FATox was evident between pre- and post-IH (+44%, P = 0.048). Although the mean Δ[HHb] values significantly increased during both IH and CH (P < 0.05), the greatest zenith of Δ[HHb] was achieved in IH compared with CH (P = 0.002). Furthermore, there was a positive correlation between Δ[HHb] and the shift in FATox measured pre- and postintervention. It is suggested that during IH, the increased bouts of muscle hypoxia, revealed by elevated Δ[HHb], coupled with cyclic periods of excess posthypoxia oxygen consumption (EPHOC, inherent to the intermittent pattern) played a significant role in driving the increase in FATox post-IH.

Succinate-mediated reactive oxygen species production in the anoxia-tolerant epaulette (Hemiscyllium ocellatum) and grey carpet (Chiloscyllium punctatum) sharks.

Anoxia/re-oxygenation (AR) results in elevated unchecked oxidative stress and mediates irreversible damage within the brain for most vertebrates. Succinate accumulation within mitochondria of the ischaemic brain appears to increase the production of reactive oxygen species (ROS) upon re-oxygenation. Two closely related elasmobranchs, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) repeatedly experience near anoxia and re-oxygenation in their habitats and have adapted to survive AR at tropical temperatures without significant brain injuries. However, these anoxia-tolerant species display contrasting strategies to survive AR, with only H. ocellatum having the capacity to supress metabolism and H. ocellatum mitochondria the capacity to depress succinate oxidation post-AR. We measured oxygen consumption alongside ROS production mediated by elevated succinate in mitochondria of permeabilized cerebellum from both shark species. Although mitochondrial respiration remained similar for both species, the ROS production in H. ocellatum was half that of C. punctatum in phosphorylating and non-phosphorylating mitochondria. Maximum ROS production in H. ocellatum was mediated by succinate loads 10-fold higher than in C. punctatum mitochondria. The contrasting survival strategies of anoxia-tolerant sharks reveal the significance of mitigating ROS production under elevated succinate load during AR, shedding light on potential mechanisms to mitigate brain injury.

Electron transfer and ROS production in brain mitochondria of intertidal and subtidal triplefin fish (Tripterygiidae).

While oxygen is essential for oxidative phosphorylation, O2 can form reactive species (ROS) when interacting with electrons of mitochondrial electron transport system. ROS is dependent on O2 pressure (PO2) and has traditionally been assessed in O2 saturated media, PO2 at which mitochondria do not typically function in vivo. Mitochondrial ROS can be significantly elevated by the respiratory complex II substrate succinate, which can accumulate within hypoxic tissues, and this is exacerbated further with reoxygenation. Intertidal species are repetitively exposed to extreme O2 fluctuations, and have likely evolved strategies to avoid excess ROS production. We evaluated mitochondrial electron leakage and ROS production in permeabilized brain of intertidal and subtidal triplefin fish species from hyperoxia to anoxia, and assessed the effect of anoxia reoxygenation and the influence of increasing succinate concentrations. At typical intracellular PO2, net ROS production was similar among all species; however at elevated PO2, brain tissues of the intertidal triplefin fish released less ROS than subtidal species. In addition, following in vitro anoxia reoxygenation, electron transfer mediated by succinate titration was better directed to respiration, and not to ROS production for intertidal species. Overall, these data indicate that intertidal triplefin fish species better manage electrons within the ETS, from hypoxic-hyperoxic transitions.

Pre-acclimation to altitude in young adults: choosing a hypoxic pattern at sea level which provokes significant haematological adaptations.

