Acute (<96 h) exposure to elevated environmental CO2 (hypercarbia) induces a pH disturbance in fishes that is often compensated by concurrent recovery of intracellular and extracellular pH (pHi and pHe, respectively; coupled pH regulation). However, coupled pH regulation may be limited at CO2 partial pressure (PCO2) tensions far below levels that some fishes naturally encounter. Previously, four hypercarbia-tolerant fishes had been shown to completely and rapidly regulate heart, brain, liver and white muscle pHi during acute exposure to >4 kPa PCO2 (preferential pHi regulation) before pHe compensation was observed. Here, we test the hypothesis that preferential pHi regulation is a widespread strategy of acid–base regulation among fish by measuring pHi regulation in 10 different fish species that are broadly phylogenetically separated, spanning six orders, eight families and 10 genera. Contrary to previous views, we show that preferential pHi regulation is the most common strategy for acid–base regulation within these fishes during exposure to severe acute hypercarbia and that this strategy is associated with increased hypercarbia tolerance. This suggests that preferential pHi regulation may confer tolerance to the respiratory acidosis associated with hypercarbia, and we propose that it is an exaptation that facilitated key evolutionary transitions in vertebrate evolution, such as the evolution of air breathing.

Freshwater environments are often characterized by high CO2 (hypercarbia) (Cole et al., 1994; Hasler et al., 2016; Park et al., 1969; Raymond et al., 2013). Hypercarbia is often associated with various behavioral and physiological effects in fishes owing to the associated acidosis (Heuer and Grosell, 2014; Heuer et al., 2019; Ou et al., 2015; Schneider et al., 2018). Hypercarbia tolerance may have been selected for as fishes expanded into new hypercarbic habitats, which are particularly common in tropical systems (Heisler, 1984; Li et al., 2013; Rasera et al., 2013; Scofield et al., 2016). Aquatic environments with high CO2 do not seem to have restricted the proliferation of fishes, as these environments are often very speciose (De Pinna, 2005).

Aquatic hypercarbia can arise by a number of factors, including presence of fishes and herbivores (Atwood et al., 2013, 2014), percolation of source water through carbonate deposits (e.g. limestone; Wetzel, 2002), microbial respiration (Heisler, 1984) and organic decay (Furch and Junk, 1997). The CO2 partial pressure (PCO2) in many aquatic water bodies can be significantly higher than the atmospheric PCO2 of 0.04 kPa (409 ppm; NOAA September 2019, https://www.esrl.noaa.gov/gmd/ccgg/trends/full.html). The global average PCO2 of stream and river systems is ca. 0.3 kPa (∼3100 μatm), approximately 8-fold above current atmospheric levels (Raymond et al., 2013). Some aquatic environments experience hypercarbia that greatly exceeds this; for example, PCO2 values may reach values as high as 2–8 kPa (20,249–79,000 μatm) in tropical systems (Cole et al., 1994; Heisler, 1984; Rasera et al., 2013), 2–4 kPa in aquaculture ponds (Damsgaard et al., 2015), 0.5–2 kPa (10–40 mg l−1) in recirculating aquaculture systems (Fivelstad, 2013; Mota et al., 2019), and 5−11 kPa (100–200 mg l−1) where CO2 is used as a barrier for invasive species (Kates et al., 2012). However, these environments are often heterogeneous, where fishes can experience large changes in CO2 levels spatially or temporally. Thus, fishes that reside in these systems must be able to respond to these changes in CO2 rapidly and effectively.

When exposed to severe acute hypercarbia, fishes may experience a large reduction in blood and intracellular tissue pH (Brauner and Baker, 2009; Brauner et al., 2019). As pH changes alter protein structure and function, and ultimately will affect whole-animal performance, acid–base regulation is one of the most tightly regulated physiological systems (Occhipinti and Boron, 2015). Consequently, hypercarbic freshwater environments represent an important system to investigate adaptations in acid–base regulation in fishes, and indeed, hypercarbia has been proposed to be an important driver not only of the evolution of air breathing in fishes, but also in the transition of vertebrates from water to land (Brauner and Baker, 2009; Ultsch, 1987).

