Module 9

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ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS:

REGULATION AND COMPENSATION IN RESPONSE
TO ELEVATED ENVIRONMENTAL CO2
COLIN J. BRAUNER*,1
RYAN B. SHARTAU*,†
CHRISTIAN DAMSGAARD*
ANDREW J. ESBAUGH‡
ROD W. WILSON§
MARTIN GROSELL¶
*Department of Zoology, University of British Columbia, Vancouver, BC, Canada
†
Pacific Biological Station, Fisheries and Oceans Canada, Nanaimo, BC, Canada
‡
Department of Marine Science, University of Texas Marine Science Institute, Port Aransas, TX,
United States
§
Biosciences, University of Exeter, Exeter, United Kingdom
¶
Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Fl, United States
1
Corresponding author: brauner@zoology.ubc.ca
1. Introduction
2. CO2 Transport and Homeostasis
2.1. CO2 Transport in the Blood
2.2. CO2 Transfer Across the Gills
2.3. Non-Teleost Models of CO2 Excretion
2.4. Air-Breathing Fish
3. Elevated Environmental CO2 and Extracellular pH Regulation
3.1. Effects of Ambient Ion Composition and Salinity
3.2. Organ Systems Used in Acid-Base Regulation and Compensation
4. Natural and Anthropogenic Elevations Environmental CO2
4.1. Ocean and Freshwater Acidification Relevant CO2 Levels
4.2. Aquaculture-Relevant CO2 Levels
4.3. Appropriate Sampling Methods to Assess Acid-Base Status in Elevated CO2
1
Carbon Dioxide Copyright # 2019 Elsevier Inc. All rights reserved
FISH PHYSIOLOGY DOI: https://doi.org/10.1016/bs.fp.2019.08.003
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2 COLIN J. BRAUNER ET AL.
5. Intracellular pH Regulation
5.1. Consequences of Intracellular pH Dysregulation
5.2. Cellular Mechanisms of pH Regulation
5.3. Preferential Intracellular pH Regulation
6. Conclusions and Future Directions
References
Acid-base balance is one of the most tightly regulated physiological processes.

Accumulation of metabolic CO2 produced at the tissues causes extra- or intra-
cellular acidosis that can disrupt cellular processes. Consequently, fish have a
well-developed system for CO2 transport and excretion; however, the system
varies significantly among fish groups, which we review in this chapter. Ele-
vated environmental CO2 that occurs naturally or due to anthropogenic
factors (e.g., climate change and in aquaculture), in both freshwater and sea-
water, induces a rapid acid-base disturbance in fish. The resulting acidosis is
compensated by a net elevation in plasma ½HCO3 in exchange for [Cl],
primarily through processes at the gills, but also the kidney. The rate and com-
pleteness of acid-base compensation during CO2 exposure is affected by water
ion composition, and at high CO2 levels, there appears to be an upper limit to
the increase in plasma ½HCO3 . Fish that naturally live in such high CO2
environments appear to have an exceptional capacity for intracellular pH
regulation. While it has long been thought that fish would not be affected by
climate change relevant CO2 levels, negative physiological effects are seen.
The effect of fluctuating CO2 levels in both marine and freshwater environments
may be especially problematic, and an area where more research is required.
1. INTRODUCTION
Acid-base regulation in vertebrates is tightly controlled and crucial for sur-

vival due to the pronounced effects of pH on protein charge that can ultimately
impact molecular, cellular, organ and whole animal performance (Madshus,
1988; Occhipinti and Boron, 2015; Putnam and Roos, 1997). Carbon dioxide
(CO2), the waste product of aerobic metabolism, is a weak acid that rapidly
affects the acid-base status of an organism. Therefore, blood CO2 levels are
tightly controlled through a complex physiological system to ensure adequate
excretion from the tissues to the environment. Section 2 gives an overview of
CO2 transport and homeostasis among different fish groups, highlighting the
similarities and differences in patterns of CO2 transport and excretion that
exist among fishes, which represent almost half of all vertebrate species.
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ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 3
Due to the low solubility of O2 in water, gill ventilation volume in fish is

high to secure sufficient O2 uptake, and because the solubility of CO2 is almost
30 times higher than O2, and because blood and water flow in the gills are
counter-current, CO2 is efficiently removed from the blood (Heisler, 1993).
Consequently, the blood arterial partial pressure of CO2 (PaCO2) in fish
is much lower than in air breathers, with values around 2 mmHg in fish
(Bayley et al., 2019; Claiborne, 1998; Evans et al., 2005; Perry and
Gilmour, 2006) compared to 40 mmHg in humans. PaCO2 values in fish are
primarily determined by environmental water CO2 levels but remain slightly
higher. Thus, when fish are exposed to elevated environmental CO2 levels,
either natural or anthropogenic in origin, this results in an elevation in PaCO2
and an associated acid-base disturbance. Changes in ventilation can never
reduce PaCO2 values below environmental levels, which combined with the
high density and viscosity of water makes ventilatory adjustments of acid-base
balance largely ineffective in fish, unlike in air breathers (Truchot, 1987).
Thus, fish regulate acid-base status primarily through differential regulation
of net H+ and HCO3 transfer between the animal and the environment
(Cameron, 1978; Evans et al., 2005; Heisler, 1984, 1986; Marshall and
Grosell, 2006; Perry and Gilmour, 2006). Section 3 reviews the effect of ele-
vated environmental CO2 on blood pH, also referred to as extracellular pH
(pHe), and the dynamics and limitations of pHe recovery. Furthermore, it
explores the role of environmental water chemistry in determining the rate
and completeness of pH compensation and the role of different organs
(the gills, kidney, and gastrointestinal tract) in acid-base compensation during
exposure to elevated CO2.
In general, fish are very effective acid-base regulators capable of defending
pHe during a range of acid-base disturbances (Truchot, 1987). In the 1970s and
80s, a great deal of research was conducted using elevated environmental CO2
as a tool to study the specific mechanisms of acid-base regulation in fish
(Cameron, 1978, 1980; Cameron and Iwama, 1987; Claiborne and Heisler,
1986; Heisler, 1984, 1986, 1988; Toews et al., 1983). This work demonstrated
that fish have a well-developed capacity to compensate for CO2 induced
acid-base disturbances and led to the early assumption that fish would be rel-
atively unaffected by natural and anthropogenic elevations in environmental
CO2. However, recent data discussed in this volume (Chapter 5, Vol 37: Heuer
et al., 2019; Chapter 6, Vol 37: Lefevre, 2019; Chapter 9, Vol 37: Munday
et al., 2019a) indicate physiological and behavioral effects are sometimes
reported at very low CO2 levels (see Heuer and Grosell, 2014) consistent
with those related to ocean acidification (OA; Caldeira and Wickett,
2003). While OA, caused by the equilibration of elevated atmospheric
CO2 with marine waters, is relatively well studied, the elevation in atmo-
spheric CO2 will also affect freshwater systems (freshwater acidification)
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and its effects on fish have so-far received little attention (Hasler et al., 2016;
Ou et al., 2015). Section 4 discusses current and projected CO2 levels
associated with ocean and freshwater acidification due to both natural
(i.e., coastal upwelling and tropical systems) and anthropogenic (climate
change and aquaculture related) causes and how these CO2 levels affect
the acid-base status of fish.
Far more is known about how environmental CO2 affects the extracellular
acid-base status than intracellular acid-base status in fish; however, the latter is
crucial to gain a comprehensive understanding of how fish are affected by
CO2. The final section of the chapter, Section 5, is a discussion of the effects
of intracellular acid-base dysregulation, cellular mechanisms involved in
acid-base compensation and a unique pattern of acid-base regulation in
fishes, where some fish have a tremendous capacity to completely and rapidly
regulate intracellular pH.
Researchers that study CO2 effects in fish encompass a broad range of
disciplines from basic physiology (to identify specific mechanisms of acid-base
regulation), to environmental physiology (to understand how fish have
evolved to deal with naturally high levels of environmental CO2) to climate
change biology (to understand how climate change relevant CO2 affects fish)
to aquaculture (to understand how CO2 affects growth and welfare in intensive
fish culture). Different disciplines express the concentration and PCO2 using
different units, which can cause confusion, especially when transitioning
between fields. To address this, we have generated a nomogram of the main
units used among the different disciplines (Fig. 1) for two water types (fresh-
water and seawater) at two temperatures (10 °C and 30 °C). In this chapter, we
report CO2 in the units that were used in the literature from which it was
retrieved, but where possible, convert and report them in additional units to
reduce confusion. We hope Fig. 1 will be useful in the interconversion between
different units used in different disciplines.
2. CO2 TRANSPORT AND HOMEOSTASIS
2.1. CO2 Transport in the Blood
Metabolic CO2 is produced in the mitochondria and must diffuse down its
PCO2 gradient through the intracellular space, across organelle and tissue
membranes (e.g., sarcolemma), through the interstitial space and finally across
the capillary wall before reaching convective blood flow. From there, CO2 is
transported by the blood to the gills, where CO2 diffuses across the gill plasma
membrane, through the interstitial space of the branchial cells and finally
across the gill epithelium into the ventilated water. Each step in this transport
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8
A
0.15
6
3
ml l−1 (ATPS)
0.10
mmol l−1
[CO2]
mg l−1
4
2
0.05
2
1
0.00
0
0.0 0.5 1.0 1.5 2.0 2.5

mmHg or torr
0 1000 2000 3000

µ atm
0 0.1 0.2 0.3

kPa
0 0.1 0.2 0.3

%
PCO2
100
B
4
150
80
3
ml l−1 (ATPS)
60
100
mmol l−1
[CO2]
mg l−1
2
40
50
1
20
0
0 10 20 30 40 50 60
mmHg or torr
0 20000 40000 60000 80000