PURPOSE: This single-blind, repeated measures study evaluated adaptive and maladaptive responses to continuous and intermittent hypoxic patterns in young adults. METHODS: Changes in haematological profile, stress and cardiac damage were measured in ten healthy young participants during three phases: (1) breathing normoxic air (baseline); (2) breathing normoxic air via a mask (Sham-controls); (3) breathing intermittent hypoxia (IH) via a mask, mean peripheral oxygen saturation (SpO2) of 85% ~ 70 min of hypoxia. After a 5-month washout period, participants repeated this three-phase protocol with phase, (4) consisting of continuous hypoxia (CH), mean SpO2 = 85%, ~ 70 min of hypoxia. Measures of the red blood cell count (RBCc), haemoglobin concentration ([Hb]), haematocrit (Hct), percentage of reticulocytes (% Retics), secretory immunoglobulin A (S-IgA), cortisol, cardiac troponin T (cTnT) and the erythropoietic stimulation index (calculated OFF-score) were compared across treatments. RESULTS: Despite identical hypoxic durations at the same fixed SpO2, no significant effects were observed in either CH or Sham-CH control, compared to baseline. While IH and Sham-IH controls demonstrated significant increases in: RBCc; [Hb]; Hct; and the erythropoietic stimulation index. Notably, the % Retics decreased significantly in response to IH (-31.9%) or Sham-IH control (-23.6%), highlighting the importance of including Sham-controls. No difference was observed in S-IgA, cortisol or cTnT. CONCLUSION: The IH but not CH pattern significantly increased key adaptive haematological responses, without maladaptive increases in S-IgA, cortisol or cTnT, indicating that the IH hypoxic pattern would be the best method to boost haematological profiles prior to ascent to altitude.

Intermittent not continuous hypoxia provoked haematological adaptations in healthy seniors: hypoxic pattern may hold the key.

PURPOSE: The purpose of this single-blind, repeated measures study was to investigate the effect of two hypoxic patterns, continuous or intermittent on key markers of haematological adaptation, stress and cardiac damage in healthy senior participants. METHODS: Fifteen healthy senior participants each followed a three-phase protocol over 3 consecutive weeks: (1) 5 consecutive days of breathing room air without a mask (2) 5 days of normoxic mask breathing (sham, FiO2 = 21%) (3) 5 days of intermittent hypoxia (IH) tailored to achieve a mean peripheral oxygen saturation (SpO2) of 85% during ~ 70 min of cumulative exposure to hypoxia. After a 5-month washout period, participants were recalled to undertake continuous hypoxia (CH, SpO2 = 85%, ~ 70 min). The red blood cell count (RBCc), haemoglobin concentration ([Hb]), haematocrit (Hct), percentage of reticulocytes (% Retics), secretory immunoglobulin A (S-IgA), cortisol, cardiac troponin T (cTnT) and the OFF-score (i.e. [Formula: see text]) were measured. RESULTS: RBCc only increased by day 5 of IH treatment compared to day 5 baseline values (+ 7.7%, p < 0.01) and day 5 Sham values (+ 12.9%, p < 0.01). [Hb] only increased by day 5 of IH treatment compared to day 5 baseline values (+ 14.7%, p < 0.01) and day 5 Sham values (+ 14.3%, p < 0.01). Hct (+ 12.7%, p < 0.01) and the OFF-score (p < 0.05) increased only during the final day of IH treatment. No difference was observed in S-IgA, cortisol or cTnT following IH or CH. CONCLUSION: These results revealed that inherent differences in the IH and CH hypoxic patterns could provide crucial components required to trigger hematological changes in senior individuals, without eliciting immunological stress responses or damaging the myocardium.

Mitochondrial plasticity in the cerebellum of two anoxia-tolerant sharks: contrasting responses to anoxia/re-oxygenation.

