The biotic ligand model as a promising tool to predict Cu toxicity in amazon blackwaters.

The Rio Negro basin of Amazonia (Brazil) is a hotspot of fish biodiversity that is under threat from copper (Cu) pollution. The very ion-poor blackwaters have a high dissolved organic carbon (DOC) concentration. We investigated the Cu sensitivity of nine Amazonian fish species in their natural blackwaters (Rio Negro). The acute lethal concentration of Cu (96 h LC50) was determined at different dilutions of Rio Negro water (RNW) in ion-poor well water (IPW), ranging from 0 to 100%. The IPW was similar to RNW in pH and ionic composition but deficient in DOC, allowing this parameter to vary 20-fold from 0.4 to 8.3 mg/L in tests. The Biotic Ligand Model (BLM; Windward version 3.41.2.45) was used to model Cu speciation and toxicity over the range of tested water compositions, and to estimate lethal Cu accumulations on the gills (LA50). The modeling predicted a high relative abundance of Cu complexes with DOC in test waters. As these complexes became more abundant with increasing RNW content, a concomitant decrease in free Cu2+ was observed. In agreement with this modeling, acute Cu toxicity decreased (i.e. 96 h LC50 values increase) with increasing RNW content. The three most sensitive species (Hemigrammus rhodostomus, Carnegiella strigatta and Hyphessobrycon socolofi) were Characiformes, whereas Corydoras schwartzi (Siluriformes) and Apistogramma agassizii (Cichliformes) were the most tolerant. These sensitivity differences were reflected in the BLM-predicted lethal gill copper accumulation (LA50), which were generally lower in Characiformes than in Cichliformes. Using these newly estimated LA50 values in the BLM allowed for accurate prediction of acute Cu toxicity in the nine Amazonian fish. Our data emphasize that the BLM approach is a promising tool for assessing Cu risk to Amazonian fish species in blackwater conditions characterized by very low concentrations of major ions but high concentrations of DOC.

Influence of hypoxia on biochemical aspects and on expression of genes related to oxygen-homeostasis of the Amazonian cichlid Astronotus ocellatus (Agassiz, 1831).

Variations in dissolved oxygen levels are common in the Amazonian aquatic environments and the aquatic organisms that inhabit these environments developed a variety of adaptive responses to deal with such conditions. Some Amazonian fish species are tolerant to low oxygen levels and the cichlid Astronotus ocellatus is one of the most hypoxia-tolerant species. Herein, we aimed to unveil the biochemical and molecular responses that A. ocellatus presents when submitted to hypoxia. Hypoxia indicators were measured, such as plasma glucose, plasma lactate, hepatic glycogen and relative transcript levels of prolyl hydroxylase 2 (phd2) and hypoxia-inducible factor-1α (hif-1α) in juveniles of approximately 50 g exposed to 1, 3, and 5 hours of hypoxia (0.7 mg O2.L-1), followed by 3 hours of recovery in normoxia (6 mg O2.L-1). Fish exposed to hypoxia reduced liver glycogen levels within 3 hours of hypoxia, when comparing with 1 hour, and increased plasma glucose and lactate. Under the same condition, phd2 transcripts levels increased in gills, but decreased in liver. In contrast, hypoxia did not affect relative gene expression of hif-1α in both tissues. Based on the transcription pattern of phd2, these results showed that liver and gills of A. ocellatus have different molecular strategies to cope with environmental hypoxia.

Ecological adaptations of Amazonian fishes acquired during evolution under environmental variations in dissolved oxygen: A review of responses to hypoxia in fishes, featuring the hypoxia-tolerant Astronotus spp.

