Simulated climate change and atrazine contamination can synergistically impair zebrafish testicular function.

Elements that interfere with reproductive processes can have profound impacts on population and the equilibrium of ecosystems. Global warming represents the major environmental challenge of the 21st century, as it will affect all forms of life in the coming decades. Another coexisting concern is the persistent pollution by pesticides, particularly the herbicide Atrazine (ATZ), which is responsible for a significant number of contamination incidents in surface waters worldwide. While it is hypothesized that climate changes will significantly enhance the toxic effects of pesticides, the actual impact of these phenomena remain largely unexplored. Here, we conducted a climate-controlled room experiment to assess the interactive effects of the projected 2100 climate scenario and environmentally realistic ATZ exposures on the reproductive function of male zebrafish. The gonadosomatic index significantly decreased in fish kept in the extreme scenario. Cellular alterations across spermatogenesis phases led to synergic decreased sperm production and increased germ cell sloughing and death. ATZ exposure alone or combined with climate change effects, disrupted the transcription levels of key genes involved in steroidogenesis, hormone signaling and spermatogenesis regulation. An additive modulation with decreased 11-KT production and increased E2 levels was also evidenced, intensifying the effects of androgen/estrogen imbalance. Moreover, climate change and ATZ independently induced oxidative stress, upregulation of proapoptotic gene and DNA damage in post-meiotic germ cell, but the negative effects of ATZ were greater at extreme scenario. Ultimately, exposure to simulated climate changes severely impaired fertilization capacity, due to a drastic reduction in sperm motility and/or viability. These findings indicate that the future climate conditions have the potential to considerably enhance the toxicity of ATZ at low concentrations, leading to significant deleterious consequences for fish reproductive function and fertility. These may provide relevant information to supporting healthcare and environmental managers in decision-making related to climate changes and herbicide regulation.

Impairment on fillet fatty acid profile and oxidative damage in pirarucu, Arapaima gigas, acutely exposed to extreme ambient temperature.

Climatic events are affecting the Amazon basin and according to projections it is predicted the intensification of climate changes through increases in temperature and carbon dioxide (CO2). Recent evidence has revealed that exposure to an extreme climate scenario elicits oxidative damage in some fish species, impairing their metabolism and physiology, contributing to their susceptibility. Thus, the comprehension of physiological alterations in Arapaima gigas (pirarucu) to the climatic changes forecasted for the next 100 years is important to evaluate its capability to deal with oxidative stress. The objective of this work was to determine whether antioxidant defense system is able to prevent muscle oxidative damage of pirarucu exposed 96 h to extreme climate scenario, as well as the effects of this exposition on muscle fatty acid levels. Lipid peroxidation and reactive oxygen species significantly increase in the muscle of pirarucus exposed to an extreme climate scenario compared to control, while muscle superoxide dismutase, catalase, and glutathione peroxidase were significantly lower. Total amount of saturated fatty acids (SFAs) was significantly higher in pirarucu exposed to an extreme climate scenario compared to control, while total content of monounsaturated (MUFAs) and polyunsaturated fatty acids (PUFAs) was significantly lower. Exposure to an extreme climate scenario causes muscular oxidative stress and that the antioxidant systems are inefficient to avoid oxidative damage. In addition, the increase of total SFAs and the decrease of MUFAs and PUFAs probably intend to maintain membrane fluidity while facing high temperature and CO2 levels.

Effects of climate change and mixtures of pesticides on the Amazonian fish Colossoma macropomum.