Initial exposure to hypercarbia acidifies the blood, and the most common response observed in fishes is that extracellular blood pH (pHe) is compensated by a net increase in plasma [HCO3] in exchange for Cl over the following 24–72 h, with the gills playing the primary role in compensation (Brauner and Baker, 2009). Changes in pHe are often associated with qualitatively similar changes in intracellular pH (pHi), as the two have generally been shown to be tightly coupled, referred to as ‘coupled pH regulation’ (Fig. 1A,C; Shartau et al., 2016a). Although pHi recovery is more rapid than pHe recovery, complete pHi recovery takes hours to days and is usually associated with a significant degree of pHe recovery (Baker et al., 2015; Larsen et al., 1997; Shartau et al., 2016a, 2019; Wood and LeMoigne, 1991) in coupled pH regulation. In some fishes, the increase in plasma [HCO3] (in exchange for Cl) associated with pHe recovery during acute hypercarbia appears to be limited to approximately 40 mmol l−1, which corresponds with complete pHe compensation at a PCO2 of approximately 2 kPa (Shartau et al., 2019).

Importantly, some groups of fishes are capable of tolerating severe acute hypercarbia (>3–6 kPa PCO2), well in excess of this PCO2 value (i.e. ca. 2 kPa PCO2) without achieving complete pHe recovery. Complete pHe compensation at 6 kPa PCO2 would require an increase of plasma [HCO3] of ca. 100–140 mmol l−1, which is not possible (Shartau et al., 2019). In these fishes, hypercarbia tolerance appears to be associated with the ability to completely and rapidly (minutes to hours) regulate pHi in tissues such as the heart, brain, liver and muscle, despite a large, often uncompensated, reduction in pHe. This strategy, termed ‘preferential pHi regulation’, where pHi is rapidly and tightly regulated despite a reduction in pHe (Fig. 1B,C; Brauner et al., 2004; Shartau et al., 2016a), was first documented by Heisler (1982) in the marbled swamp eel (Synbranchus marmoratus) when he stated that ‘this particular strategy of acid–base regulation provides a constant milieu for the intracellular structures and demonstrates the prevalence of intracellular over extracellular acid–base regulation’. He also observed a similar pattern in salamanders exposed to aquatic hypercarbia (Heisler et al., 1982). Later, this strategy of preferential pHi regulation was observed in armored catfish (Pterygoplichthys pardalis; Brauner et al., 2004), white sturgeon (Acipenser transmontanus; Baker et al., 2009a) and striped catfish (Pangasianodon hypophthalmus; Sackville et al., 2018), and was hypothesized to be associated with acute hypercarbia tolerance (Brauner and Baker, 2009). However, it is not known whether preferential pHi regulation is restricted to a few specialized fishes, or whether it represents a more common pattern among fishes.

In this study, we address the hypothesis that preferential pHi regulation is relatively widespread among fishes. To investigate this, we conducted a survey of the presence or absence of preferential pHi regulation amongst 10 phylogenetically separated fish species, spanning six orders, eight families and 10 genera (Betancur-R et al., 2013) endemic to three continents (North America, South America and Africa). Initial experiments were conducted on a coupled pH regulator, rainbow trout (Oncorhynchus mykiss) and a preferential pHi regulator, white sturgeon, to determine the rate of PCO2 increase used to assess acute hypercarbia tolerance in fishes used in the following hypercarbia exposures. Thereafter, 10 fish species were exposed to severe acute hypercarbia ranging from 1.5 to 6 kPa PCO2, depending upon the acute hypercarbia tolerance, and the strategy for acid–base regulation was examined by terminal sampling. As preferential pHi regulation was hypothesized to be rare (Brauner et al., 2004), species selected for pH measurements were chosen from primarily hypercarbic-prone environments to increase the chance of finding fishes using this strategy of pH regulation; species from various orders were selected to assess the phylogenetic distribution of preferential pHi regulation. The results demonstrate the widespread usage of preferential pHi regulation amongst fishes, revealing that this physiological strategy is common and likely associated with tolerance of a hypercarbia-induced respiratory acidosis in fishes.

Animal acquisition and holding

In this study, 11 species of fish were used and experiments were conducted as follows. From September 2012 to November 2013, measurements were made on the following species at the University of British Columbia (UBC), Vancouver, BC, Canada: rainbow trout (Oncorhynchus mykiss; 250–400 g) from Miracle Springs Inc. (Mission, BC, Canada) and tilapia hybrid (Oreochromis niloticus×mossambicus×hornorum; ∼300–400 g) from Redfish Ranch (Courtenay, BC, Canada). Experiments with white sturgeon (Acipenser transmontanus; ∼200 g) were conducted at the International Centre for Sturgeon Studies, Vancouver Island University (VIU), Nanaimo, BC, Canada; these fish were progeny of wild-caught brood stock from the Fraser River reared at the facility since 1991.

