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.

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 (10-14 days) in both normoxia and hypoxia (10-20 Torr, 4 h). Routine oxygen consumption rates (O2) 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 10-14 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.

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.

Low-O2 acclimation shifts the hypoxia avoidance behaviour of snapper (Pagrus auratus) with only subtle changes in aerobic and anaerobic function.

It was hypothesised that chronic hypoxia acclimation (preconditioning) would alter the behavioural low-O(2) avoidance strategy of fish as a result of both aerobic and anaerobic physiological adaptations. Avoidance and physiological responses of juvenile snapper (Pagrus auratus) were therefore investigated following a 6 week period of moderate hypoxia exposure (10.2-12.1 kPa P(O(2)), 21 ± 1 °C) and compared with those of normoxic controls (P(O(2))=20-21 kPa, 21 ± 1 °C). The critical oxygen pressure (P(crit)) limit of both groups was unchanged at ~7 kPa, as were standard, routine and maximum metabolic rates. However, hypoxia-acclimated fish showed increased tolerances to hypoxia in behavioural choice chambers by avoiding lower P(O(2)) levels (3.3 ± 0.7 vs 5.3 ± 1.1 kPa) without displaying greater perturbations of lactate or glucose. This behavioural change was associated with unexpected physiological adjustments. For example, a decrease in blood O(2) carrying capacity was observed after hypoxia acclimation. Also unexpected was an increase in whole-blood P(50) following acclimation to low O(2), perhaps facilitating Hb-O(2) off-loading to tissues. In addition, cardiac mitochondria measured in situ using permeabilised fibres showed improved O(2) uptake efficiencies. The proportion of the anaerobic enzyme lactate dehydrogenase, at least relative to the aerobic marker enzyme citrate synthase, also increased in heart and skeletal red muscle, indicating enhanced anaerobic potential, or in situ lactate metabolism, in these tissues. Overall, these data suggest that a prioritization of O(2) delivery and O(2) utilisation over O(2) uptake during long-term hypoxia may convey a significant survival benefit to snapper in terms of behavioural low-O(2) tolerance.

In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil, erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes.

Inhibition of mammalian cytochrome P450 enzymes (CYPs) is well characterized; major hepatic CYPs can be inhibited by drugs and other environmental contaminants. CYP function and inhibition has not yet been well established in fish yet these studies are important for several reasons. First, such studies will provide functional information for non-mammalian CYPs. Second, specific inhibitors can be used as a diagnostic tool for studying CYP-mediated reactions. Lastly, pharmaceutical mixtures are found in the aquatic environment and adverse effects associated with drug-drug interactions, including CYP inhibition by pharmaceuticals may be of concern. Using liver microsomes from untreated and ß-naphthoflavone (BNF)-treated rainbow trout, eight fluorescent CYP-mediated catalytic assays were used to assess in vitro CYP inhibition by four pharmaceuticals: fluoxetine, ciprofloxacin, gemfibrozil and erythromycin. Expressed zebrafish CYP1 proteins (CYP1A, CYP1B1, CYP1C1 and CYP1C2) were assessed for inhibition with selected substrates. All pharmaceuticals decreased the metabolism of a number of substrates. Fluoxetine was the strongest and most broad inhibitor of CYP-mediated reactions in liver microsomes. Zebrafish CYP1s were strongly inhibited by erythromycin and fluoxetine. Although the pharmaceuticals are selective CYP inhibitors in mammals, inhibition across a number of substrates suggests they are broad inhibitors in fish. These data demonstrate that in vitro hepatic CYP inhibition by pharmaceuticals is possible in fish and the patterns seen here are different than what would be expected based on CYP inhibition in mammals.

The ionoregulatory responses to hypoxia in the freshwater rainbow trout Oncorhynchus mykiss.

We utilized the rainbow trout, a hypoxia-intolerant freshwater teleost, to examine ionoregulatory changes at the gills during hypoxia. Progressive mild hypoxia led first to a significant elevation (by 21%) in J(Na)(influx) (measured with 22Na), but at 4-h hypoxia when PCO2 reached approximately 110 mmHg, there was a 79% depression in J(Na)(influx). Influx remained depressed during the first hour of normoxic recovery but was restored back to control rates thereafter; there were no significant changes in J(Na)(efflux) or J(Na)(net). A more prolonged (8 h) and severe hypoxic (approximately 80 mmHg) exposure induced a triphasic response whereby J(Na)(influx) was significantly elevated during the first hour, as during mild hypoxia, but returned to control rates during the subsequent 3 h. Thereafter, rates started to gradually increase and remained significantly elevated by about 38% through to 8 h of hypoxia. A similar triphasic trend was observed with J(Na)(efflux) but with larger changes than in J(Na)(influx), such that negative Na+ balance occurred during the hypoxic exposure. Net K+ loss rates to the water approximately doubled. There were no significant alterations in ammonia excretion rates in either of the hypoxia regimes. Branchial Na+/K+-ATPase activity did not change during 4 h at PO2 approximately 80 mmHg or return to normoxia; H+-ATPase activity also did not change during hypoxia but was significantly depressed by approximately 75% after 6 h of normoxic recovery. Scanning electron microscopy revealed that within 1 h of exposure to PO2 approximately 80 mmHg, exposed mitochondria-rich cell (MRC) numbers increased by 30%, while individual MRC exposed surface area and total MRC surface area both increased by three- to fourfold. MRC numbers had decreased below control levels by 4 h of hypoxia, but surface exposure remained elevated by approximately twofold, a response that persisted through 6 h of normoxic recovery. Environmental hypoxia induces complex changes in gill ionoregulatory function in this hypoxia-intolerant species that are very different from those recently reported in the hypoxia-tolerant Amazonian oscar.