Several studies highlighted the complexity of mixing pesticides present in Amazonian aquatic environments today. There is evidence that indicates that ongoing climate change can alter the pattern of pesticide use, increasing the concentration and frequency of pesticide applications. It is known that the combination of thermal and chemical stress can induce interactive effects in aquatic biota, which accentuates cell and molecular damage. However, considering that the effects of climate change go beyond the increase in temperature the objective of this study was to evaluate the effect of climate change scenarios proposed by 6 th IPCC report and a mixture of pesticides on the tambaqui (Colossoma macropomum). The hypothesis of this study is that the negative effects will be accentuated by the combination of an extreme climate changes scenario and a mixture of pesticides. To test the hypothesis, juvenile tambaqui were exposed to a combination of four pesticides (chlorpyrifos, malathion, carbendazim and atrazine) in two scenarios, one that simulates current environmental conditions and another that predicted the environmental scenario for the year 2100. Fish were subjected to the experimental conditions for 96 h. At the end of the experiment, samples of blood, gills, liver, brain, and muscle were obtained for hematological, genotoxic, biochemical, and histopathological analyses. The results demonstrate that environmentally realistic concentrations of pesticides, when mixed, can alter the biochemical responses of tambaqui. The extreme scenario promotes hematological adjustments, but impairs branchial antioxidant enzymes. There is an interaction between the mixture of pesticides and the extreme scenario, accentuating liver tissue damage, which demonstrates that even increased activity of antioxidant and biotransformation enzymes were not sufficient to prevent liver damage.

Oil spill in an amazon blackwater environment: Biochemical and physiological responses of local fish species.

The accidental spill of petroleum asphalt cement (PAC) in São Raimundo (SR Harbor, located on the Rio Negro (Manaus, Amazonas, Brazil) was monitored through the analysis of polyciclic aromatic hydrocarbons (PAHs) in water and a set of biomarkers in fishes (exposure biomarkes: PAHs-type metabolites concentrations in bile; the activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in liver. Effect biomarkers: lipid peroxidation concentration (LPO) in liver, acetylcholinesterase activity in brain, and genotoxic DNA damage in erythrocytes). Two fish species, Acarichthys heckelii and Satanoperca jurupari, were collected 10, 45, and 90 days after the PAC spill in São Raimundo. At the same time, fish were collected from the Tupé Sustainable Development Reserve (Tupé) which served as a reference area. The sampling periods were related to the rising waters of the natural flood pulse of the Rio Negro. Higher concentrations of PAHs in water were observed at 10 and 45 days and returned to the values of TP 90 days after the PAC spill, a period in which harbor waters rose about 0.2 m. Unlike the PAHs in water, biomarker responses in both fish species significantly increased following the PAC spill in SR. Hepatic ethoxyresorufin-O-deethylase (EROD), PAH-like metabolites in bile, and erythrocyte DNA damage increases, together with inhibition of acetylcholinesterase (AChE) activity in the brain were the most evident responses for both fish species. The calculated pyrolytic index showed mixed sources of PAHs (petrogenic and pyrolytic). The applied PCA-FA indicated important relationships between dissolved organic carbon (DOC) and PAHs concentrations in water, where DOC and PAHs concentrations contributed to biomarkers responses for both fish species in all collection periods.

In vivo exposure to high temperature compromises quality of the sperm in Colossoma macropomum.

Increases in temperature can affect the reproduction of fish by decreasing the quality of gametes for fertilization. Therefore, this study aimed to evaluate the in vivo effect of temperature on the production and sperm quality of Colossoma macropomum, which is an economically relevant species for Brazil, and other countries. Broodstock were exposed for 10 days at temperatures of 29 °C (n = 4) and 35 °C (n = 4). After exposure, semen was collected and sperm quality was evaluated for kinetic, biochemical, morphological parameters, membrane integrity, and oxygen consumption. The sperm quality of males of specimens of C. macropomum exposed in vivo to the higher temperature was compromised and showed a difference in all the analyses (P < 0.05). Sperm showed decreased motility (51.9 ± 11.6 s) compared to the control (61.3 ± 5.5 s); curvilinear (CLV), straight line (SLV), and average path (APV) velocities decreased, and straightness (STR) and beat cross frequency (BCF) increased; oxygen consumption decreased (32.8 ± 3.4 pmol. (s.ml)-1, picomol per second per ml) compared to the control (46.6 ± 3.5 pmol. (s.ml)-1); and the activity of catalase (CAT) and glutathione S-transferase (GST) enzymes increased and decreased, respectively, in sperm from fish exposed to high temperatures. Furthermore, an increase in non-viable sperm with damaged membranes and sperm with altered morphology was observed. The observed alterations indicate that C. macropomum confined in environments with high temperatures possibly produces more sperm that are unviable for the fertilization process.

