High capacity for extracellular acid–base regulation in the air-breathing fish Pangasianodon hypophthalmus

In most water-breathing fish species, respiratory acidosis (i.e. an acidosis caused by elevated CO₂) is compensated by transepithelial exchange of acid–base equivalents with the environment, i.e. excretion of H⁺ or retention of HCO₃⁻, such that extracellular pH (pH_e) is restored in the face of the increased partial pressure of carbon dioxide in the arterial blood (Pa_CO2) (Claiborne et al., 2002; Evans et al., 2005; Perry and Gilmour, 2006). However, the gills of air-breathing fishes are generally reduced in size – an adaptation that is presumed to aid in avoiding branchial O₂ loss in hypoxic water – but have retained their ancestral function in acid–base and ion regulation (Graham, 1997; Tamura and Moriyama, 1976). It has therefore been suggested that the reduced surface area of the gills of air-breathing fishes places limitations on transepithelial ion exchange, thus constraining the branchial capacity for acid–base regulation (Brauner and Baker, 2009; Shartau and Brauner, 2014). Therefore, all studies on acid–base regulation in air-breathing fishes to date indicate a low capacity for exchange of acid–base equivalents in pH_e regulation and a preferential regulation of intracellular pH, during a respiratory acidosis (Brauner and Baker, 2009; Brauner et al., 2004; Harter et al., 2014b; Heisler, 1982; Shartau and Brauner, 2014).

Pangasianodon hypophthalmus is a facultative air-breathing fish, which uses a modified and highly vascularized swim bladder for air-breathing during environmental hypoxia (Lefevre et al., 2011a). In contrast to some air-breathing fishes, P. hypophthalmus appears to have larger gills (Graham, 1997; Lefevre et al., 2011a; Tamura and Moriyama, 1976) that may allow for considerable branchial ion exchange, and thus confer high capacity for pH_e regulation during hypercapnia [increased water partial pressure of carbon dioxide (Pw_CO2)]. P. hypophthalmus inhabits tropical freshwater plains within the Mekong River Delta in South-East Asia. Such ecosystems are characterized by frequent high organic loading resulting in combined hypoxia and hypercapnia with Pw_CO2 reaching 65 mmHg (Furch and Junk, 1997; Ultsch, 1987; Willmer, 1934). Furthermore, it is farmed extensively in severely hypoxic (Lefevre et al., 2011b) and hypercapnic (this study) water throughout South-East Asia.

To investigate the ability of P. hypophthalmus to compensate pH_e during a respiratory acidosis, we measured pH_e, Pa_CO2 and accumulation of HCO₃⁻ in cannulated fishes during exposure to two levels of hypercapnia (7 and 22 mmHg CO₂) in normoxic water at 28–30°C. In addition, we also investigated how P. hypophthalmus deals with the combined severe hypercapnia and hypoxia it experiences in natural tropical waters (Furch and Junk, 1997; Willmer, 1934) by exposing cannulated individuals to water circulated from a local aquaculture pond. We hypothesized that the large gills of P. hypophthalmus confer greater capacity to regulate pH_e during hypercapnia than observed in other air-breathing fishes with a more reduced gill size.

Within the first 3 h of each hypercapnia exposure, Pa_CO2 rose and remained stable for the following 72 h (Fig. 1A). The initial rise in Pa_CO2 was attended by a reduction in pH_e (Fig. 1B), while the calculated [HCO₃⁻]_plasma increased along the in vitro non-bicarbonate buffer line (β_NB) of −18.3±5.2 slykes [i.e. pH_e fell with a slope of −16.5±2.4 and −22.7±4.1 slykes (mmol l⁻¹ HCO₃⁻ pH_e⁻¹) in the 22 mmHg CO₂ and pond treatments, respectively] (Fig. 2A). Hematocrit (Hct) was 15.3±1.5, 18.7±2.6, 17.2±0.9 and 22.4±1.6% in fishes exposed to control, 7 mmHg, 22 mmHg and pond water, respectively, and fell slightly after 72 h to 13.9±1.0, 18.4±4.2, 13.2±1.1 and 22.9±3.5% in the respective groups.

