Preferentially regulating intracellular pH (pHi) confers exceptional CO2 tolerance on fish, but is often associated with reductions in extracellular pH (pHe) compensation. It is unknown whether these reductions are due to intrinsically lower capacities for pHe compensation, hypercarbia-induced reductions in water pH or other factors. To test how water pH affects capacities and strategies for pH compensation, we exposed the CO2-tolerant fish Pangasianodon hypophthalmus to 3 kPa PCO2 for 20 h at an ecologically relevant water pH of 4.5 or 5.8. Brain, heart and liver pHi was preferentially regulated in both treatments. However, blood pHe compensation was severely reduced at water pH 4.5 but not 5.8. This suggests that low water pH limits acute pHe but not pHi compensation in fishes preferentially regulating pHi. Hypercarbia-induced reductions in water pH might therefore underlie the unexplained reductions to pHe compensation in fishes preferentially regulating pHi, and may increase selection for preferential pHi regulation.
The aquatic partial pressure of carbon dioxide (PCO2) in tropical river basins can be driven above 6 kPa daily by microbial respiration and organic decay (Furch and Junk, 1997). These rapid elevations in PCO2 exceed atmospheric levels by over 200-fold, and impose a severe acute respiratory acidosis on fish as CO2 diffuses from the water into their blood and tissue (Heisler, 1984). Despite the extreme nature of these rapid acidoses, many fishes routinely endure this challenge, as evidenced by the high levels of species richness and abundance in these environments (Dudgeon et al., 2006).
Coupled pH regulation (pHcoupled) and preferential intracellular pH regulation (pHpi) are two strategies fish use to compensate for an acute respiratory acidosis (Shartau et al., 2016). These strategies represent endpoints of a continuum along which rates and degrees of intracellular pH (pHi) and extracellular pH (pHe) compensation vary.
In pHcoupled, tissue pHi is coupled to blood pHe. During an acidosis event, pHi and pHe both fall and recover together along similar trajectories within 24–48 h. Coupled recovery of pHi and pHe requires transepithelial exchange of acid–base relevant ions for net acid excretion and/or base uptake (Stewart, 1978; Claiborne et al., 2002). The exchange of chloride for bicarbonate and/or sodium for protons is believed to primarily drive this recovery, but full compensation is generally associated with an increase in plasma bicarbonate balanced by an equimolar reduction in plasma chloride (Heisler, 1984; Brauner and Baker, 2009).
In pHpi, pHi is not coupled to pHe. Within minutes of CO2 exposure, pHi is at or above control levels despite large reductions in pHe (Baker, 2010). Additionally, pHe recovery is often incomplete or absent within 24–48 h (Brauner et al., 2004). Here, pHi is maintained by the exchange of acid–base relevant ions between intracellular and extracellular compartments whether pHe compensation occurs or not (Brauner and Baker, 2009; Occhipinti and Boron, 2015), and reductions in the rate and degree of acute pHe compensation remain unexplained.
Why fishes express pHcoupled or pHpi is unclear. However, severe acute hypercarbia is hypothesized to select for pHpi by exceeding the capacity and/or limiting the rate of acute pHe compensation required for pHcoupled to defend pHi (Shartau et al., 2016). Indeed, full pHe compensation within 24–48 h of hypercarbia is limited to ∼2 kPa PCO2 in most freshwater fishes tested, while many fishes expressing pHpi can robustly defend pHi above 6 kPa PCO2 without pHe compensation (Brauner and Baker, 2009; Shartau et al., 2016). One hypothesis for this apparent limit to acute pHe compensation suggests many fishes are unable to elevate plasma bicarbonate above the ∼25–30 mmol l−1 required for full pHe recovery at ∼2 kPa PCO2, let alone the ∼100–150 mmol l−1 required at ∼6 kPa (Heisler, 1984; Brauner and Baker, 2009). A second hypothesis posits that water ion composition reduces the rate and/or degree of pHe compensation by creating unfavourable trans-epithelial gradients for acid–base relevant ion exchange (Larsen and Jensen, 1997). Indeed, most CO2 exposures exceeding the capacity for acute pHe compensation in freshwater fishes also reduce water pH below 5.3, which is proposed to thermodynamically inhibit net proton excretion in rainbow trout at ambient PCO2 (Lin and Randall, 1995). Despite supporting evidence for both hypotheses, neither has been directly tested for a role in limiting pHe compensation and selecting for pHpi during acute hypercarbia.