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.

Gut microbiota of an Amazonian fish in a heterogeneous riverscape: integrating genotype, environment, and parasitic infections.

A number of key factors can structure the gut microbiota of fish such as environment, diet, health state, and genotype. Mesonauta festivus, an Amazonian cichlid, is a relevant model organism to study the relative contribution of these factors on the community structure of fish gut microbiota. M. festivus has well-studied genetic populations and thrives in rivers with drastically divergent physicochemical characteristics. Here, we collected 167 fish from 12 study sites and used 16S and 18S rRNA metabarcoding approaches to characterize the gut microbiome structure of M. festivus. These data sets were analyzed in light of the host fish genotypes (genotyping-by-sequencing) and an extensive characterization of environmental physico-chemical parameters. We explored the relative contribution of environmental dissimilarity, the presence of parasitic taxa, and phylogenetic relatedness on structuring the gut microbiota. We documented occurrences of Nyctotherus sp. infecting a fish and linked its presence to a dysbiosis of the host gut microbiota. Moreover, we detected the presence of helminths which had a minor impact on the gut microbiota of their host. In addition, our results support a higher impact of the phylogenetic relatedness between fish rather than environmental similarity between sites of study on structuring the gut microbiota for this Amazonian cichlid. Our study in a heterogeneous riverscape integrates a wide range of factors known to structure fish gut microbiomes. It significantly improves understanding of the complex relationship between fish, their parasites, their microbiota, and the environment. IMPORTANCE The gut microbiota is known to play important roles in its host immunity, metabolism, and comportment. Its taxonomic composition is modulated by a complex interplay of factors that are hard to study simultaneously in natural systems. Mesonauta festivus, an Amazonian cichlid, is an interesting model to simultaneously study the influence of multiple variables on the gut microbiota. In this study, we explored the relative contribution of the environmental conditions, the presence of parasitic infections, and the genotype of the host on structuring the gut microbiota of M. festivus in Amazonia. Our results highlighted infections by a parasitic ciliate that caused a disruption of the gut microbiota and by parasitic worms that had a low impact on the microbiota. Finally, our results support a higher impact of the genotype than the environment on structuring the microbiota for this fish. These findings significantly improve understanding of the complex relationship among fish, their parasites, their microbiota, and the environment.

Important role of endogenous microbial symbionts of fish gills in the challenging but highly biodiverse Amazonian blackwaters.

Amazonian blackwaters are extremely biodiverse systems containing some of Earth's most naturally acidic, dissolved organic carbon -rich and ion-poor waters. Physiological adaptations of fish facing these ionoregulatory challenges are unresolved but could involve microbially-mediated processes. Here, we characterize the physiological response of 964 fish-microbe systems from four blackwater Teleost species along a natural hydrochemical gradient, using dual RNA-Seq and 16 S rRNA of gill samples. We find that host transcriptional responses to blackwaters are species-specific, but occasionally include the overexpression of Toll-receptors and integrins associated to interkingdom communication. Blackwater gill microbiomes are characterized by a transcriptionally-active betaproteobacterial cluster potentially interfering with epithelial permeability. We explore further blackwater fish-microbe interactions by analyzing transcriptomes of axenic zebrafish larvae exposed to sterile, non-sterile and inverted (non-native bacterioplankton) blackwater. We find that axenic zebrafish survive poorly when exposed to sterile/inverted blackwater. Overall, our results suggest a critical role for endogenous symbionts in blackwater fish physiology.

Ammonia Increases the Stress of the Amazonian Giant Arapaima gigas in a Climate Change Scenario.

