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.

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.

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.

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.

Amazon fish bacterial communities show structural convergence along widespread hydrochemical gradients.

The world's richest freshwater fish community thrives in gradients of contrasting environments in Amazonia, ranging from ion-poor acidic black waters, to ion-rich circumneutral white waters. These hydrochemical gradients structure Amazonian fish assemblages via ecological speciation events. Fish bacterial communities contain an important genetic heritage essential for their hosts' survival and are also involved in adaptive divergence via niche adaptation processes, but the extent to which they evolve in response to hydrochemical gradients in Amazonia is unknown. Here we investigated bacterial communities (gut and skin mucus) of two ecologically and phylogenetically divergent host species (Mesonauta festivus and Serrasalmus rhombeus) distributed throughout these hydrochemical gradients. The goal was to characterize intra- and interspecific Amazonian fish microbiome variations across multiple scales. Using a 16S metabarcoding approach, we investigated the microbiota of 43 wild M. festivus, 32 S. rhombeus and seven water samples, collected at seven sampling sites encompassing both water colours. Taxonomical structures of bacterial communities from both host species were significantly correlated to the environmental continua of magnesium, sodium, dissolved organic carbon, calcium, dissolved O2 , pH, potassium, hardness and chloride. Analysis of discriminating features in community structures across multiple scales demonstrated intra- and interspecific structural parallelisms in the response to the hydrochemical gradients. Together, these parallelisms suggest the action of selection on bacterial community structures along Amazonian hydrochemical gradients. Functional approaches along with reciprocal transplant experiments will provide further insights on the potential contribution of Amazonian fish microbiomes in host adaptation and ecological speciation events.

pH drop impacts differentially skin and gut microbiota of the Amazonian fish tambaqui (Colossoma macropomum).

Aquatic organisms are increasingly exposed to lowering of environmental pH due to anthropogenic pressure (e.g. acid rain, acid mine drainages). Such acute variations trigger imbalance of fish-associated microbiota, which in turn favour opportunistic diseases. We used the tambaqui (Colossoma macropomum), an Amazonian fish tolerant to significant pH variation in its natural environment, to assess the response of fish endogenous microbiota to acute short-term acid stress. We exposed 36 specimens of tambaquis to acidic water (pH 4.0) over 2 consecutive weeks and sampled cutaneous mucus, feces and water at 0, 7 &14 days. The 16S RNA hypervariable region V4 was sequenced on Illumina MiSeq. After two weeks of acidic exposure, fecal and skin microbiota taxonomic structures exhibited different patterns: skin microbiota was still exhibiting a significantly disturbed composition whereas fecal microbiota recovered a similar composition to control group, thus suggesting a stronger resilience capacity of the intestinal microbiota than cutaneous microbiota.

Protection by natural blackwater against disturbances in ion fluxes caused by low pH exposure in freshwater stingrays endemic to the Rio Negro.

Stenohaline freshwater stingrays (Potamotrygon spp.) are endemic to the very dilute (Na(+), Cl(-), Ca2(+) 300 micromol L(-1) in reference water (low DOC) to about 100 micromol L(-1) in blackwater (high DOC). In reference water, both JNain and JClin were inhibited >90%, both JNaout and JClout more than doubled, and J(Amm) did not change at pH 4.0. In blackwater, the inhibition of influxes was attenuated, the increases in outflux did not occur, and J(Amm) increased by 60% at pH 4.0. Addition of 100 micromol L(-1) Ca(2+) to reference water prevented the increases in JNaout and JClout and allowed J(Amm) to increase at pH 4.0, which demonstrates that the gills are sensitive to Ca(2+). However, addition of Ca(2+) to blackwater had no effect on the responses to pH 4.0. Addition of commercial humic acid to reference water did not duplicate the effects of natural Rio Negro blackwater at the same DOC level; instead, it greatly exacerbated the increases in JNaout and JClout at low pH and prevented any protective influence of added Ca(2+). Thus, blackwater DOC appears to be very different from commercial humic acid. Biogeochemical modeling indicated that blackwater DOC prevents Ca(2+) binding, but not H(+) binding, to the gills and that the protective effects of blackwater cannot be attributed to its higher buffer capacity or its elevated Al or Fe levels. Natural DOC may act directly at the gills at low pH to exert a protective effect and, when doing so, may override any protective action of Ca(2+) that might otherwise occur.

Safest level of tricaine methanesulfonate (ms-222) to induce anesthesia in juveniles of Matrinxã, Brycon cephalus

The use of MS-222 as an anesthetic for matrinxã juveniles was investigated. At dosage of 100 mg/L or lower fish did not achieve a complete anesthesia state. At 150 mg/L, MS-222 induced anesthesia within 36 seconds and recovered from a 10 minutes period of anesthesia within 5.2 min. Higher concentrations (200, 250 and 300 mg/L) anesthetized fish in lesser times, with the offset of mortality (16.7 and 33.3%) at the 200 and 300 mg/L MS-222 doses, respectively. The only significant differences observed in the hematological parameters, was for the glucose values in fish anesthetized with 250 and 300 mg/L. From the results, the recommended dose of MS-222 for handling matrinxã juveniles is 150 mg/L.

Investigou-se o uso de MS-222 como anestésico para juvenis de matrinxã. Concentrações de 100 mg/L ou menores não causam uma indução completa à anestesia. A 150 mg/L os peixes foram induzidos à anestesia após 36 segundos e se recuperam em 5,2 minutos após exposição ao anestésico durante 10 minutos. Em concentrações maiores (200, 250 e 300 mg/L) os peixes foram induzidos a anestesia em menor tempo, porém observou-se mortalidade de 16,7 e 33,3 % nas doses de 200 e 300 mg/L, respectivamente. Nos parâmetros hematológicos observou-se uma alteração significante nos níveis de glicose para os peixes anestesiados com doses de 250 e 300 mg/L. Os resultados obtidos indicam que 150 mg/L é a dose ideal de MS-222 para anestesiar juvenis de matrinxã.

Aspectos biológicos de peixes amazônicos. IV. Padrões eletroforéticos de hemoglobinas de 22 espécies coletadas na ilha da Marchantaria (Manaus - AM)

The hemoglobin patterns of hemolysates from 22 fish species belonging to Manchantaria island (Solimões river , Manaus, AM) had been characterized by starch gel and agarstarch gel electrophresis. High interespecific hemoglobin heterogeneity was observed, similarly to those described for sub-tropical and temperate fish species. The resolution power of the electrophoretic supports was considered. The adaptability question of the multiple hemoglobin systems was regarded.

Foram estudados hemolisados de 22 espécies de peixes coletados na Ilha da Marchantaria (rio Solimões, Manaus - AM), através de eletroforeses em gel de amido e gel de ágar-amido. Uma grande heterogeneidade hemoglobínica interespecífica foi detectada, a exemplo do observado para espécies de zona subtropical e temperada. A capacidade de resolução de dois suportes eletroforéticos é discutida. Os resultados são discutidos em função da possível adaptabilidade conferida pelos sistemas de múltiplas hemoglobinas.