The Mekong catfish Pangasianodon hypophthalmus was recently reported to fully compensate pHe at 4 kPa PCO2 (Damsgaard et al., 2015). Compensation was associated with a surprising ∼45 mmol l−1 increase in plasma bicarbonate within 48 h of exposure. This elevated capacity for acute pHe compensation suggests that P. hypophthalmus might express pHcoupled rather than pHpi to defend pHi in acute hypercarbia above 2 kPa PCO2. This would be in stark contrast to findings for 19 of 20 CO2-tolerant freshwater fishes tested (Shartau et al., 2016), including the Amazonian catfish Pterygoplichthys pardalis, which expresses pHpi and negligible pHe compensation at 1–6 kPa PCO2 (Brauner et al., 2004). However, pHi in P. hypophthalmus was not examined for preferential regulation, and water pH during hypercarbic exposure was 5.8 (Damsgaard et al., 2015). This is well above the proposed threshold water pH of 5.3 for net proton excretion in rainbow trout, and much higher than water pH in the P. pardalis study (water pH 4.5 at 4 kPa PCO2; Brauner et al., 2004).
We therefore sought to answer two questions. First, is the exceptional capacity for acute pHe compensation in P. hypophthalmus limited by a lower, more common hypercarbic water pH? Second, if pHe compensation is limited by low water pH, can P. hypophthalmus express pHpi like most other CO2-tolerant freshwater fishes tested? To address these questions, we measured pHe and pHi in P. hypophthalmus during exposure to 3 kPa PCO2 for 20 h in water artificially held at pH 4.5 or 5.8. Our results provide further insight into the factors limiting pHcoupled and selecting for pHpi.
MATERIALS AND METHODS
Pangasianodon hypophthalmus (Sauvage 1878) were obtained from a local fish supplier in Can Tho, Vietnam and kept at Can Tho University for 3 months prior to experimentation. Fish were held in aerated 3000 l tanks fitted with a recirculating biofiltration system and kept on a 12 h:12 h light:dark photoperiod. Water Cl− and pH in these holding conditions were 0.35 mmol l−1 and 7.2±0.1, respectively, which is similar to that listed for native habitat in the nearby Mekong River (in mmol l−1: [Cl−] 0.28, [Na+] 0.39, [Ca2+] 0.63, [Mg2+] 0.33, [CaCO3] 0.53, pH 7.2; Ozaki et al., 2014; Kongmeng and Larsen, 2014). Fish were fed to satiation once daily with commercial dry pellets obtained from a local supplier and held under these conditions for at least 3 weeks prior to experimentation. Fish wet mass ranged between 50 and 100 g. All husbandry and experimentation were performed in accordance with national guidelines for the protection of animal welfare in Vietnam as well as the University of British Columbia Animal Use Protocol (AUP) no. A11-0235.
Protocol and measurements
One day prior to experimentation, fish were randomly transferred from holding tanks to a 200 l aerated experimental tank kept at 28°C. On the day of experimentation, fish were exposed to 3 kPa PCO2 in water at a pH of either 5.8 or 4.5 for up to 20 h. Water pH of 5.8 was achieved by bubbling 3% CO2 into the aerated experimental water at trial onset. Water pH of 4.5 was achieved by simultaneously introducing sulfuric acid (H2SO4) into the aerated experimental water while bubbling with 3% CO2. pH 4.5 was chosen as the lower water pH because it matches that of a previous study where the Amazonian catfish P. pardalis was exposed to 3 kPa PCO2 (Brauner et al., 2004). The desired PCO2 and water pH for each treatment were reached within 15 min of trial onset. Sulfuric acid was used to avoid introducing ions, such as Na+ and Cl−, which may confound the effects of water pH on acid–base regulation. Water PCO2 and pH were monitored continuously using an Oxyguard Pacific system fitted with a G10ps CO2 probe and a K01svpld pH probe (Oxyguard International A/S, Farum, Denmark). The G10ps probe measures PCO2 independently of water pH, such that measurements are not confounded by pH changes in the experimental treatments. A mix of CO2 and air was regulated by the Oxyguard system to reach and maintain a constant water PCO2 of 3 kPa (±0.02 kPa) and full oxygen saturation. Fish were terminally sampled (see below) following 0, 3 and 20 h exposure to 3 kPa PCO2 in both water pH treatments.