Experiments were conducted at the Instituto Nacional de Pesquisas da Amazônia (INPA) (Manaus, AM, Brazil) with fishes caught in the Rio Negro near Manaus and transferred to a holding facility at INPA in 2008: tamoatá (Hoplosternum littorale; ∼70 g), matrinxã (Brycon amazonicus; ∼57 g), tambaqui (Colossoma macropomum; ∼61 g) and oscar (Astronotus ocellatus; ∼27 g). In 2013, the following fishes were obtained from local fish farms and transferred to facilities at INPA: tambaqui (∼100 g) and oscar (∼100 g).

Experiments with the following farm-reared species were conducted at the South Farm Aquaculture research facility at Mississippi State University (MSU) (Starkville, MS, USA) in March 2013: American paddlefish (Polyodon spathula; ∼150–400 g), alligator gar (Atractosteus spatula ∼400−1000 g) and channel catfish (Ictalurus punctatus; ∼100–200 g). Experiments with the following species were conducted at the University of North Texas (UNT) (Denton, TX, USA) with fishes caught from nearby lakes/rivers in November 2012: spotted gar (Lepisosteus oculatus; ∼300–700 g) and channel catfish (∼50–200 g).

Typically, animals were kept for at least 2 weeks in tanks prior to experiments under a natural photoperiod with flow-through water at the following temperatures depending on the location (UBC: 12°C for rainbow trout, 22°C for tilapia hybrid; VIU: 12°C; MSU: 18°C; UNT: 26°C; INPA: 28°C). Water used in this study at the various locations depended on the facility where experiments were performed. As we were only interested in the presence or absence of preferential pHi regulation in these fishes, as opposed to investigating how water quality affects pH regulation, these water quality differences were not of specific concern in the experimental design. Water quality parameters for each site are listed in Table 1.

Fishes were fed to satiety three times per week. Feed was withheld for at least 48 h before the start of experiments.

Experimental method to assess strategy of acid–base balance during severe acute hypercarbia

To determine the rate of CO2 increase to use in hypercarbia experiments, we first used model species that were previously known to be either a coupled pH regulator (rainbow trout; Wood and LeMoigne, 1991) or a preferential pHi regulator (white sturgeon; Baker et al., 2009a) to determine the optimal rate of hypercarbia increase for use in subsequent experiments to investigate presence or absence of preferential pHi regulation.

Fishes were randomly selected from the holding tank and placed in individual black plexiglass boxes (24 liters) with aeration in a re-circulating system (flow rate ∼3 l min−1 per box, 15°C; total water volume of system ∼320 liters) overnight prior to experiments. Fishes were then exposed to progressively increasing levels of hypercarbia at a rate of 1, 2 or 4 kPa h−1. Water PCO2 was monitored to ensure PCO2 increased at the desired rate for the duration of exposure using a thermostated (15oC) Radiometer PCO2 electrode (E5036) (output, Radiometer PHM 73). Fishes were continuously observed until the desired endpoint was reached – loss of equilibrium (LOE), which indicates the fish's acute CO2 tolerance. Immediately following this, fishes were transferred to a recovery tank for at least 48 h.

The strategy of acid–base regulation used during exposure to severe acute hypercarbia in fishes was determined at a PCO2 of 1.5, 3 or 6 kPa. The PCO2 tension used was based upon an initial CO2 exposure trial at a rate of 2 kPa h−1. This allowed us to identify the highest PCO2 test exposure that could be used to observe changes in pHi during maximal pHe depression; as pHi only changes by approximately one-third of pHe (owing to a lower starting pHi value and greater tissue buffer value), larger reductions in pHe allow for a more accurate determination of whether fishes tightly and completely regulate pHi consistent with preferential pHi regulation. Once the PCO2 tension that the fish species of interest could tolerate was determined, we performed the following experiment.

Fishes were acclimated individually for 24 h in black plexiglass boxes in the system described above; this period is sufficient to allow recovery from handling stress in sturgeon (Baker et al., 2005; Barton et al., 2000). Normocarbic fish (n=6–8) were terminally sampled immediately (see below) following this acclimation period (control group). Other fish were then exposed to 3 h hypercarbia (1.5, 3 or 6 kPa PCO2, depending on the CO2 trial above). We also examined the response of an extremely hypercarbia-tolerant species, tambaqui, to even more severe hypercarbia of 20 kPa PCO2 owing to their CO2 tolerance being extremely high in the CO2 tolerance trial.

