Genome-wide association study reveals genes associated with the absence of intermuscular bones in tambaqui (Colossoma macropomum).

The presence of intermuscular bones in fisheries products limits the consumption and commercialization potential of many fish species, including tambaqui (Colossoma macropomum). These bones have caused medical emergencies and are an undesirable characteristic for fish farming because their removal is labor-intensive during fish processing. Despite the difficulty in identifying genes related to the lack of intermuscular bone in diverse species of fish, the discovery of individuals lacking intermuscular bones in a Neotropical freshwater characiform fish has provided a unique opportunity to delve into the genetic mechanisms underlying the pathways of intermuscular bone formation. In this study, we carried out a GWAS among boneless and wt tambaqui populations to identify markers associated with a lack of intermuscular bone. After analyzing 11 416 SNPs in 360 individuals (12 boneless and 348 bony), we report 675 significant (Padj  < 0.003) associations for this trait. Of those, 13 associations were located near candidate genes related to the reduction of bone mass, promotion of bone formation, inhibition of bone resorption, central control of bone remodeling, bone mineralization and other related functions. To the best of our knowledge, for the first time, we have successfully identified genes related to a lack of intermuscular bones using GWAS in a non-model species.

Metabolic adjustment of Pyrrhulina aff. brevis exposed to different climate change scenarios.

The increases in CO2 concentrations and, consequently, temperature due to climate change are predicted to intensify. Understanding the physiological responses of Pyrrhulina aff. brevis to the climatic scenarios proposed by the IPCC (2014) for the next 100 years is of fundamental importance to determine its susceptibility. Thus, the present study aimed to evaluate the effects of the predicted climatic scenarios for the year 2100 on the metabolic adjustments of P. aff. brevis . Specifically, the rate of oxygen uptake, electron transport system capacity, glycogen and lactate content and the role of Na+K+-ATPases and H+-ATPase were evaluated. P. aff. brevis individuals were exposed for 15 days to the simulated climatic scenarios in climate scenario rooms, where temperature and CO2 in the air were controlled. Two rooms were used to simulate the climatic scenarios predicted by the IPCC (2014): moderate (RCP 6; 2.5 °C and 400 µatm CO2 above current levels) and extreme (RCP 8.5; 4.5 °C and 900 µatm CO2 above current levels), in addition to the "control room" that represents the current scenario. There was an increase in the metabolic rate (MO2) in the animals acclimated to the climate change scenarios (RCP 6 and RCP 8.5) compared to the current scenario. These responses showed a typical effect of temperature on energy demand in relation to the increase in temperature and CO2. Our data showed an increase in O2 consumption (MO2), lactate levels and H+-ATPase activity of the animals acclimated to the moderate and extreme climate change scenarios. Such adjustments presented a clear metabolic imbalance, an alteration that may imply challenges for survival, growth, distribution and reproduction in the face of the expected environmental changes for the year 2100.

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.

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.

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.

Acute hypoxia up-regulates HIF-1α and VEGF mRNA levels in Amazon hypoxia-tolerant Oscar (Astronotus ocellatus).

Amazon fish maintain oxygen uptake through a variety of strategies considered evolutionary and adaptive responses to the low water oxygen saturation, commonly found in Amazon waters. Oscar (Astronotus ocellatus) is among the most hypoxia-tolerant fish in Amazon, considering its intriguing anaerobic capacity and ability to depress oxidative metabolism. Previous studies in hypoxia-tolerant and non-tolerant fish have shown that hypoxia-inducible factor-1α (HIF-1α) gene expression is positively regulated during low oxygen exposure, affecting vascular endothelial growth factor (VEGF) transcription and fish development or tolerance in different manners. However, whether similar isoforms exists in tolerant Amazon fish and whether they are affected similarly to others physiological responses to improve hypoxia tolerance remain unknown. Here we evaluate the hepatic HIF-1α and VEGF mRNA levels after 3 h of acute hypoxia exposure (0.5 mgO2/l) and 3 h of post-hypoxia recovery. Additionally, hematological parameters and oxidative enzyme activities of citrate synthase (CS) and malate dehydrogenase (MDH) were analyzed in muscle and liver tissues. Overall, three sets of responses were detected: (1) as expected, hematocrit, hemoglobin concentration, red blood cells, and blood glucose increased, improving oxygen carrying capacity and glycolysis potential; (2) oxidative enzymes from liver decreased, corroborating the tendency to a widespread metabolic suppression; and (3) HIF-1α and VEGF increased mRNA levels in liver, revealing their role in the oxygen homeostasis through, respectively, activation of target genes and vascularization. This is the first study to investigate a hypoxia-related transcription factor in a representative Amazon hypoxia-tolerant fish and suggests that HIF-1α and VEGF mRNA regulation have an important role in enhancing hypoxia tolerance in extreme tolerant species.

Loss of genetic diversity in farmed populations of Colossoma macropomum estimated by microsatellites.