Prior to sampling, fish were rapidly transferred (<1–2 s) from experimental tanks by net to a neighbouring 20 l tank containing a lethal concentration of benzocaine (100 mg l−1 benzocaine in 3 ml of 70% ethanol), which was darkened and covered to reduce struggling. Following cessation of gill ventilation (<2 min), a 0.5 ml blood sample was collected by caudal puncture with a heparinized syringe. Blood samples were subsequently divided into two aliquots, one of which was immediately measured for pHe. The spinal cord was then severed, and tissues (heart, liver and brain) were excised, wrapped in pre-labelled aluminium foil and frozen in liquid nitrogen. This entire procedure was completed within 2 min of ventilatory arrest. The second blood aliquot was centrifuged for 3 min at 6000 rpm to separate plasma and red blood cells (RBCs). Plasma and RBCs were frozen in liquid nitrogen with the tissue samples, and all samples were subsequently transferred to −80°C for storage until further analysis.
pHe, pHi and water pH were measured with a Radiometer Analytical SAS pH electrode (GK2401C; Villeurbanne, France) connected to a Radiometer PHM84 pH meter (Copenhagen, Denmark) thermostatically set to 28°C to match the water temperature of the experiments. RBC pHi was measured according to the freeze–thaw method (Zeidler and Kim, 1977), and tissue pHi was measured according to the metabolic inhibitor tissue homogenate method (Portner et al., 1990; McKenzie et al., 2003; Baker et al., 2009b). Total CO2 (TCO2) was measured in plasma (Corning 965 CO2 analyser, Essex, UK). Blood PCO2 and plasma [HCO3−] were calculated from pHe and TCO2 with the Henderson–Hasselbalch equation. CO2 solubility (αCO2) and pK′ values were taken from Boutilier et al. (1984).
Data were analysed with Prism 5 for Mac OS X (Version 5.0a; GraphPad Software, Inc.). Means for each metric were compared within treatments and across time with one-way ANOVA and Tukey's post hoc test (P<0.05). All data are presented as means±s.e.m.
RESULTS AND DISCUSSION
After 3 h of hypercarbia, pHe fell dramatically in both treatments, as expected. The increased blood PCO2 reduced pHe from 7.79±0.02 to 7.40±0.03 and 7.45±0.012 in pH 5.8 and pH 4.5 water, respectively (P<0.01; Fig. 1). Furthermore, pHe in both treatments fell below the blood non-bicarbonate buffer line (Fig. 1). This suggests a metabolic component to the extracellular acidosis in both treatments, but plasma lactate concentration did not increase (Table 1). Thus, this metabolic component was instead probably due to a net exchange of HCO3− and/or H+ between the intracellular and extracellular compartments, which is consistent with pHpi expression (Heisler, 1982; Baker et al., 2009a).
After 20 h of hypercarbia, there was evidence for pHe compensation in pH 5.8 water but little in pH 4.5 water. In pH 5.8 water, pHe recovered by ∼40% from 3 h (Fig. 1, P<0.05) as plasma [HCO3−] doubled to exceed the blood buffer line by ∼9 mmol l−1 at the respective PCO2 (Fig. 1, P<0.01). In contrast, pHe in pH 4.5 water did not recover significantly from 3 h (Fig. 1), and plasma [HCO3−] did not exceed the blood buffer line (Fig. 1).
