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.

We measured unidirectional ion fluxes of fish collected directly from the Rio Negro, an extremely dilute, acidic blackwater tributary of the Amazon. Kinetic analysis of Na(+) uptake revealed that most species had fairly similar J(max) values, ranging from 1,150 to 1,750 nmol g(-1) h(-1), while K(m) values varied to a greater extent. Three species had K(m) values <33 micromol L(-1), while the rest had K(m) values >or=110 micromol L(-1). Because of the extremely low Na(+) concentration of Rio Negro water, the differences in K(m) values yield very different rates of Na(+) uptake. However, regardless of the rate of Na(+) uptake, measurements of Na(+) efflux show that Na(+) balance was maintained at very low Na(+) levels (<50 micromol L(-1)) by most species. Unlike other species with high K(m) values, the catfish Corydoras julii maintained high rates of Na(+) uptake in dilute waters by having a J(max) value at least 100% higher than the other species. Corydoras julii also demonstrated the ability to modulate kinetic parameters in response to changes in water chemistry. After 2 wk in 2 mmol L(-1) NaCl, J(max) fell >50%, and K(m) dropped about 70%. The unusual acclimatory drop in K(m) may represent a mechanism to ensure high rates of Na(+) uptake on return to dilute water. As well as being tolerant of extremely dilute waters, Rio Negro fish generally were fairly tolerant of low pH. Still, there were significant differences in sensitivity to pH among the species on the basis of degree of stimulation of Na(+) efflux at low pH. There were also differences in sensitivity to low pH of Na(+) uptake, and two species maintained significant rates of uptake even at pH 3.5. When fish were exposed to low pH in Rio Negro water instead of deionized water (with the same concentrations of major ions), the effects of low pH were reduced. This suggests that high concentrations of dissolved organic molecules in the water, which give it its dark tea color, may interact with the branchial epithelium in some protective manner.

No abstract

The so-called “blackwaters” of the Amazonian Rio Negro are rich in highly coloured dissolved organic carbon (DOC), but ion-poor and very acidic, conditions that would cause fatal ionoregulatory failure in most fish. However these blackwaters support 8% of the world’s ichthyofauna. We tested the hypothesis that native DOC provides protection against ionoregulatory dysfunction in this extreme environment. DOCs were isolated by reverse-osmosis from two Rio Negro sites. Physico-chemical characterization clearly indicated a terrigenous origin, with a high proportion of hydroxyl and phenolic sites, high chemical reactivity to protons, and unusual proteinaceous fluorescence. When tested using zebrafish (a model organism), Rio Negro DOC provided almost perfect protection against ionoregulatory disturbances associated with acute exposure to pH 4.0 in ion-poor water. DOC reduced diffusive losses of Na+ and Cl−, and promoted a remarkable stimulation of Na+ uptake that otherwise would have been completely inhibited. Additionally, prior acclimation to DOC at neutral pH reduced rates of branchial Na+ turnover, and provided similar protection against acid-induced ionoregulatory disturbances, even if the DOC was no longer present. These results reinforce the important roles that DOC molecules can play in the regulation of gill functions in freshwater fish, particularly in ion-poor, acidic blackwaters.

SUMMARY The transition from aquatic to aerial respiration is associated with dramatic physiological changes in relation to gas exchange, ion regulation,acid–base balance and nitrogenous waste excretion. Arapaima gigas is one of the most obligate extant air-breathing fishes,representing a remarkable model system to investigate (1) how the transition from aquatic to aerial respiration affects gill design and (2) the relocation of physiological processes from the gills to the kidney during the evolution of air-breathing. Arapaima gigas undergoes a transition from water-to air-breathing during development, resulting in striking changes in gill morphology. In small fish (10 g), the gills are qualitatively similar in appearance to another closely related water-breathing fish (Osteoglossum bicirrhosum); however, as fish grow (100–1000 g), the inter-lamellar spaces become filled with cells, including mitochondria-rich(MR) cells, leaving only column-shaped filaments. At this stage, there is a high density of MR cells and strong immunolocalization of Na+/K+-ATPase along the outer cell layer of the gill filament. Despite the greatly reduced overall gill surface area, which is typical of obligate air-breathing fish, the gills may remain an important site for ionoregulation and acid–base regulation. The kidney is greatly enlarged in A. gigas relative to that in O. bicirrhosum and may comprise a significant pathway for nitrogenous waste excretion. Quantification of the physiological role of the gill and the kidney in A. gigas during development and in adults will yield important insights into developmental physiology and the evolution of air-breathing.