Ammonia is toxic to fish, and when associated with global warming, it can cause losses in aquaculture. In this study, we investigated the physiological and zootechnical responses of Arapaima gigas to the current scenarios and to RCP8.5, a scenario predicted by the IPCC for the year 2100 which is associated with high concentrations of environmental ammonia (HEA). Forty-eight chipped juvenile A. gigas were distributed in two experimental rooms (current scenario and RCP8.5) in aquariums with and without the addition of ammonia (0.0 mM and 2.44 mM) for a period of 30 days. The HEA, the RCP8.5 scenario, and the association of these factors affects the zootechnical performance, the ionic regulation pattern, and the levels of ammonia, glucose, triglycerides, sodium, and potassium in pirarucu plasma. The branchial activity of H+-ATPase was reduced and AChE activity increased, indicating that the species uses available biological resources to prevent ammonia intoxication. Thus, measures such as monitoring water quality in regard to production, densities, and the feed supplied need to be more rigorous and frequent in daily management in order to avoid the accumulation of ammonia in water, which, in itself, proved harmful and more stressful to the animals subjected to a climate change scenario.

Bacterioplankton Communities in Dissolved Organic Carbon-Rich Amazonian Black Water.

The Amazon River basin sustains dramatic hydrochemical gradients defined by three water types: white, clear, and black waters. In black water, important loads of allochthonous humic dissolved organic matter (DOM) result from the bacterioplankton degradation of plant lignin. However, the bacterial taxa involved in this process remain unknown, since Amazonian bacterioplankton has been poorly studied. Its characterization could lead to a better understanding of the carbon cycle in one of the Earth's most productive hydrological systems. Our study characterized the taxonomic structure and functions of Amazonian bacterioplankton to better understand the interplay between this community and humic DOM. We conducted a field sampling campaign comprising 15 sites distributed across the three main Amazonian water types (representing a gradient of humic DOM), and a 16S rRNA metabarcoding analysis based on bacterioplankton DNA and RNA extracts. Bacterioplankton functions were inferred using 16S rRNA data in combination with a tailored functional database from 90 Amazonian basin shotgun metagenomes from the literature. We discovered that the relative abundances of fluorescent DOM fractions (humic-, fulvic-, and protein-like) were major drivers of bacterioplankton structure. We identified 36 genera for which the relative abundance was significantly correlated with humic DOM. The strongest correlations were found in the Polynucleobacter, Methylobacterium, and Acinetobacter genera, three low abundant but omnipresent taxa that possessed several genes involved in the main steps of the ß-aryl ether enzymatic degradation pathway of diaryl humic DOM residues. Overall, this study identified key taxa with DOM degradation genomic potential, the involvement of which in allochthonous Amazonian carbon transformation and sequestration merits further investigation. IMPORTANCE The Amazon basin discharge carries an important load of terrestrially derived dissolved organic matter (DOM) to the ocean. The bacterioplankton from this basin potentially plays important roles in transforming this allochthonous carbon, which has consequences on marine primary productivity and global carbon sequestration. However, the structure and function of Amazonian bacterioplanktonic communities remain poorly studied, and their interactions with DOM are unresolved. In this study, we (i) sampled bacterioplankton in all the main Amazon tributaries, (ii) combined information from the taxonomic structure and functional repertory of Amazonian bacterioplankton communities to understand their dynamics, (iii) identified the main physicochemical parameters shaping bacterioplanktonic communities among a set of >30 measured environmental parameters, and (iv) characterized how bacterioplankton structure varies according to the relative abundance of humic compounds, a by-product from the bacterial degradation process of allochthonous DOM.

Emersion and recovery alter oxygen consumption, ammonia and urea excretion, and oxidative stress parameters, but not diffusive water exchange or transepithelial potential in the green crab (Carcinus maenas).

