Acute CO2 tolerance in fishes is associated with air breathing but not the Root effect, red cell ßNHE, or habitat.

High CO2 (hypercapnia) can impose significant physiological challenges associated with acid-base regulation in fishes, impairing whole animal performance and survival. Unlike other environmental conditions such as temperature and O2, the acute CO2 tolerance thresholds of fishes are not understood. While some fish species are highly tolerant, the extent of acute CO2 tolerance and the associated physiological and ecological traits remain largely unknown. To investigate this, we used a recently developed ramping assay, termed the Carbon Dioxide maximum (CDmax), that increases CO2 exposure until loss of equilibrium (LOE) is observed. We investigated if there was a relationship between CO2 tolerance and the Root effect, ß-adrenergic sodium proton exchanger (ßNHE), air-breathing, and fish habitat in 17 species. We hypothesized that CO2 tolerance would be higher in fishes that lack both a Root effect and ßNHE, breathe air, and reside in tropical habitats. Our results showed that CDmax ranged from 2.7 to 26.7 kPa, while LOE was never reached in four species at the maximum PCO2 we could measure (26.7 kPa); CO2 tolerance was only associated with air-breathing, but not the presence of a Root effect or a red blood cell (RBC) ßNHE, or fish habitat. This study demonstrates that the diverse group of fishes investigated here are incredibly tolerant of CO2 and that although this tolerance is associated with air-breathing, further investigations are required to understand the basis for CO2 tolerance.

Preferential intracellular pH regulation is a common trait amongst fishes exposed to high environmental CO2.

Acute (<96 h) exposure to elevated environmental CO2 (hypercarbia) induces a pH disturbance in fishes that is often compensated by concurrent recovery of intracellular and extracellular pH (pHi and pHe, respectively; coupled pH regulation). However, coupled pH regulation may be limited at CO2 partial pressure (PCO2 ) tensions far below levels that some fishes naturally encounter. Previously, four hypercarbia-tolerant fishes had been shown to completely and rapidly regulate heart, brain, liver and white muscle pHi during acute exposure to >4 kPa PCO2  (preferential pHi regulation) before pHe compensation was observed. Here, we test the hypothesis that preferential pHi regulation is a widespread strategy of acid-base regulation among fish by measuring pHi regulation in 10 different fish species that are broadly phylogenetically separated, spanning six orders, eight families and 10 genera. Contrary to previous views, we show that preferential pHi regulation is the most common strategy for acid-base regulation within these fishes during exposure to severe acute hypercarbia and that this strategy is associated with increased hypercarbia tolerance. This suggests that preferential pHi regulation may confer tolerance to the respiratory acidosis associated with hypercarbia, and we propose that it is an exaptation that facilitated key evolutionary transitions in vertebrate evolution, such as the evolution of air breathing.

Tambaqui (Colossoma macropomum) acclimated to different tropical waters from the Amazon basin shows specific acute-stress responses.

Stress responses in teleosts include the release of hormones into the bloodstream. Their effects depend on the species and on the environmental conditions. The Amazon basin collects waters of diverse chemical composition, and some fish are able to inhabit several of them. However, the effects of these waters on the stress axis are still unknown. Here we show how acute air-exposure differently affects stress biomarkers in tambaqui (Colossoma macropomum), a tropical model species, when acclimated to two Amazonian waters (Rio Negro -RN- water rich in humic acids and poor in ions, and groundwater -IG- with no humic acids and higher concentration of ions). This study described primary and secondary stress responses after air exposure including plasma cortisol, energy metabolites, pH and ions, skin mucus energy metabolites, as well as gills and kidney Na+/K+-ATPase and Na+/H+-exchanger (NHE) activities. Several differences were described in these stress biomarkers due to the acclimation water. The most remarkable ones include increased mucus glucose only in RN-fish, and mucus lactate only in IG-fish after air exposure. Moreover, an inverse relationship between plasma cortisol and Na+ concentrations as well as a direct relationship between plasma ammonia and branchial NHE activity were observed only in RN-fish. Our results demonstrate how important is to study stress responses in fish acclimated to different environments, as physiological differences can be magnified during episodes of high energy expenditure. In addition to having a direct application in aquaculture, this study will improve the management of critical ecosystems such as the Amazon.