µ atm
0 2 4 6 8
kPa
0 2 4 6 8
%
PCO2
Fig. 1. Nomogram for dissolved CO2 in freshwater (solid lines) and seawater (dashed lines) at
10 °C (blue) and 30 °C (red) for CO2 tensions relevant to ocean acidification (A) and tropical water
and aquaculture systems (B) reporting the most commonly used units. The concentration of
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dissolved CO2 in water ([CO2]) was calculated as the product of the solubility (αCO2) and partial
pressure of carbon dioxide (PCO2) (Henry’s law). Solubilities used were 0.07061 (freshwater, 10 °C),
0.03925 (freshwater, 30 °C), 0.06080 (seawater, 10 °C), and 0.03457 mmol L1 mmHg1
(Boutilier et al., 1984). Interconversions between units of partial pressure were 1 mmHg ¼
0.133 kPa ¼ 1316 μatm ¼ 0.1316%. Interconversions between concentrations were 1 mmol L1
¼ 44.1 mg L1 ¼ 44.1 ppm ¼ 24.0 mL O2 L1 (ambient temperature pressure saturated). The
latter is calculated using the ideal gas equation at 20 °C and 760 mmHg, and results in a 3% error
when converted to 10 °C or 30 °C.
cascade from the tissues to the water is aided by distinct pools of the enzyme
carbonic anhydrase (CA), which rapidly catalyzes the conversion of CO2 to
HCO3 (referred to as CO2 hydration), and the reverse (referred to as
HCO3 dehydration; see reviews by Esbaugh and Tufts, 2006a; Geers and
Gros, 2000; Henry and Heming, 1998), according to the following simplified
equation:
CO2 + H2 O Ð H + + HCO
3 (1)
The significance of this reaction lies in the fact that the CO2 permeability
across membranes is much greater than that of HCO3 , and that the PCO2
gradient across membranes is independent of ½HCO3 . Furthermore, CO2
acts as a weak acid in water, which at in vivo relevant pH values, results in
the majority of the total CO2 load existing as HCO3 in intracellular and
extracellular fluids. Finally, the uncatalyzed rate of HCO3 dehydration is
very slow (25–90 s; Swenson, 2000) in relation to blood flow. As such, specific
intra and extracellular pools of CA are integral to mobilizing HCO3 stores
and augmenting CO2 diffusion across membranes.
At the site of CO2 production, metabolically active tissues, such as muscle,
possess intracellular pools of cytoplasmic CA (CAc). This was first thought to
be paradoxical, as it was theorized that such a CA pool would only serve to
trap metabolic CO2 in the intracellular space (Roughton, 1935). Instead, this
pool has been found to contribute to the facilitated diffusion of CO2 in the
intracellular space, which was demonstrated to increase CO2 transport by a
factor of 2–3.7 depending on the muscle type (Geers and Gros, 2000). While
most of this work has been conducted on mammalian systems, it seems likely
that a similar function occurs in fish. It is well known that fish muscle contains
a cytoplasmic CA pool (Esbaugh and Tufts, 2004, 2006b; Esbaugh et al., 2005;
Henry et al., 1993), and fish cardiac muscle contains a cytoplasmic CA pool
that is absent in mammals (Esbaugh and Tufts, 2004; Esbaugh et al., 2004).
The concept of facilitated diffusion is rooted in the fact that diffusion of
CO2 and HCO3 occurs in parallel, with a greater role for facilitation by
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CA when the HCO3 :CO2 ratio in the cytoplasm is high (Geers and Gros,
2000). Measured values of intracellular pH (pHi) in fish muscle typically range
from 7.2 to 7.3 (Esbaugh et al., 2009a, 2012; P€ ortner et al., 1990), which would
result in a HCO3 :CO2 ratio of between 13 and 17, a value consistent with that
of mammals. The role of CAc in facilitating CO2 removal should be more
important at rest than following intense exercise where the pHi of muscle
can drop as low as 6.4 (Esbaugh et al., 2009a) and the HCO3 :CO2 ratio falls
to almost 1 (Geers and Gros, 2000). However, the only empirical evidence
from fish indicates that CAc may be more important following intense exer-
cise. In a series of experiments utilizing a perfused tail preparation from rain-
bow trout (Oncorhynchus mykiss) selective inhibition of CAc resulted in no
effect on CO2 excretion at rest (Henry et al., 1997a), but significantly impaired
excretion during simulated exercise (Wang et al., 1998).
The second pool of membrane-bound plasma-accessible CA has been
implicated in the transport of CO2 across the sarcolemma and capillary walls.
This pool functions to hydrate CO2 to HCO3 as it diffuses across the respec-
tive epithelium, which maximizes the PCO2 gradient, and thus CO2 diffusion,
between the fluid compartments. In fish, this has been verified for the white
muscle sarcolemma using a membrane impermeable inhibitor—quaternary
ammonium sulfanilamide (QAS)—which significantly reduced CO2 efflux
from a perfused tail preparation (Henry et al., 1997a). This pool of CA
appeared to be more important under resting conditions, as the effects of
QAS were reduced under simulated exercise conditions (Wang et al., 1998).
The evidence for a similar role in the capillaries is more ambiguous. The obser-
vation that plasma CA infusion eliminated the venous pH disequilibrium in
rainbow trout (Currie et al., 1995; Perry et al., 1997) was taken as evidence
that little plasma-accessible CA activity was present in the venous capillaries
(Henry and Heming, 1998). This differs in other regions of the cardiovascular
system, where recent evidence from the red muscle and heart lumen indicates
the presence of plasma-accessible CA that is sufficient to aid oxygen unloading
from the red blood cell through short-circuiting of a Na+/H+ exchanger (NHE)
and acidifying the red blood cell (Alderman et al., 2016; Harter and Brauner,
2017; Randall et al., 2014; Rummer et al., 2013). This acidification then reduces
the affinity of hemoglobin (Hb) for oxygen (Bohr effect) to an even greater
degree than occurs in most vertebrates due to metabolic CO2 production alone
(Harter and Brauner, 2017; Rummer and Brauner, 2011, see also Chapter 10,
Vol 37: Munday et al., 2019b). Presumably, this pool of CA could also contrib-
ute to CO2 removal from the tissues; however, direct evidence is lacking. The
pool of CA that is usually attributed to CO2 efflux and pH equilibration at the
tissues (capillary and sarcolemma) is a CA-IV-like isoform, which is bound to
the membrane by a glycophosphatidylinositol (GPI) linkage. The available
evidence in fish at present is almost exclusively transcriptomic with evidence
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for muscle and/or heart expression of CA-IV from a variety of fish species
(Alderman et al., 2016; Esbaugh et al., 2009a,b; Gilmour et al., 2007a); how-
ever, no specific cellular localization of CA-IV has been performed outside of
the gill which is an important consideration as multiple membrane-associated
CA isoforms (IX, XII, and XIV) have been shown to be expressed in muscle
and heart tissue (Esbaugh and Tufts, 2004; Esbaugh et al., 2009a,b).
Once CO2 diffuses into the red blood cell, it is quickly hydrated to form
HCO3 and H+ by a blood specific CA isoform that appears to differ from
cytosolic forms. The HCO3 is removed from the red blood cell in exchange
for Cl by the anion exchanger band 3 (AE; SLC4a1), and some of the H+ s are
buffered by Hb or bound to Hb as O2 is released at the tissues (Haldane effect).
The H+ s that are not buffered by Hb reduce the pH of the red blood cell and
induce the Bohr effect, further facilitating O2 delivery. The Haldane effect and
Bohr effect inextricably link CO2 and O2 transport where CO2 removal from
the tissues enhances O2 delivery, and O2 unloading facilitates CO2 removal
from the tissues (see review by Brauner and Randall, 1998).
The general model for all fishes is that at physiological pH (which is much
higher than the pK of Eq. 1) over 90% of the total CO2 load in the blood is
transported as HCO3 (Boutilier et al., 1984) with the bulk found in the
plasma (Perry et al., 1996); however, there are notable lineage-specific excep-
tions (Fig. 2). Unlike tetrapods, the contribution of oxygenation dependent
CO2 that directly binds to Hb (carbamino CO2) is negligible in fish
(Heming et al., 1986). The CO2 capacitance (△[TCO2]/△PCO2), which refers
to the change in total blood CO2 content relative to the change in PCO2, is greatly
influenced by the buffer value of Hb which binds H+ released upon CO2 hydra-
tion, and the magnitude of the Haldane effect (where Hb binds H+ s upon
deoxygenation). In combination with the red blood cell Cl/HCO3 exchanger,
this results in dual end-product removal from the red cell and further promotes
CO2 hydration. Importantly, this pattern of CO2 capacitance varies among fish
groups, as discussed below (see Fig. 2).
2.2. CO2 Transfer Across the Gills

The general model for CO2 excretion across the gills of teleost fishes is well
defined and is largely consistent with the pathway in the lungs of terrestrial
vertebrates (see previous reviews Esbaugh and Tufts, 2006a; Perry et al.,
2009; Tufts and Perry, 1998; Fig. 2). The process begins with the simple dif-
fusion of gaseous CO2 and O2 in opposite directions across the branchial epi-
thelium. The reduction in red blood cell PCO2 drives the dehydration of
intracellular HCO3 in the presence of CA (see Section 2.1). This coincides
with the release of H+ from Hb following oxygenation via the Haldane
effect (Christianson et al., 1914), which further facilitates CO2 excretion.
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Fig. 2. Model of general patterns of CO2 excretion in different fish groups: agnathans (hagfish
(A) and lamprey (B)), sharks (C) and teleosts (D, E). In agnathans, red blood cells lack
Cl/HCO3 exchange. Hagfish (A), possess plasma-accessible carbonic anhydrase (CA;
CA-IV-like isoform) permitting HCO3 dehydration to CO2 from both the plasma and red blood
cell compartments. Lamprey (B), lack the CA-IV-like isoform and all HCO3 dehydration is
restricted to the red blood cell and dependent upon hemoglobin (Hb) oxygenation to provide
H+ s through a large Haldane effect. In sharks (C), a CA-IV-like isoform permits HCO3 dehy-
dration to CO2 from the plasma and red blood cell compartments, where the latter is highly depen-
dent upon red blood cell Cl/HCO–3 exchange. In most teleosts (D), a CA-IV-like isoform is absent
and all HCO3 dehydration to CO2 is restricted to the red blood cell with a tight coupling of O2
uptake and CO2 excretion through a large Bohr and Haldane effect. Icefish (E), represent a teleost
exception where there are no red blood cells, and a CA-IV-like isoform permits all HCO3 dehy-
dration to CO2 from the plasma compartment. Figure prepared by Jacelyn Shu and modified from
Nikinmaa et al. (2019) with permission.
The depletion of red blood cell intracellular HCO3 drives the uptake of
plasma HCO3 through the AE (SLC4a1) in exchange for Cl, where it
can be rapidly dehydrated to CO2 in the presence of CA. This allows for sub-
stantial dehydration of the plasma HCO3 pool which otherwise could not
occur as plasma-accessible CA is generally thought to be absent in the gills
of teleosts (see reviews by Harter and Brauner, 2017; Randall et al., 2014).
In fact, a single pass of blood through the gills is sufficient to eliminate up
to 35% of the total CO2 load from the plasma (Perry, 1986) despite the short
gill resident time of <2.5 s (Cameron and Polhemus, 1974). An interesting
exception to this within teleosts is the icefish (Champsocephalus gunnari) which
completely lack Hb and red blood cells in their circulatory system. The icefish
has recently been shown to possess plasma-accessible CA that may completely
compensate for the lack of red blood cell CA, permitting all CO2 to be excreted
directly from the plasma compartment, a pattern unlike any other vertebrate
(Harter et al., 2018; Fig. 2).
CO2 excretion in teleosts operates with an apparent diffusion limitation
whereby the efficiency of CO2 exchange is reduced as gill blood flow increases
(Desforges et al., 2002; Perry, 1986). The limitations of the system are defined
by the ability to dehydrate the plasma HCO3 pool that must enter the red
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blood cell. When uncatalyzed, the dehydration reaction has a half time of
between 25 and 90 s (Swenson, 2000), but this is dramatically increased by
the presence of red blood cell CA. In fact, teleost red blood cells have been
shown to contain between 11,000 and 22,000 CA enzyme units per milliliter
when measured at 4 °C, where a single unit will double the uncatalyzed dehy-
dration rate (calculated from Esbaugh et al., 2005). The importance of CA has
been demonstrated for fish on numerous occasions, including observations
of elevated PCO2 following acetazolamide infusion (Hoffert and Fromm,
1973)—a potent CA inhibitor—as well as reduced PCO2 following plasma
infusion of bovine CA (Desforges et al., 2001; Esbaugh et al., 2012;
Tzaneva et al., 2011; Wood and Munger, 1994). Yet the apparent diffusion
limitations are not actually defined by the amount of red blood cell CA, which
is thought to be present in excess of physiological function in most vertebrates
(Henry and Swenson, 2000; Swenson, 2000; Swenson and Maren, 1978).
Instead, the rate-limiting step in CO2 excretion is the red blood cell band
3 AE. This was best demonstrated in fish by the observation that in vivo
red cell lysis, and thus liberation of red blood cell CA, resulted in reduced
blood PCO2 in rainbow trout (Perry and Gilmour, 1993).
While it is generally accepted that red blood cell band 3 AE is the rate-
limiting step for CO2 excretion under typical circumstances, other character-
istics of the branchial epithelium can also impact CO2 excretion efficiency.
More simply, parameters that increase the capacity for molecular CO2 diffu-
sion across the gill can contribute to a relative decline in plasma PCO2 and
include changes in gill perfusion or changes in gill anatomical surface area
and diffusion distance. The latter is especially exemplified in teleost fish that
increase blood water diffusion distance and reduce lamellar surface area of the
gill when the demand for O2 is low through the development of an inter-
lamellar cell mass (ILCM; Matey et al., 2008; Phuong et al., 2017; Sollid
et al., 2003). Interestingly, studies on goldfish (Carassius auratus) have shown
that the presence of ILCM does not affect arterial PO2, but significantly
increases arterial PCO2 (Tzaneva et al., 2011). The significance of these find-
ings is magnified by the fact that the presence of ILCM was associated with
low temperature (7 °C), and thus a lower rate of metabolic CO2 production
than ILCM-free fish (25 °C). There is also some support that fish may manip-
ulate diffusion distance specifically in response to elevated CO2 (Esbaugh
et al., 2016) in an analogous fashion to observations following hypoxia expo-
sure (Henriksson et al., 2008; Mitrovic et al., 2009).
A final factor that has been of interest in recent years is that of channel-
mediated CO2 diffusion across lipid bilayers. The importance of gas channels
for increasing membrane NH3 and CO2 permeability was first discussed in the
context of mammalian systems following the observation that the gastric
gland epithelium isolated from rabbit showed low NH3 and CO2 permeability
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(Waisbren et al., 1994). It was subsequently demonstrated that members of the