The green crab (Carcinus maenas) is an inshore species affected by intertidal zonation patterns, facing periods of emersion during low tide and submersion during high tide. During these periods of air and subsequent water exposure, these species can face physiological challenges. We examined changes in O2 consumption rate (MO2), and ammonia and urea excretion rates over sequential 14 h periods in seawater (32 ppt, control), in air and during recovery in seawater after air exposure (13°C throughout). At the end of each exposure, the anterior (5th) and posterior (8th) gills and the hepatopancreas were removed for measurements of oxidative stress parameters (TBARs and catalase in the gills and hepatopancreas, and protein carbonyls in the gills). MO2 remained unchanged during air exposure, but increased greatly (3.4-fold above control levels) during the recovery period. Ammonia and urea net fluxes were reduced by 98% during air exposure, but rebounded during recovery to >2-fold the control rates. Exchangeable water pools, rate constants of diffusive water exchange, unidirectional diffusive water flux rates (using tritiated water) and transepithelial potential were also measured during control and recovery treatments, but exhibited no significant changes. Damage to proteins was not observed in either gill. However, lipid damage occurred in the anterior (respiratory) gill after the air exposure but not in the posterior (ionoregulatory) gill or hepatopancreas. Catalase activity also decreased significantly in recovery relative to levels during air exposure in both the anterior gill and hepatopancreas, but not in the posterior gill. The crabs did not modify water metabolism or permeability. We conclude that MO2 was maintained but not enhanced during air exposure, while ammonia and urea-N excretion were impaired. As a result, all of these parameters increase greatly during re-immersion recovery, and oxidative stress also occurs. Clearly, emersion is not without physiological costs.

Correlation between caffeine and coprostanol in contrasting Amazonian water bodies.

The evaluation of contamination by domestic sewage is relevant in the Amazon region; however, it has neither been well-developed nor accompanied by research or monitoring programs. In this study, caffeine and coprostanol as indicators of sewage were investigated in water samples from Amazonian water bodies that crisscross the city of Manaus (Amazonas state, Brazil) and cover regions with distinct main land uses such as high-density residential, low-density residential, commercial, industrial, and environmental protection areas. Thirty-one water samples were studied based on their dissolved and particulate organic matter (DOM and POM) fractions. Quantitative determination of both caffeine and coprostanol was carried out using LC-MS/MS with APCI in the positive ionization mode. The streams of the urban area of Manaus had the highest concentrations of caffeine (1.47-69.65 µg L-1) and coprostanol (2.88-46.92 µg L-1). Samples from the peri-urban Tarumã-Açu stream and from the streams in the Adolpho Ducke Forest Reserve showed much lower concentrations of caffeine (20.20-165.78 ng L-1) and coprostanol (31.49-120.44 ng L-1). Samples from the Negro River showed a wider range of concentrations of caffeine (20.59-873.59 ng L-1) and coprostanol (31.72-706.46 ng L-1), with the highest values found in the outfalls of the urban streams. Levels of caffeine and coprostanol were significantly positively correlated in the different organic matter fractions. The coprostanol/(coprostanol + cholestanol) ratio proved to be a more suitable parameter than the coprostanol/cholesterol one in low-density residential areas. Proximity to densely populated areas and the flow of water bodies appear to influence the caffeine and coprostanol concentrations, which was observed in their clustering in the multivariate analysis. The results indicate that caffeine and coprostanol can be detected even in water bodies that receive very low domestic sewage input. Therefore, this study revealed that both caffeine in DOM and coprostanol in POM represent viable alternatives for use in studies and monitoring programs even in remote areas of the Amazon, where microbiological analyses are often unfeasible.

Vitellogenin Ab structure of the amazonian Arapaima gigas.