Tissue pHi of brain, heart and liver was preferentially regulated in both pH 5.8 and pH 4.5 water (Fig. 2), but there was variation between tissues and treatments. Brain pHi increased from control after 3 h of hypercarbia in both treatments (P<0.05) and remained elevated at 20 h (Fig. 2). In contrast, heart and liver pHi did not differ significantly from controls in either treatment at any time. However, heart and liver pHi did differ within their respective tissues between 3 and 20 h in the pH 5.8 water treatment (Fig. 2, P<0.05). Thus, brain pHi appears more robustly defended than that of heart and liver, and heart and liver pHi appears more tightly regulated in pH 4.5 water than in pH 5.8 water. The latter difference could be attributed to a greater acidosis associated with higher in vivo PCO2 in pH 5.8 water (Fig. 1), but this remains unknown. RBC pHi fell with pHe at 3 h in both treatments (Fig. 2), and did not recover within 20 h despite significantly increasing in pH 4.5 water. Lack of RBC pHi regulation has been observed in all fishes expressing pHpi to date (Shartau et al., 2016) and is consistent with the absence of β-adrenergically stimulated Na+−H+ exchange in Siluriformes (Berenbrink et al., 2005; Phuong et al., 2017). Despite this variation, the observed patterns in pHi across all tissues in both treatments were typical of pHpi expression (Shartau et al., 2016), and are corroborated by the reduction in plasma [HCO3−] below the blood buffer line observed after 3 h of hypercarbia in both treatments.
Our results show that the exceptional rate and degree of acute pHe compensation in P. hypophthalmus is severely limited at a water pH of 4.5. Furthermore, P. hypophthalmus expresses pHpi rather than pHcoupled whether pHe compensation occurs or not. As discussed below, this suggests hypercarbia-induced reductions in water pH may underlie previously unexplained reductions to the rate and degree of pHe compensation in fishes expressing pHpi. Variation in buffering capacity of the surrounding water might therefore mask higher, more similar rates and degrees of acute pHe compensation across teleosts than previously believed, and low water buffering capacity may increase selection for pHpi at PCO2 normally within the limits of acute pHe compensation and pHcoupled.
Impaired pHe compensation in P. hypophthalmus at a water pH of 4.5 is associated with an absence of net transepithelial exchange of acid–base relevant ions. Low water pH is hypothesized to inhibit bicarbonate uptake and proton excretion by creating unfavourable transepithelial gradients for ion transport machinery (Parks et al., 2010) and/or directly impairing transporter structure–function (Kwong et al., 2014). Indeed, inhibition of transepithelial ion flux by low water pH at ambient PCO2 has been shown in several fishes (Freda and McDonald, 1988; Shartau et al., 2017; Ultsch, 1988). Although not tested here, similar thermodynamic and/or structure–function effects on ion transport could be limiting pHe compensation in P. hypophthalmus. However, many fishes adapted to low pH environments still regulate plasma ions (Kwong et al., 2014). Thus, determining whether and how these fishes might compensate pHe at low water pH also merits future study.
Surprisingly, this study is the first to directly test the isolated effects of water pH on acid–base regulation in fishes during acute hypercarbia. Previous studies have shown that acute pHe compensation is also affected to a lesser degree by variation in water hardness and ion composition (Larsen and Jensen, 1997; Tovey and Brauner, 2018). However, logistical constraints precluded manipulating individual ions and controlling for pH in these studies. As a result, water pH differed by 1.5 units between treatments in some cases, and higher water pH was always associated with higher rates and degrees of pHe compensation. In light of our findings, revisiting these experiments while controlling for water pH would be of interest, helping to further disentangle the effects of pH from those of other ions on acid–base regulation in fish.