Protein synthesis is lowered by 4EBP1 and eIF2-α signaling while protein degradation may be maintained in fasting, hypoxic Amazonian cichlids Astronotus ocellatus.

The Amazonian cichlid Astronotus ocellatus is highly tolerant to hypoxia, and is known to reduce its metabolic rate by reducing the activity of energetically expensive metabolic processes when oxygen is lacking in its environment. Our objectives were to determine how protein metabolism is regulated in A. ocellatus during hypoxia. Fish were exposed to a stepwise decrease in air saturation (100%, 20%, 10% and 5%) for 2 h at each level, and sampled throughout the experiment. A flooding dose technique using a stable isotope allowed us to observe an overall decrease in protein synthesis during hypoxia in liver, muscle, gill and heart. We estimate that this decrease in rates of protein synthesis accounts for a 20 to 36% decrease in metabolic rate, which would enable oscars to maintain stable levels of ATP and prolong survival. It was also determined for the first time in fish that a decrease in protein synthesis during hypoxia is likely controlled by signaling molecules (4EBP1 and eIF2-α), and not simply due to a lack of ATP. We could not detect any effects of hypoxia on protein degradation as the levels of NH4 excretion, indicators of the ubiquitin proteasome pathway, and enzymatic activities of lysosomal and non-lysosomal proteolytic enzymes were maintained throughout the experiment.

The influence of lifestyle and swimming behavior on metabolic rate and thermal tolerance of twelve Amazon forest stream fish species.

The metabolism of fishes is profoundly affected by environmental factors such as temperature, oxygen concentration, and pH levels. Also, biotic elements, for instance, activity levels of species, have been suggested to affect the energy demand, driving their capacity to support environmental challenges. The present work aims to investigate the effects of the lifestyle and swimming activities levels of fishes living in Amazon forest stream on the aerobic metabolism and thermal tolerance. Intermittent flow respirometry was used to measure routine metabolic rate and thermal maximum metabolic rate with a thermal ramp methodology. Critical thermal tolerance, thermal aerobic scope, and thermal factorial aerobic scope were calculated for twelve species belonging to different families. Our findings showed a correlation between routine and thermal maximum metabolic rate and, between metabolic rate and activity levels. Species belonging to Characidae and Crenuchidae families have high resting metabolic rates, which decrease their factorial aerobic scope and reduce their abilities to cope with warming events. Therefore, these species have low thermal tolerance. Instead, species from families Rivulidae and Cichlidae showed opposite metabolic results and larger thermal windows. We hypothesize that these responses are related to an evolutionary trade-off between lifestyle and energetic requirements and warming will favor species with low activity performance.

In vitro effects of increased temperature and decreased pH on blood oxygen affinity of 10 fish species of the Amazon.

Blood-O2 affinities (P50 ) were measured over a physiologically relevant pH range at 31 (highest temperature average of Rio Negro over the last 8 years), 33 and 35° C for 10 species of the Rio Negro, aiming to test the acute effects of temperature foreseen by the IPCC (Intergovernmental Panel on Climate Change) for coming years. The animals were collected during an expedition to the Anavilhanas Islands of the Rio Negro, 110 km upstream from Manaus (2° 23' 41″ S; 60° 55' 14″ W). Hoplias malabaricus showed higher blood-O2 sensitivity to pH changes (Bohr effect, Φ = Δlog10 P50 ΔpH(-1) ) at both 31° C (Φ = -0·44) and 35° C (Φ = -0·26) compared to Osteoglossum bicirrhosum (Φ = -0·54 at 31° C and Φ = -0·58 at 35° C), but lower P50 under most conditions, and a greater sensitivity of P50 to temperature. Two out of the 10 analysed species had significant increases of P50 (lower blood-O2 affinity) at the highest temperature throughout the pH range tested. For all other species, a minor increase of P50 over the assay-tested temperatures was observed, although all presented a normal Bohr effect. Overall, a diversity of intensities of pH and temperature effects on blood-O2 affinities was observed, which seems to be connected to the biological characteristics of the analysed species. Thermal disturbances in their habitats, likely to occur due to the global warming, would impair blood-O2 binding and unloading in some of the analysed fish species. Copyright © 2016 John Wiley & Sons, Ltd.