The vitellogenin is composed by polypeptides that are precursors of egg yolk proteins that provides embryo and larvae nutrition. The mRNA encoding for vitellogenin Ab (Vtg-Ab; 4,536 bp long and 1,512 amino acids) were obtained by RNA-Seq library sequencing of pirarucu gonads. The Vtg-Ab sequences had high homology with Vtgs of other three teleosts species of the order Osteoglossiformes. The transcript of ovarian Vtg was identified based on structural criteria, and so we classify the Vtg of pirarucu as Vtg-Ab due to the truncated or shortened phosvitin (N-terminal end) and phosvitinless domain (C-terminal end). The Vtg-Ab of pirarucu present two major deletions with 133 amino acids in the Lipovitellin I domain and 89 amino acids in the truncated or shortened Phosvitin domain, both located in the N-terminal end region. The three-dimensional (3-D) structure Vtg-Ab protein shows the presence of a typical 4α-helices bundle protein that runs in anti-parallel. In general, the characterization of Vtg-Ab may be the useful elucidation of the hormonal regulation of vitellogenesis and improve the production of pirarucu for broodstock management in aquaculture and preparation of Vtg antibody production (species-specific) for sex identification.

Tissue distribution of appetite regulation genes and their expression in the Amazon fish Colossoma macropomum exposed to climate change scenario.

Climate change leads to an increase in water acidification and temperature, two environmental factors that can change fish appetite and metabolism, affecting fish population in both wild and aquaculture facilities. Therefore, our study tested if climate change affects gene expression levels of two appetite-regulating peptides - Neuropeptide Y (NPY) and Cholecystokinin (CCK) - in the brain of tambaqui, Colossoma macropomum. Additionally, we show the distribution of these genes throughout the body. Amino acid sequences of CCK and NPY of tambaqui showed high similarity with other Characiformes, with the closely related order Cypriniformes, and even with the more distantly related order Salmoniformes. High apparent levels of both peptides were expressed in all brain areas, while expression levels varied for peripheral tissues. NPY and CCK mRNA were detected in all peripheral tissues but cephalic kidney for CCK. As for the effects of climate change, we found that fish exposed to extreme climate scenario (800 ppm CO2 and 4.5 °C above current climate scenario) had higher expression levels of NPY and lower expression levels of CCK in the telencephalon. The extreme climate scenario also increased food intake, weight gain, and body length. These results suggest that the telencephalon is probably responsible for sensing the metabolic status of the organism and controlling feeding behavior through NPY, likely an orexigenic hormone, and CCK, which may act as an anorexigenic hormone. To our knowledge, this is the first study showing the effects of climate change on the endocrine regulation of appetite in an endemic and economically important fish from the Amazon. Our results can help us predict the impact of climate change on both wild and farmed fish populations, thus contributing to the elaboration of future policies regarding their conservation and sustainable use.

Genomic and Environmental Factors Shape the Active Gill Bacterial Community of an Amazonian Teleost Holobiont.

Fish bacterial communities provide functions critical for their host's survival in contrasting environments. These communities are sensitive to environmental-specific factors (i.e., physicochemical parameters, bacterioplankton), and host-specific factors (i.e., host genetic background). The relative contribution of these factors shaping Amazonian fish bacterial communities is largely unknown. Here, we investigated this topic by analyzing the gill bacterial communities of 240 wild flag cichlids (Mesonauta festivus) from 4 different populations (genetic clusters) distributed across 12 sites in 2 contrasting water types (ion-poor/acidic black water and ion-rich/circumneutral white water). Transcriptionally active gill bacterial communities were characterized by a 16S rRNA metabarcoding approach carried on RNA extractions. They were analyzed using comprehensive data sets from the hosts genetic background (Genotyping-By-Sequencing), the bacterioplankton (16S rRNA) and a set of 34 environmental parameters. Results show that the taxonomic structure of 16S rRNA gene transcripts libraries were significantly different between the 4 genetic clusters and also between the 2 water types. However, results suggest that the contribution of the host's genetic background was relatively weak in comparison to the environment-related factors in structuring the relative abundance of different active gill bacteria species. This finding was also confirmed by a mixed-effects modeling analysis, which indicated that the dissimilarity between the taxonomic structure of bacterioplanktonic communities possessed the best explicative power regarding the dissimilarity between gill bacterial communities' structure, while pairwise fixation indexes (FST) from the hosts' genetic data only had a weak explicative power. We discuss these results in terms of bacterial community assembly processes and flag cichlid fish ecology. IMPORTANCE Host-associated microbial communities respond to factors specific to the host physiology, genetic backgrounds, and life history. However, these communities also show different degrees of sensitivity to environment-dependent factors, such as abiotic physico-chemical parameters and ecological interactions. The relative importance of host- versus environment-associated factors in shaping teleost bacterial communities is still understudied and is paramount for their conservation and aquaculture. Here, we studied the relative importance of host- and environment-associated factors structuring teleost bacterial communities using gill samples from a wild Amazonian teleost model (Mesonauta festivus) sampled in contrasting habitats along a 1500 km section of the Amazonian basin, thus ensuring high genetic diversity. Results showed that the contribution of the host's genetic background was weak compared to environment-related bacterioplanktonic communities in shaping gill bacterial assemblages, thereby suggesting that our understanding of teleost microbiome assembly could benefit from further studies focused on the ecological interplay between host-associated and free-living communities.