The Amazon Basin presents a dynamic regime of dissolved oxygen (DO) oscillations, which varies among habitats within the basin, including spatially, daily, and seasonally. Fish species inhabiting these environments have developed many physiological adaptations to deal with the frequent and periodic events of low (hypoxia), or no (anoxia) DO in the water. Cichlid fishes, especially the genus Astronotus (A. ocellatus and A. crassipinnis), are hypoxic-tolerant species that can survive in very low DO levels for long periods, while adults often inhabit places where DO is close to zero. The present review will focus on some metabolic adjustments that Amazonian fish use in response to hypoxic conditions, which include many strategies from behavioral, morphological, physiological, and biochemical strategies. These strategies include ASR (aerial surface respiration), lip expansion, branchial tissue remodeling, increases in glycolytic metabolism with the increase of blood glucose levels, and increases in anaerobic metabolism with increases of plasma lactate levels. Other groups over evolutionary time developed obligate aerial respiration with changes in pharyngeal and swim bladder vascularization as well as the development of a true lung. However, most species are water-breathing species, such as A. ocellatus and A. crassipinnis, which are detailed in this study because they are used as hypoxia-tolerant model fish. Herein, we draw together the literature data of the physiological mechanisms by which these species decrease aerobic metabolism and increase anaerobic metabolism to survive hypoxia. This is the first attempt to synthesize the physiological mechanisms of the hypoxia-tolerant Astronotus species.

The effects of dissolved organic carbon on the reflex ventilatory responses of the neotropical teleost (Colossoma macropomum) to hypoxia or hypercapnia.

The tambaqui (Colossoma macropomum), migrates annually between whitewater and blackwater rivers of the Amazon. Unlike the whitewater, blackwater is characterized by higher levels of dissolved organic carbon (DOC), including humic acids (HA). Because humic substances impair sensory processes, the current study tested the hypothesis that O2 and/or CO2 chemoreception is impeded in blackwater owing to the presence of HA. Thus, the ventilatory responses of tambaqui to hypoxia or hypercapnia were assessed in well water transported from Manaus, local blackwater, and in well water containing HA either extracted from Rio Negro water or obtained commercially (Sigma Aldrich; SA). In well water, tambaqui exhibited typical hyperventilatory responses to hypoxia or hypercapnia. These responses were prevented by simultaneously exposing fish to SA HA (20 mg l-1). The negative effects of SA HA on ventilation were prevented when natural DOC (30 mg l-1; extracted from Rio Negro water after first removing the endogenous HA fraction) was added concurrently, indicating a protective effect of this non-humic acid DOC fraction. The hyperventilatory responses were unaffected during acute exposure or after acclimation of fish to Rio Negro water. HA extracted from Rio Negro water did not impair the hyperventilatory responses to hypoxia or hypercapnia. This study, while demonstrating a negative effect of SA HA derived from peat (coal) on the control of breathing in tambaqui, failed to reveal any detrimental consequences of HA (derived from the decomposition of a variety of lignin-rich plants) naturally occurring in the blackwaters of the Rio Negro.

Boron Oxide Nanoparticles Exhibit Minor, Species-Specific Acute Toxicity to North-Temperate and Amazonian Freshwater Fishes.

Boron oxide nanoparticles (nB2O3) are manufactured for structural, propellant, and clinical applications and also form spontaneously through the degradation of bulk boron compounds. Bulk boron is not toxic to vertebrates but the distinctive properties of its nanostructured equivalent may alter its biocompatibility. Few studies have addressed this possibility, thus our goal was to gain an initial understanding of the potential acute toxicity of nB2O3 to freshwater fish and we used a variety of model systems to achieve this. Bioactivity was investigated in rainbow trout (Oncorhynchus mykiss) hepatocytes and at the whole animal level in three other North and South American fish species using indicators of aerobic metabolism, behavior, oxidative stress, neurotoxicity, and ionoregulation. nB2O3 reduced O. mykiss hepatocyte oxygen consumption (MO2) by 35% at high doses but whole animal MO2 was not affected in any species. Spontaneous activity was assessed using MO2 frequency distribution plots from live fish. nB2O3 increased the frequency of high MO2 events in the Amazonian fish Paracheirodon axelrodi, suggesting exposure enhanced spontaneous aerobic activity. MO2 frequency distributions were not affected in the other species examined. Liver lactate accumulation and significant changes in cardiac acetylcholinesterase and gill Na+/K+-ATPase activity were noted in the north-temperate Fundulus diaphanus exposed to nB2O3, but not in the Amazonian Apistogramma agassizii or P. axelrodi. nB2O3 did not induce oxidative stress in any of the species studied. Overall, nB2O3 exhibited modest, species-specific bioactivity but only at doses exceeding predicted environmental relevance. Chronic, low dose exposure studies are required for confirmation, but our data suggest that, like bulk boron, nB2O3 is relatively non-toxic to aquatic vertebrates and thus represents a promising formulation for further development.