Exposure to anoxia leads to rapid ATP depletion, alters metabolic pathways and exacerbates succinate accumulation. Upon re-oxygenation, the preferential oxidation of accumulated succinate most often impairs mitochondrial function. Few species can survive prolonged periods of hypoxia and anoxia at tropical temperatures and those that do may rely on mitochondria plasticity in response to disruptions to oxygen availability. Two carpet sharks, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) display different adaptive responses to prolonged anoxia: while H. ocellatum enters energy-conserving metabolic depression, C. punctatum temporarily elevates its haematocrit, prolonging oxygen delivery. High-resolution respirometry was used to investigate mitochondrial function in the cerebellum, a highly metabolically active organ that is oxygen sensitive and vulnerable to injury after anoxia/re-oxygenation (AR). Succinate was titrated into cerebellar preparations in vitro, with or without pre-exposure to AR, then the activity of mitochondrial complexes was examined. As in most vertebrates, C. punctatum mitochondria significantly increased succinate oxidation rates, with impaired complex I function post-AR. In contrast, H. ocellatum mitochondria inhibited succinate oxidation rates and both complex I and II capacities were conserved, resulting in preservation of oxidative phosphorylation capacity post-AR. Divergent mitochondrial plasticity elicited by elevated succinate post-AR parallels the inherently divergent physiological adaptations of these animals to prolonged anoxia, namely the absence (C. punctatum) and presence (H. ocellatum) of metabolic depression. As anoxia tolerance in these species also occurs at temperatures close to that for humans, examining their mitochondrial responses to AR could provide insights for novel interventions in clinical settings.

Acidosis Maintains the Function of Brain Mitochondria in Hypoxia-Tolerant Triplefin Fish: A Strategy to Survive Acute Hypoxic Exposure?

The vertebrate brain is generally very sensitive to acidosis, so a hypoxia-induced decrease in pH is likely to have an effect on brain mitochondria (mt). Mitochondrial respiration (JO2) is required to generate an electrical gradient (ΔΨm) and a pH gradient to power ATP synthesis, yet the impact of pH modulation on brain mt function remains largely unexplored. As intertidal fishes within rock pools routinely experience hypoxia and reoxygenation, they would most likely experience changes in cellular pH. We hence compared four New Zealand triplefin fish species ranging from intertidal hypoxia-tolerant species (HTS) to subtidal hypoxia-sensitive species (HSS). We predicted that HTS would tolerate acidosis better than HSS in terms of sustaining mt structure and function. Using respirometers coupled to fluorimeters and pH electrodes, we titrated lactic-acid to decrease the pH of the media, and simultaneously recorded JO2, ΔΨm, and H+ buffering capacities within permeabilized brain and swelling of mt isolated from non-permeabilized brains. We then measured ATP synthesis rates in the most HTS (Bellapiscus medius) and the HSS (Forsterygion varium) at pH 7.25 and 6.65. Mitochondria from HTS brain did have greater H+ buffering capacities than HSS mt (∼10 mU pH.mgprotein -1). HTS mt swelled by 40% when exposed to a decrease of 1.5 pH units, and JO2 was depressed by up to 15% in HTS. However, HTS were able to maintain ΔΨm near -120 mV. Estimates of work, in terms of charges moved across the mt inner-membrane, suggested that with acidosis, HTS mt may in part harness extra-mt H+ to maintain ΔΨm, and could therefore support ATP production. This was confirmed with elevated ATP synthesis rates and enhanced P:O ratios at pH 6.65 relative to pH 7.25. In contrast, mt volumes and ΔΨm decreased downward pH 6.9 in HSS mt and paradoxically, JO2 increased (∼25%) but ATP synthesis and P:O ratios were depressed at pH 6.65. This indicates a loss of coupling in the HSS with acidosis. Overall, the mt of these intertidal fish have adaptations that enhance ATP synthesis efficiency under acidic conditions such as those that occur in hypoxic or reoxygenated brain.

Will ocean acidification affect the early ontogeny of a tropical oviparous elasmobranch (Hemiscyllium ocellatum)?