The genetic variability of four Colossoma macropomum broodstocks, three from fish farms in different regions and one from the natural environment in Brazil, was analyzed using microsatellite markers. The wild progeny (n = 30) were caught in the Solimões-Amazonas River, at the varzea lakes; this location is used to mature the fish from larvae to juveniles. The three fish farms were selected according to the age of their lineages and broodstock availability: DNOCS (n = 21) is located in the Ceará State, representing the oldest lineage of cultivated tambaqui in Brazil; Balbina (n = 30) is located in the Amazonas State, representing the youngest stocks of tambaqui farmed in Brazil (approximately 15 years); and UFRPE (n = 30) is located in the Pernambuco State and is considered to be a mixed stock formed from the DNOCS and Balbina lineages. The analysis of 13 microsatellite loci indicated the occurrence of a variability reduction in the farmed populations; the UFRPE stock was the population with the highest diversity level. Low values of molecular coancestry were found in these populations. Additionally, significant differences in the RST values among the populations were detected, as was the occurrence of genetic structure. The genetic loss found in these populations may have been influenced by the founder effect. Because no breeding programs were during the entire production period and no pedigree records were kept for these broodstocks, we suggest that a wild population might be used as an important genetic resource to increase the genetic diversity of renewal stock lineages.

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.

Ion fluxes and hematological parameters of two teleosts from the Rio Negro, Amazon, exposed to hypoxia.

The aim of this study was to describe the effect of hypoxia on whole body ion fluxes and hematological parameters in two Amazonian teleosts: Serrasalmus eigenmanni and Metynnis hypsauchen. The increase of Na+ and Cl- effluxes on M. hypsauchen exposed to hypoxia may be related to an increase of gill ventilation and effective respiratory surface area, to avoid a reduction in the oxygen uptake, and/or with the decrease of pHe, that could inhibit Na+ and Cl- transporters and, therefore, reduce influx of these ions. Effluxes of Na+ and Cl- were lower in hypoxia than in normoxia for S. eigenmanni, possibly because in hypoxia this species would reduce gill ventilation and oxygen uptake, which would lead to a decrease of gill ion efflux and, consequently, reducing ion loss. The increase on hematocrit (Ht) during hypoxia in M. hypsauchen probably was caused by an increase of the red blood cell volume (MCV). For S. eigenmanni the increase on glucose possibly results from the usage of glucose reserve mobilization. Metynnis hypsauchen showed to be more sensitive to hypoxia than Serrasalmus eigenmanni, since the first presented more significant alterations on these osmoregulatory and hematological parameters. Nevertheless, the alterations observed for both species are strategies adopted by fishes to preserve oxygen supply to metabolizing tissues during exposure to hypoxia.

Ion fluxes and hematological parameters of two teleosts from the Rio Negro, Amazon, exposed to hypoxia / Fluxos iônicos e parâmetros hematológicos em duas espécies de teleósteos do Rio Negro, Amazônia, expostos à hipoxia

The aim of this study was to describe the effect of hypoxia on whole body ion fluxes and hematological parameters in two Amazonian teleosts: Serrasalmus eigenmanni and Metynnis hypsauchen. The increase of Na+ and Cl- effluxes on M. hypsauchen exposed to hypoxia may be related to an increase of gill ventilation and effective respiratory surface area, to avoid a reduction in the oxygen uptake, and/or with the decrease of pHe, that could inhibit Na+ and Cl- transporters and, therefore, reduce influx of these ions. Effluxes of Na+ and Cl- were lower in hypoxia than in normoxia for S. eigenmanni, possibly because in hypoxia this species would reduce gill ventilation and oxygen uptake, which would lead to a decrease of gill ion efflux and, consequently, reducing ion loss. The increase on hematocrit (Ht) during hypoxia in M. hypsauchen probably was caused by an increase of the red blood cell volume (MCV). For S. eigenmanni the increase on glucose possibly results from the usage of glucose reserve mobilization. Metynnis hypsauchen showed to be more sensitive to hypoxia than Serrasalmus eigenmanni, since the first presented more significant alterations on these osmoregulatory and hematological parameters. Nevertheless, the alterations observed for both species are strategies adopted by fishes to preserve oxygen supply to metabolizing tissues during exposure to hypoxia.