Climate vulnerability of South American freshwater fish: Thermal tolerance and acclimation.

Freshwater fish are restricted by their physiology to rivers and lakes, and are generally limited in their capacity to disperse across basins. As a result, there is often a close match between the evolutionary history of river basins and their natural history. Thus, the regional landscape and ecological features, such as temperature, have shaped the evolution and adaptation of local fish assemblages. Climate change is expected to affect fish diversity and increase extinction, especially in low latitudes, and it has been suggested that species that inhabit low latitude species are more susceptible since they live close to their maximum thermal limits and have low capacity for acclimation. To understand the mechanisms of variation in thermal tolerance across a broad-scale of South American fishes is fundamental to be able to assess the vulnerability of species and habitat to global warming. Herein, we present the first attempt to analyze the vulnerability of South American freshwater fish species, based on the review of upper thermal limits of 106 species from a broad range of latitudinal habitats. Our findings show that upper thermal limits decrease with latitude, while the thermal safety margin (TSM) increase. Furthermore, the latitude has little effects on the acclimation response ratio, and the TSM decreased with rising temperatures. These data suggest that thermal phenotypic acclimation has low potential for mitigating global warming. These results indicate that South American fish species living in tropical areas are more susceptible to global warming since they are already living close to their maximum habitat temperature.

Mechanisms of toxic action of copper and copper nanoparticles in two Amazon fish species: Dwarf cichlid (Apistogramma agassizii) and cardinal tetra (Paracheirodon axelrodi).

Copper oxide nanoparticles (nCuO) are widely used in boat antifouling paints and are released into the environment, potentially inducing toxicity to aquatic organisms. The present study aimed to understand the effects of nCuO and dissolved copper (Cu) on two ornamental Amazon fish species: dwarf cichlid (Apistogramma agassizii) and cardinal tetra (Paracheirodon axelrodi). Fish were exposed to 50% of the LC50 for nCuO (dwarf cichlid 58.31µgL-1 and cardinal tetra 69.6µgL-1) and Cu (dwarf cichlid 20µgL-1 and cardinal tetra 22.9µgL-1) for 24, 48, 72 and 96h. Following exposure, aerobic metabolic rate (MO2), gill osmoregulatory physiology and mitochondrial function, oxidative stress markers, and morphological damage were evaluated. Our results revealed species specificity in metabolic stress responses. An increase of MO2 was noted in cardinal tetra exposed to Cu, but not nCuO, whereas MO2 in dwarf cichlid showed little change with either treatment. In contrast, mitochondria from dwarf cichlid exhibited increased proton leak and a resulting decrease in respiratory control ratios in response to nCuO and Cu exposure. This uncoupling was directly related to an increase in reactive oxygen species (ROS) levels. Our findings reveal different metabolic responses between these two species in response to nCuO and Cu, which are probably caused by the differences between species natural histories, indicating that different mechanisms of toxic action of the contaminants are associated to differential osmoregulatory strategies among species.

Different ecophysiological responses of freshwater fish to warming and acidification.