aquaporin (Cooper and Boron, 1998; Nakhoul et al., 1998) and Rhesus (Rh)
channel (Endeward et al., 2008; Kustu and Inwood, 2006) gene families were
able to mediate NH3 and CO2 diffusion. Interestingly, the aquaporin family
shows clear selectivity for NH3 and CO2 with human AQP5 showing
higher permeability for CO2 than AQP1 and AQ4 (Musa-Aziz et al., 2009).
Conversely, the Rh channels show little selectivity for CO2 with all demon-
strating similar ranges of permeability (Geyer et al., 2013). These observations
have also been extended to zebrafish (Danio rerio) where evidence has been
provided for dual NH3 and CO2 transport by Rhbg, Rhcg1 (Perry et al.,
2010a) and AQP1a1 (Chen et al., 2010; Talbot et al., 2015). Despite the evi-
dence that aquaporin and Rh channels can provide additional permeability
through a lipid membrane, there is still debate regarding the physiological
significance of this finding, particularly for CO2. It has been argued that
the natural background permeability of CO2 is sufficiently high to maintain
physiological function, owing to the high oil:water partition coefficient
(Hulikova and Swietach, 2014). This was supported by assessing CO2 perme-
ation rates in multicellular tissue growths of three different cell lines, which
showed no change in CO2 diffusivity following treatment with channel
blockers (Hg2+ and p-chloromercuribenzoic acid; Hulikova and Swietach,
2014). Nonetheless, the available data from fish are more supportive of the
physiological significance of Rh and AQP1a1 in CO2 transport, at least with
respect to the zebrafish. Larval zebrafish showed a significant reduction in the
respiratory exchange ratio ðṀ CO2 =Ṁ O2 Þ following morpholino knockdown
of Rhcg1, Rhbg (Perry et al., 2010a) as well as AQP1a1 (Talbot et al., 2015).
These data suggest that in the absence of gas channels, the apparent diffusion
limitations on CO2 excretion are exacerbated.
2.3. Non-Teleost Models of CO2 Excretion

The teleost model of CO2 transport conforms to the general pattern
observed in tetrapods with about 90% of the total CO2 being carried in the
plasma, and access to red blood cell CA provided by an anion exchange trans-
porter (Fig. 2). Yet this does not appear to be the case for the more basal fish
lineages. For example, the available evidence from sea lamprey (Petromyzon
marinus; Agnatha) suggests that the red blood cell carries over 20% of the total
CO2 in venous blood (Ferguson et al., 1992; Tufts and Boutilier, 1989; Tufts
et al., 1992). The primary reason for this is an absence of red blood cell anion
exchange activity (Nikinmaa and Railo, 1987; and see review by Tufts and
Perry, 1998), which means that the metabolic CO2 that enters the red blood
cell at the tissues is retained throughout the circulation. The carrying capacity
for CO2 is maintained in part because of a large Bohr and Haldane effect that
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offers high H+ buffering at the tissue (Ferguson et al., 1992; Nikinmaa and
Mattsoff, 1992; Fig. 2). This is aided by the presence of red blood cell NHE
that provides additional intracellular pH regulation (Nikinmaa, 1986;
Nikinmaa et al., 1986; Tufts, 1992). At the gills, the large Haldane effect in
lamprey initiates intracellular acidification during Hb-oxygenation that
drives HCO3 dehydration, with 62% of the total arterial-venous difference
occurring within the red blood cell (Tufts et al., 1992). Curiously, there is
no evidence of plasma-accessible CA in the gills of lamprey (Henry et al.,
1993; Fig. 2), which would mean that approximately 80% of the total CO2
in lamprey blood is at the mercy of the slow uncatalyzed HCO3 dehydration
rate, and thus effectively trapped in the plasma. Furthermore, the red blood cell
CA activity in lamprey is an order of magnitude lower than that found in tel-
eosts (Esbaugh and Tufts, 2006b; Henry et al., 1993) despite the fact that this
is the only known scenario where red blood cell CA activity would represent
the rate-limiting step in CO2 excretion.
A very different scenario occurs in the other group of agnathans, the hag-
fish. The available evidence from these species suggests that the CO2 carriage
patterns are similar to teleosts, with nearly 90% of the total CO2 load found in
the plasma (Esbaugh et al., 2009b; Tufts et al., 1998). Yet these species also
lack appreciable red blood cell anion exchange ability (Ellory et al., 1987;
Esbaugh et al., 2009b), have a small Haldane effect, and have very low red
blood cell CA activity (Esbaugh et al., 2009b). Furthermore, hagfish generally
have low Hb buffer values (Esbaugh et al., 2009b; Tufts et al., 1998) and little
NHE activity (Nikinmaa et al., 1993). Taken together, this would suggest that
the red blood cell should play a limited role in blood CO2 transport. Instead,
there is evidence that hagfish possess several pools of plasma-accessible CA
that can participate in CO2 transport outside of the red blood cell (Fig. 2).
These include (1) membrane-bound CA-IV in the gills; (2) membrane-bound
CA-IV and XV found in the red blood cells; (3) circulating CA activity in
the plasma (Esbaugh et al., 2009b). While it has yet to be empirically demon-
strated, it seems reasonable to hypothesize that O2 and CO2 transport in this
group is spatially separated with the red blood cell driving O2 transport, and
the plasma driving CO2 transport. This also means that CO2 capacitance in hag-
fish is defined by plasma non-bicarbonate buffering (βNB). While the separated
plasma βNB is higher than that of most teleosts (Esbaugh et al., 2009b; Tufts
et al., 1998), the whole blood βNB may be much lower and possibly represent
a constraint on CO2 capacitance. For example, Eptatretus stoutii has a true
plasma βNB of only 4.8 mM pH1 (Esbaugh et al., 2009b) as compared to
values ranging from 7 to 14.3 mM pH1 in teleosts (Tufts and Perry, 1998).
The elasmobranch model of CO2 transport seems to represent an interme-
diate between hagfish and teleosts (see reviews by Gilmour and Perry, 2009b;
Morrison et al., 2016; Nikinmaa et al., 2019; Fig. 2). Like teleosts, elasmo-
branchs generally carry the majority of CO2 in the plasma and contain red
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blood cell anion exchange activity (Obaid et al., 1979). Yet these species also
lack a large Haldane effect (Perry et al., 1996), have relatively low red blood
cell CA activity (Henry et al., 1997b; Maren et al., 1980), contain plasma-
accessible CA-IV in their gills (Gilmour et al., 2002, 2007a), and circulating
CA in the plasma (Henry et al., 1997b), as well as high levels of separated
plasma βNB (Gilmour et al., 2002). This combination of characteristics would
seem to support the presence of two distinct pathways for CO2 excretion in
elasmobranchs. An as yet unresolved question is how much each of these path-
ways is relied upon under different scenarios. Using the slowly membrane-
permeable CA inhibitor benzolamide, Swenson and Maren (1987) demon-
strated that elimination of >98.5% of the branchial CA activity had no effect
on plasma PCO2 in dogfish (Squalus acanthias). The permeable inhibitor
methazolamide resulted in elevated PCO2 after only 1 h post-infusion. These
results suggest that the red blood cell pool drives the majority of CO2 excretion
under resting conditions. Conversely, Gilmour et al. (2001) used benzolamide
to demonstrate a significant decrease in the arterial-venous difference in total
CO2, which indicated reduced CO2 excretion when branchial CA activity
was inhibited. This result was not observed in rainbow trout using similar
experimental conditions. Similar support for a role of branchial CA in CO2
excretion was provided by the observation that reducing hematocrit by 62%
had no effect on PCO2, despite a significant increase in cardiac output
(Gilmour and Perry, 2004). A significant increase in PCO2 was observed
following infusion with the membrane impermeable inhibitor F3500. Taken
together, these data support a role for both plasma and red blood cell mediated
CO2 excretion in elasmobranchs under resting conditions (Fig. 2). An impor-
tant caveat is that the physiological studies have been performed on only a
single species. While branchial and plasma CA activity is found in other elas-
mobranch species, as well as at least one chimaeran (Gilmour et al., 2002), it
remains to be seen if the patterns of CO2 excretion observed in dogfish are
broadly representative of elasmobranchs; however, a recent study investigat-
ing 13 different chondrichthyan species confirms that, for the most part, this
appears to be the case (McMillan et al., 2019).
2.4. Air-Breathing Fish
The model of teleost CO2 transport described above is complicated some-

what in air-breathing fish owing to the dual respiratory epithelia: the gills and
the air-breathing organ. Air-breathing fish are generally classified as either
obligate or facultative, whereby the former requires access to air to survive
while the latter selectively uses air-breathing but can sustain vital function
using only their gills (Graham, 1997). It is important to note that this classi-
fication relates to O2 transport and not CO2 transport, as many obligate
air breathers primarily use their gills to eliminate CO2 (see Graham, 1997).
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For example, the obligate air-breathing African lungfish (Protopterus

annectens) eliminates <15% of metabolic CO2 through its air-breathing
organ (Gilmour et al., 2007b). However, the relative contribution of the air-
breathing organ increases following an acidosis, suggesting that the impor-
tance of air-breathing to CO2 transport is dynamic in some air-breathing fish
(Gilmour et al., 2007b).
As the majority of CO2 is excreted across the gills, the pathways for CO2 are
largely similar to those of teleosts with a few additional considerations. First is
the relative importance of the Haldane effect in offloading CO2 at the gills,
which presumably is reduced as the gills are a negligible site of O2 uptake—
at least in obligate air breathers (Brauner et al., 2004a). The available evidence
suggests that the gills of air-breathing fish are similar to water-breathing teleosts
in that they do not have plasma-accessible CA (Gervais and Tufts, 1998). Thus,
the red blood cell must remain the primary route for CO2 excretion. The lack of
an appreciable Haldane effect during branchial CO2 excretion may be offset by
the intrinsically higher levels of PCO2 carried in the blood of air breathers
(Brauner et al., 2004a), many of which have levels in excess of 10mmHg
(e.g., Gilmour et al., 2007b; Graham, 1997). This would result in a much greater
PCO2 gradient between the blood and the water and increase the driving force
for CO2 diffusion. In contrast to the gills, the air-breathing organ of at least one
air-breather (Amia calva) does have membrane-bound CA, which is presumably
plasma oriented (Gervais and Tufts, 1998). Yet it remains unclear if this isoform
plays a significant role in CO2 excretion into the air-breathing organ, or if its role
is more akin to that of mammals where it primarily acts to alleviate pH disequi-
librium in the pulmonary capillaries to facilitate proper ventilatory control
(see review by Esbaugh and Tufts, 2006a).
3. ELEVATED ENVIRONMENTAL CO2 AND EXTRACELLULAR