Atmospheric CO2 is increasing due to anthropogenic causes. Approximately 30% of this CO2 is being absorbed by the oceans and is causing ocean acidification (OA). The effects of OA on calcifying organisms are starting to be understood, but less is known about the effects on non-calcifying organisms, notably elasmobranchs. One of the few elasmobranch species that has been studied with respect to OA is the epaulette shark, Hemiscyllium ocellatum. Mature epaulette sharks can physiologically and behaviourally tolerate prolonged exposure to elevated CO2, and this is thought to be because they are routinely exposed to diurnal decreases in O2 and probably concomitant increases in CO2 in their coral reef habitats. It follows that H. ocellatum embryos, while developing in ovo on the reefs, would have to be equally if not more tolerant than adults because they would not be able to escape such conditions. Epaulette shark eggs were exposed to either present-day control conditions (420 µatm) or elevated CO2 (945 µatm) and observed every 3 days from 10 days post-fertilization until 30 days post-hatching. Growth (in square centimetres per day), yolk usage (as a percentage), tail oscillations (per minute), gill movements (per minute) and survival were not significantly different in embryos reared in control conditions when compared with those reared in elevated CO2 conditions. Overall, these findings emphasize the importance of investigating early life-history stages, as the consequences are expected to transfer not only to the success of an individual but also to populations and their distribution patterns.

Could thermal sensitivity of mitochondria determine species distribution in a changing climate?

For many aquatic species, the upper thermal limit (Tmax) and the heart failure temperature (THF) are only a few degrees away from the species' current environmental temperatures. While the mechanisms mediating temperature-induced heart failure (HF) remain unresolved, energy flow and/or oxygen supply disruptions to cardiac mitochondria may be impacted by heat stress. Recent work using a New Zealand wrasse (Notolabrus celidotus) found that ATP synthesis capacity of cardiac mitochondria collapses prior to T(HF). However, whether this effect is limited to one species from one thermal habitat remains unknown. The present study confirmed that cardiac mitochondrial dysfunction contributes to heat stress-induced HF in two additional wrasses that occupy cold temperate (Notolabrus fucicola) and tropical (Thalassoma lunare) habitats. With exposure to heat stress, T. lunare had the least scope to maintain heart function with increasing temperature. Heat-exposed fish of all species showed elevated plasma succinate, and the heart mitochondria from the cold temperate N. fucicola showed decreased phosphorylation efficiencies (depressed respiratory control ratio, RCR), cytochrome c oxidase (CCO) flux and electron transport system (ETS) flux. In situ assays conducted across a range of temperatures using naive tissues showed depressed complex II (CII) and CCO capacity, limited ETS reserve capacities and lowered efficiencies of pyruvate uptake in T. lunare and N. celidotus. Notably, alterations of mitochondrial function were detectable at saturating oxygen levels, indicating that cardiac mitochondrial insufficiency can occur prior to HF without oxygen limitation. Our data support the view that species distribution may be related to the thermal limits of mitochondrial stability and function, which will be important as oceans continue to warm.

Revisiting redox-active antioxidant defenses in response to hypoxic challenge in both hypoxia-tolerant and hypoxia-sensitive fish species.

It is not known whether changes in antioxidant levels always occur in fish in response to the oxidative stress that usually accompanies a hypoxic challenge. The studies of antioxidant responses to hypoxia in fish have mostly focused on very anoxia-tolerant species and indicate that there is an enhancement of antioxidant defenses. Here we present new data on redox-active antioxidants from three species, which range in their tolerance to hypoxia: the epaulette shark, threespine stickleback, and rainbow trout, together with a compilation of results from other studies that have measured oxidative stress parameters in hypoxia-exposed fish. The results suggest that in general, fish do not show an increase in redox-active antioxidant defense in response to oxidative stress associated with hypoxia. Rather, the changes in antioxidant defenses during hypoxia are very much species- and tissue-specific and are not linked to the level of hypoxia tolerance of the fish species.

Transcriptional responses to hypoxia are enhanced by recurrent hypoxia (hypoxic preconditioning) in the epaulette shark.