Impact of high temperature, CO2 and parasitic infection on inflammation, immunodepression and programmed cell death in Colossoma macropomum at the transcriptional level.

The production of tambaqui Colossoma macropomum has recently reached a milestone, being considered the main native species produced in South American continental waters. Despite the importance of this fish, its immunity is poorly understood, and global warming could pose severe risks to its health as increasing water temperature leads to an increase in the incidence of parasitic diseases. In an experimental context based on the high-emission scenario of the 5th Intergovernmental Panel on Climate Change (IPCC) report, we evaluated the synergistic effect of exposure to the extreme climate change scenario (RCP8.5) during two exposure periods (7 and 30 days) and two levels of parasitism by monogeneans (low and high). The goal was to understand how the tambaqui immune system will react to this challenge. To achieve this goal, we analyzed the expression of nine immunity-related genes (jak3, stat3, il-10, socs1, casp1, il-1ß, tp53, bcl2, and hif-1α) in the spleen. Our main findings showed downregulation in the jak3/stat3 pathway, genes related to the control of inflammation and apoptosis, in addition to upregulation of proinflammatory genes and those related to pyroptosis during the first 7 days of exposure to the extreme climate scenario, also indicating a stage of immunodepression in these animals. After 30 days of exposure, all genes tended to return to similar levels in the current scenario, possibly due to the decrease in parasite load caused by chronic exposure to the extreme scenario. Our data strongly suggest that the increase in parasitism intensity caused by the extreme climate change scenario is responsible for disturbances in the host's immune system. However, more studies are needed to clarify this poorly understood cascade of events.

Investigating the mechanisms of dissolved organic matter protection against copper toxicity in fish of Amazon's black waters.

We investigated how natural dissolved organic matter (DOM) of the Rio Negro (Amazon) affects acute copper (Cu) toxicity to local fish: the cardinal tetra (Paracheirodon axelrodi) and the dwarf cichlid (Apistogramma agassizii). It is established that Cu2+ complexation with DOM decreases Cu bioavailability (and thus toxicity) to aquatic organisms, as conceptualized by the Biotic Ligand Model (BLM). However, we also know that Rio Negro's DOM can interact with fish gills and have a beneficial effect on Na+ homeostasis, the main target of acute Cu toxicity in freshwater animals. We aimed to tease apart these potential protective effects of DOM against Cu-induced Na+ imbalances in fish. In the laboratory, we acclimated fish to Rio Negro water (10 mg L-1 DOC) and to a low-DOM water (1.4 mg L-1 DOC) with similar ion composition and pH (5.9). We measured 3-h Cu uptake in gills and unidirectional and net Na+ physiological fluxes across a range of Cu concentrations in both waters. Various DOM pre-acclimation times (0, 1 and 5 days) were evaluated in experiments with P. axelrodi. Copper exposure led to similar levels of net Na+ loss in the two fish, but with distinct effects on Na+ influx and efflux rates reflecting their different ionoregulation strategies. Rio Negro DOM protected against Cu uptake and toxicity in the two fish species. Both Cu uptake in fish gills and Na+ regulation disturbances were relatively well predicted by the modelled aqueous free Cu2+ ion concentration. These findings suggest that protection by DOM occurs mainly from Cu complexation under the tested conditions. The prevalence of this geochemical-type protection over a physiological-type protection agrees with the BLM conceptual framework, supporting the use of the BLM to assess the risk of Cu in these Amazonian waters.