pH REGULATION
From the above section, it is clear that fish have an intricate system to trans-
port metabolically produced CO2 from its source of production in the tissues to
the environment. Blood PCO2 and pH are tightly controlled to ensure metabolic
homeostasis despite different patterns of CO2 transport and excretion in different
fish groups (Fig. 2). However, this system can operate bi-directionally, and thus
exposure to elevated environmental CO2 can lead to large physiological distur-
bances. Environmental CO2 levels can fluctuate due to many factors, however,
we focus on those related to OA (up to 1000–2000μatm; 0.75–1.5 mmHg), fresh-
water acidification (with temperate lakes and streams up to 4000 μatm; 3 mmHg
and some tropical systems reaching 78,000μatm; 60mmHg) and in intensive
aquaculture (up to 28,000μatm; 22.5 mmHg; 40 mg/L). See Section 4 for
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elaboration upon these conditions. The physiological disturbances associated

with elevated environmental CO2 levels in the aforementioned ranges are dis-
cussed next, followed by the influence of water ionic composition (see
Section 3.1) and the role of different organ systems on acid-base compensation
(see Section 3.2).
During exposure to elevated environmental CO2, internally elevated CO2
(hypercapnia) hydrates to H+ and HCO3 and acidifies bodily fluids (Eq. 1).
This acid-base disturbance is to some degree buffered by internal buffer sys-
tems in the blood and extracellular space serving as an initial line of defense
against the acidosis. However, the protons that are not buffered lead to a
reduction in pHe. In general, elevated sustained environmental CO2 results
in a transient reduction in pHe and tissue pHi (but see Sections 3 and 5.3).
The resulting acidoses directly affect the acid-base status, but may indirectly
affect O2 transport characteristics, and this may represent the greatest initial
stressor at high CO2 levels. A reduction in red blood cell pHi not only reduces
Hb-O2 affinity via the Bohr effect, but it may also reduce Hb-O2 carrying
capacity, known as the Root effect. Most teleosts possess a Root effect (see
Berenbrink et al., 2005) and thus, exposure to elevated environmental CO2
can result in a reduction in internal O2 levels, hypoxemia. Many fish that pos-
sess a Root effect can protect red blood cell pHi through the release of cate-
cholamines and adrenergic stimulation of NHE (see Berenbrink et al., 2005
and Chapter 10, Vol 37: Munday et al., 2019b). However, this protective effect
may be overwhelmed at high CO2 tensions.
The CO2 induced acidosis, compensation, and eventual pHe recovery have
been well studied in fish, and the reader is referred to several excellent reviews
(Claiborne et al., 2002; Evans et al., 2005; Heisler, 1984, 1986, 1993; Truchot,
1987). The dynamics of the acid-base disturbance and recovery are often rep-
resented using a pH/HCO3 plot (also known as a Davenport diagram;
Davenport, 1974), which allows the simultaneous visualization of plasma
½HCO3 , pH, and PCO2 of the blood. For example, in Fig. 3, exposure to
elevated environmental CO2 initially induces a reduction in pHe along a
non-bicarbonate buffer line (a respiratory acidosis) represented by the arrow
connecting numbers 1 and 2. Subsequent pHe recovery is accomplished
through active net H+ excretion (see below and Claiborne et al., 2002;
Evans et al., 2005; Heisler, 1984, 1986, 1993; Perry and Gilmour, 2006), which
is associated with a net increase in plasma ½HCO3 and an equimolar reduc-
tion in plasma [Cl]. The increase in pHe occurs along the respective PCO2-
isopleth (metabolic alkalosis) set by the equilibrium between CO2 and H+
(the Henderson-Hasselbalch equation, Eq. 2).

HCO 3
pH ¼ pK + log 10 (2)
αCO2 PCO2
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60 mmHg
154 (−0.91)
150
[HCO3] (mM)
100
−
18 mmHg
50 41.5 (−0.54)
7.5 mmHg
13.4 (−0.29)
1.34 (−0.05) 3.0 mmHg

2.5 mmHg
0
6.75 7.00 7.25 7.50 7.75
pH
Fig. 3. Predicted changes in acid-base status upon exposure to different levels of environmental
CO2. Changes in acid-base status were calculated for a fish with a control arterial blood PCO2
of 2.5 mmHg (point 1) that was elevated to 3.0, 7.5, 18, or 60 mmHg, representative of exposures
to ocean acidification (OA), temperate and tropical aquaculture, and tropical freshwater swamps,
respectively. After exposure to CO2, pH and ½HCO3 changes along the dashed non-bicarbonate
blood buffer line (from point 1 to 2), and blood pH subsequently recovers in association with
an increase in plasma ½HCO3 along their corresponding PCO2 isopleths (dotted curved lines).
The required elevation in plasma ½HCO3 for blood pH compensation, and the initial reduction
in blood pH (in brackets) are shown in the top right corner of each trace. The figure shows that the
initial pH disturbance is modest in an OA scenario requiring a small change in net acid excretion to
compensate pH, but that the pH disturbance associated with exposure to high environmental CO2
typical of tropical swamps is very large requiring unrealistic changes in net acid excretion to com-
pensate pH (i.e., an increase in plasma ½HCO3 of 154 mM). See Fig. 5 for a magnified version of
the OA scenario with different starting blood PCO2 levels. PCO2-isopleths and the model were
generated using Eq. (2) with αCO2 and pK0 from Boutilier et al. (1984), temperature ¼ 25 °C
and a non-bicarbonate buffering capacity of 10 slykes.
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The PCO2 isopleths are much steeper at high PCO2 compared to low
PCO2, and therefore proportionally larger increases in ½HCO3 are required
to compensate pH when challenged by high PCO2 (Eq. 3; Fig. 3).

Δ HCO 3 ¼ ΔPCO2 αCO2 10
pHpK
(3)
This suggests that fish exposed to future OA scenarios, where internal PCO2
may change by 0.5 mmHg (i.e., from 2.5 to 3.0 mmHg; 3330–4000 μatm), would
require a 1.34 mM increase in ½HCO3 to fully compensate pH. In more
extreme scenarios, such as in aquaculture systems where water PCO2 levels
of 7–18 mmHg (10,000–24,000 μatm) have been observed (Damsgaard et al.,
2015; Ellis et al., 2017), full pHe compensation would require a 13–42mM
increases in plasma ½HCO3 (Fig. 3). However, the most extreme CO2 condi-
tions are found in tropical swamps with dense vegetation, where water PCO2
may reach values as high as 60mmHg (80,000 μatm; Furch and Junk, 1997).
Complete pHe compensation at this PCO2 would require an increase in plasma
½HCO3 of over 150 mM to compensate pHe fully. The latter is not physiolog-
ically possible as it would require complete replacement of all extracellular
anions (in particular, plasma Cl) with ½HCO3 if normal osmotic status
was to be maintained.
A recent analysis suggests that the ability to actively increase net proton
excretion (which does not distinguish between H+ excretion into the water
and/or HCO3 uptake from the water) is the single most important factor that
determines the capacity for a fish to compensate pHe under elevated environ-
mental CO2 while non-bicarbonate buffering effectively plays a comparably
minor role (Shartau et al., 2019). Some fish examined, however, do not possess
the ability to elevate plasma ½HCO3 beyond an apparent 30 mM threshold
in the blood during exposure to elevated environmental CO2 which limits the
water PCO2 where fish can fully compensate pHe to about 15 mmHg (Brauner
and Baker, 2009; Heisler, 1988; Shartau et al., 2019; Fig. 4). This limitation
was first observed by Heisler (1984), which he referred to as an “apparent
bicarbonate concentration threshold.” Most studies that investigate acid-base
regulatory responses to elevated environmental CO2 are conducted at different
temperatures, in water of different ionic composition, with different PCO2 levels
and exposure durations and different life histories. Clearly, more systematic
studies are required to fully characterize the nature of the “apparent bicarbonate
concentration threshold” (Heisler, 1984; Shartau et al., 2019). While there are
some freshwater species that may exceed this apparent 30 mM ½HCO3 thresh-
old, the largest increases in plasma ½HCO3 from control resting values during
elevated PCO2 in a teleost is 50mM (McKenzie et al., 2003 and see Shartau
et al., 2019) which would limit complete pHe compensation to an environmental
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A 100
% pH compensation
80
60
40
20
B
150
Required Δ[HCO3] (mM)
−
100
50
0
0 10 20 30 40 50 60
PCO2 (torr)
Fig. 4. The effect of a 30 mM plasma ½HCO3 limit on the capacity for pH compensation (A) and
the elevation in plasma ½HCO3 that would be required for complete blood pH compensation
(B) at increasing environmental PCO2 values. If fish are unable to elevate ½HCO3 by
>30 mM (after non-bicarbonate buffering), full blood pH compensation is limited to 15 mmHg
(A). For species that can elevate plasma ½HCO3 beyond that, very large increases in plasma
[HCO 3 ] would be required. This model was generated using the Henderson-Hasselbalch equation
(Eq. 2), an initial blood PCO2 and pH of 2.5 mmHg and 7.8, respectively, a body temperature of
25 °C, non-bicarbonate buffering of 10 slykes, and αCO2 and pK0 from Boutilier et al. (1984).
Adapted from Shartau et al. (2019).
PCO2 of about 25 mmHg (33,000 μatm; Fig. 4). So how do fish live in naturally
high CO2 waters of tropical systems that can reach PCO2 levels of 60 mmHg
(80,000 μatm; Figs. 3 and 4)? The solution, in part, appears to lie in an excep-
tional capacity to regulate tissue pHi during exposure to these extremely high
environmental CO2 levels, which is discussed in Section 5.3.
A range of physiological, anatomical, and ecological factors may limit
the ability of fish to compensate pHe during elevated environmental CO2.
The ionic composition of the water has an influence on the rate and complete-
ness of recovery from a respiratory acidosis which is discussed next, as do the
organ systems involved in compensation which is discussed immediately
following.
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3.1. Effects of Ambient Ion Composition and Salinity
An increase in the ion content of water increases the rate and completeness
of pHe compensation during exposure to CO2, and may explain a large pro-
portion of the variation in acid-base responses that have been observed in fish
under different water conditions (Larsen and Jensen, 1997). While the positive
effect of ion content on acid-base regulation in fish has been known for a long
time, it is largely based upon interspecific comparisons where many factors
differ (see reviews by Heisler, 1984, 1986; Truchot, 1987). Relatively little
work has been done directly comparing the dynamics of acid-base regulation
during exposure to elevated CO2 in a single species in freshwater relative to
seawater or in freshwater of differing ionic strengths. In rainbow trout accli-
mated to soft water (low ionic strength), hard water (high ionic strength) and
seawater, and then exposed to 10,000 μatm CO2, pHe recovery at rest was
similar in hard water and seawater, both of which were more rapid than fish
in soft water. Interestingly, in all conditions, sustained exercise increased the
rate of pHe recovery over resting rates, indicating the large effect of activity
level on the dynamics of acid-base regulation (Tovey and Brauner, 2018).
Given that fish in the wild are rarely at rest, activity level may play an impor-
tant role in influencing acid-base status, a topic that has received little attention
but clearly worthy of further study. In barramundi (Lates calcarifer) acclimated
to salinities of 0, 7.5, 15, 22.5 and 32 ppt, exposure to 10,000 μatm CO2 resulted
in an increase in net H+ excretion in all salinities, with a more rapid response
in fish in 22.5 and 32 ppt (Weakley et al., 2012). Thus, in general, acid-base dis-
turbances appear to be corrected more rapidly in fish in hard freshwater and
seawater relative to soft freshwater water (Larsen and Jensen, 1997; Tovey
and Brauner, 2018; Weakley et al., 2012), but interestingly the dynamics
may not be that different between hard freshwater and seawater despite the
large differences in water ionic composition.
The effect of ionic composition on the rate and completeness of acid-base
recovery may be directly due to differences in low counter-ion availability for
acid-base relevant ion exchange (Larsen and Jensen, 1997), or due to indirect
effects. For example, low water ion concentrations are typically associated
with a low water buffer capacity, which will result in a lower water pH at a
given PCO2 compared to water with higher ion concentrations. This should
set a thermodynamic constraint on pH compensation, as the electrochemical
gradients for H+ excretion become unfavorable (Lin and Randall, 1995).
The idea that a reduction in water pH (all other parameters remaining
constant) should reduce the capacity for pH regulation during elevated envi-
ronmental CO2 was tested in striped catfish, Pangasianodon hypophthalmus.
A reduction in water pH from 5.8 to 4.5, virtually eliminated blood pHe
recovery and changes in plasma ½HCO3 following 20 h of continuous
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exposure to 3 kPa CO2 (Sackville et al., 2018). The interaction between water
pH and elevated environmental CO2 is clearly important in understanding
the dynamics of acid-base regulation in different water types and worthy of
further investigation.
3.2. Organ Systems Used in Acid-Base Regulation and Compensation