In this study, we were interested in assessing the presence or absence of preferential pHi regulation in fishes. As such, we specifically chose 3 h as the sampling time point because we wanted to measure pHi when the reduction in pHe was at its maximum. At 3 h, all fishes (whether coupled or preferential pHi regulators) will experience a reduction in pHe during hypercarbia; this has been demonstrated in several species including white sturgeon (Baker et al., 2009a), Pacific hagfish (Eptatretus stoutii; Baker et al., 2015), armored catfish (Brauner et al., 2004), rainbow trout (Tovey and Brauner, 2018) and striped catfish (Damsgaard et al., 2015). Fishes using coupled pH regulation will experience a reduction in tissue pHi values at this time point as pHi recovery takes considerably longer than 3 h (Shartau et al., 2016a). In contrast, fishes using preferential pHi will exhibit no change or an increase in pHi values at 3 h of hypercarbia relative to controls despite the large reduction in pHe at this time point. It should be emphasized that at 3 h of hypercarbia, minimal pHe recovery would be expected in any fish species, and this protocol was used to assess the ability to rapidly and completely regulate pHi as predicted by preferential pHi regulation. This protocol does not allow for the interpretation of pHe regulatory ability, which was not the focus of this study.

Within the recirculating system, target CO2 tensions were achieved by bubbling a reservoir tank with preset rates of air and 100% CO2 using Sierra Instruments mass flow controllers. Water was then pumped from the reservoir tank to a header tank and then gravity-fed into the experimental tanks where water PCO2 was measured with a PCO2 electrode to confirm target CO2 tensions, which was reached within approximately 10 min once the trial started; water O2 levels remained >80% saturation. Water from the experimental tanks drained into the reservoir tank. Fishes were allowed access to normocarbic air during hypercarbia.

At the time of sampling, each box was isolated from the recirculation and anesthetic (MS-222; tricaine methanesulfonate; 0.3 g l−1 buffered with NaHCO3) was added to the water while maintaining the respective water PCO2. Once ventilation ceased (<3 min), each fish was turned ventral side up, while gills remained submerged in the aerated water and blood (∼2–3 ml) was drawn from the caudal vein into a syringe (3 ml syringe, 23 G 1¼ inch needle) flushed with heparinized saline (10 i.u. ml−1, lithium heparin; Sigma-Aldrich H0878) and placed on ice. By rapidly taking blood samples with the gills submerged and disturbances to the fish minimized, caudal puncture is a valid method for assessing blood acid–base status (Brauner et al., 2019).

Following this procedure, fishes were killed via cephalic concussion and cervical dislocation and tissues (0.5–1.0 g) were removed within 2–3 min, wrapped in aluminium foil and immediately flash-frozen in liquid N2 and stored at −80°C until pHi was determined. Tissues were sampled in the following order: heart (gently squeezed and patted dry to remove any excess blood), liver, dorsal white muscle (left side, just posterior of the dorsal fin; skin and red muscle removed), and brain. Blood was divided into two aliquots. Blood pH and hematocrit were measured from one aliquot; the other aliquot was centrifuged (3 min at 11,200 g) and plasma was removed for measurement of [Cl].

Blood pH was measured using a Radiometer PHM 84 (Copenhagen, Denmark) connected to a thermostatted (at the temperature at which the fish was sampled) Radiometer Analytical SAS pH electrode (GK2401C, Cedex, France). Red blood cell (RBC) pHi was measured using the freeze–thaw method as described by Zeidler and Kim (1977). Tissue pHi was measured using the metabolic inhibitor tissue homogenate method (MITH) (Pörtner et al., 1990) and validated for use in fishes by Baker et al. (2009b). Plasma Cl (HBI model 4425000; digital chloridometer) and osmolarity (model 5520; Westcor Vapor Pressure Osmometer) were measured; not all samples were measured owing to loss of plasma samples during transport.

Calculations and statistical analysis

Data were compared by Welch's two-tailed t-test, or where multiple treatments were evaluated, data were analyzed by ANOVA, followed by Tukey's post hoc test. If the data did not meet the assumptions of normality (Shapiro–Wilk normality test) or equal variance (Bartlett's test), a Kruskal–Wallis test followed by Dunn's multiple comparison test was used (P<0.05). Cohen's d was calculated in R to determine effect size using the effsize package (https://CRAN.R-project.org/package=effsize); this provides information about the size of differences between group means. GraphPad Prism (v.5) and R version 3.5.1 (https://www.r-project.org/) were used for statistical analyses and preparation of figures. All values are expressed as means±s.e.m. throughout.