Ion uptake in naturally acidic water.

The first studies on ion regulation in fish exposed to low pH, which were inspired by the Acid Rain environmental crisis, seemed to indicate that ion transport at the gills was completely and irreversibly inhibited at pH 4.0-4.5 and below. However, work on characid fish native to the Rio Negro, a naturally acidic, blackwater tributary of the Amazon River, found that they possess ion transport mechanisms that are completely insensitive to pHs as low as 3.25. As more species were examined it appeared that pH-insensitive transport was a trait shared by many, if not most, species in the Order Characiformes. Subsequently, a few other species of fish have been shown to be able to transport ions at low pH, in particular zebrafish (Danio rerio), which show rapid recovery of Na+ uptake at pH 4.0 after initial inhibition. Measurements of rates of Na+ transport during exposure to pharmacological agents that inhibit various transport proteins suggested that characiform fish do not utilize the generally accepted mechanisms for Na+ transport that rely on some form of H+ extrusion. Examination of zebrafish transport at low pH suggest the rapid recovery may be due to a novel Na+/K+ exchanger, but after longer term exposure they may rely on a coupling of Na+/H+ exchangers and NH3 excretion. Further work is needed to clarify these mechanisms of transport and to find other acid-tolerant species to fully gain an appreciation of the diversity of physiological mechansisms involved.

Overcoming the coupled climate and biodiversity crises and their societal impacts.

Earth's biodiversity and human societies face pollution, overconsumption of natural resources, urbanization, demographic shifts, social and economic inequalities, and habitat loss, many of which are exacerbated by climate change. Here, we review links among climate, biodiversity, and society and develop a roadmap toward sustainability. These include limiting warming to 1.5°C and effectively conserving and restoring functional ecosystems on 30 to 50% of land, freshwater, and ocean "scapes." We envision a mosaic of interconnected protected and shared spaces, including intensively used spaces, to strengthen self-sustaining biodiversity, the capacity of people and nature to adapt to and mitigate climate change, and nature's contributions to people. Fostering interlinked human, ecosystem, and planetary health for a livable future urgently requires bold implementation of transformative policy interventions through interconnected institutions, governance, and social systems from local to global levels.

Exposure to pH 3.5 water has no effect on the gills of the Amazonian tambaqui (Colossoma macropomum).

Tambaqui (Colossoma macropomum) are a model species for tropical fish physiology, but details are lacking about their ionoregulatory response to acid waters. To provide specifics, we measured unidirectional Na+ fluxes in low pH waters. Sodium influx ([Formula: see text]) was uninhibited during acute exposure to pH 4.5 and 3.5, and Na efflux ([Formula: see text]) rose only slightly at pH 3.5; net Na+ flux ([Formula: see text]) remained positive at all pH. Similarly, during 24 h transfer to pH 3.5 [Formula: see text], [Formula: see text], and [Formula: see text] were unchanged at all times. Taking a closer look at the mechanism of Na+ transport in the gills of tambaqui we found that [Formula: see text] was uninhibited by HMA, a Na+/H+-exchanger blocker, and Benzamil, a Na+-channel inhibitor, casting doubt on their role in Na+ uptake in this fish. Measurement of Na+/K+-ATPase (NKA) and H+-ATPase (VHA) activity showed that neither changed at low pH compared to measurements at pH 6.5. Western blot analysis of ATPase expression saw no changes in amount of NKA and VHA at low pH, and immunohistochemistry showed expression of both NKA and VHA on lamellae and interlamellar region of tambaqui gills and that both proteins co-localized to the same gill cells. Location of expression also did not change in low pH water. Amazingly, tambaqui seem unaffected by pH 3.5 water, making them one of the most acid-tolerant fish species examined so far. In addition, they appear to share key ionoregulatory traits with other fish of the order Characiformes, which suggest a common origin for the ionoregulatory attributes.