The gill and the kidney have long been ascribed roles in acid-base balance
regulation (Heisler, 1986, 1988, 1993; Truchot, 1987). More recently, the gas-
trointestinal system has also been recognized due to its contributions to overall
acid-base status in marine teleosts (Wilson et al., 1996). In the following, con-
tributions of organ systems and specific transport mechanisms associated with
the regulation of pHe for unfed fish are discussed. The impacts of feeding on
acid-base balance are discussed elsewhere (Chapter 7, Vol 37: Wood, 2019).
3.2.1. BRANCHIAL MECHANISMS

The gills are the dominant site for the dynamic exchange of acid-base
equivalents with the environment, with many excellent reviews on this topic
(Claiborne et al., 2002; Dymowska et al., 2012; Evans et al., 2005; Gilmour
and Perry, 2009a; Heisler, 1984; Hwang et al., 2011; Kumai and Perry,
2012). The gills can accomplish net acid secretion during elevated environmen-
tal CO2 either by direct H+ excretion (Heisler, 1993), usually in exchange or
associated with Na+ uptake, or HCO3 uptake or retention in exchange for
Cl (Esbaugh et al., 2012; Evans et al., 2005; Perry et al., 1992) at specific gill
ionocytes, also known as mitochondrion rich cells (MRC). The morphology
and physiology of ionocytes associated with ion and acid-base regulation have
been reviewed in detail elsewhere and vary dramatically between species and
habitat salinity and ion composition (see Evans et al., 2005; Hwang et al.,
2011; Marshall and Grosell, 2006; Perry and Gilmour, 2006). In general, gill
surface area and ionocyte composition greatly affect the capacity for acid-base
regulation in fish. Thus, any reduction in gill surface area compared to similar-
sized species, as well as reduced ionocyte density, would be expected to have
negative effects on the capacity for acid-base regulation. For example, many
air-breathing fish, that use the same basic principles for acid-base regulation,
have reduced gill surface area compared to similar-sized water-breathing
fish (Graham, 1997) and they display accordingly reduced capacities for pH
regulation compared to many water-breathing fish (reviewed by Bayley
et al., 2019; Shartau and Brauner, 2014).
A range of transporters within branchial ionocytes are involved with the net
excretion of acid and associated ion transport (see Evans et al., 2005; Hwang
et al., 2011; Marshall and Grosell, 2006; Perry and Gilmour, 2006). These
consist of Na+/H+ exchangers (NHE), vacuolar-type H+-ATPase (VHA),
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Na+-coupled HCO3 transporters (NBC), and AE which have all been shown
to be involved to some degree during acid-base regulation in rainbow trout
(Gilmour and Perry, 2009a; Perry and Gilmour, 2006), dogfish (Gilmour and
Perry, 2009a; Tresguerres et al., 2006), tilapia (Oreochromis mossambicus;
Hwang et al., 2011), zebrafish (Gilmour and Perry, 2009a; Kwong et al.,
2014), medaka (Hsu et al., 2014), lungfish (P. annectens; Gilmour et al.,
2007b), hagfish (Tresguerres et al., 2007), and white sturgeon (Acipenser trans-
montanus; Baker et al., 2009; Shartau et al., 2017b). Assisting with the move-
ment of acid-base equivalents are Na+-K+-2Cl cotransporter (NKCC),
Na+, K+-ATPase (NKA), and Na+-Cl cotransporter (NCC) which help main-
tain the electrochemical gradient and allow Na+-dependent transporters to
function (Hwang et al., 2011). Additionally, CA plays an important role in
acid-base regulation, and more recently, Rhesus proteins have been implicated
in the movement of NH3 across the branchial membrane in several fish species
(e.g., zebrafish (Kumai and Perry, 2011) and pupfish (Cyprinodon variegatus;
Brix and Grosell, 2012)), implying a role in acid-base regulation (Hwang
et al., 2011). The following sections describe some of the more recent advances
that help to elucidate the pathways involved in acid-base regulation of the gill, in
particular during exposure to elevated environmental CO2.
3.2.1.1. Carbonic Anhydrase. The branchial epithelium CAc (Esbaugh

et al., 2012; Georgalis et al., 2006a; Lin et al., 2008) provides H+ and
HCO3 for differentially regulated apical and basolateral extrusion from
the gill epithelial cells, and thus assumes a central role in acid-base balance.
As would be expected, CAc responds to changes in ambient CO2. Levels of
mRNA for CAc in gulf toadfish (Opsanus beta) exposed to 1900 μatm and
in medaka (Oryzias latipes) embryos and larvae exposed to >1200 and
4200 μatm, respectively, were reduced (Esbaugh et al., 2012; Tseng et al.,
2013). The downregulation of CAc at lower water PCO2 levels supports the
observation that increased HCO3 uptake, rather than H+ extrusion may
facilitate pH compensation in seawater. In contrast, exposure to 8000 μatm
(6 mmHg) lead to elevated CAc mRNA and protein in freshwater rainbow
trout (Georgalis et al., 2006a), and exposure to 50,000 μatm (37.5 mmHg) lead
to increased CA protein abundance and enzymatic activity in the marine mid-
shipman (Porichthys notatus; Perry et al., 2010b). These results may reflect a
requirement for direct H+ extrusion under these more extreme conditions.
Interestingly, a metabolic acidosis induced by elevated salinity exposure in
the gulf toadfish (Genz et al., 2008), was compensated at the gill and associ-
ated with elevated branchial CAc (Sattin et al., 2010). Additional work is
required to better understand the interactions between CA and HCO–3 uptake
versus H+ extrusion under different levels of environmental CO2.
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3.2.1.2. Bicarbonate Transport. Based on the limited number of studies con-

ducted to date, HCO3 uptake from the water may be important for pHe com-
pensation during elevated environmental CO2 (Esbaugh et al., 2012; Tseng
et al., 2013). A diversity of HCO3 transporters from the SLC4 (a1 (AE1),
a2 and a4 (NBC)) and SLC26 (a3, a4 (pendrin), and a6) families have been
demonstrated in fish gills (Bayaa et al., 2009; Esbaugh et al., 2012; Grosell
et al., 2009; Kurita et al., 2008; Lee et al., 2011; Piermarini et al., 2002;
Taylor and Grosell, 2008). So far, no reports have demonstrated transcrip-
tional responses by any SLC26 family members in response to elevated envi-
ronmental CO2 and neither SLC26a3 nor a6 were impacted in the gills of
toadfish exposed to 1900 μatm (Esbaugh et al., 2012). In contrast, mRNA
of the SLC4 members examined to date all change in response to environmen-
tal CO2. The upregulation of two AE1 isoforms in the gills of medaka exposed
to 7000 μatm (Tseng et al., 2013) suggests basolateral localization of these pro-
teins to assist HCO3 movement from the gill to the blood. In support of this
hypothesis are studies on zebrafish, which show that this anion exchanger is
responsible for HCO3 movement across the basolateral membrane in some
MRCs (Lee et al., 2011). SLC4 AE2 expression in gill tissue of toadfish was
reduced after exposure to 1900 μatm, suggesting an apical localization and role
in HCO3 excretion. Although this transporter is most commonly found in the
basolateral membrane in other vertebrate epithelia (Romero et al., 2004),
some epithelia have apical AE2 localization (Aranda et al., 2004) supporting
their proposed roles in reduced net gill HCO3 excretion (and thus retention)
during elevated environmental CO2. Last, SLC4a4 (NBC1), ascribed to the
basolateral membrane to extrude HCO3 , appears to respond differently to
environmental CO2 depending on species, life stage as well as exposure
duration and intensity. NBC1 mRNA levels remained unchanged in gills of
toadfish exposed to 1900 μatm (Esbaugh et al., 2012), suggesting that other
transporters are rate-limiting for HCO3 uptake in this species. The eelpout
(Zoarces viviparus) exposed to 10,000 μatm (7.5 mmHg) shows an initial reduc-
tion followed by an increase in NBC expression after 42 days of exposure
(Deigweiher et al., 2008) consistent with a need for HCO3 uptake. In
medaka, there is also evidence for a role in both NBC1a and NBC1b in
pHe regulation (Tseng et al., 2013).
While in some fish there is evidence for uptake of HCO3 from the water
during compensation for a respiratory acidosis, other fish species compensate
in a very different way. In the freshwater brown bullhead (Ictalurus nebulosus),
elevated environmental CO2 resulted in a covering of the MRCs (referred to as
chloride cells in that study) reducing their fractional surface area by up to 95%
(Goss et al., 1992). This was thought to reduce the rate of HCO3 loss and
associated Cl uptake by reducing the number of Cl/HCO3 exchangers
exposed to the water. The net result is consistent with retention of plasma
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HCO3 that occurs in conjunction with a reduction in plasma Cl during com-
pensation for a respiratory acidosis. This pattern has also been observed in
white sturgeon (Baker et al., 2009) and to a lesser degree in rainbow trout
(Goss and Perry, 1993). How widespread this pattern is among fish species
during elevated environmental CO2 remains to be investigated.
3.2.1.3. Proton Transport. At least three NHEs (NHE1, NHE2, and NHE3)
and VHA are relevant for branchial contributions to acid-base balance
(Hwang et al., 2011). The NHE1 isoform (NHE1a and NHE1b) in gills is down-
regulated in response to high environmental CO2 levels (10,000–52,000 μatm;
7.5–39mmHg; Deigweiher et al., 2008; Edwards et al., 2005; Rimoldi et al.,
2009) consistent with basolateral localization of NHE1 and the need to reduce
proton retention by transport from the gill epithelium to the blood under these
conditions. For NHE3, an apical isoform, exposure to 10,000 μatm (7.5 mmHg)
resulted in elevated gill protein abundance (Edwards et al., 2005). Further,
elevated mRNA expression was observed in earlier life stages after exposure
to CO2 levels as low as 1200 μatm (Tseng et al., 2013). These responses support
a role for NHE3 in acid secretion during elevated environmental CO2. The
transporter NHE2 is also present in the gill of freshwater and marine teleosts
(Edwards et al., 2005, 2010; Larsen et al., 2014), as well as elasmobranchs
(Guffey et al., 2015). In rainbow trout exposed to elevated CO2, NHE2 mRNA
levels were increased in the peanut lectin agglutinin binding (PNA+) MRCs
indicating NHE2 may play a role in pHe regulation during a respiratory acidosis
(Ivanis et al., 2008).
The VHA appears to be present in the apical membrane of freshwater fish
gills (Boisen et al., 2003; Bury and Wood, 1999; Horng et al., 2007, 2009).
While we are aware of just one study demonstrating a role for the VHA in
response to elevated environmental CO2 in freshwater fish (Seidelin et al.,
2001), it is involved in acid secretion and may be stimulated by low ambient
pH (Horng et al., 2007, 2009). In contrast with freshwater fish, the VHA
appears to be preferentially basolateral in euryhaline and marine fish
(Katoh et al., 2003). In the euryhaline barramundi acclimated to a salinity
of 15 ppt and exposed to 10,000 μatm (7.5 mmHg) CO2, Weakley et al.
(2012) observed an increase in VHA expression that was correlated with whole
animal net H+ excretion rates. However, two studies examining proton pump
responses to elevated CO2 in seawater found downregulation of mRNA
(Esbaugh et al., 2012; Tseng et al., 2013) which, given a basolateral localization
of the proton pump, may be associated with an increased net acid secretion by
the gills.
3.2.1.4. Na+/K+-ATPase. Although not an acid-base transporter per se,