For inter-specific data analysis, phylogenetically informed statistical analyses were used to account for phylogenetic non-independence. First, a composite, time-calibrated phylogeny was generated using divergence time obtained from the literature (Aschliman et al., 2012; Betancur-R et al., 2013, 2015; Blair, 2005). A Pagel test for binary character evolution was used to test whether the evolution of preferential pH regulation affected the evolution of air-breathing (Pagel, 1994).

To infer the evolutionary history of pH regulatory strategy, we first compared two different evolutionary models for the strategy for pH regulation using the two states ‘coupled pH regulation’ and ‘preferential pHi regulation’. Here, we fitted a continuous-time Markov model using maximum likelihood using either the same forward and reverse transition rates between the two states (lnL=−9.98, AICc=22.2, AICw=0.68) or different forward and reverse transition rates between the two states (lnL=−9.72, AICc=24.2, AICw=0.32). This was done using the fitContinuous()-function in the geiger package (Harmon et al., 2007). Based on AICw, the first model was used to infer the evolutionary history of the strategy for pH regulation. Next, we generated 10,000 stochastic character maps of the evolution of pH regulatory strategy on the phylogeny using the make.simmap function in the phytools package (Bollback, 2006; Revell, 2011), and summarized the probability of the pH regulatory strategy being preferential pHi regulation in all internal branches in the phylogeny using the describe.simmap function in the phytools package (Bollback, 2006; Revell, 2011).

Within species, the PCO2 at loss of equilibrium (LOE; defined as the inability of fishes to maintain an upright position within the water column) recorded for fishes exposed to the highest rate of increase in environmental hypercarbia (4 kPa h−1) was lower than that of fish exposed to lower rates (P<0.01); however, there was no difference between the rates of 1 and 2 kPa h−1 for either species. In rainbow trout, mean PCO2 values at LOE were 5.5±0.3, 4.8±0.3 and 2.7±0.2 kPa at exposure rates of 1, 2 and 4 kPa h−1, respectively. In white sturgeon, mean PCO2 values at LOE were 22.1±2.2, 14.6±2.3 and 6.3±1.8 kPa, respectively (Fig. 2). Comparison between species at the different rates of hypercarbia increase indicated that white sturgeon had a higher PCO2 at LOE for 1 and 2 kPa h−1, but not 4 kPa h−1 (P<0.01). No mortalities occurred in the 72 h following LOE in fishes allowed to subsequently recover in normocarbia.

Exposure of rainbow trout, a strict coupled pH regulator (Shartau et al., 2016a), to 3 kPa PCO2 for 3 h resulted in the expected reductions in pHe, RBC pHi and pHi of heart, liver, brain and white muscle (Table 2, Fig. 3). Hypercarbia exposure for 3 h to 1.5 kPa PCO2 in American paddlefish, 4 kPa PCO2 in tamoatá, matrinxã and tambaqui, and 6 kPa PCO2 in spotted gar, alligator gar, channel catfish and tilapia hybrid reduced both pHe and RBC pHi as expected, but not in oscar exposed to 4 kPa PCO2, although the statistical power of this analysis was low owing to a limited sample size (for oscar only, n=2). In contrast to rainbow trout, all other species investigated exhibited an increase or no statistically significant change in pHi of the non-RBC tissues during 3 h of hypercarbia, indicative of preferential pHi regulation. The pHi of the heart increased in spotted gar, tamoatá, matrinxã, tambaqui and oscar (P<0.05). Other tissues also exhibited increases in pHi: liver of alligator gar, brain of channel catfish, and brain and white muscle of tilapia hybrid (Table 2, Fig. 3); no other statistically significant changes were observed in other tissues despite the severity of the extracellular and RBC acidosis. Similarly, exposure of tambaqui to 20 kPa PCO2 severely reduced pHe and RBC pHi, yet induced no changes in heart, liver or brain pHi, although white muscle pH was reduced (P<0.05; Table 2, Fig. 3).