All animals require molecular oxygen for aerobic energy production, and oxygen availability has played a particularly important role in the evolution of aquatic animals. This study investigates how previous exposure to hypoxia (preconditioning) primes protective transcriptional responses in a hypoxia-tolerant vertebrate species, the epaulette shark (Hemiscyllium ocellatum). The epaulette shark is a basal cartilaginous fish that in its natural environment experiences cyclic hypoxic periods. We evaluated whether the transcription of a set of crucial prosurvival genes is affected differently by a single short-term (2 h) exposure to sublethal hypoxia compared with eight such successive hypoxia exposures (hypoxia preconditioning). We discovered that hypoxia preconditioning amplifies transcriptional responses compared with animals that experienced a single hypoxic bout. In the heart we observed that hypoxic preconditioning, but not a single hypoxic exposure, resulted in higher transcript levels of genes that regulate oxygen and energy homeostasis, including those of hypoxia-inducible factor-1 alpha, adenosine signaling pathway components, and genes affecting circulation [prostaglandin synthetase 2 (cox-2) and natriuretic peptide C]. This suggests that in a single short-term hypoxic bout, the responses to low oxygen are regulated at the level of pre-existing proteins or translational and posttranslational machinery, whereas transcriptional responses are induced in experiments that parallel the natural environmental cycles of oxygen availability. These findings have general implications for understanding how vertebrates regulate protective gene expression upon physiological stress.

Hypoxia tolerance in elasmobranchs. I. Critical oxygen tension as a measure of blood oxygen transport during hypoxia exposure.

The critical O(2) tension of whole-animal O(2) consumption rate (M(O2)), or P(crit), is the water P(O2) (Pw(O(2))) at which an animal transitions from an oxyregulator to an oxyconformer. Although P(crit) is a popular measure of hypoxia tolerance in fishes because it reflects the capacity for O(2) uptake from the environment at low Pw(O(2)), little is known about the interrelationships between P(crit) and blood O(2) transport characteristics and increased use of anaerobic metabolism during hypoxia exposure in fishes, especially elasmobranchs. We addressed this knowledge gap using progressive hypoxia exposures of two elasmobranch species with differing hypoxia tolerance. The P(crit) of the hypoxia-tolerant epaulette shark (Hemiscyllium ocellatum, 5.10±0.37 kPa) was significantly lower than that of the comparatively hypoxia-sensitive shovelnose ray (Aptychotrema rostrata, 7.23±0.40 kPa). Plasma [lactate] was elevated above normoxic values at around P(crit) in epaulette sharks, but increased relative to normoxic values at Pw(O(2)) below P(crit) in shovelnose rays, providing equivocal support for the hypothesis that P(crit) is associated with increased anaerobic metabolism. The M(O2), arterial P(O2) and arterial blood O(2) content (Ca(O(2))) were similar between the two species under normoxia and decreased in both species with progressive hypoxia, but as Pw(O(2)) declined, epaulette sharks had a consistently higher M(O2) and Ca(O(2)) than shovelnose rays, probably due to their significantly greater in vivo haemoglobin (Hb)-O(2) binding affinity (in vivo Hb-O(2) P(50)=4.27±0.57 kPa for epaulette sharks vs 6.35±0.34 kPa for shovelnose rays). However, at Pw(O(2)) values representing the same percentage of each species' P(crit) (up to ∼175% of P(crit)), Hb-O(2) saturation and Ca(O(2)) were similar between species. These data support the hypothesis that Hb-O(2) P(50) is an important determinant of P(crit) and suggest that P(crit) can predict Hb-O(2) saturation and Ca(O(2)) during hypoxia exposure, with a lower P(crit) being associated with greater O(2) supply at a given Pw(O(2)) and consequently better hypoxia tolerance. Thus, P(crit) is a valuable predictor of environmental hypoxia tolerance and hypoxia exposures standardized at a given percentage of P(crit) will yield comparable levels of arterial hypoxaemia, facilitating cross-species comparisons of responses to hypoxia.

Hypoxia tolerance in elasmobranchs. II. Cardiovascular function and tissue metabolic responses during progressive and relative hypoxia exposures.