Future climate change scenarios predict threatening outcomes to biodiversity. Available empirical data concerning biological response of freshwater fish to climate change remains scarce. In this study, we investigated the physiological and biochemical responses of two Iberian freshwater fish species (Squalius carolitertii and the endangered S. torgalensis), inhabiting different climatic conditions, to projected future scenarios of warming (+3°C) and acidification (ΔpH=-0.4). Herein, metabolic enzyme activities of glycolytic (citrate synthase - CS, lactate dehydrogenase - LDH) and antioxidant (glutathione S-transferase, catalase and superoxide dismutase) pathways, as well as the heat shock response (HSR) and lipid peroxidation were determined. Our results show that, under current water pH, warming causes differential interspecific changes on LDH activity, increasing and decreasing its activity in S. carolitertii and in S. torgalensis, respectively. Furthermore, the synergistic effect of warming and acidification caused an increase in LDH activity of S. torgalensis, comparing with the warming condition. As for CS activity, acidification significantly decreased its activity in S. carolitertii whereas in S. torgalensis no significant effect was observed. These results suggest that S. carolitertii is more vulnerable to climate change, possibly as the result of its evolutionary acclimatization to milder climatic condition, while S. torgalensis evolved in the warmer Mediterranean climate. However, significant changes in HSR were observed under the combined warming and acidification (S. carolitertii) or under acidification (S. torgalensis). Our results underlie the importance of conducting experimental studies and address species endpoint responses under projected climate change scenarios to improve conservation strategies, and to safeguard endangered freshwater fish.

Protein analysis and gene expression indicate differential vulnerability of Iberian fish species under a climate change scenario.

Current knowledge on the biological responses of freshwater fish under projected scenarios of climate change remains limited. Here, we examine differences in the protein configuration of two endemic Iberian freshwater fish species, Squalius carolitertii and the critically endangered S. torgalensis that inhabit in the Atlantic-type northern and in the Mediterranean-type southwestern regions, respectively. We performed protein structure modeling of fourteen genes linked to protein folding, energy metabolism, circadian rhythms and immune responses. Structural differences in proteins between the two species were found for HSC70, FKBP52, HIF1α and GPB1. For S. torgalensis, besides structural differences, we found higher thermostability for two proteins (HSP90 and GBP1), which can be advantageous in a warmer environment. Additionally, we investigated how these species might respond to projected scenarios of 3° climate change warming, acidification (ΔpH = -0.4), and their combined effects. Significant changes in gene expression were observed in response to all treatments, particularly under the combined warming and acidification. While S. carolitertii presented changes in gene expression for multiple proteins related to folding (hsp90aa1, hsc70, fkbp4 and stip1), only one such gene was altered in S. torgalensis (stip1). However, S. torgalensis showed a greater capacity for energy production under both the acidification and combined scenarios by increasing cs gene expression and maintaining ldha gene expression in muscle. Overall, these findings suggest that S. torgalensis is better prepared to cope with projected climate change. Worryingly, under the simulated scenarios, disturbances to circadian rhythm and immune system genes (cry1aa, per1a and gbp1) raise concerns for the persistence of both species, highlighting the need to consider multi-stressor effects when evaluating climate change impacts upon fish. This work also highlights that assessments of the potential of endangered freshwater species to cope with environmental change are crucial to help decision-makers adopt future conservation strategies.

Air breathing and aquatic gas exchange during hypoxia in armoured catfish.

Air breathing in fish is commonly believed to have arisen as an adaptation to aquatic hypoxia. The effectiveness of air breathing for tissue O2 supply depends on the ability to avoid O2 loss as oxygenated blood from the air-breathing organ passes through the gills. Here, we evaluated whether the armoured catfish (Hypostomus aff. pyreneusi)-a facultative air breather-can avoid branchial O2 loss while air breathing in aquatic hypoxia, and we measured various other respiratory and metabolic traits important for O2 supply and utilization. Fish were instrumented with opercular catheters to measure the O2 tension (PO2) of expired water, and air breathing and aquatic respiration were measured during progressive stepwise hypoxia in the water. Armoured catfish exhibited relatively low rates of O2 consumption and gill ventilation, and gill ventilation increased in hypoxia due primarily to increases in ventilatory stroke volume. Armoured catfish began air breathing at a water PO2 of 2.5 kPa, and both air-breathing frequency and hypoxia tolerance (as reflected by PO2 at loss of equilibrium, LOE) was greater in individuals with a larger body mass. Branchial O2 loss, as reflected by higher PO2 in expired than in inspired water, was observed in a minority (4/11) of individuals as water PO2 approached that at LOE. Armoured catfish also exhibited a gill morphology characterized by short filaments bearing short fused lamellae, large interlamellar cell masses, low surface area, and a thick epithelium that increased water-to-blood diffusion distance. Armoured catfish had a relatively low blood-O2 binding affinity when sampled in normoxia (P50 of 3.1 kPa at pH 7.4), but were able to rapidly increase binding affinity during progressive hypoxia exposure (to a P50 of 1.8 kPa). Armoured catfish also had low activities of several metabolic enzymes in white muscle, liver, and brain. Therefore, low rates of metabolism and gill ventilation, and a reduction in branchial gas-exchange capacity, may help minimize branchial O2 loss in armoured catfish while air breathing in aquatic hypoxia.