O objetivo deste trabalho foi descrever o efeito da hipoxia no fluxo iônico corporal e nos parâmetros hematológicos em duas espécies de teleósteos da Amazônia: Serrasalmus eigenmanni e Metynnis hypsauchen. O aumento dos efluxos de Na+ e Cl- em M. hypsauchen expostos à hipoxia pode estar relacionado ao aumento da ventilação branquial e da eficiência da área da superfície respiratória, a fim de evitar redução na captação de oxigênio; e/ou com a diminuição do pHe, que pode inibir os transportadores de Na+ e Cl- e, então, reduzir o influxo destes íons. Os efluxos de Na+ e Cl- foram menores em hipoxia do que em normoxia para a espécie S. eigenmanni, possivelmente porque esta espécie em hipoxia poderia reduzir a ventilação branquial e a captação de oxigênio, a qual levaria a uma diminuição do efluxo branquial de íons e, conseqüentemente, à redução da perda de íons. O aumento do hematócrito (Ht) durante hipoxia em M. hypsauchen provavelmente foi causado pelo aumento do volume das células vermelhas do sangue (MCV). Para a espécie S. eigenmanni, o aumento da glicose possivelmente foi resultado do uso da mobilização da reserva de glicose. A espécie Metynnis hypsauchen mostrou ser mais sensível à hipoxia do que a espécie Serrasalmus eigenmanni, uma vez que a primeira espécie apresentou mais alterações significativas em seus parâmetros osmorregulatórios e hematológicos. Contudo, as alterações observadas em ambas as espécies são estratégias adotadas pelos peixes a fim de preservar o suprimento de oxigênio para metabolização nos tecidos durante exposição à hipoxia.

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

Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by (3)H-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 degrees 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.

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.

Specialized metabolism and biochemical suppression during aestivation of the extant South American lungfish -- Lepidosiren paradoxa

Lepidosiren paradoxa (pirambóia) is the single representative of Dipnoan (lungfish) in South America. This species is considered a living fossil, in spite of some reports describing this fish as having a very specialized life style. It aestivates during the dry season, and has developed metabolic adaptations to cope with both flooding and drought. The literature describing its tissue ultra-structure shows high glycogen stored in the muscle, suggesting a strong dependence on anaerobic glycolysis. The present paper reports tissue enzyme levels of LDH, MDH, and CS, and isozymic tissue distribution of LDH, MDH, ADH, PGI, SOD, and PGM of 7 aestivating specimens from Lago do Canteiro in the Amazonas River. Animals were caught while burrowed in mud during the aestivation period. Our findings reveal high anaerobic capacity of both skeletal and heart muscles, even during the aestivation period, when enzymes showed suppressed levels compared to those of non-aestivating animals (data from the literature). Isozymic patterns suggest loss of duplicate condition in most analyzed loci, a characteristic that occurs mainly in higher vertebrate categories. These data indicate that, compared to the fish group, lungfish may be considered advanced, despite retaining primitive morphological characters

Specialized metabolism and biochemical suppression during aestivation of the extant South American lungfish--Lepidosiren paradoxa.

Lepidosiren paradoxa (pirambóia) is the single representative of Dipnoan (lungfish) in South America. This species is considered a living fossil, in spite of some reports describing this fish as having a very specialized life style. It aestivates during the dry season, and has developed metabolic adaptations to cope with both flooding and drought. The literature describing its tissue ultra-structure shows high glycogen stored in the muscle, suggesting a strong dependence on anaerobic glycolysis. The present paper reports tissue enzyme levels of LDH, MDH, and CS, and isozymic tissue distribution of LDH, MDH, ADH, PGI, SOD, and PGM of 7 aestivating specimens from Lago do Canteiro in the Amazonas River. Animals were caught while burrowed in mud during the aestivation period. Our findings reveal high anaerobic capacity of both skeletal and heart muscles, even during the aestivation period, when enzymes showed suppressed levels compared to those of non-aestivating animals (data from the literature). Isozymic patterns suggest loss of duplicate condition in most analyzed loci, a characteristic that occurs mainly in higher vertebrate categories. These data indicate that, compared to the fish group, lungfish may be considered advanced, despite retaining primitive morphological characters.

Karyological, biochemical, and physiological aspects of Callophysus macropterus (Siluriformes, Pimelodidae) from the Solimöes and Negro Rivers (Central Amazon)

Karyological characteristics, i.e., diploid number, chromosome morphology and nucleolus organizer regions (NORs), biochemical characteristics, i.e., electrophoretic analysis of blood hemoglobin and the tissue enzymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), and phosphoglucose isomerase (PGI), and physiological characteristics, i.e., relative concentration of hemoglobin and intraerythrocytic concentrations of organic phosphates were analyzed for the species Callophysus macropterus collected from Marchantaria Island (white water system - Solimöes River) and Anavilhanas Archipelago (black water system - Negro River). Karyological and biochemical data did not reveal significant differences between specimens collected at the two sites. However, the relative distribution of hemoglobin bands I and III (I = 16.33 ñ 1.05 and III = 37.20 ñ 1.32 for Marchantaria specimens and I = 6.33 ñ 1.32 and III = 48.05 ñ 1.55 for Anavilhanas specimens) and levels of intraerythrocytic GTP (1.32 ñ 0.16 and 2.76 ñ 0.18 for Marchantaria and Anavilhanas specimens, respectively), but not ATP or total phosphate, were significantly different, indicating a physiological adaptation to the environmental conditions of these habitats. It is suggested that C. macropterus specimens from the two collecting sites belong to a single population, and that they adjusted some physiological characteristics to adapt to local environmental conditions.

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.