Fish and aquatic habitat conservation in South America: a continental overview with emphasis on neotropical systems.

Fish conservation in South America is a pressing issue. The biodiversity of fishes, just as with all other groups of plants and animals, is far from fully known. Continuing habitat loss may result in biodiversity losses before full species diversity is known. In this review, the main river basins of South America (Magdalena, Orinoco, Amazon and Paraná-La Plata system), together with key aquatic habitats (mangrove-fringed estuaries of the tropical humid, tropical semi-arid and subtropical regions) are analysed in terms of their characteristics and main concerns. Habitat loss was the main concern identified for all South American ecosystems. It may be caused by damming of rivers, deforestation, water pollution, mining, poor agricultural practice or inadequate management practice. Habitat loss has a direct consequence, which is a decrease in the availability of living resources, a serious social and economic issue, especially for South American nations which are all developing countries. The introduction of exotic species and overfishing were also identified as widespread across the continent and its main freshwater, coastal and marine ecosystems. Finally, suggestions are made to find ways to overcome these problems. The main suggestion is a change of paradigm and a new design for conservation actions, starting with integrated research and aiming at the co-ordinated and harmonized management of the main transboundary waters of the continent. The actions would be focused on habitat conservation and social rescue of the less well-off populations of indigenous and non-indigenous peoples. Energy and freshwater demands will also have to be rescaled in order to control habitat loss.

Digestive enzymes of eight Amazonian teleosts with different feeding habits.

The levels of amylase, maltase, lipase and alkaline protease from eight fish species of the Amazon were analysed. The enzyme levels are not related to fish feeding habits, reflecting their ever-changing habitats and their opportunistic feeding behaviour.

Cadmium-induced disruption of environmental exploration and chemical communication in matrinxã, Brycon amazonicus.

The effects of cadmium exposure on both environment exploration and behavioral responses induced by alarm substance in matrinxã (Brycon amazonicus), a fish species endemic to the Amazon basin, were investigated. Fish exposed to 9.04+/-0.07 microg/L waterborne cadmium for 96h followed by 24h depuration period in clean water, were video-recorded for 15 min, followed by immediate introduction of conspecific skin extract to the tank and a new 30 min period of fish video-recording. Cd-exposed matrinxã showed a significantly lowered locomotor activity (t-test t(12)=2.7; p=0.025) and spatial distribution (t-test t(12)=2.4; p=0.03) relative to the unexposed control fish prior to the alarm substance introduction, and did not present any significant reaction when the skin extract was introduced. The control fish, in opposite, showed a higher level of activity and spatial distribution prior the skin extract contact and significantly decreased their response after the chemical stimulus (locomotion-repeated-measure ANOVA F(1,11)=5.6; p=0.04; spatial distribution F(1,11)=19.4; p=0.001). In conclusion, exposure to a low level of cadmium affects both the environment exploration performance and the conspecific chemical communication in matrinxã. If the reduced environmental exploration performance of Cd-exposed fish is an adjustment to the compromised chemical communication or an independent effect of cadmium is the next step to be investigated.

Dietary tissue cadmium accumulation in an amazonian teleost (Tambaqui, Colossoma macropomum Cuvier, 1818).