Roundup® exposure promotes gills and liver impairments, DNA damage and inhibition of brain cholinergic activity in the Amazon teleost fish Colossoma macropomum.

Roundup Original® (RD) is a glyphosate-based herbicide used to control weeds in agriculture. Contamination of Amazon waters has increased as a consequence of anthropogenic pressure, including the use of herbicides as RD. The central goal of this study was to evaluate the toxic effects of RD on juveniles of tambaqui (Colossoma macropomum). Our findings show that biomarkers in tambaqui are organ specific and dependent on RD concentration. Alterations in gills structural and respiratory epithelium were followed by changes in hematological parameters such as concentration of hemoglobin, particularly in fish exposed to the higher concentration tested (75% of RD LC50 96 h). In addition, both RD concentrations affected the biotransformation process in gills of tambaqui negatively. Instead, liver responses suggest that a production of reactive oxygen species (ROS) occurred in fish exposed to RD, particularly in the animals exposed to 75% RD, as seen by imbalances in biotransformation and antioxidant systems. The increased DNA damage observed in red blood cells of tambaqui exposed to RD is in agreement with this hypothesis. Finally, both tested sub-lethal concentrations of RD markedly inhibited the cholinesterase activity in fish brain. Thus, we can suggest that RD is potentially toxic to tambaqui and possibly to other tropical fish species.

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.

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.

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.

Intracellular glucose and binding of hexokinase and phosphofructokinase to particulate fractions increase under hypoxia in heart of the amazonian armored catfish (Liposarcus pardalis).

Armored catfish (Liposarcus pardalis), indigenous to the Amazon basin, have hearts that are extremely tolerant of oxygen limitation. Here we test the hypothesis that resistance to hypoxia is associated with increases in binding of selected glycolytic enzymes to subcellular fractions. Preparations of isolated ventricular sheets were subjected to 2 h of either oxygenated or hypoxic (via nitrogen gassing) treatment during which time the muscle was stimulated to contract. The bathing medium contained 5 mM glucose and was maintained at 25 degrees C. Initial experiments revealed increases in anaerobic metabolism. There was no measurable decrease in glycogen level; however, hypoxic treatment led to a twofold increase in heart glucose and a 10-fold increase in lactate content. It is suggested that the increase in heart glucose content is a result of an enhanced rate of facilitated glucose transport that exceeds the rate of phosphorylation of glucose. Further experiments assessed activities of metabolic enzymes in crude homogenates and subsequently tracked the degree of enzyme binding associated with subcellular fractions. Total maximal activities of glycolytic enzymes (hexokinase [HK], phosphofructokinase [PFK], aldolase, pyruvate kinase, lactate dehydrogenase), and a mitochondrial marker, citrate synthase, were not altered with the hypoxic treatment. A substantial portion (>/=50%) of HK is permanently bound to mitochondria, and this level increases under hypoxia. The amount of HK that is bound to the mitochondrial fraction is at least fourfold higher in hearts of L. pardalis than in rat hearts. Hypoxia also resulted in increased binding of PFK to a particulate fraction, and the degree of binding is higher in hypoxia-tolerant fish than in hypoxia-sensitive mammalian hearts. Such binding may be associated with increased glycolytic flux rates through modulation of enzyme-specific kinetics. The binding of HK and PFK occurs before any significant decrease in glycogen level.

Rapid regulation of Na+ fluxes and ammonia excretion in response to acute environmental hypoxia in the Amazonian oscar, Astronotus ocellatus.

