Interactions between hypoxia tolerance and food deprivation in Amazonian oscars, Astronotus ocellatus

Oscars are often subjected to a combination of low levels of oxygen and fasting during nest-guarding on Amazonian floodplains. We questioned whether this anorexia would aggravate the osmo-respiratory compromise. We compared fed and fasted oscars (1014 days) in both normoxia and hypoxia (1020 Torr, 4 h). Routine oxygen consumption rates (MO2) were increased by 75% in fasted fish, reflecting behavioural differences, whereas fasting improved hypoxia resistance and critical oxygen tensions (Pcrit) lowered from 54 Torr in fed fish to 34 Torr when fasting. In fed fish, hypoxia reduced liver lipid stores by approximately 50% and total liver energy content by 30%. Fasted fish had a 50% lower hepatosomatic index, resulting in lower total liver protein, glycogen and lipid energy stores under normoxia. Compared with hypoxic fed fish, hypoxic fasted fish only showed reduced liver protein levels and even gained glycogen (+50%) on a per gram basis. This confirms the hypothesis that hypoxia-tolerant fish protect their glycogen stores as much as possible as a safeguard for more prolonged hypoxic events. In general, fasted fish showed lower hydroxyacylCoA dehydrogenase activities compared with fed fish, although this effect was only significant in hypoxic fasted fish. Energy stores and activities of enzymes related to energy metabolism in muscle or gills were not affected. Branchial Na+ uptake rates were more than two times lower in fed fish, whereas Na+ efflux was similar. Fed and fasted fish quickly reduced Na+ uptake and efflux during hypoxia, with fasting fish responding more rapidly. Ammonia excretion and K+ efflux were reduced under hypoxia, indicating decreased transcellular permeability. Fasted fish had more mitochondria-rich cells (MRC), with larger crypts, indicating the increased importance of the branchial uptake route when feeding is limited. Gill MRC density and surface area were greatly reduced under hypoxia, possibly to reduce ion uptake and efflux rates. Density of mucous cells of normoxic fasted fish was approximately fourfold of that in fed fish. Overall, a 1014 day fasting period had no negative effects on hypoxia tolerance in oscars, as fasted fish were able to respond more quickly to lower oxygen levels, and reduced branchial permeability effectively. © 2013. Published by The Company of Biologists Ltd

Regulation of gill transcellular permeability and renal function during acute hypoxia in the Amazonian oscar (Astronotus ocellatus): New angles to the osmorespiratory compromise

Earlier studies demonstrated that oscars, endemic to ion-poor Amazonian waters, are extremely hypoxia tolerant, and exhibit a marked reduction in active unidirectional Na+ uptake rate (measured directly) but unchanged net Na+ balance during acute exposure to low Po2, indicating a comparable reduction in whole body Na+ efflux rate. However, branchial O2 transfer factor does not fall. The present study focused on the nature of the efflux reduction in the face of maintained gill O 2 permeability. Direct measurements of 22Na appearance in the water from bladder-catheterized fish confirmed a rapid 55% fall in unidirectional Na+ efflux rate across the gills upon acute exposure to hypoxia (PO2=10-20torr; 1 torr=133.3 Pa), which was quickly reversed upon return to normoxia. An exchange diffusion mechanism for Na + is not present, so the reduction in efflux was not directly linked to the reduction in Na+ influx. A quickly developing bradycardia occurred during hypoxia. Transepithelial potential, which was sensitive to water [Ca2+], became markedly less negative during hypoxia and was restored upon return to normoxia. Ammonia excretion, net K+ loss rates, and 3H2O exchange rates (diffusive water efflux rates) across the gills fell by 55-75% during hypoxia, with recovery during normoxia. Osmotic permeability to water also declined, but the fall (30%) was less than that in diffusive water permeability (70%). In total, these observations indicate a reduction in gill transcellular permeability during hypoxia, a conclusion supported by unchanged branchial efflux rates of the paracellular marker [3H]PEG-4000 during hypoxia and normoxic recovery. At the kidney, glomerular filtration rate, urine flow rate, and tubular Na+ reabsorption rate fell in parallel by 70% during hypoxia, facilitating additional reductions in costs and in urinary Na+, K+ and ammonia excretion rates. Scanning electron microscopy of the gill epithelium revealed no remodelling at a macro-level, but pronounced changes in surface morphology. Under normoxia, mitochondria-rich cells were exposed only through small apical crypts, and these decreased in number by 47% and in individual area by 65% during 3 h hypoxia. We suggest that a rapid closure of transcellular channels, perhaps effected by pavement cell coverage of the crypts, allows conservation of ions and reduction of ionoregulatory costs without compromise of O2 exchange capacity during acute hypoxia, a response very different from the traditional osmorespiratory compromise

Do mitochondria limit hot fish hearts? Understanding the role of mitochondrial function with heat stress in Notolabrus celidotus.