Genomics of Serrasalmidae teleosts through the lens of microbiome fingerprinting.

Associations between host genotype and host-associated microbiomes have been shown in a variety of animal clades, but studies on teleosts mostly show weak associations. Our study aimed to explore these relationships in four sympatric Serrasalmidae (i.e., piranha) teleosts from an Amazonian lake, using data sets from the hosts genomes (single nucleotide polymorphisms from genotyping by sequencing), skin and gut microbiomes (16S rRNA gene metataxonomics) and diets (COI metabarcoding) from the same fish individuals. First, we investigated whether there were significant covariations of microbiome and fish genotypes at the inter- and intraspecific levels. We also assessed the extent of covariation between Serrasalmidae diet and microbiome, to isolate genotypic from dietary effects on community structure. We observed a significant covariation of skin microbiomes and host genotypes at interspecific (R2  = 24.4%) and intraspecific (R2  = 6.2%) levels, whereas gut microbiomes correlated poorly with host genotypes. Serrasalmidae diet composition was significantly correlated to fish genotype only at the interspecific level (R2  = 5.4%), but did not covary with gut microbiome composition (Mantel R = -.04). Second, we investigated whether the study of interspecific differentiation could benefit from considering host-associated microbial communities in addition to host genotypes. By using a nonmetric multidimensional scaling (NMDS) ordination-based approach, we observed that ordinations from skin- and gut species-specific bacterial biomarkers identified through a random forest algorithm could significantly increase the average interspecific differentiation detected through host genotype data alone. Although future studies encompassing additional species and environments are needed, our results suggest Serrasalmidae microbiomes could constitute an insightful trait to be considered when studying the interspecific differences between members of this clade.

Global change and physiological challenges for fish of the Amazon today and in the near future.

Amazonia is home to 15% (>2700, in 18 orders) of all the freshwater fish species of the world, many endemic to the region, has 65 million years of evolutionary history and accounts for 20% of all freshwater discharge to the oceans. These characteristics make Amazonia a unique region in the world. We review the geological history of the environment, its current biogeochemistry and the evolutionary forces that led to the present endemic fish species that are distributed amongst three very different water types: black waters [acidic, ion-poor, rich in dissolved organic carbon (DOC)], white waters (circumneutral, particle-rich) and clear waters (circumneutral, ion-poor, DOC-poor). The annual flood pulse is the major ecological driver for fish, providing feeding, breeding and migration opportunities, and profoundly affecting O2, CO2 and DOC regimes. Owing to climate change and other anthropogenic pressures such as deforestation, pollution and governmental mismanagement, Amazonia is now in crisis. The environment is becoming hotter and drier, and more intense and frequent flood pulses are now occurring, with greater variation between high and low water levels. Current projections are that Amazon waters of the near future will be even hotter, more acidic, darker (i.e. more DOC, more suspended particles), higher in ions, higher in CO2 and lower in O2, with many synergistic effects. We review current physiological information on Amazon fish, focusing on temperature tolerance and ionoregulatory strategies for dealing with acidic and ion-poor environments. We also discuss the influences of DOC and particles on gill function, the effects of high dissolved CO2 and low dissolved O2, with emphasis on water- versus air-breathing mechanisms, and strategies for pH compensation. We conclude that future elevations in water temperature will be the most critical factor, eliminating many species. Climate change will likely favour predominantly water-breathing species with low routine metabolic rates, low temperature sensitivity of routine metabolic rates, high anaerobic capacity, high hypoxia tolerance and high thermal tolerance.