Understanding the effects of metal contamination in the Amazon basin is important because of the potential impact on this region of high biodiversity. In addition, the significance of fish as the primary source of protein for the local human population (living either alongside the Amazon River or in the city of Manaus) highlights the need for information on the metal transfer through the food chain. Bioaccumulation of metals in fish can occur at significant rates through the dietary route, without necessarily resulting in death of the organism. The goal of this work was to expose an economic relevant species from the Amazon basin (tambaqui, Colossoma macropomum) to dietary cadmium (Cd) at concentrations of 0, 50, 100, 200, and 400 microg.g-1 dry food. Fish were sampled on days 15, 30, and 45 of the feeding trials. Tissues were collected for analysis of Cd concentration using graphite furnace atomic absorption spectrophotometry. Cd accumulation in the tissues occurred in the following order: kidney > liver > gills > muscle. Relative to other freshwater fish (e.g., rainbow trout, tilapia), tambaqui accumulated remarkably high levels of Cd in their tissues. Although Cd is known to affect Ca2+ homeostasis, no mortality or growth impairment occurred during feeding trials.

Metabolic adjustments in two Amazonian cichlids exposed to hypoxia and anoxia.

The effects of graded hypoxia on the physiological and biochemical responses were examined in two closely related species of cichlids of the Amazon: Astronotus crassipinnis and Symphysodon aequifasciatus. Ten fish of each species were exposed to graded hypoxia for 8 h in seven oxygen concentrations (5.92, 3.15, 1.54, 0.79, 0.60, 0.34, and 0.06 mg O(2) L(-)(1)), with the aim to evaluate hypoxia tolerance and metabolic adjustments, where plasma glucose and lactate levels, hepatic and muscle glycogen contents, and maximum enzyme activities (PK, LDH, MDH and CS) in skeletal and cardiac muscles were measured. Another experimental set was done to quantify oxygen consumption (MO(2)) and opercular movements in two oxygen concentrations. Hypoxia tolerance differed between the two species. Astronotus crassipinnis was able to tolerate anoxia for 178 min while S. aequifasciatus was able to withstand 222 min exposure in deep hypoxia (0.75 mg O(2) L(-)(1)). Suppressed MO(2) was observed during exposure to 0.34 (A. crassipinnis) and 0.79 mg O(2) L(-)(1) (S. aequifasciatus), while opercular movements increased in both species exposed to hypoxia. Higher levels of muscle and liver glycogen and larger hypoxia-induced increases in plasma glucose and lactate were observed in A. crassipinnis, which showed a higher degree of hypoxia tolerance. Changes in enzyme levels were tissue-specific and differed between species suggesting differential abilities in down-regulating oxidative pathways and increasing anaerobic metabolism. Based on the present data, we conclude that these animals are good anaerobes and highly adapted to their environment, which is allowed by their abilities to regulate metabolic pathways and adjust their enzyme levels.

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.

Oxygen transfer in fish: morphological and molecular adjustments.

A wide range of organismic, physiological and biochemical adjustments to improve oxygen transfer is observed in fish exposed to environmental hypoxia and during anemia. Many fish species of the Amazon obtain oxygen directly from air when water oxygen is low. The accessory air-breathing organs include modifications of the gills, mouth, stomach and intestine, and swimbladder vascularization. Other species extend the lower lip and skim to improve oxygen uptake from the oxygen-rich surface layer of the water. The amount of oxygen uptake from air was estimated for Hoplosternum littorale and Lipossarcus pardalis. In addition, the oxygen uptake from the water surface was estimated for Colossoma macropomum. Blood oxygen content was reduced by 30% in Hoplosternum littorale and Colossoma macropomum and 70% in Lipossarcus pardalis if they were denied access to air. Adjustments of intraerythrocytic levels of ATP and GTP significantly improve oxygen transfer in fish during environmental hypoxia and anemia. In contrast to environmental hypoxia, intraerythrocytic levels of ATP and GTP increase during anemia in fish facilitating oxygen unloading to the tissues. It is suggested that the increase in ATP and GTP levels during anemia occurs because the conditions required to increase the activity of adenylate and guanylate phosphate synthetic pathways are similar.

Low pH and calcium effects on net Na+ and K+ fluxes in two catfish species from the Amazon River (Corydoras: Callichthyidae).

