The evolution of accessory air-breathing structures is typically associated with reduction of the gills, although branchial ion transport remains pivotal for acid–base and ion regulation. Therefore, air-breathing fishes are believed to have a low capacity for extracellular pH regulation during a respiratory acidosis. In the present study, we investigated acid–base regulation during hypercapnia in the air-breathing fish Pangasianodon hypophthalmus in normoxic and hypoxic water at 28–30°C. Contrary to previous studies, we show that this air-breathing fish has a pronounced ability to regulate extracellular pH (pHe) during hypercapnia, with complete metabolic compensation of pHe within 72 h of exposure to hypoxic hypercapnia with CO2 levels above 34 mmHg. The high capacity for pHe regulation relies on a pronounced ability to increase levels of HCO3 in the plasma. Our study illustrates the diversity in the physiology of air-breathing fishes, such that generalizations across phylogenies may be difficult.

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 (pHe) is restored in the face of the increased partial pressure of carbon dioxide in the arterial blood (PaCO2) (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 pHe 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 pHe regulation during hypercapnia [increased water partial pressure of carbon dioxide (PwCO2)]. 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 PwCO2 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 pHe during a respiratory acidosis, we measured pHe, PaCO2 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 pHe during hypercapnia than observed in other air-breathing fishes with a more reduced gill size.

Acid–base regulation during hypercapnia

Within the first 3 h of each hypercapnia exposure, PaCO2 rose and remained stable for the following 72 h (Fig. 1A). The initial rise in PaCO2 was attended by a reduction in pHe (Fig. 1B), while the calculated [HCO3]plasma increased along the in vitro non-bicarbonate buffer line (βNB) of −18.3±5.2 slykes [i.e. pHe fell with a slope of −16.5±2.4 and −22.7±4.1 slykes (mmol l−1 HCO3 pHe−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.

Fig. 1.

Arterial plasma values in Pangasianodon hypophthalmus during exposure to hypercapnia. Arterial partial pressure of carbon dioxide (A), plasma pH (pHe) (B), plasma [HCO3] (C) and plasma [Cl] (D) in cannulated P. hypophthalmus during exposure to 7 mmHg CO2, 22 mmHg CO2, pond water (34 mmHg CO2) and normocapnic water (control). Parameters at time zero were measured in well-aerated water just prior to exposure to experimental conditions. Data are means±s.e.m. (N=6).

Fig. 1.

Arterial plasma values in Pangasianodon hypophthalmus during exposure to hypercapnia. Arterial partial pressure of carbon dioxide (A), plasma pH (pHe) (B), plasma [HCO3] (C) and plasma [Cl] (D) in cannulated P. hypophthalmus during exposure to 7 mmHg CO2, 22 mmHg CO2, pond water (34 mmHg CO2) and normocapnic water (control). Parameters at time zero were measured in well-aerated water just prior to exposure to experimental conditions. Data are means±s.e.m. (N=6).

Fig. 2.

Changes in acid-base parameters and [Cl]plasma during exposure to hypercapnia in cannulated Pangasianodon hypophthalmus. Fish were exposed to 7 mmHg CO2, 22 mmHg CO2 and pond water and blood parameters were determined at 0, 3, 6, 24, 48 and 72 h. (A) Davenport diagram with CO2 isopleths at PaCO2 upon exposure to 7 mmHg CO2, 22 mmHg CO2 and pond water, respectively. Dashed line indicates non-bicarbonate buffer effect determined in vitro. (B) Individual changes in [Cl]plasma and [HCO3]plasma after exposure to hypercapnia. Data are means±s.e.m. (N=6).

Fig. 2.

Changes in acid-base parameters and [Cl]plasma during exposure to hypercapnia in cannulated Pangasianodon hypophthalmus. Fish were exposed to 7 mmHg CO2, 22 mmHg CO2 and pond water and blood parameters were determined at 0, 3, 6, 24, 48 and 72 h. (A) Davenport diagram with CO2 isopleths at PaCO2 upon exposure to 7 mmHg CO2, 22 mmHg CO2 and pond water, respectively. Dashed line indicates non-bicarbonate buffer effect determined in vitro. (B) Individual changes in [Cl]plasma and [HCO3]plasma after exposure to hypercapnia. Data are means±s.e.m. (N=6).