NKA drives anion exchangers such as NBCs and NHEs, by establishing
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and maintaining Na+ and Cl gradients required for their activity. Conse-
quently, elevated environmental CO2 influences enzymatic activity and mRNA
expression of NKA subunits during exposures to 1900 μatm (1.5 mmHg;
Esbaugh et al., 2012), 6000 μatm (4.5 mmHg; Melzner et al., 2009) and
10,000 μatm (7.5 mmHg; Deigweiher et al., 2008). Although a transient reduc-
tion in enzymatic activities was observed in toadfish, studies on cod (Gadus
morhua) and eelpout indicate an increased demand for NKA activity during
exposure to elevated environmental CO2. While such an increased NKA
demand may be solely in support of acid-base regulation (implying an energetic
cost associated with elevated environmental CO2; see Chapter 6, Vol 37:
Lefevre, 2019), it may also support a compensatory response to changes in intes-
tinal transport in marine fish (see Section 2.3.; Heuer et al., 2012).
3.2.2. RENAL MECHANISMS
Net renal contribution to acid-base balance is generally assumed to be in the
order of 5% (Heisler, 1993), however, a recent study in the freshwater swamp eel
(Monopterus albus; Thinh et al., 2019), an obligate air-breather, shows it can be
much higher in some species. In general, renal contribution is likely even
reduced in marine teleosts with much lower urine flow rates (0.3 mL kg1 h1)
compared to freshwater teleosts with flow rates of 3.0 mL kg1 h1 (Larsen
et al., 2014; Marshall and Grosell, 2006). However, the kidney of freshwater
teleosts plays a significant role in the reabsorption of HCO3 initially lost by
glomerular filtration (Georgalis et al., 2006b; Perry and Gilmour, 2006;
Wheatly et al., 1984). During exposure to high levels of environmental
CO2, plasma may reach levels between 30 and 50 mM (see Section 3 and
reviews by Heisler, 1993; Shartau et al., 2019) which, with a glomerular filtra-
tion rate of 4 mL kg1 h1, could translate to a glomerular base loss of
200 μEqv kg1 h1. To prevent this, there is considerable acid secretion by
the renal tubules for tubular reabsorption of HCO3 but this acid secretion
does not contribute much to net acid excretion from the animal to the environ-
ment. Indeed, studies of renal and branchial contributions in rainbow trout
experiencing hypercapnia due to hyperoxia exposures revealed renal acid
secretion rates of 122 μEqv kg1 h1 (Wheatly et al., 1984), compared to peak
branchial excretion rates of 214 μEqv kg1 h1 (Wood et al., 1984). These stud-
ies illustrate that renal processes contribute significantly to acid-base balance,
even if not evident from net renal acid excretion.
The mechanisms of tubular HCO3 reabsorption in teleost fish is assumed
to be similar to that of mammals where acid secretion into the tubule is respon-
sible for the reabsorption. The CO2 resulting from acid secretion and HCO3
in the filtrate enters the tubular cells where it is converted to HCO3 (and H+)
which is transported back to the extracellular fluids by NBC (reviewed by
Perry et al., 2003; Petochi et al., 2011). This process could limit the ability
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to elevate plasma ½HCO3 and limit pHe compensation during exposure to

elevated environmental CO2 (see Section 3), an area clearly worthy of
further study.
In both rainbow trout and American eel (Anguilla rostrata), exposure to a
PCO2 of 10,000 μatm resulted in an increased renal H+ pump and NBC expres-
sion (Perry et al., 2003). An elegant study on rainbow trout exposed to
8000 μatm CO2 revealed a role for the membrane-bound, extracellular
CA-IV in renal HCO3 reabsorption (Georgalis et al., 2006b). CA-IV acts
to catalyze the conversion of HCO3 to CO2 upon H+ secretion and inhibition
of this enzyme resulted in increased renal base loss (Georgalis et al., 2006b).
Further, a role for CAc in compensation for hypercapnia in catalyzing the
conversion of CO2 to HCO3 after entering the tubular epithelial cells has
been shown (Georgalis et al., 2006b). Most of the studies conducted on the
mechanisms associated with tubular HCO3 reabsorption have been con-
ducted at relatively high CO2 levels, and we are unaware of studies on renal
adjustments during climate change and current day relevant environmental
CO2 levels, also an area worthy of further investigation.
3.2.3. ROLE OF THE GASTROINTESTINAL TRACT

In contrast to the kidney which contributes to acid-base balance mainly in
freshwater teleosts, the gastrointestinal tract of unfed fish only contributes to
acid-base balance of teleosts in salinities hyperosmotic to their plasma
(Table 1). Marine teleosts drink to combat osmotic fluid loss to their concen-
trated environment (Larsen et al., 2014) and absorb fluid across the intestinal
epithelium. Fluid absorption is driven in part by Na+ and Cl absorption but
is also facilitated by precipitation of CaCO3 in the intestinal lumen, which acts
to reduce luminal osmotic pressure (Wilson et al., 2002) and thereby permits
water absorption (Genz et al., 2011; Whittamore et al., 2010; Wilson et al.,
2002). The substrate for this CaCO3 formation is derived from Ca2+ entering
via ingested seawater and from HCO3 secreted by the intestinal epithelium as
reviewed elsewhere (Grosell, 2006, 2011; Grosell et al., 2009; see Chapter 4,
Vol 37: Grosell, 2019).
The importance of intestinal base secretion and rectal base excretion for
whole animal acid-base balance is illustrated by two studies: The first study
found that gulf toadfish acclimated to different salinities displayed vastly
different rectal base excretion rates. These results demonstrated that pHe
was defended, despite the increase rectal base loss at higher salinity, by com-
pensatory branchial acid excretion. The second study in European flounder
(Platichthys flesus), showed that perfusing the intestine with two elevated
Ca2+ concentrations caused dramatic and proportional increases in rectal
base excretion, together with similar compensatory increases in net acid
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Table 1
Rectal base excretion rates by marine and euryhaline teleosts during normal drinking or during
intestinal perfusion
Drinking/ Rectal base

perfusion Temperature Environmental excretion
Species (ml kg1 h1) (Cº) variables (μEqv kg1 h1) References
Rainbow Drinking: 11 32 ppt 114 15 Wilson et al.

trout 3.12 0.48 (1996)
Rainbow Perfused: 80 11 32 ppt 552 66 Wilson et al.
trout (1996)
Lemon sole Perfused: 15 14 30 ppt 200a Grosell et al.
(1999)
European Perfused: 3.8 13 30 ppt 145a Wilson et al.
flounder (2002)
Gulf toadfish Drinking: 22–26 9 ppt 9.3 2.7 Genz et al.
1.38 0.30 (2008)
2.60 0.92 (2008)
3.82 0.58 (2008)
European Perfusion: 5.04; 12.5 33 ppt 230a Cooper et al.
flounder 10 mM Ca2+ (2010)
Gulf toadfish Drinking: 22–25 32–35 ppt 48.1 5.9 Heuer et al.
1.20 0.14 (2012)
Gulf toadfish Drinking: 22–25 32–35 ppt; 64.2 6.6 Heuer et al.
1.45 0.14 1900 μatm (2012)
CO2
a
denotes values estimated from figures in the cited publications.
excretion by the gills, although a transient blood acidosis was observed

(Cooper et al., 2010). While base excretion from rectal fluids of marine fish
represents a substantial base loss, it does not appear to be modulated to correct
organismal acid-base balance disturbances.
If the gastrointestinal tract played a role in acid-base compensation during
exposure to elevated environmental CO2, rectal base excretion would be
expected to be reduced during CO2 exposure to compensate for the respiratory
acidosis. However, if anything, the opposite is observed. In the gulf toadfish
exposed to climate change/current day CO2 levels (1900 μatm) an increase
in rectal base excretion was observed (Table 1; Heuer et al., 2012) and in
the plainfin midshipman (Porichthys notatus) exposed to 50,000 μatm
(37.5 mmHg) an increase in rectal fluid total CO2 concentration was observed
(Perry et al., 2010b). Similar responses have been observed in fish during
longer-term acclimation and in isolated intestinal epithelia (Heuer and
Grosell, 2016; Perry et al., 2010b). In the intestinal epithelia, the rate-limiting
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step for HCO3 secretion is the basolateral NBC (Taylor et al., 2010) which
is not downregulated during elevated environmental CO2 exposure (see
Chapter 4, Vol 37: Grosell, 2019; Heuer et al., 2012). Thus, although the
gastrointestinal tract plays an important role in acid-base regulation of marine
fish, it does not appear to play a significant role during exposure to elevated
environmental CO2.
4. NATURAL AND ANTHROPOGENIC ELEVATIONS

ENVIRONMENTAL CO2
Below we discuss the range of elevated CO2 levels that have been reported
in relation to OA and coastal upwelling, freshwater acidification of temperate
lakes and streams as well as tropical systems, and finally in intensive aquacul-
ture. This will highlight the spatial and temporal variations in CO2 levels
within and among these systems.
We report the effects on the acid-base status of fish where known and pro-
pose a refinement of sampling techniques for assessing acid-base status in fish.
4.1. Ocean and Freshwater Acidification Relevant CO2 Levels