The present study analyzes Na+ and K+ disturbances caused by low pH in two catfish species from the Amazon River. Corydoras adolfoi inhabits ion-poor, black-stained, low pH (3.5-4.0) waters, while C. schwartzi is native to ion-rich waters at circumneutral pH. Fish were exposed to pH 3.5 Ca2+-free, and Ca2+-enriched (approximately 500 micromol/l) water to determine the protective effects of calcium. Net Na+ and K+ fluxes were measured in the water collected from the fish experimental chambers. C. adolfoi was unable to control the Na+ efflux at low pH, exhibiting Na+ loss up to -594 +/- 84 nmol g(-1) h(-1) during the first hour. After 3 and 6 h, net Na+ flux increased by 7- and 23-fold, respectively. In C. schwartzi, at pH 3.5, the initial high Na+ loss (-1,063 +/- 73 nmol g(-1) h(-1)) was gradually attenuated. A K+ loss occurred in both species, but remained relatively constant throughout exposure. High [Ca2+] affected ion losses in both species. C. adolfoi had 70% loss attenuation, indicating incapacity to control Na+ efflux. In C. schwartzi, elevated [Ca2+] completely prevented the Na+ losses caused by exposure to low pH. Rather different patterns were seen for K+ fluxes, with C. adolfoi showing no K+ disruption when exposed to low pH/high [Ca2+]. Thus, C. adolfoi loses Na+ during acid exposure, but has the ability to control K+ loss, while C. schwartzi controls diffusive Na+ loss but exhibits a slightly higher K+ loss. Ion balance was influenced by [Ca2+] at low pH in C. schwartzi but not in C. adolfoi.

Effects of temperature on leucocytes of Colossoma macropomum and Hoplosternum littorale (Pisces).

1. The effects of different thermal regimens on qualitative and quantitative characteristics of leucocytes were evaluated in two fish species of the Amazon region. 2. The proportion of circulating types of leucocytes changed significantly in Colossoma macropomum (tambaqui) but not in Hoplosternum littorale (tamoatá) exposed for four-week terms to 20, 25, 30, 35, and 40 degrees C. 3. The proportion of circulating lymphocytes decreased significantly in tambaquis exposed to 30, 35, and 40 degrees C. No changes in lymphocyte proportions were observed in tamoatás. 4. Neutrophils were almost absent in tambaquis, except in animals exposed to 40 degrees C. No significant changes in circulating neutrophils were observed in tamoatás. 5. The circulating leucocytes of Colossoma macropomum and Hoplosternum littorale are affected in different ways by temperature changes, suggesting species-specific adjustments to this parameter.

Biochemical adjustments to hypoxia by Amazon cichlids.

The isozyme distribution of cichlid lactate dehydrogenase (LDH) is related to species environmental preferences. Cichlasoma amazonarum occurs in different environments and presents LDH tissue distribution patterns that correlate with oxygen tension at the capture location. Cichlasoma amazonarum was exposed to long-term severe hypoxia (51 days at 36.4 +/- 5.9 mmHg), tissue LDH isozyme distribution was analyzed by electrophoresis and enzyme activities were measured by monitoring the oxidation of NADH as pyruvate was reduced to lactate. The exposure of Cichlasoma amazonarum to long-term severe hypoxia resulted in changes in the tissue distribution of LDH isozymes. The major changes in response to hypoxia occurred in heart, liver and brain: isozyme A4 was activated in heart and brain, whereas isozyme B4 was activated in liver. The most significant quantitative change occurred in brain LDH of hypoxia-exposed animals which adopted muscle type kinetics, reflecting a new LDH isozyme distribution. LDH activity was significantly reduced (P < 0.05) in animals exposed to hypoxia (N = 8), suggesting an overall LDH suppression. Pyruvate inhibition decreased in all hypoxia-exposed tissues. Thus, the ability of Cichlasoma amazonarum to regulate LDH tissue expression according to oxygen availability allows the animal to survive chronic hypoxic environments. This phenotypic plasticity may occur in other hypoxia-tolerant fish species.