Cardiovascular function and metabolic responses of the heart and other tissues during hypoxia exposure were compared between the hypoxia-tolerant epaulette shark (Hemiscyllium ocellatum) and the hypoxia-sensitive shovelnose ray (Aptychotrema rostrata). In both species, progressive hypoxia exposure caused increases in stroke volume and decreases in heart rate, cardiac output, cardiac power output (CPO, an assessment of cardiac energy demand) and dorsal aortic blood pressure, all of which occurred at or below each species' critical P(O2) for whole-animal O(2) consumption rate, M(O2) (P(crit)). In epaulette sharks, which have a lower P(crit) than shovelnose rays, routine levels of cardiovascular function were maintained to lower water P(O2) levels and the changes from routine levels during hypoxia exposure were smaller compared with those for the shovelnose ray. The maintenance rather than depression of cardiovascular function during hypoxia exposure may contribute to the superior hypoxia tolerance of the epaulette shark, presumably by improving O(2) delivery and waste removal. Compared with shovelnose rays, epaulette sharks were also better able to maintain a stable cardiac high-energy phosphate pool and to minimize metabolic acidosis and lactate accumulation in the heart (despite higher CPO) and other tissues during a 4 h exposure to 40% of their respective P(crit) (referred to as a relative hypoxia exposure), which results in similar hypoxaemia in the two species (∼16% Hb-O(2) saturation). These different metabolic responses to relative hypoxia exposure suggest that variation in hypoxia tolerance among species is not solely dictated by differences in O(2) uptake and transport but also by tissue-specific metabolic responses. In particular, lower tissue [lactate] accumulation in epaulette sharks than in shovelnose rays during relative hypoxia exposure suggests that enhanced extra-cardiac metabolic depression occurs in the former species. This could facilitate strategic utilization of available O(2) for vital organs such as the heart, potentially explaining the greater hypoxic cardiovascular function of epaulette sharks.

Forecasting elasmobranch survival following exposure to severe stressors.

Current fishing practices and habitat degradation in most of the world's oceans pose significant threats to marine fish including elasmobranchs. The accurate prediction of survival probability for elasmobranchs subjected to prolonged immobilisation and diminished oxygen availability during capture and a vulnerable state post-release, is reliant on selecting a reliable set of biomarkers to profile as well as using them to design pre-release interventions which minimise elasmobranch death. The purpose of this review is: i) to make a case for the need to develop new biomarkers to use in conjunction with blood chemistry; ii) to briefly present the survival strategies used by other vertebrates subjected to diminished oxygen iii) to discuss new approaches to forecasting the effect that altered physiological and biochemical markers have on long-term survival with a particular emphasis on oxidative stress, the adenylate energy charge, heat shock protein expression and the capacity for repair, so that a more detailed profile of the qualities of elasmobranch survivorship can be constructed. In addition, the review will discuss the relevance of biomarkers to field samples as well as their incorporation into laboratory based research, aimed at providing physiological and biochemical data to inform conservation management.

A radical approach to beating hypoxia: depressed free radical release from heart fibres of the hypoxia-tolerant epaulette shark (Hemiscyllum ocellatum).

Hypoxia and warm ischemia are primary concerns in ischemic heart disease and transplant and trauma. Hypoxia impacts tissue ATP supply and can induce mitochondrial dysfunction that elevates reactive species release. The epaulette shark, Hemiscyllum ocellatum, is remarkably tolerant of severe hypoxia at temperatures up to 34 °C, and therefore provides a valuable model to study warm hypoxia tolerance. Mitochondrial function was tested in saponin permeabilised ventricle fibres using high-resolution respirometry coupled with purpose-built fluorospectrometers. Ventricular mitochondrial function, stability and reactive species production of the epaulette shark was compared with that of the hypoxia-sensitive shovelnose ray, Aptychotrema rostrata. Fibres were prepared from each species acclimated to normoxic water conditions, or following a 2 h, acute hypoxic exposure at levels representing 40% of each species' critical oxygen tension. Although mitochondrial respiratory fluxes for normoxia-acclimated animals were similar for both species, reactive species production in the epaulette shark was approximately half that of the shovelnose ray under normoxic conditions, even when normalised to tissue oxidative phosphorylation flux. The hypoxia-sensitive shovelnose ray halved oxidative phosphorylation flux and cytochrome c oxidase flux was depressed by 34% following hypoxic stress. In contrast, oxidative phosphorylation flux of the epaulette shark ventricular fibres isolated from acute hypoxia exposed the animals remained similar to those from normoxia-acclimated animals. However, uncoupling of respiration revealed depressed electron transport systems in both species following hypoxia exposure. Overall, the epaulette shark ventricular mitochondria showed greater oxidative phosphorylation stability and lower reactive species outputs with hypoxic exposure, and this may protect cardiac bioenergetic function in hypoxic tropical waters.