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.

SUMMARY Environmental hypercapnia induces a respiratory acidosis that is usually compensated within 24-96 h in freshwater fish. Water ionic composition has a large influence on both the rate and degree of pH recovery during hypercapnia. Waters of the Amazon are characteristically dilute in ions, which may have consequences for acid-base regulation during environmental hypercapnia in endemic fishes. The armoured catfish Liposarcus pardalis, from the Amazon, was exposed to a water PCO2 of 7, 14 or 42 mmHg in soft water (in μmol l-1: Na+, 15,Cl-, 16, K+, 9, Ca2+, 9, Mg2+, 2). Blood pH fell within 2 h from a normocapnic value of 7.90±0.03 to 7.56±0.04, 7.34±0.05 and 6.99±0.02, respectively. Only minor extracellular pH (pHe) recovery was observed in the subsequent 24-96 h. Despite the pronounced extracellular acidosis,intracellular pH (pHi) of the heart, liver and white muscle was tightly regulated within 6 h (the earliest time at which these parameters were measured) via a rapid accumulation of intracellular HCO3-. While most fish regulate pHi during exposure to environmental hypercapnia, the time course for this is usually similar to that for pHe regulation. The degree of extracellular acidosis tolerated by L. pardalis, and the ability to regulate pHi in the face of an extracellular acidosis, are the greatest reported to date in a teleost fish. The preferential regulation of pHi in the face of a largely uncompensated extracellular acidosis in L. pardalis is rare among vertebrates, and it is not known whether this is associated with the ability to air-breathe and tolerate aerial exposure, or living in water dilute in counter ions, or with other environmental or evolutionary selective pressures. The ubiquity of this strategy among Amazonian fishes and the mechanisms employed by L. pardalis are clearly worthy of further study.

Our goal was to compare the internal physiological responses to acid challenge in an acidophilic tropical teleost endemic to dilute low-pH waters with those in nonacidophilic temperate species such as salmonids, which have been the subjects of most previous investigations. The Amazonian tambaqui (Colossoma macropomum), which migrates between circumneutral water and dilute acidic "blackwater" of the Rio Negro, was exposed to a graded low-pH and recovery regime in representative soft water (Na+ = 15, Cl- = 16, Ca2+ = 20 mumol L-1). Fish were fitted with arterial catheters for repetitive blood sampling. Water pH was altered from 6.5 (control) to 5.0, 4.0, 3.0, and back to 6.5 (recovery) on successive days. Some deaths occurred at pH 3.0. Throughout the regime, there were no disturbances of blood gases (O2 and CO2 tensions and contents) or lactate levels, and only very minor changes in acid-base status of plasma and red cells. However, erythrocytic guanylate and adenylate levels increased at pH's less than or equal to 5.0. Down to pH 4.0, plasma glucose, cortisol, and total ammonia levels remained constant, but all increased at pH 3.0, denoting a stress response. Plasma Na+ and Cl- levels declined and plasma protein concentration increased at pH 3.0, indicative of ionoregulatory and fluid volume disturbance, and neither recovered upon return to pH 6.5. Cortisol and ammonia elevations also persisted. Transepithelial potential changed progressively from highly negative values (inside) at pH 6.5 to highly positive values at pH 3.0; these alterations were fully reversible. Experimental elevations in water calcium levels drove the transepithelial potential positive at circumneutral pH, attenuated or prevented changes in transepithelial potential at low pH, and reduced Na+ and Cl- loss rates to the water during acute low-pH challenges. In general, tambaqui exhibited responses to low pH that were qualitatively similar but quantitatively more resistant than those previously documented in salmonids.