During continued hypercapnia, [HCO₃⁻]_plasma increased with a concurrent restoration of pH_e (Fig. 1B,C) along the Pa_CO2 isopleths of the Davenport diagram (Fig. 2A), indicating that pH_e regulation is primarily metabolic involving a combination of HCO₃⁻ retention and/or H⁺ excretion. There was a slight tendency for the Pa_CO2−Pw_CO2 difference to decrease at higher Pw_CO2, possibly indicating an increased ABO efflux of CO₂ at increasing levels of hypercapnia, as also observed in South American lungfish (Sanchez et al., 2005). The rate of HCO₃⁻ accumulation was faster in fishes exposed to pond water, where the hypercapnia was most severe. pH_e was fully compensated within 24 h at 7 mmHg CO₂ and within 72 h at 22 mmHg CO₂ and exposure to pond water. This reveals a pronounced capacity for metabolic pH_e regulation during hypercapnia in P. hypophthalmus. This is in stark contrast to all other air-breathing fishes studied to date that have a low capacity for pH_e regulation during a respiratory acidosis (Shartau and Brauner, 2014). Thus, the armored catfish Liposarcus pardalis only recovered 8% and 22% of the pH_e disturbance when exposed to ∼7 mmHg CO₂ and 42 mmHg CO₂, respectively for 96 h (Brauner et al., 2004). Similarly, the bowfin Amia calva regulated 28% and 24% of the pH_e disturbance at 11 and 45 mmHg CO₂, respectively, after 24 h (Brauner and Baker, 2009) and Arapaima gigas only partly compensated the pH_e disturbance incurred after exposure to 40 mmHg CO₂ for 72 h (Gonzalez et al., 2010). No indication of extracellular acid–base regulation was detected in the South American lungfish (Lepidosiren paradoxa) over 50 h at 49 mmHg CO₂ (Sanchez et al., 2005). The ability to regulate pH_e during hypercapnia in P. hypophthalmus is also highly developed compared with several water-breathing fishes, such as the common carp Cyprinus carpio, which recovered 50% of the initial pH_e disturbance after 48 h exposure to 8 mmHg CO₂ (Claiborne and Heisler, 1984) and the white sturgeon Acipenser transmontanus, which only compensated 35% of pH_e after exposure to 28 mmHg for 72 h (Crocker and Cech, 1998). The European eel exhibits a medium capacity for acid–base regulation, compensating 75% of its disturbance after 6 weeks at a P_CO2 of 15 mmHg (McKenzie et al., 2003). P. hypophthalmus thus shows an acid–base regulatory capacity during a respiratory acidosis in line with active water-breathers such as cod and rainbow trout, which compensate the full pH_e disturbance within 24 h of exposure to 7.5 and 15 mmHg, respectively (Eddy et al., 1977; Larsen et al., 1997).

The metabolic compensation of pH_e in P. hypophthalmus includes an accumulation of plasma HCO₃⁻ through HCO₃⁻ retention and/or H⁺ excretion, which is generally thought to involve equimolar exchange of HCO₃⁻ with Cl⁻ and H⁺ with Na⁺, respectively (Claiborne et al., 2002; Evans et al., 2005; Perry and Gilmour, 2006). Here, [Cl⁻]_plasma decreased during all hypercapnia treatments (Fig. 1D) and individual reductions in [Cl⁻]_plasma were mirrored by increases in [HCO₃⁻]_plasma, suggesting involvement of a HCO₃⁻/Cl⁻ exchanger in pH_e regulation during hypercapnia (Fig. 2B). However, in response to the most severe hypercapnia (34 mmHg CO₂), [HCO₃⁻]_plasma increased by approximately 30 mmol l⁻¹ after 72 h, but was only accompanied by a ∼12 mmol l⁻¹ reduction in [Cl⁻]_plasma. This suggests that over this longer time course other mechanisms for ion exchange than an equimolar HCO₃⁻/Cl⁻ anion exchanger are involved in pH_e regulation during hypercapnia. Alternatively, it has been proposed that freshwater fishes possess non-stoichiometric, HCO₃⁻/Cl⁻ anion exchangers, exchanging multiple HCO₃⁻/Cl⁻ ions, which might explain the non-stoichiometric changes in [HCO₃⁻]_plasma relative to [Cl⁻]_plasma (Grosell et al., 2009).

The metabolic acid–base compensation during hypercapnia seemed unaffected by hypoxia. During hypoxia, P. hypophthalmus decreases gill ventilation and the resulting reduced water flow might impose limitations on branchial ion exchange (Lefevre et al., 2011a). Thus, the acid–base regulation seen here either indicates a lack of limitation of ventilation or that there is a pronounced renal–intestinal ion exchange, which has also been suggested to be a general trait for air-breathing fishes (Graham, 1997; Perry and Gilmour, 2006).