Hypercarbia exposure did not alter plasma Cl or osmolarity (when measured) (Table 3). Hematocrit remained unchanged except in American paddlefish and rainbow trout. In American paddlefish, hematocrit was reduced from 27±2% to 18±2% following 3 h exposure to 1.5 kPa PCO2. Rainbow trout hematocrit was elevated from the control value of 27±2% to 35±2% following 3 h exposure to 1.5 kPa PCO2 and to 51±4% following 3 h exposure to 3 kPa PCO2 (Table 3). No changes in plasma osmolarity were observed (Table 3).

The phylogenetic model indicates that preferential pHi regulation was the most likely strategy for pH regulation amongst the earliest actinopterygian fishes (Bayesian posterior probability of 0.55, 0.67 and 0.87 for the most recent common ancestor of vertebrates, jawed vertebrates and ray-finned fishes, respectively; Fig. 4), and it was lost by three species. Preferential pHi regulation was not specifically associated with air-breathing fishes (P=0.36).

The objective of this study was to investigate the presence or absence of preferential pHi regulation in phylogenetically separated fishes. Amongst the basal and derived bony euteleostomi fishes investigated, nine out of 10 species studied exhibited preferential pHi regulation. Fishes using preferential pHi regulation were, with the exception of American paddlefish, tolerant of hypercarbia levels well above the value at which pHe compensation can be achieved in prolonged exposure (ca. 2 kPa PCO2; Shartau et al., 2019), indicating it may represent a ubiquitous strategy of acid–base regulation in hypercarbia-tolerant fishes (Figs 3 and 4).

Acid–base regulation during hypercarbia

During hypercarbia, all species examined except rainbow trout tightly regulated pHi despite a pHe reduction (Fig. 3); these species are considered to be preferential pHi regulators. Most previous studies measuring both pHi and pHe demonstrate that following hypercarbia exposure, pHi and pHe are initially reduced, and both are compensated in the subsequent hours to days with recovery of pHi occurring faster (Shartau et al., 2016a; coupled pH regulation). In the present study, all nine preferential pHi regulating species experienced no reduction in heart, liver or brain following 3 h exposure to water PCO2 ranging from 1.5 to 20 kPa. White muscle was also well regulated, with reduced white muscle pHi only occurring in tilapia hybrid exposed to 6 kPa PCO2 and tambaqui exposed to 20 kPa PCO2; white muscle was not reduced in tambaqui exposed to 4 kPa PCO2 (Table 2, Fig. 3). Notably, in heart, liver and brain there were several observed increases in pHi despite the reduction in pHe; it is unclear why these tissues overcompensated pHi, but it may be that their ability to finely regulate pHi is less well developed than other tissues, and it has been observed in other studies (e.g. Baker et al., 2009a). The reduction in white muscle pHi may be a consequence of the large size of this tissue and the fact that pHi reductions in white muscle are less damaging than those to heart, liver or brain. In contrast to those four tissues, RBC pHi was reduced in all species. This reduction in RBC pHi has been observed in other species, including those demonstrating preferential pHi regulation (Baker et al., 2009a; Brauner et al., 2004; Shartau et al., 2016a); these reductions in RBC are expected given their limited capacity for pH regulation. Although many teleosts have the capacity to rapidly regulate RBC pHi using beta-adrenergic Na+/H+ exchange (β-NHE) (Berenbrink et al., 2005), this ability is short-lived and provides limited protection during severe acidoses such as hypercarbia (Shartau et al., 2019), as is observed in rainbow trout RBC pHi in particular, which is well studied in relation to RBC β-NHE.

Most studies where fishes have been exposed to acute hypercarbia have used PCO2 <2 kPa and observe concurrent pHe and pHi reductions; there are only a few studies at higher PCO2 levels where both pHe and pHi have been measured throughout CO2 exposure (Brauner and Baker, 2009; Shartau et al., 2016a). Species that utilize coupled pH regulation appear unable to completely compensate blood pHe during acute exposure to water PCO2 >2 kPa (Heisler, 1984), possibly to prevent hypochloremia associated with net increase in plasma HCO3 in equimolar exchange for plasma Cl (Baker et al., 2015; Brauner and Baker, 2009). This relationship between pHe compensation and plasma Cl during hypercarbia exposure was assessed in the present study. Not surprisingly, as pHe recovery was absent, there were no changes to plasma Cl (Table 3) in those species measured. This is consistent with findings from armored catfish (Brauner et al., 2004), but differs from those from white sturgeon, which exhibit significant reductions in plasma Cl following 6 h exposure to 1.5, 6 and 12 kPa PCO2 despite no pHe compensation (Baker and Brauner, 2012; Baker et al., 2009a).

