Vera Maria Fonseca de Almeida-Val

Hypoxia tolerance of Amazon fish: Respirometry and energy metabolism of the cichlid Astronotus Ocellatus

Abstract As a result of regular flood pulses, the Amazon basin exhibits large annual changes in its chemical and physical parameters. Ecology and distribution of fish communities seem to be directed by seasonal and diurnal oxygen level oscillations. Amazon fish have developed strategies to thrive under these varying conditions. Astronotus ocellatus normally survives large fluctuations in oxygen availability in varzea lakes. Respirometric and metabolic changes in A. ocellatus were studied during exposure to stepwise declining oxygen levels. Respiration rates were continuously recorded. Haematologic and metabolic parameters (lactate, glucose, cortisol and free fatty acids (FFA)) were determined. A. ocellatus was found to be hypoxia tolerant; it survives more than 16 h of severe hypoxia (pO 2 ≤0.4 mg l −1 ) and even 4 h of complete anoxia at 28°C. Its routine metabolic rate is 10.8 mg O 2 h −1 per 100 g fresh weight. A significant decrease in standard metabolic rate (SMR) starts at 20% air saturation, whereas a significant change of blood lactate does not start until 6% air saturation in water. We suggest therefore that A. ocellatus responses to environmental hypoxia are based mainly on suppressed metabolic rate, whereas under deep hypoxia or anoxia partial compensation is obtained from anaerobic glycolysis.

Scaling effects on hypoxia tolerance in the Amazon fish Astronotus ocellatus (Perciformes: Cichlidae): contribution of tissue enzyme levels

Astronotus ocellatus is one of the most hypoxia tolerant fish of the Amazon; adult animals can tolerate up to 6 h of anoxia at 28 degrees C. Changes in energy metabolism during growth have been reported in many fish species and may reflect the way organisms deal with environmental constraints. We have analyzed enzyme levels (lactate dehydrogenase, LDH: EC 1.1.1.27; and malate dehydrogenase, MDH: EC 1.1.1.37) in four different tissues (white muscle, heart, liver, and brain) from different-sized animals. Both enzymes correlate with body size, increasing the anaerobic potential positively with growth. To our knowledge, this is the first description of scaling effects on hypoxia tolerance and it is interesting to explore the fact that hypoxia survivorship increases due to combining effects of suppressing metabolic rates and increasing anaerobic power as fish grow.

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

SUMMARY The physiological and behavioural responses of two size groups of oscar (Astronotus ocellatus) to hypoxia were studied. The physiological responses were tested by measuring ṀO2 during decreasing environmental oxygen tensions. Larger oscars were better able to maintain oxygen consumption during a decrease in PO2, regulating routine ṀO2 to a significantly lower PO2 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 ṀO2 was seen (50 mmHg). Interestingly, smaller oscars postponed ASR to around 22 mmHg, well beyond the PO2 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.

Enzymes of cardiac energy metabolism in Amazonian teleosts and the fresh‐water stingray (Potamotrygon hystrix)

The maximal in vitro activity of key enzymes of energy metabolism was determined in heart from three Amazonian teleosts, matrincha (Brycon cephalus), acara acu (Astronaotus ocellatus) and tambaqui (Collossoma macropomum), as well as an elasmobranch, the fresh-water stingray (Potamotrygon hystrix). All species are obligate water breathers. Hearts of Amazonian teleosts have activity levels of the glycolytic enzymes hexokinase (HK), phosphofructokinase (PFG), pyruvate kinase (PK), and lactate dehydrogenase (LDH) similar to north temperate and Antarctic species when comparisons are made within the usual body temperature range. In contrast, activity level of enzymes required for aerobic oxidation of fatty acids, citrate synthase (CS), carnitine palmitoyl transferase (CPT), and 3-hydroxyacyl CoA dehydrogenase (HOAD) were all substantially lower in the Amazonian teleosts compared to other teleosts. The enzyme profile suggests that 1) activity levels of enzymes of carbohydrate metabolism are conserved over a wide range of body temperatures, and 2) Amazonian teleosts have a much greater reliance upon anaerobic metabolism from glucose than aerobic metabolism to sustain energy production. The heart of fresh-water stingray has high levels of CS, HK, PFK, and PK, implying an aerobic metabolism which is glucose based. In contrast to marine elasmobranchs, the fresh-water stingray has detectable levels of CPT and HOAD, suggestive of a capacity for low-level fatty acid catabolism. As such, the inability of muscle of marine elasmobranchs to utilize fatty acids as an energy source may not be common feature of all elasmobranchs.