Our study does not identify the anatomical, cellular or molecular components underlying the high capacity for pH_e regulation, but this might be associated with the large gills and hence ion-exchange surface area in P. hypophthalmus (Lefevre et al., 2011a). It might also be a consequence of a low HCO₃⁻/Cl⁻-exchanger activity, augmenting HCO₃⁻ retention, which would be consistent with the proposed low Cl⁻ permeability of the gill of P. hypophthalmus, as indicated by a low uptake rate of nitrite (Lefevre et al., 2011c). Both characteristics contrast with those of other air-breathing fishes (Graham, 1997; Lefevre et al., 2014; Tamura and Moriyama, 1976). Other mechanisms may contribute to the high regulatory capacity for pH_e regulation in P. hypophthalmus, such as H⁺/Na⁺ exchange, greater renal or intestinal contribution in acid–base regulation, the ability to excrete CO₂ across the air-breathing organ etc.

We provide the first documentation of very high Pw_CO2 in P. hypophthalmus aquaculture (supplementary material Fig. S1), and confirm previous studies of severe hypoxia in these ponds (Lefevre et al., 2011b). The severe hypercapnia observed here is in line with many other observations from naturally hypoxic tropical waters (Furch and Junk, 1997; Willmer, 1934) and exceeds the level of respiratory acidosis that fishes are believed to be able fully to compensate for metabolically (Heisler, 1982; Larsen and Jensen, 1997; Crocker and Cech, 1998; Brauner et al., 2004). Under hypercapnic and hypoxic conditions (occurring frequently in both natural and aquaculture water in the tropics), P. hypophthalmus must live with an extensive metabolic compensation and a strongly elevated [HCO₃⁻]_plasma and thus suppressed Cl⁻ uptake. While the ability to regulate pH_e seems beneficial for tropical freshwater species, the physiological consequences of long-term maintenance of a pronounced metabolic compensation are unknown and should be a subject for future research.

In summary, the air-breathing P. hypophthalmus is endowed with ample capacity for pH_e regulation during hypercapnia, which contradicts the trend that air-breathing fishes are inefficient in extracellular acid–base regulation during a respiratory acidosis. The diversity of air-breathing fishes is considerable, with more than 400 species and 65 separate evolutionary events and it is therefore unsurprising that physiological challenges are accommodated using different physiological and anatomical building blocks in different groups. It may thus prove difficult to generalize on the capacity to cope with physiological challenges across the phylogenetically distinct air-breathing fishes.

Pangasianodon hypophthalmus Sauvage 1878 were purchased from a local fish supplier and transferred to Can Tho University (Vietnam), where they were held in large well-aerated tanks at 27°C for 4 months prior to experimentation. The fishes were fed daily with commercially purchased dry pellets and water was changed every third day. All experiments were performed in accordance with national guidelines for the protection of animal welfare in Vietnam.

A total of 22 fishes (mass, 831±66 g; length, 43±1 cm; means±s.e.m.) were anesthetized in 0.1 g l⁻¹ benzocaine and a polyethylene PE50 catheter was inserted into the dorsal aorta through the dorsal side of the mouth (Soivio et al., 1975) while the gills were irrigated with well-oxygenated water with 0.05 g l⁻¹ benzocaine. After recovery for ∼24 h in well-aerated water, a normocapnic blood sample was taken and the fishes were exposed to normoxic water (P_O2∼120 mmHg) at 28–30°C containing either 7 mmHg or 22 mmHg CO₂, supplied from a 500 l recirculating tank. Pw_CO2 was monitored using an Oxyguard Pacific system coupled with a G10ps CO₂ probe and a K01svpld pH probe (Oxyguard International A/S, Farum, Denmark), which supplied CO₂ to the water when pH increased above a value corresponding to the desired Pw_CO2. At each CO₂ level, 1 ml blood was sampled from the catheter for immediate determination of blood gases at 0, 3, 6, 24, 48 and 72 h. Pa_CO2 and pH_e were measured using a handheld iStat with a G3+ cartridge (Abbot Point of Care Inc., Princeton, NJ, USA). Hct was measured as the fractional volume of red blood cells in blood after centrifugation at 12,000 r.p.m. for 3 min. Total plasma CO₂ concentration ([CO₂]_plasma) was measured according to Cameron, 1971. Hemoglobin (Hb) concentration was measured spectrophotometrically after conversion to metHb. [Cl⁻]_plasma was measured using a chloride titrator (Sherwood model 926S MK II Chloride analyzer). The concentration of NH₃ in the water was measured using the phenol–hypochlorite method; water NO₂⁻ was measured using the Griess reaction and water NO₃⁻ levels were measured using the salicylate method.