The capacity for pHe compensation is influenced by exposure time and water quality, as fishes chronically exposed to gradual hypercarbia can compensate pHe beyond their capacity during acute exposure (McKenzie et al., 2003; Smart et al., 1979). Furthermore, the rate and degree of pHe compensation during acute hypercarbia exposure is also affected by water salinity (Tovey and Brauner, 2018), ion composition (Larsen and Jensen, 1997) and pH (Sackville et al., 2018). However, survival during severe acute hypercarbia may depend on protecting pHi (Shartau et al., 2017a; Vandenberg et al., 1994; Wood et al., 1983; Yoshikawa et al., 1994) more so than pHe. Mortality in marine fishes during severe acute hypercarbia may be due to reduced heart pHi (Hayashi et al., 2004). Reduced cardiac pHi is hypothesized to lower cardiac contractility, which reduces cardiac output and O2 supply (Vandenberg et al., 1994). Protection of cardiac pHi may guard against reduced cardiac performance, as an in situ perfused heart preparation demonstrated cardiac output, power output and stroke volume did not change during acute hypercarbia (5 kPa PCO2) in the preferentially pHi-regulating armored catfish (Hanson et al., 2009) and white sturgeon (Baker et al., 2011). The ability to protect pHi in various tissues may allow those species to be resilient to a variety of respiratory and metabolic acidoses (Harter et al., 2014; Shartau et al., 2017a) and provide them with a window for pHe to recover following the development of an acidosis as time and/or conditions permit. Nevertheless, preferential pHi regulation may not be sufficient in itself to confer tolerance to severe acute hypercarbia. This is demonstrated in American paddlefish, which fully protected pHi despite their relative sensitivity to hypercarbia based on an initial assessment of CO2 tolerance, and were only exposed to a PCO2 of 1.5 kPa when assessed for pH regulation owing to them experiencing LOE at 2.7±0.5 kPa PCO2; the basis for this sensitivity is unknown.

Although preferential pHi regulation may be important for tolerating acute exposure to hypercarbia levels at which pHe compensation would be limited, several fish species that exhibit preferential pHi regulation also possess the ability to regulate pHe. During exposure to 4 kPa PCO2, complete or partial pHe recovery occurs over 24 h in the preferentially pHi-regulating tamoatá, matrinxã, tambaqui and oscar (data not shown). This is similar to the response observed in the striped catfish of the Mekong, which compensate pHe by 48 h at ca. 4 kPa PCO2 (Damsgaard et al., 2015) while preferentially regulating pHi (Sackville et al., 2018). In the striped catfish, the degree of pHe regulation was significantly reduced at lower water pH despite complete preferential pHi regulation (Sackville et al., 2018), indicating that water pH also has a direct effect on the ability to compensate pHe. Thus, preferential pHi regulation does not preclude pHe regulation, but results in a far more rapid and robust pHi regulatory response at times when pHe is depressed during initial exposure to hypercarbia.

The cellular and molecular mechanisms involved in this ability for pHi regulation remain largely unknown; however, in tissues that preferentially regulate pHi, there must be a net transport of acid–base equivalents across the plasma membrane into the extracellular fluid surrounding the tissues. Tissue buffering may play a role in minimizing any reduction to pHi immediately following the onset of the acidosis; the relationship between the non-bicarbonate tissue buffer capacity and preferential pHi regulation is uncertain. Preferential pHi regulation is dependent on active regulation of acid–base equivalents; the specific transporters are not known, but are assumed to involve common acid–base transporters such as sodium proton exchangers (NHEs) and Cl/HCO3 transporters (e.g. AE). The putative mechanisms of acid–base regulation in response to CO2-induced respiratory acidosis are reviewed in Brauner et al. (2019) and remain an exciting area of future research.

Preferential pHi regulation: a strategy for expansion into hypercarbic environments?

Preferential pHi regulation is observed in a phylogenetically separated group of fishes, ranging from basal to derived, that includes both water and air breathers found in habitats ranging from temperate to tropical (Fig. 4). Reconstruction of the evolution of preferential pHi regulation on a vertebrate phylogeny indicates that preferential pHi regulation was likely the ancestral state for pH regulation in adult actinopterygians (Fig. 4); however, this conclusion should be interpreted cautiously given the limited number of species investigated in a very diverse group. It would be interesting to investigate the presence or absence of preferential pHi regulation more thoroughly in more basal vertebrate and invertebrate groups (i.e. Chondrichthyes, Dipnoi, Cephalochordata, Hemichordata, Urochordata and Echinodermata) to provide a more comprehensive picture of the early evolution of preferential pHi regulation.

