Dissolved organic carbon from the upper Rio Negro protects zebrafish (Danio rerio) against ionoregulatory disturbances caused by low pH exposure

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.

Figure 1. Unidirectional sodium influx (J^Na_in, upward positive solid bar), unidirectional sodium efflux (J^Na_out, downward negative solid bars) and net sodium flux rates (J^Na_net, cross-hatched bars) of adult zebrafish in ion-poor water (IPW).

Means ± 1 SEM (N = 10 in each treatment). In the left-hand panel, the first three sets of bars represent fish initially tested (0-3 h) under the same control condition (no DOC) to which they were all acclimated (IPW – pH 7.0), and then in the middle panel acutely exposed (3–6 h) to either IPW + DOC – pH 7.0, or IPW – pH 4.0, or IPW + DOC – pH 4.0, followed in the right-hand panel by a recovery period (6–9 h) in which all fish were again exposed to the common acclimation condition (IPW – pH7.0). In addition, the fourth and fifth bars represent sodium flux rates of zebrafish which had been acclimated to DOC at pH 7.0 for two weeks prior to test. In the left-hand panel, these fish were initially tested (0–3 h) under their common acclimation condition (IPW + DOC – pH 7.0), and then in the middle panel acutely exposed (3–6 h) to either IPW – pH 4.0, or IPW + DOC – pH 4.0, followed in the right-hand panel by a recovery period (6–9 h) in which both groups were again exposed to their common acclimation condition (IPW + DOC – pH 7.0). Upper case letters represent significant differences (p < 0.05) in J^Na_in or J^Na_out among fish under different exposure regimes (different shading schemes) within the same flux period. Lower case letters represent significant differences (p < 0.05) in J^Na_in or J^Na_out of animals in the same regime of exposure (bars with same shading scheme), among different flux periods. Bars sharing the same letter are not significantly different.

Figure 2. Net chloride flux rates (J^Cl_net) of adult zebrafish in ion-poor water (IPW).

Means ± 1 SEM (N = 10 in each treatment). In the left-hand panel, the first three bars represent fish initially tested (0-3 h) under the same control condition (no DOC) to which they were all acclimated (IPW – pH 7.0), and then in the middle panel acutely exposed (3–6 h) to either IPW + DOC – pH 7.0, or IPW – pH 4.0, or IPW + DOC – pH 4.0, followed in the right-hand panel by a recovery period (6–9 h) in which all fish were again exposed to the common acclimation condition (IPW – pH7.0). In addition, the fourth and fifth bars represent J^Cl_net values of zebrafish which had been acclimated to DOC at pH 7.0 for two weeks prior to test. In the left-hand panel, these fish were initially tested (0–3 h) under their common acclimation condition (IPW + DOC – pH 7.0), and then in the middle panel acutely exposed (3–6 h) to either IPW – pH 4.0, or IPW + DOC – pH 4.0, followed in the right-hand panel by a recovery period (6–9 h) in which both groups were again exposed to their common acclimation condition (IPW + DOC – pH 7.0). Statistical significance (p < 0.05) is shown as in Fig. 1.

Figure 3. Net ammonia flux rates (J^Amm_net) of adult zebrafish in ion-poor water (IPW).

Means ± 1 SEM (N = 10 in each treatment). In the left-hand panel, the first three bars represent fish initially tested (0–3 h) under the same control condition (no DOC) to which they were all acclimated (IPW – pH 7.0), and then in the middle panel acutely exposed (3–6 h) to either: IPW + DOC – pH 7.0, or IPW – pH 4.0, or IPW + DOC – pH 4.0, followed in the right-hand panel by a recovery period (6–9 h) in which all fish were again exposed to the common acclimation condition (IPW – pH 7.0). In addition, the fourth and fifth bars represent J^Amm_net values of zebrafish which had been acclimated to DOC at pH 7.0 for two weeks prior to test. In the left-hand panel, these fish were initially tested (0–3 h) under their common acclimation condition (IPW + DOC – pH 7.0), and then in the middle panel acutely exposed (3–6 h) to either IPW – pH 4.0, or IPW + DOC – pH 4.0, followed in the right-hand panel by a recovery period (6–9 h) in which both groups were again exposed to their common acclimation condition (IPW + DOC – pH 7.0). Statistical significance (p < 0.05) is shown as in Fig. 1.

Figure 4. The relationship between net ammonia flux rates (J^Amm_net) and unidirectional Na⁺ uptake rates (J^Na_in) of adult zebrafish in different exposure conditions in ion-poor water (IPW).

Means ± 1 SEM. Gray circles represents flux rates of both J^Amm_net and J^Na_in of zebrafish under the acclimation conditions (i.e. IPW – pH 7.0 or IPW + DOC – pH7.0; 0–3 h), and then acutely exposed to either IPW + DOC – pH 7.0, or IPW – pH 4.0 or IPW + DOC – pH 4.0 (3–6 h), followed by a recovery period in which they were again exposed to their acclimation condition (IPW – pH 7.0 or IPW + DOC – pH 7.0). Note that the two black circles (not used in the regression) represent data from fish acutely exposed to IPW – pH 4.0 (no DOC), and these same fish during the recovery period at IPW – pH 7.0, where J^Amm_net was entirely uncoupled from J^Na_in. Nonlinear regression analysis was performed using Sigma Plot v 11.0. r² = 0.639; p = 0.001, F = 19.531.