Hearts are the first organs to fail in animals exposed to heat stress. Predictions of climate change mediated increases in ocean temperatures suggest that the ectothermic heart may place tight constraints on the diversity and distribution of marine species with cardiovascular systems. For many such species, their upper temperature limits (Tmax) and respective heart failure (HF) temperature (T(HF)) are only a few degrees from current environmental temperatures. While the ectothermic cardiovascular system acts as an "ecological thermometer," the exact mechanism that mediates HF remains unresolved. We propose that heat-stressed cardiac mitochondria drive HF. Using a common New Zealand fish, Notolabrus celidotus, we determined the THF (27.5°C). Haemoglobin oxygen saturation appeared to be unaltered in the blood surrounding and within heat stressed hearts. Using high resolution respirometry coupled to fluorimeters, we explored temperature-mediated changes in respiration, ROS and ATP production, and overlaid these changes with T(HF). Even at saturating oxygen levels several mitochondrial components were compromised before T(HF). Importantly, the capacity to efficiently produce ATP in the heart is limited at 25°C, and this is prior to the acute T(HF) for N. celidotus. Membrane leakiness increased significantly at 25°C, as did cytochrome c release and permeability to NADH. Maximal flux rates and the capacity for the electron transport system to uncouple were also altered at 25°C. These data indicate that mitochondrial membrane integrity is lost, depressing ATP synthesis capacity and promoting cytochrome c release, prior to T(HF). Mitochondria can mediate HF in heat stressed hearts in fish and play a significant role in thermal stress tolerance, and perhaps limit species distributions by contributing to HF

ROS production by permeabilized heart fibres in (fmol H₂O₂ (mg. s) ⁻¹) with increasing temperature.

<p>(A) ROS production in the Leak-I state; (B) ROS production in the OXP-I state; (C) ROS production in the OXP-I, II state; (D) ROS production in the ETS uncoupled state. A dose-dependent agonist analysis curves were fitted for all states (black daggered lines). Values are means ± S.E.M for <i>N</i> = 8. Means sharing the same letter are not significantly different from one another at <i>p</i>≤0.05.</p

Glycolytic intermediates and enzymes in control and experimental fish cardiac tissue.

<p>Unit for lactate and enzymes is µmol. min⁻¹. mg protein⁻¹.</p><p>Values are means ± S.E.M (<i>N</i> = 4<i>).</i></p>*<p>denotes significant change at <i>p</i>≤0.05.</p

In permeabilized heart fibres from control (grey bars) and experimental (black bars) fish (<i>N</i> = 8) (A) mean respirational flux (insert) RCR values for control vs experimental mitochondrial respiration and (B) ROS production.

<p>See methods for mitochondrial respiration state details. Values are means ± S.E.M. The asterisks denote a significant difference between control and experimental fish at a given respiration state at <i>p</i>≤0.05.</p

Ratios based on mitochondrial respirational flux in <i>N. celidotus</i> at 15°C, 17.5°C, 20°C, 25°C, 27.5°C, 30°C and 32.5°C. OXP-I, II/Leak-I, II (also termed RCR 2, [50]) ratio is a simple proxy of inner membrane permeability.

<p>The Leak-I, II/ETS (termed flux control ratio, FCR) provides a measure of ETS capacity relative to the leak respiration state when phosphorylation is inhibited by atractyloside. CCO/OXP-I, II and CCO/ETS are measures of the capacity of cytochrome <i>c</i> oxidase (CCO) relative to maximum phosphorylation (OXP-I, II) or the ETS respectively.</p><p>Values are means ± S.E.M. (<i>N</i> = 8 at each temperature).</p><p>Means with the same letter of the same case are not significantly different from one another <i>(p≤</i>0.05).</p

Pyruvate affinity in cardiac fibres of <i>N. celidotus</i> examined at 20°C, 25°C, 27.5°C, 30°C and 32.5°C.

<p>(A) Pyruvate concentration giving half maximal respiration rate (K_{m app}); (B) maximal pyruvate stimulated respiratory flux rate (V_max) (C) ratio of V_max/K_{m app} as an indicator of substrate efficiency. Values are mean s± S.E.M for <i>N</i> = 8. Means sharing the same letter are not significantly different from one another at <i>p</i>≤0.05.</p

Understanding thermal limits of HF.

<p>Previous studies showed that mitochondria were robust beyond temperatures at which the heart fails (green dashed line). This study questions whether cardiac mitochondria fail before (a causal mechanism) or after heart failure (an effect, red line)?</p

Changes in haemoglobin oxygen saturation in <i>N. celidotus</i> expressed as the change of infrared (940 nm) to red (600 nm) absorbance ratio in control (<i>N</i> = 4, grey x) and experimental (<i>N = 6,</i> black x) fish.

<p>Values are expressed for individual fish and linear regression was fitted for control (grey line) and experimental (black line) data with 95% CI in daggered lines. Control fish did not experience changes in temperature; therefore regressions were performed on absorbance ratios relative to time (top x-axis). Regression analysis for experimental fish were performed on absorbance ratios relative to temperature (bottom x-axis). Goodness of fit is given as R².</p