During continued hypercapnia, [HCO3]plasma increased with a concurrent restoration of pHe (Fig. 1B,C) along the PaCO2 isopleths of the Davenport diagram (Fig. 2A), indicating that pHe regulation is primarily metabolic involving a combination of HCO3 retention and/or H+ excretion. There was a slight tendency for the PaCO2PwCO2 difference to decrease at higher PwCO2, 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. pHe 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 pHe 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 pHe regulation during a respiratory acidosis (Shartau and Brauner, 2014). Thus, the armored catfish Liposarcus pardalis only recovered 8% and 22% of the pHe 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 pHe disturbance at 11 and 45 mmHg CO2, respectively, after 24 h (Brauner and Baker, 2009) and Arapaima gigas only partly compensated the pHe 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 pHe 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 pHe disturbance after 48 h exposure to 8 mmHg CO2 (Claiborne and Heisler, 1984) and the white sturgeon Acipenser transmontanus, which only compensated 35% of pHe 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 PCO2 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 pHe disturbance within 24 h of exposure to 7.5 and 15 mmHg, respectively (Eddy et al., 1977; Larsen et al., 1997).

List of symbols and abbreviations

     
  • [CO2]total

    total plasma CO2 concentration

  •  
  • Hct

    hematocrit

  •  
  • PaCO2

    arterial partial pressure of carbon dioxide

  •  
  • pHe

    plasma pH

  •  
  • pK

    acid dissociation constant for CO2 hydration

  •  
  • PwCO2

    water partial pressure of carbon dioxide

  •  
  • PwO2

    water partial pressure of oxygen

  •  
  • αCO2

    CO2 solubility in trout plasma

  •  
  • βNB

    non-bicarbonate buffer effect

The metabolic compensation of pHe 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 pHe 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 pHe 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 pHe 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 pHe 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 PwCO2 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 pHe 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 pHe 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.

Animal handling

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.

Experimental protocols

Series I: acid–base regulation during normoxia

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 (PO2∼120 mmHg) at 28–30°C containing either 7 mmHg or 22 mmHg CO2, supplied from a 500 l recirculating tank. PwCO2 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 PwCO2. 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. PaCO2 and pHe 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.

Series II: acid–base regulation during hypoxia

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 PaCO2, pHe, 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; PwO2=16.8±13.4 mmHg; PwCO2=34.3±3.3 (means±s.d.).

Blood tonometry: non-bicarbonate buffering and calibration of iStat PaCO2

The effect of βNB 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 βNB was calculated as Δ[HCO3]total×ΔpHe−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 PCO2 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 PCO2 by the iStat compared with true PCO2 delivered by the Wösthoff pumps (supplementary material Fig. S2A). We corrected all measurements of PaCO2 according to the regression presented in supplementary material Fig. S2A.

Calculations

Blood acid dissociation constant for CO2 hydration (pK) was calculated from the Henderson–Hasselbach equation:
(1)
In Series I and in blood tonometry, [HCO3]plasma was calculated by subtracting physically dissolved CO2 from [CO2]total using temperature-compensated CO2 solubility in trout plasma (αCO2) from Boutilier et al. (1985).

In Series II, [HCO3]plasma was calculated from Eqn 1 (supplementary material Fig. S2B) using PaCO2 and pHe from iStat, pK (pK=4.83+0.17 pHe) from Series I and αCO2 from Boutilier et al. (1985).

Partial pressures of oxygen and carbon dioxide in aquaculture ponds

PwCO2, PwO2, pHw 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 PCO2 probe and an YSI ProODO optical dissolved oxygen meter (YSI Inc., Yellow Springs, OH, USA). PwCO2 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 (PwCO2=18.3 mmHg+2.76 mmHg m−1; F1,19=4.53, P<0.05, R2=0.15). pHw 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 PwO2 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 (PwO2=109 mmHg−16.3 mmHg m−1; F1,20=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.

Author contributions

C.D., L.T.H.G., D.D.T. and P.V.T. performed experiments; C.D. analyzed data and prepared figures; C.D., T.W. and M.B. designed the study, interpreted results and edited, revised and drafted manuscript; all authors approved final version of manuscript.

Funding

The research was funded by the Danish Ministry of Foreign Affairs (DANIDA) [DFC no. 12-014AU].

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Competing interests

The authors declare no competing or financial interests.

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