Fish expressing pHpi often exhibit reduced rates and degrees of acute pHe compensation relative to those expressing pHcoupled (Shartau et al., 2016). Furthermore, the approximate limit of 2 kPa PCO2 for acute pHe compensation observed in many freshwater teleosts expressing pHcoupled (Heisler, 1984; Brauner and Baker, 2009) is much less than the 3–4 kPa limit observed in many marine teleosts (Hayashi et al., 2004; Perry et al., 2010). However, we show that low water pH during hypercarbia inhibits acute pHe compensation in a freshwater fish expressing pHpi to a rate and degree equal to that of marine teleosts. This suggests low water pH might underlie previously observed reductions in the rate and degree of acute pHe compensation in other fishes expressing pHpi. Further, it suggests that all teleosts, whether expressing pHpi or pHcoupled and whether freshwater or marine, might possess similarly high capacities for acute pHe compensation. Indeed, differences in water buffering capacity could underlie much of the observed variation in these traits. Most fishes expressing pHpi are investigated in the poorly buffered waters of their native tropical river basins (Shartau and Brauner, 2014), where modest hypercarbia dramatically reduces water pH (pH 4.5 at 3 kPa PCO2, Rio Blanco, Brazil; Gonzalez et al., 2005). These tropical waters are more poorly buffered than those in which fishes expressing pHcoupled are typically tested (pH 5.5 at 3 kPa PCO2 in Vancouver city water, Canada; Shartau et al., 2017), and both have a lower pH than seawater (pH 6.9 at 3 kPa PCO2; Hayashi et al., 2004). Other studies further support this hypothesis. For example, freshwater rainbow trout express pHcoupled and typically have a limit of ∼2 kPa PCO2 for acute pHe compensation (Wood and LeMoigne, 1991; Brauner and Baker, 2009). However, rainbow trout exposed to hypercarbia in water at pH 6.9 fully compensated pHe at ∼3 kPa PCO2 within 24–48 h (Dimberg, 1988; Larsen and Jensen, 1997). This was accomplished by a net 45 mmol l−1 increase in plasma bicarbonate, matching that observed in P. hypophthalmus and marine teleosts. Thus, low water buffering capacity may mask shared, higher capacities for acute pHe compensation closer to 3–4 kPa PCO2 across teleosts.
We are also the first to observe pHpi expression in the presence and absence of acute pHe compensation at the same PCO2 in one species. This preference to regulate pHe despite the ability to independently maintain pHi suggests that even fishes expressing pHpi may incur performance costs in the absence of pHe compensation. The nature of these costs remains unknown, but if low water pH inhibits transepithelial ion transport as discussed, other vital processes relying on the same ion transport pathways could be impacted (e.g. osmoregulation, ammonia excretion, RBC function, etc.). This finding suggests that fishes expressing pHpi in low water pH during hypercarbia might incur additional performance costs relative to those expressing pHpi in high water pH. Thus, at PCO2 within the limits of pHe compensation, water buffering capacity might be an important layer of habitat complexity that affects the performance and distribution of fishes regardless of whether they express pHcoupled or pHpi.
Our findings highlight an important role for water pH in determining the rate and degree of acute pHe compensation in P. hypophthalmus specifically, and perhaps in fishes generally. This suggests hypercarbia-induced reductions in water pH may underlie previously unexplained reductions to the rate and degree of pHe compensation in fishes expressing pHpi. Based on these results, we suggest a higher limit for acute pHe compensation closer to 3–4 kPa PCO2 might be shared across teleosts when uninhibited by water pH. Low water buffering capacity might therefore be an important selective pressure for pHpi at CO2 tensions normally within the limits of acute pHe compensation and pHcoupled.
We thank two anonymous reviewers for comments that greatly improved the manuscript.
Conceptualization: M.A.S., R.B.S., C.D., C.J.B., M.H., L.M.P.; Methodology: M.A.S., R.B.S., C.J.B.; Formal analysis: M.A.S., R.B.S., C.D., M.H., L.M.P.; Investigation: M.A.S., R.B.S., C.D., M.H., L.M.P.; Resources: T.W., M.B., D.T.T.H., N.T.P.; Writing - original draft: M.A.S., R.B.S.; Writing - review & editing: M.A.S., R.B.S., C.D., M.H., L.M.P., T.W., M.B., C.J.B.; Supervision: T.W., M.B., C.J.B.; Project administration: T.W., M.B., D.T.T.H., N.T.P., C.J.B.; Funding acquisition: T.W., M.B., D.T.T.H., N.T.P., C.J.B.
This study was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Accelerator Supplement (446005-13) and Discovery Grant (261924-13) to C.J.B. and by the Danish Ministry of Foreign Affairs (DANIDA) [DFC no: 12-014AU9]. M.A.S. and R.B.S. were generously supported by NSERC Canada Graduate Scholarships and the American Physiological Society, and C.D. by the Carlsberg Foundation.
The authors declare no competing or financial interests.