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

SUMMARY 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 O2 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=10–20 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 [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.

Responses to hypoxia and recovery: repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid, Astronotus ocellatus.

SUMMARY Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by 3H-labelled phenylalanine incorporation rates, were determined in Astronotus ocellatus to investigate the cellular mechanisms behind hypoxia-induced metabolic depression and recovery. RoMR was significantly depressed, by approximately 50%, when dissolved oxygen levels reached 10% saturation (0.67±0.01 mg l–1 at 28±1°C). This depression in RoMR was accompanied by a 50–60% decrease in liver, heart and gill protein synthesis, but only a 30% decrease in brain protein synthesis. During recovery from hypoxia, an overshoot in RoMR to 270% of the normoxic rate was observed, indicating the accumulation of an oxygen debt during hypoxia. This conclusion was consistent with significant increase in plasma lactate levels during the hypoxic exposure, and the fact that lactate levels rapidly returned to pre-hypoxic levels. In contrast, a hyperactivation of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is attributed to an increase in cellular processes other than protein synthesis.

Hypoxia adaptation in fish of the Amazon: a never-ending task

In addition to seasonal long-term changes in dissolved oxygen and carbon dioxide, water bodies of the Amazon present periodic short-term episodes of hypoxia and even anoxia. To preserve gas exchange and acid base balance, fish of the Amazon have developed multiple adaptive solutions which occur at all biological levels. These solutions are thought to represent adaptive convergence rather than phylogenetic relatedness. Fish of the Amazon exposed to different experimental conditions adjust, for example, several parameters to improve oxygen transfer from the gas-exchange site to the tissues. These parameters include morphological changes such as the development of the lower lip in Colossoma, changes in ventilation rates, changes in circulatory parameters, increased circulating red blood cells, decreased levels of intraerythrocytic phosphates, and adjustments of intraerythrocytic pH (pHi). These adjustments that allow fish to survive both short- and long-term hypoxia occur in different degrees in different fi...

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.

Evolutionary trends of LDH isozymes in fishes

Abstract 1. 1. LDH characteristics (loci numbers, number of isozymes constituted by A and B subunits, and tissue specificity of LDH-C4) were reviewed for 245 fish species. 2. 2. The occurrence of different isozyme numbers among species of a single family (e.g. 3, 4 and 5 banded patterns), plus the existence of alleles generating 4 or 5 banded patterns in a single population indicate that the number of LDH isozymes constituted by A and B subunits might not be a character under selective pressure. 3. 3. The regulatory pattern of LDH-C ∗ gene, i.e. its occurrence in several tissues of primitive fishes plus its highly restricted tissue distribution in advanced teleost, and even its absence in some intermediate groups (e.g. Characiformes and Siluriformes) suggest a possible selective pressure upon an ancient gene. 4. 4. Four evolutionary trends are suggested considering isozyme tissue distribution as well as LDH physiological role: (i) convergence of B to A subunit as in good anaerobes; (ii) restriction of B subunits in advanced teleost, resulting in the same pattern as in (i); (iii) non-divergence between A and B subunit as occurs in some Amazon fishes which are obligatory aerobes and (iv) the restriction of C subunit to specific tissues in advanced teleost reaching a high specialization degree, which physiological meaning is not explained yet.

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.