The role of size in synchronous air breathing of Hoplosternum littorale.

Synchronized air breathing may have evolved as a way of minimizing the predation risk known to be associated with air breathing in fish. Little is known about how the size of individuals affects synchronized air breathing and whether some individuals are required to surface earlier than necessary in support of conspecifics, while others delay air intake. Here, the air-breathing behavior of Hoplosternum littorale held in groups or in isolation was investigated in relation to body mass, oxygen tensions, and a variety of other physiological parameters (plasma lactate, hepatic glycogen, hematocrit, hemoglobin, and size of heart, branchial basket, liver, and air-breathing organ [ABO]). A mass-specific relationship with oxygen tension of first surfacing was seen when fish were held in isolation; smaller individuals surfaced at higher oxygen tensions. However, this relationship was lost when the same individuals were held in social groups of four, where synchronous air breathing was observed. In isolation, 62% of fish first surfaced at an oxygen tension lower than the calculated P(crit) (8.13 kPa), but in the group environment this was reduced to 38% of individuals. Higher oxygen tensions at first surfacing in the group environment were related to higher levels of activity rather than any of the physiological parameters measured. In fish held in isolation but denied access to the water surface for 12 h before behavioral testing, there was no mass-specific relationship with oxygen tension at first surfacing. Larger individuals with a greater capacity to store air in their ABOs may, therefore, remain in hypoxic waters for longer periods than smaller individuals when held in isolation unless prior access to the air is prevented. This study highlights how social interaction can affect air-breathing behaviors and the importance of considering both behavioral and physiological responses of fish to hypoxia to understand the survival mechanisms they employ.

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 P(O(2)), indicating a comparable reduction in whole body Na(+) efflux rate. However, branchial O(2) 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 (22)Na 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 (P(O(2))=10-20 torr; 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 [Ca(2+)], became markedly less negative during hypoxia and was restored upon return to normoxia. Ammonia excretion, net K(+) loss rates, and (3)H(2)O 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 [(3)H]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 O(2) exchange capacity during acute hypoxia, a response very different from the traditional osmorespiratory compromise.

Water balance and renal function in two species of African lungfish Protopterus dolloi and Protopterus annectens.

The basic physiology of water balance and kidney function was characterized in two species of African lungfish, Protopterus dolloi and Protopterus annectens. Diffusive water efflux rate constants were low (0.13 h(-1)-0.38 h(-1) in various series) relative to values in freshwater teleost fish. Efflux rate constants increased approximately 3-fold after feeding in both species, and were greatly decreased after 8 months terrestrialization (P. dolloi only tested). Urine flow rates (UFR, 3.9-5.2 mL kg(-1) h(-1)) and glomerular filtration rates (GFR, 6.6-9.3 mL kg(-1) h(-1)) were quite high relative to values in most freshwater teleosts. However urinary ion excretion rates were low, with net re-absorption of >99% Na(+), >98% Cl(-), and >78% Ca(2+) from the primary filtrate, comparable to teleosts. Net water re-absorption was significantly greater in P. dolloi (56%) than in P. annectens (23%). We conclude that renal function in lungfish is similar to that in other primitive freshwater fish, but there is an interesting dichotomy between diffusive and osmotic permeabilities. Aquatic lungfish have low diffusive water permeability, an important pre-adaptation to life on land, and in accord with greatly reduced gill areas and low metabolic rates. However osmotic permeability is high, 4-12 times greater than diffusive permeability. A role for aquaporins in this dichotomy is speculated.

Respiratory responses to progressive hypoxia in the Amazonian oscar, Astronotus ocellatus.

This study determined the respiratory responses to progressive hypoxia in oscar, an extremely hypoxia-tolerant Amazonian cichlid. Oscar depressed oxygen consumption rates (MO2), beginning at a critical O2 tension (Pcrit) of 46Torr, to only 14% of normoxic rates at 10Torr. Total ventilation (Vw) increased up to 4-fold, entirely due to a rise in ventilatory stroke volume (no change in ventilatory frequency), and water convection requirement (Vw/MO2) increased substantially (up to 15-fold). Gill O2 extraction fell steadily, from 60% down to 40%. Although O2 transfer factor (an index of gill O2 diffusion capacity) increased transiently in moderate hypoxia, it decreased at 10Torr, which may have caused the increased expired-arterial PO2 difference. Venous PO2 was always very low (< or =7Torr). Anaerobic metabolism made a significant contribution to ATP supply, indicated by a 3-fold increase in plasma lactate that resulted in an uncompensated metabolic acidosis. Respiration of isolated gill cells was not inhibited until below 5Torr; because gill water PO2 always exceeded this value, hypoxic ion flux arrest in oscars [Wood et al., Am. J. Physiol. Reg. Integr. Comp. Physiol. 292, R2048-R2058, 2007] is probably not caused by O2 limitation in ionocytes. We conclude that metabolic depression and tolerance of anaerobic bi-products, rather than a superior capacity for O2 supply, allow oscar to thrive in extreme hypoxia in the Amazon.