4.1.1. OCEAN ACIDIFICATION
Surface waters of the open ocean have relatively stable CO2 levels, which are
close to equilibrium with the atmosphere and thus, as water PCO2 increases,
there is a corresponding increase in OA (Doney et al., 2009; see Chapter 1,
Vol 37: McNeil and Matsumoto, 2019). Current atmospheric and open ocean
PCO2 is close to 410 μatm and is predicted to reach 1000 μatm (0.8 mmHg)
by the year 2100 (Meinshausen et al., 2011). This prediction has stimulated
research on the impact of increases in PCO2 within this range (i.e.,
400–1000 μatm—see Chapter 5, Vol 37: Heuer et al., 2019; Chapter 9, Vol
37: Munday et al., 2019a) on the physiological, behavioral, and ecological
effects on marine fish. However, coastal systems naturally exhibit much larger
fluctuations in PCO2 (and higher maximum CO2) than the open ocean on a diel,
tidal, and seasonal basis, due to a generally higher biological activity (both res-
piration and photosynthesis; Cloern et al., 2016) and particularly in temperate
areas due to seasonal stratification and upwelling events (Cai et al., 2011; Feely
et al., 2008). Some experimental studies on the effects of OA on fish have there-
fore targeted higher PCO2 levels of 1800–2000 μatm (1.5 mmHg; e.g., Esbaugh
et al., 2012; Heuer et al., 2012, 2016; Strobel et al., 2012), as this is currently
relevant in certain coastal and upwelling zones (Cai et al., 2011; Feely et al.,
2008). Such high levels are expected to become more widespread in the future
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and are predicted to occur globally in the open ocean by the year 2200
(Meinshausen et al., 2011). More recently, Baumann (2019) has highlighted
that even more extreme short-term fluctuations (over hours, days or months)
of between 200 and 5000 μatm (0.2–3.75 mmHg) already occur commonly in
coastal marine environments (Baumann et al., 2015; Wallace et al., 2014;
Wang and Cai, 2004). Interestingly, this existing variability is more than twice
the most extreme prediction for the rise in open ocean PCO2 by the year 2300.
Furthermore, the variability encountered, and the frequency of reaching the
highest levels of CO2 is predicted to intensify as atmospheric PCO2 rises expo-
nentially in the future (McNeil and Sasse, 2016), with the monthly range of sea
surface PCO2 levels quadrupling by the year 2100. It is therefore increasingly
important to understand how the natural fluctuations in PCO2 influence biota
including fish (e.g., Jarrold et al., 2017), rather than just a constant, elevated
CO2 level often used in experiments.
While CO2 levels consistent with current upwelling events and future pre-
dictions associated with climate change will induce acid-base disturbances in
fish, the magnitude of the acidosis is transient in nature (see Fig. 3). A study on
the gulf toadfish revealed a △pHe of 0.12 peaking at 30 min after the onset of
exposure to 1900 μatm which was fully compensated by 2 h (Esbaugh et al.,
2012). This time course for pHe compensation is also supported by measures
of net H+ excretion rates, which returned to control values by 2–4 h following
exposure to 1000–5000 μatm CO2 (0.8–3.8 mmHg; Allmon and Esbaugh,
2017). Several studies have provided evidence of metabolic compensation to
OA relevant CO2 levels at later time points, as evidenced by elevated plasma
½HCO3 and PCO2 but unaffected pHe (Ern and Esbaugh, 2016; Esbaugh
et al., 2016; Green and Jutfelt, 2014).
Although metabolic adjustments of acid-base balance are prevalent for
reasons discussed above, there is evidence for some level of respiratory com-
pensation. In the marine red drum (Sciaenops ocellatus) a 32% reduction in
branchial diffusive distance was observed following exposure to 1200 μatm
(0.9 mmHg) for 14 days (Esbaugh et al., 2016) which could reduce the
PCO2 gradient between the blood and water and would be expected to reduce
the magnitude of the respiratory acidosis. However, in this study, it was not
sufficient to correct acid-base status, so the role of respiratory compensation
for even low CO2 levels consistent with OA appears relatively minor. Respi-
ratory compensation can also come in the form of elevated ventilatory volume
(e.g., Ern and Esbaugh, 2016; Gilmour, 2001; Perry and Abdallah, 2012).
While such a response would not be able to correct an inward CO2 diffusion
gradient during extreme exposures, it can act to reduce the difference in PCO2
between the blood and water (as occurs during hypoxia-induced hyperventi-
lation) and thus reduce the magnitude of the associated metabolic adjust-
ments. Such a response takes on greater significance when considering OA
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relevant CO2 exposures. The ventilatory response that accompanies exposures

to PCO2 values of 1000 and 5000 μatm have been calculated to reduce the need
for metabolic HCO3 retention by 38% (Ern and Esbaugh, 2016), an area wor-
thy of further study.
4.1.2. FRESHWATER ACIDIFICATION
Inland freshwaters are very different compared to marine systems in terms
of dissolved CO2. Firstly, average PCO2 levels tend to be considerably higher
than atmospheric equilibrium and hence surface waters in the open ocean. The
average for over 6700 streams and rivers reported by Raymond et al. (2013)
was 2300 or 3100 μatm (1.7–2.3 mmHg; depending on whether potential biases
were discounted or not), which were both within the range (1300–4300 μatm;
1–3.2 mmHg) found by earlier regional and global studies (Alin et al., 2011;
Aufdenkampe et al., 2011; Cole and Caraco, 2001; Richey et al., 2002). How-
ever, equally intriguing and relevant to the physiology of freshwater fish is
the large variability in PCO2, both diel and seasonal, within individual freshwa-
ter habitats. For example, over a one-year cycle Maberly (1996) measured PCO2
values in a productive UK lake from close to zero (during summer
phytoplankton blooms) to 6.9-fold higher than atmospheric levels following
the overturn in late autumn (i.e., >2400μatm, 1.8 mmHg). Changes of a similar
magnitude can even occur over a diel cycle in inland lakes (e.g., reducing CO2
from 2400 μatm to effectively zero between dawn and dusk; Xu et al., 2019).
A further difference compared to marine ecosystems is the rate of increase
of dissolved CO2 in freshwaters as atmospheric CO2 rises globally. Weiss et al.
(2018) reported an increase of 16 μatm per year in four large freshwater reser-
voirs (average depth 40 m), which is about eight times faster than the current
increase in atmospheric and open ocean PCO2. The explanation for this dis-
crepancy appears to be the relative changes in terrestrial primary productivity
and soil respiration caused by climate change (warming and rising atmo-
spheric CO2). The net effect is an increase in both dissolved organic carbon
and dissolved inorganic carbon in groundwater and run-off into freshwater
ecosystems (Golub, 2016; Hasler et al., 2016; Wetzel, 2001).
There are a few interesting, though more isolated, extreme examples of
high CO2 in freshwater ecosystems. For example, in groundwater fed river sys-
tems, PCO2 values up to 43 times higher than atmospheric (i.e., >17,000 μatm,
13 mmHg) were observed in a field study of three major temperate rivers
in Germany (Demars and Tremolières, 2009). As mentioned previously
(see Section 3) dense vegetation in tropical swamps can result in extreme
CO2 levels in excess of 78,000 μatm (60 mmHg; Furch and Junk, 1997). Even
more rarely, natural freshwater environments can exceed even the highest levels
reported within intensive aquaculture (see below and Chapter 8, Vol 37:
Skov, 2019). For example, Blinn and Sanderson (1989) reported a travertine
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(limestone) warm water spring that feeds the Montezuma Well in Arizona
(average depth of 6.7 m), which achieves dissolved CO2 levels of 550 mg/L
(i.e., CO2 of 330,000 μatm at 21°C, 250 mmHg). Although fish are absent,
57 taxa of invertebrates were found which raises intriguing questions of their
physiology and acid-base regulation in particular.
While there are many studies on how fish regulate blood acid-base status at
high CO2 tensions in freshwater (see Section 3), there are few studies at lower
CO2 tensions that are consistent with future predictions associated with cli-
mate change in freshwater systems (Ou et al., 2015). The higher average, large
temporal variability, and faster rate of increase associated with climate
change, all contribute to the fascinating issue of how freshwater fish and their
physiology is being impacted (and will be in the future) compared to marine
fish which have received far more attention, given the global focus on OA.
This is clearly an area worthy of further investigation, especially considering
that 40% of the world’s fish live in freshwater.
4.1.3. POTENTIAL BASIS FOR INCREASED SENSITIVITY OF YOUNG FISH TO

ELEVATED CO2
In general, larval and early juvenile fish appear to be more affected by
exposure to OA relevant CO2 levels than adult fish (see Chapter 5, Vol 37:
Heuer et al., 2019; Chapter 9, Vol 37: Munday et al., 2019a). Some fish have
been observed to display maladaptive changes in behavior during exposure to
OA relevant CO2 levels (see Chapter 5, Vol 37: Heuer et al., 2019). There are
also studies showing organ damage (Frommel et al., 2012, 2014, 2016),
increased mortality, and reduced growth in larval fish exposed to OA relevant
CO2 levels that may be due to incomplete and/or inadequate acid-base regu-
lation, or elevated metabolic costs associated with acid-base regulation
(see Chapter 9, Vol 37: Munday et al., 2019a). The maladaptive behavior
has been proposed to be associated with the changes in ion- and acid-base
status associated with pH compensation in response to elevated environmental
CO2, where plasma HCO3 increases in exchange for Cl. The changes in
ion-concentration have been proposed to change the reversal potential of the
GABA receptor in the brain of fish and alter behavioral responses (see Chap-
ter 5, Vol 37: Heuer et al., 2019; Heuer et al., 2012, Heuer et al., 2012; Nilsson
et al., 2012; Nilsson and Lefevre, 2016). What could make larval and early juve-
nile fish more susceptible than adults when exposed to the same changes in envi-
ronmental PCO2? Interestingly, the resting membrane potentials decrease as
neurons mature (see Chapter 5, Vol 37: Heuer et al., 2019) which may play a
role; however, it may also have to do with resting blood PCO2 levels. Here,
we used Eqs. (2) and (3) to predict the magnitude of the acidosis and the increase
in plasma ½HCO3 associated with pH compensation during exposure to end-
of-century predicted levels of 1000 μatm (0.8 mmHg). If we assume that under
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control conditions (CO2 ¼ 400 μatm, 0.3 mmHg) an adult fish has a blood
PaCO2 of 2.5 mmHg (Brauner et al., 2000) during exposure to 1000 μatm, arte-
rial PCO2 may be expected to increase up to 0.5 mmHg. This would result in a
0.05 pH unit acidosis that could be compensated through a 1.34mM increase
in ½HCO3 , which would represent a 20% increase in resting plasma ½HCO3
(Fig. 5). However, in a larval fish, blood PCO2 may be much lower than in adult
fish due to the much larger surface area: volume ratio and thus is it conceivable
that blood PCO2 would be much closer to that of the environment. The PaCO2
in larval fish has never been measured due to technical challenges, but assuming
it was similar to environmental levels under control conditions (i.e., 0.3 mmHg),
then exposure to 1000 μatm (and an increase in 0.5 mmHg) would almost double
the magnitude of the acidosis. Although pHe compensation would also be asso-
ciated with a 1.34mM increase in plasma ½HCO3 as in the adult fish (Fig. 5),
this would correspond to a 167% increase in plasma ½HCO3 . While PaCO2 in
A B
3.0 mmHg
% change in [HCO3] (mM)
1.34, 20% 150