Twelve fishes (mass=1152±56 g; length=45±0.7 cm) with arterial catheters were allowed to recover for ∼24 h in well-aerated water and then exposed to water from a Vietnamese aquaculture pond pumped from 2 m depth (pond was 3.5 m deep). A 300 µl blood sample was taken from each fish through the catheter at 0, 3, 6, 24, 48 and 72 h for immediate determination of Pa_CO2, pH_e, Hct, [Hb] and [Cl⁻]_plasma. Pond water conditions were: [NH₃]=0.058 µmol l⁻¹; [NO₂⁻]=1.9 µmol l⁻¹; [NO₃⁻]=1.3 µmol l⁻¹; pH=6.29; Temp=30.8±0.4; Pw_O2=16.8±13.4 mmHg; Pw_CO2=34.3±3.3 (means±s.d.).

The effect of β_NB was found by equilibrating 5 ml blood from 11 individual fishes in an Eschweiler tonometer with humidified gas mixtures provided from two serial-linked gas mixing pumps (Wösthoff, Bochum, Germany). Oxygen-saturated blood was equilibrated with 7 and 22 mmHg CO₂ for at least 30 min to allow for full equilibration of the blood with the gas. [CO₂]_total was measured as described above and β_NB was calculated as Δ[HCO₃⁻]_total×ΔpH_e⁻¹ assuming a linear relationship (Nightingale and Fedde, 1972). Prior to the experiments, all gas mixing pumps were validated using a Servomex 570A Oxygen Analyzer.

Harter et al. (2014a) recently criticized the reliability of the iStat for blood gas measurements in fish at 10 and 20°C. To validate our iSTAT measurements from blood at 30°C, it was necessary to check for errors associated with the problems in temperature compensation (Malte et al., 2014). Thus, we measured P_CO2 of blood samples equilibrated to 7, 22 and 37 mmHg CO₂ in tonometers receiving water saturated gas mixtures delivered by Wösthoff pumps. This direct comparison revealed a slight, but consistent underestimation of P_CO2 by the iStat compared with true P_CO2 delivered by the Wösthoff pumps (supplementary material Fig. S2A). We corrected all measurements of Pa_CO2 according to the regression presented in supplementary material Fig. S2A.

In Series II, [HCO₃⁻]_plasma was calculated from Eqn 1 (supplementary material Fig. S2B) using Pa_CO2 and pH_e from iStat, pK (pK=4.83+0.17 pH_e) from Series I and α_CO2 from Boutilier et al. (1985).

Pw_CO2, Pw_O2, pH_w and temperature were measured in 12 Vietnamese aquaculture ponds at 0, 1, 2 and 3 meters depth with either small (30–50 g) or large fishes (400–1000 g) using an Oxyguard Pacific Commander box fitted with a pH and P_CO2 probe and an YSI ProODO optical dissolved oxygen meter (YSI Inc., Yellow Springs, OH, USA). Pw_CO2 at the surface was significantly higher in ponds with large fish (18.0±1.8 mmHg and 2.9±0.8 mmHg, respectively; 1 mmHg=133 Pa; supplementary material Fig. S1A) and increased with water depth (Pw_CO2=18.3 mmHg+2.76 mmHg m⁻¹; F_1,19=4.53, P<0.05, R²=0.15). pH_w at the surface was correspondingly lower in ponds with large fish compared with small fish (6.37±0.03 and 7.10±0.11, respectively; supplementary material Fig. S1B). The corresponding Pw_O2 was higher in ponds with small fish compared with large fish (108±13 and 44±13 mmHg, respectively; supplementary material Fig. S1C) and decreased significantly with depth in ponds with small fish (Pw_O2=109 mmHg−16.3 mmHg m⁻¹; F_1,20=7.408, P<0.05, R²=0.27).

We would like to thank The Danish Research Council. In addition, we would like to thank Do Thanh Long for allowing us to perform experiments at his farm.