The physiological tolerance of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxic exposure at three seasonal temperatures.

The epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) are commonly found in periodically hypoxic environments. The ecophysiological time available for these animals to safely exploit these niches during different seasonal temperatures was examined. The time to loss of righting reflex (T (LRR)) was examined in response to an open ended anoxic challenge at three seasonal temperatures (23, 25 and 27°C). Ventilation rates were measured in an open ended anoxic challenge at 23°C and during 1.5 h of anoxia followed by 2 h of re-oxygenation at 23 and 25°C. The mean T (LRR) of epaulette and grey carpet sharks was inversely proportional to temperature. The T (LRR) was similar between species at 23°C; however, grey carpet sharks had significantly reduced T (LRR) at higher temperatures. During the standardised anoxic challenge, epaulette sharks entered into ventilatory depression significantly earlier at 25°C. During re-oxygenation, epaulette sharks exposed to anoxia at 23°C had no significant increase in ventilation rates. However, after anoxic challenge and re-oxygenation at 25°C, epaulette sharks showed a significant increase in ventilation rates during re-oxygenation. Grey carpet sharks displayed no evidence of ventilatory depression during anoxia. However, during re-oxygenation, grey carpet sharks had significantly elevated ventilation rates above pre-experimental levels and control animals. These data demonstrate that the anoxia tolerance times of both species were temperature dependent, with a significant reduction in the T (LRR) occurring at higher temperatures. Epaulette sharks had a significantly greater T (LRR) at higher temperatures than grey carpet sharks, which did not enter into a ventilatory depression.

Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks.

BACKGROUND: Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum). RESULTS: Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart). We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were eukaryotic translation elongation factor 1 beta (eef1b), ubiquitin (ubq) and polymerase (RNA) II (DNA directed) polypeptide F (polr2f). The performance of the ribosomal protein L6 (rpl6) was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software. CONCLUSIONS: Our results indicate that eef1b and ubq are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation.

Compensatory proteome adjustments imply tissue-specific structural and metabolic reorganization following episodic hypoxia or anoxia in the epaulette shark (Hemiscyllium ocellatum).

The epaulette shark (Hemiscyllium ocellatum) represents an ancestral vertebrate model of episodic hypoxia and anoxia tolerance at tropical temperatures. We used two-dimensional gel electrophoresis and mass spectrometry-based proteomics approaches, combined with a suite of physiological measures, to characterize this species' responses to 1) one episode of anoxia plus normoxic recovery, 2) one episode of severe hypoxia plus recovery, or 3) two episodes of severe hypoxia plus recovery. We examined these responses in the cerebellum and rectal gland, two tissues with high ATP requirements. Sharks maintained plasma ionic homeostasis following all treatments, and activities of Na(+)/K(+)-ATPase and caspase 3/7 in both tissues were unchanged. Oxygen lack and reoxygenation elicited subtle adjustments in the proteome. Hypoxia led to more extensive proteome responses than anoxia in both tissues. The cerebellum and rectal gland exhibited treatment-specific responses to oxygen limitation consistent with one or more of several strategies: 1) neurotransmitter and receptor downregulation in cerebellum to prevent excitotoxicity, 2) cytoskeletal/membrane reorganization, 3) metabolic reorganization and more efficient intracellular energy shuttling that are more consistent with sustained ATP turnover than with long-term metabolic depression, 4) detoxification of metabolic byproducts and oxidative stress in light of continued metabolic activity, particularly following hypoxia in rectal gland, and 5) activation of prosurvival signaling. We hypothesize that neuronal morphological changes facilitate prolonged protection from excitotoxicity via dendritic spine remodeling in cerebellum (i.e., synaptic structural plasticity). These results recapitulate several highly conserved themes in the anoxia and hypoxia tolerance, preconditioning, and oxidative stress literature in a single system. In addition, several of the identified pathways and proteins suggest potentially novel mechanisms for enhancing anoxia or hypoxia tolerance in vertebrates. Overall, our data show that episodic hypoxic or anoxic exposure and recovery in the epaulette shark amplifies a constitutive suite of compensatory mechanisms that further prepares them for subsequent insults.