The Amazonian oscar is extremely resistant to hypoxia, and tolerance scales with size. Overall, ionoregulatory responses of small ( approximately 15 g) and large oscars ( approximately 200 g) to hypoxia were qualitatively similar, but the latter were more effective. Large oscars exhibited a rapid reduction in unidirectional Na(+) uptake rate at the gills during acute hypoxia (Po(2) approximately 10 mmHg), which intensified with time (7 or 8 h); Na(+) efflux rates were also reduced, so net balance was little affected. The inhibitions were virtually immediate (1st h) and preceded a later 60% reduction (at 3 h) in gill Na(+)-K(+)-ATPase activity, reflected in a 60% reduction in maximum Na(+) uptake capacity without change in affinity (Km) for Na(+). Upon acute restoration of normoxia, recovery of Na(+) uptake was delayed for 1 h. These data suggest that dual mechanisms may be involved (e.g., immediate effects of O(2) availability on transporters, channels, or permeability, slower effects of Na(+)-K(+)-ATPase regulation). Ammonia excretion appeared to be linked indirectly to Na(+) uptake, exhibiting a Michaelis-Menten relationship with external [Na(+)], but the Km was less than for Na(+) uptake. During hypoxia, ammonia excretion fell in a similar manner to Na(+) fluxes, with a delayed recovery upon normoxia restoration, but the relationship with [Na(+)] was blocked. Reductions in ammonia excretion were greater than in urea excretion. Plasma ammonia rose moderately over 3 h hypoxia, suggesting that inhibition of excretion was greater than inhibition of ammonia production. Overall, the oscar maintains excellent homeostasis of ionoregulation and N-balance during severe hypoxia.

Tribute to R. G. Boutilier: the effect of size on the physiological and behavioural responses of oscar, Astronotus ocellatus, to hypoxia.

The physiological and behavioural responses of two size groups of oscar (Astronotus ocellatus) to hypoxia were studied. The physiological responses were tested by measuring M(O(2)) during decreasing environmental oxygen tensions. Larger oscars were better able to maintain oxygen consumption during a decrease in P(O(2)), regulating routine M(O(2)) to a significantly lower P(O(2)) threshold (50 mmHg) than smaller oscars (70 mmHg). Previous studies have also demonstrated a longer survival time of large oscars exposed to extreme hypoxia, coupled with a greater anaerobic enzymatic capability. Large oscars began aquatic surface respiration (ASR) at the oxygen tension at which the first significant decrease in M(O(2)) was seen (50 mmHg). Interestingly, smaller oscars postponed ASR to around 22 mmHg, well beyond the P(O(2)) at which they switched from oxyregulation to oxyconformation. Additionally, when given the choice between an hypoxic environment containing aquatic macrophyte shelter and an open normoxic environment, small fish showed a greater preference for the hypoxic environment. Thus shelter from predators appears particularly important for juveniles, who may accept a greater physiological compromise in exchange for safety. In response to hypoxia without available shelter, larger fish reduced their level of activity (with the exception of aggressive encounters) to aid metabolic suppression whereas smaller oscars increased their activity, with the potential benefit of finding oxygen-rich areas.

Mitochondrial K(ATP) channels and sarcoplasmic reticulum influence cardiac force development under anoxia in the Amazonian armored catfish Liposarcus pardalis.

The contribution of alterations in mitochondrial K(ATP) channel activity and the sarcoplasmic reticulum (SR) to anaerobic cardiac function in the anoxia tolerant armored catfish Liposarcus pardalis were assessed. K(ATP) channels contribute to hypoxic cardioprotection in mammals, but little is known of their action in more hypoxia tolerant animals. Anoxia resulted in a decrease in force in isometrically contracting ventricle strips to approximately 40% of the pre-anoxic level. This was maintained for at least 2 h. Upon reoxygenation, hearts recovered to the same level as control preparations. Treatment with 5-hydroxydecanoic acid (5HD), a specific mitochondrial K(ATP) blocker significantly increased force in preparations during anoxia and caused hypercontracture at reoxygenation. Ryanodine, a specific inhibitor of SR function, significantly increased force loss in ventricle preparations under anoxia. Results show that mitochondrial K(ATP) channel activity and SR function are important in anaerobic and post-anaerobic contractility in armored catfish heart.