Recently, preferential pHi regulation has been hypothesized to be an embryonic trait that is either retained or lost in adults (Shartau et al., 2016a), as studies in embryonic reptiles indicate use of preferential pHi regulation during development with a gradual loss to the adults (Shartau et al., 2018, 2016b). In embryonic/larval fishes, acid–base regulation is not well studied, but early-stage zebrafish embryos exposed to hypercarbia can rapidly regulate pHi, suggesting that some degree of preferential pHi regulation may occur (Molich and Heisler, 2005). Fishes inhabiting environments prone to severe acid–base challenges could retain the embryonic preferential pHi regulatory strategy to successfully tolerate these challenges as adults; this may extend to fishes that experience severe metabolic acid–base disturbances as armored catfish (Harter et al., 2014) and white sturgeon (Shartau et al., 2017a) protect pHi during severe exercise. In contrast, fishes inhabiting less stressful environments may lose this trait, which may explain, at least in part, the absence of preferential pHi regulation (and use of coupled pH regulation) in temperate species such as rainbow trout and Coho salmon, which are unlikely to encounter severe hypercarbia (Ou et al., 2015) (Fig. 4); this remains to be investigated further.

Hypercarbia tolerance may have been a selective pressure for adult fishes expanding into certain CO2-rich environments. Additionally, Ultsch (1987, 1996) has postulated that aquatic hypercarbia was a selective pressure, along with aquatic hypoxia, for the evolution of air breathing. Aerial respiration in fishes is often associated with increased blood PCO2 because (1) hypoxic waters are often simultaneously hypercarbic, and (2) CO2 release still largely occurs at the gills, but gill ventilation is typically reduced during air breathing (Shartau and Brauner, 2014). CO2 tolerance conferred by preferential pHi regulation, which leads to rapid and complete pHi regulation, may have been a beneficial adaptation that was co-opted for the evolution of air breathing, and thus could be considered an exaptation for this important evolutionary transition in vertebrates.

In summary, prior to this study, only four fish species were known to exhibit preferential pHi regulation; we identified an additional nine species using this strategy, and now there are a total of 13 species that span a broad phylogenetical range, from the basal actinopterygians to derived teleosts, known to use preferential pHi regulation. Here, we demonstrate that preferential pHi regulation may be a widely used strategy to survive and tolerate CO2 tensions ranging from 1.5 to 20 kPa PCO2. As acid–base regulation is intimately associated with routine physiological functioning and, ultimately, survival, understanding how preferential pHi regulation allows fishes, and other vertebrates, to tolerate severe acid–base disturbances may provide insight into key evolutionary transitions in vertebrates, such as the evolution of air breathing.

We thank Fernanda Dragan and Nazare Paula da Silva at the Instituto Nacional de Pesquisas da Amazônia, Mack Fondren and the staff of the Mississippi State University South Farm Aquaculture Facility, Dave Switzer and Gord Edmondson at the International Center for Sturgeon Studies at Vancouver Island University, as well as the numerous individuals who assisted with fish care, and provided assistance with experiments and feedback. We also thank the two anonymous reviewers for comments that greatly improved the manuscript.

Author contributions

Conceptualization: R.B.S., D.W.B., C.J.B.; Methodology: R.B.S.; Validation: R.B.S.; Formal analysis: R.B.S.; Investigation: R.B.S., D.W.B., T.H., D.L.A., Z.K.; Resources: D.L.A., P.J.A., A.V., D.A.C., M.S.H., C.J.B.; Data curation: R.B.S., C.D.; Writing - original draft: R.B.S., C.J.B.; Writing - review & editing: R.B.S., T.H., D.L.A., P.J.A., A.V., D.A.C., Z.K., M.S.H., C.D., C.J.B.; Visualization: C.D.; Supervision: R.B.S., C.J.B.; Project administration: C.J.B.; Funding acquisition: C.J.B.

Funding

The project was supported by Natural Sciences and Engineering Research Council of Canada funding to R.B.S. and C.J.B. C.D. is supported by the Carlsberg Foundation.

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Competing interests

The authors declare no competing or financial interests.