Hematological responses of the grey carpet shark (Chiloscyllium punctatum) and the epaulette shark (Hemiscyllium ocellatum) to anoxia and re-oxygenation.

We compared the hematological responses of wild and captive populations of two closely related sharks to a standardized anoxic challenge and during a 12 hr recovery period in normoxia: the epaulette shark (Hemiscyllium ocellatum, Bonnaterre, 1788) and the grey carpet shark (Chiloscyllium punctatum, Müller and Henle, 1838). Compared to normoxic controls, a significant increase in hematocrit (captive 22.3%; wild 35.9%) coupled with a decline in mean corpuscular hemoglobin concentration occurred in epaulette sharks indicating erythrocyte swelling in response to anoxia. However, the grey carpet shark had a significantly increased hematocrit (captive 27.2%; wild 29.3%), erythrocyte count (captive 37.6%; wild 46.3%) and hemoglobin concentration (captive 31.9%; wild 31.5%), suggesting a release of erythrocytes into the circulation and/or hemoconcentration in response to anoxia. Plasma glucose concentrations were maintained in both wild and captive epaulette sharks and in wild grey carpet sharks during anoxia but increased significantly after 2 hr of re-oxygenation (epaulette: captive 55.8%; wild 50.1%; grey carpet shark: wild 70.3%) and remained elevated for 12 hr. Captive grey carpet sharks had an immediate increase in plasma glucose concentrations after anoxia (96.4%), which was sustained for 12 hr of re-oxygenation. Lactate concentrations significantly increased in captive and wild animals of both species after anoxia, reaching a peak at 2 hr of re-oxygenation. Both species showed significant, yet divergent, hematological changes in response to anoxia and re-oxygenation, which may not only prolong their survival and assist in recovery but also reflect their respective ecophysiological adaptations to the extreme environments that they inhabit.

GABA is not elevated during neuroprotective neuronal depression in the hypoxic epaulette shark (Hemiscyllium ocellatum).

Prolonged hypoxic exposure results in cell failure, glutamate excitotoxicity and apoptosis in the brain. The epaulette shark can withstand prolonged hypoxic exposure without brain injury, while maintaining normal function and activity at tropical temperatures. We examined whether the inhibitory neurotransmitter GABA was involved in hypoxia tolerance and neuroprotection during hypoxic preconditioning. Sharks were exposed to either cyclic hypoxic preconditioning or normoxic conditions. Whole brain GABA concentration was determined using high performance liquid chromatography; GABA distribution in neuronal structures was localised with immunohistochemistry and quantified. While the overall brain level of GABA was not significantly different, there was a significant heterogeneous change in GABA distribution. GABA immunoreactivity was elevated in key motor and sensory nuclei from preconditioned animals, including the nucleus motorius nervi vagi and the cerebellar crest (p<0.001), corresponding to areas of previously reported neuronal hypometabolism. Since the neuroprotection in all other hypoxia and anoxia tolerant species examined so far relies in part on significant elevations in GABA and the phylogenetically older epaulette shark does not, it is reasonable to assume that further research in this unique animal model may yield clues to new key modulators of neuroprotection. Understanding such mechanisms may facilitate the development of therapeutic interventions in the treatment of transient ischaemic attacks, strokes and traumatic brain injury.