7.5
−
[HCO3] (mM)
−0.05 2.5 mmHg
100
5.0
−
50
0.8 mmHg
2.5 1.34, 167%
−0.09 0.3 mmHg 0
7.65 7.70 7.75 7.80 0.3 1.0 3.0 10.0 30.0
pH Starting PCO2 (mmHg)
Fig. 5. Predicted changes in acid/base status upon exposure to ocean acidification (OA) relevant
CO2 levels. (A) Acid/base status in a typical adult water-breathing fish (blood PCO2 ¼ 2.5 mmHg
and blood pH 7.8); (A) and that in a fish with much lower blood PCO2 levels (blood PCO2 ¼
0.3 mmHg and pH 7.8; possibly representative of larval fishes); (B) during changes in environmental
CO2 from 400 to 1000 μatm (0.3–0.8 mmHg, and thus an increase in blood PCO2 of 0.5 mmHg). This
shows that the initial pH disturbance due to an increase in blood PCO2 of 0.5 mmHg (¼600 μatm) is
double in fish with a low starting PCO2 (0.09) relative to a higher starting PCO2 (0.05), but the
required absolute increases in ½HCO3 to compensate is identical (1.34 mmol L1 HCO3 ). PCO2
isopleths are 0.3, 0.8, 2.5, and 3 mmHg (from bottom to top). (B) Relative changes in ½HCO3
required to compensate pH after CO2 induced acidification of the blood in animals with different
starting blood PCO2 values. The relative change in ½HCO3 is much greater in fish with a low blood
PCO2 relative to those with a high PCO2. PCO2-isopleths in (A) and models in (A) and (B) were
generated using Eq. (2) with αCO2 and pK0 from Boutilier et al. (1984), temperature ¼ 25 °C and
a non-bicarbonate buffering capacity of 10 slykes.
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larval fish will be higher than the environment to permit CO2 excretion, it is
not known how much higher it would be. Fig. 5B shows the relative increase
in plasma ½HCO3 that would be required to compensate for a 0.5 mmHg
increase in blood PCO2 from different starting PCO2 values. This exercise serves
to show that different starting blood PCO2 values between adult and larval fish,
or even between different fish species, may potentially underlie differences in
CO2 sensitivity associated with OA relevant increases in CO2. Whether larval
fish have lower blood PCO2 values than adult fish would be an interesting first
step to determine whether this may be the basis of increased sensitivity to CO2.
4.2. Aquaculture-Relevant CO2 Levels
Intensive aquaculture has involved long-term rearing of fish at high CO2

levels (well above those predicted by OA) long before the term “ocean
acidification” became common parlance. Current guidelines for safe CO2
levels in intensive aquaculture ranges from 10 to 40 mg/L (Blancheton,
2000; Fivelstad et al., 1999; Petochi et al., 2011). When accounting for the
effects of temperature and salinity on the solubility of CO2 in water, this equa-
tes to PCO2 ranging from >7000 to >28,000 μatm (i.e., 5–21 mmHg, 17.5–70
times higher than current atmospheric conditions, see Fig. 1). Furthermore,
these high levels are not limited to finfish production in recirculating aquacul-
ture systems (RAS—see below), indeed elevated PCO2 appears to be an
unavoidable consequence of intensive aquaculture settings in general. For
example, over 40% of Norwegian salmon smolt hatcheries (flow-through
and RAS) report CO2 levels >5400 μatm (4 mmHg; Noble et al., 2012), shrimp
ponds in Bangladesh have CO2 levels averaging >17,000 μatm (12.8 mmHg;
Sahu et al., 2013; Saksena et al., 2006), and striped catfish and swamp eel
aquaculture ponds in Vietnam, dissolved CO2 can reach 45,000 μatm
(30–35 mmHg, Damsgaard et al., 2015; Thinh et al., 2019). Acutely, CO2
can accumulate to even higher levels, for example during short-term (10 h) live
transportation of Atlantic salmon (Salmo salar) in well boats, CO2 reached
130 mg/L (Orellana and Wecker, 2014) which is equivalent to >70,000 μatm
(53 mmHg) assuming a typical salinity (35 ppt) and temperature (12 °C) for
salmon in sea cages.
Why is CO2 so high in aquaculture? The cause is two-fold. Firstly, the inev-
itably high rates of oxygen consumption and hence CO2 production when
growing animals at high stocking densities (e.g., commonly 10–35 kg/m3 for
salmon; Turnbull et al., 2005) and specific practices where density exceeds
100 kg/m3, such as during short-term transportation of live fish (Farrell,
2006; Orellana and Wecker, 2014; Tang et al., 2009). Secondly, the very
different physicochemical properties of CO2 compared to O2 that affect gas
equilibration. At high biomass densities, dissolved O2 can be rapidly depleted
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and thus must be monitored and managed very well in modern aquaculture
systems (Summerfelt et al., 2000). However, the solubility of CO2 in water
is about 30 times higher than for O2, and consequently, CO2 removal is much
more difficult than O2 addition. If gas tensions are restored with air equilibra-
tion, atmospheric PO2 can be restored in minutes while atmospheric PCO2
levels require hours to be restored entirely (e.g., Hoppe et al., 2012). CO2
removal is further complicated with the use of pure O2, which increases the
O2 diffusion gradient almost fivefold, requiring a fifth of the total gas flow
to restore water O2 levels. The latter reduces CO2 removal fivefold unless addi-
tional CO2 stripping technology is employed (e.g., Noble et al., 2012). Another
limitation of CO2 removal is that unlike O2, which simply dissolves in water,
dissolved CO2 dissociates to H+ and HCO3 (Eq. 1). At typical pH values in
aquaculture (generally well above pH 6, the pK for this reaction) the majority
of total CO2 exists as HCO3 . Thus, to remove metabolically produced CO2
this large pool of HCO–3 must first be converted back into CO2 before it can be
removed by gas equilibration. However, the dehydration reaction is so slow
that the removal of CO2 will always lag behind the restoration of dissolved
O2 values in aquaculture settings. Within RAS, this problem is exacerbated
slightly on each pass of the water through the water treatment systems. Very
high CO2 levels are therefore currently an unavoidable consequence of the
water recycling process in RAS (see Chapter 8, Vol 37: Skov, 2019).
Fish exposed to these elevated CO2 tensions will compensate for the respi-
ratory acidosis as described in Section 3, which has been well described in
fish. However, the difference here is that not only are PCO2 levels very high,
but they may vary both spatially and temporally within the respective systems.
Thus fish will be forced to constantly re-adjust their acid-base status to
compensate. Given that acid-base regulation is ultimately driven by active
ionoregulation, which has a metabolic cost, oscillating PCO2 levels may affect
the growth and condition of the fish. Most studies to date conducted on the
effect of different CO2 levels in aquaculture rear fish at constant CO2 tensions,
while the effect of oscillating CO2 tensions has received little attention to date
(an exception is Ou et al., 2015) but is clearly an important area for further
research in intensive aquaculture (see Chapter 8, Vol 37: Skov, 2019). The met-
abolic cost of acid-base regulation remains largely unknown and is another
area worthy of investigation.
4.3. Appropriate Sampling Methods to Assess Acid-Base Status

in Elevated CO2
There is increasing interest in quantifying the effect of elevations in envi-
ronmental CO2 (i.e., due to ocean and freshwater acidification and RAS)
on the blood acid-base status of exposed fish. However, acid-base relevant
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parameters (i.e., blood and intracellular pH, PCO2, total CO2, and HCO3 )
are very susceptible to artifacts associated with methods of sampling and
the analyzers used for blood measurement. The following are some sugges-
tions for best practices along with some criticisms of the methodology that
is often used.
The most common method for acquiring blood samples involves anesthe-
tizing and/or humanely stunning/killing the fish, removing it from the water
and puncturing a blood vessel that is easily located without prior dissection,
often referred to as the “grab n’ stab” method. Blood is most commonly drawn
from the caudal vessel. For the majority of analyses carried out on blood sam-
ples, this can be appropriate, but for assessment of acid-base status, it is often
problematic. In all water-breathing fish, as soon as the animal is removed from
water the gills collapse and gas exchange is impaired. Thus, blood O2 rapidly
declines (resulting in anaerobiosis and a metabolic acidosis) and CO2 increases
(resulting in a respiratory acidosis). Thus, the longer the fish is air-exposed,
the less likely measured blood gases and acid-base status values will be repre-
sentative of in vivo conditions. A further serious complication occurs if fish
struggle during the capture process, recruiting anaerobic white muscle in a
stress/escape response. Wood et al. (1983) went as far as stating that the phys-
iological disturbance associated with such sampling methods is sufficient to
render blood acid-base data meaningless due to the perturbed blood lactate
in particular. The addition of a metabolic acid reduces pH itself but also
titrates existing HCO3 in the plasma (which is the vast majority of plasma
total CO2) which can dramatically elevate blood PCO2. For this reason, it
is preferable to surgically implant a cannula into an artery or vein under anes-
thesia, followed by 24–48 h recovery of the fish (Soivio et al., 1975). This
allows the experimenter to remove blood samples via the cannula without
removing the fish from the water or inducing a struggle.
Numerous studies have investigated the impact of elevated environmental
CO2 relevant to salmonids grown in RAS, producing valuable data regarding
the effects of CO2 on growth and health status (e.g., Fivelstad et al., 2003a,b,
2015; Good et al., 2010, 2018; Khan et al., 2018; Martens et al., 2006; Mota
et al., 2019; Summerfelt et al., 2000; see Chapter 8, Vol 37: Skov, 2019). How-
ever, it is notoriously difficult to obtain useful blood gas and acid-base data
from fish held in groups in large tanks where cannulation is not possible.
As a cautionary tale for interpreting data obtained using the grab n’ stab sam-
pling method under such conditions, it is worth noting that some studies have
shown an apparent trend for increasing blood pH (i.e., alkalosis) as ambient
CO2 rises (Fivelstad, 2013; Fivelstad et al., 1998; Petochi et al., 2011). This
clearly contradicts studies on cannulated fish, in which elevated environmental
CO2 results in an initial respiratory acidosis lasting a few hours, with
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subsequent complete recovery of blood pH due to a compensatory elevation of

plasma ½HCO3 at constant, elevated PCO2 (see Section 3). The most likely
explanation for an apparent blood alkalosis in fish acclimated over weeks to
elevated ambient CO2 is based on the grab n’ stab method causing a metabolic
acidosis (i.e., driven by lactic acid production during capture and sampling).
However, compared to control CO2 conditions the fish acclimated to elevated
CO2 experience a smaller reduction in blood pH during the sampling process
because the plasma ½HCO3 starts at a higher level (e.g., up to four times higher
in Fivelstad, 2013 and in Petochi et al., 2011) and therefore provides substantial
additional buffering of the metabolic acidosis. Thus, the conclusion that blood
pH is actually higher in fish acclimated to elevated ambient CO2 is probably an
artifact of the sampling technique, rather than reflecting the actual in vivo blood
acid-base status, and would be misleading if accepted at face value.
Surgical techniques to cannulate blood vessels in fish (particularly the dor-
sal aorta) have been successfully used since the 1960s (Axelsson and Fritsche,
1994; Iwama and Ishimatsu, 1994; Smith and Bell, 1964; Soivio et al., 1975).
Unfortunately, cannulation of blood vessels in fish is only applicable to species
or life stages that are large enough (most studies have used fish >150 g) and for
researchers with appropriate training and access to suitable surgical labora-
tory facilities. Thus the ability to obtain precise blood gas and acid-base data
from fish exposed to changes in environmental CO2 is quite limited to partic-
ular species, body sizes, and researchers. However, a non-surgical technique
that allows successful blood sampling for valid blood gas and acid-base status
is feasible if care is taken to avoid lactic acid production due to struggling, and
reduced gas exchange caused by removing fish from the water. Furthermore, it
can be used on fish that are too small to cannulate. This technique involves
careful anesthetizing of individual fish within their home tank (without dis-
turbing the animal during the process), followed by transfer to a gill irrigation
system containing a maintenance dose of anesthetic. The latter allows artificial
ventilation of the gills with realistic rates of gill water flow to match those
occurring in vivo. By doing so, it is feasible to consistently measure blood
gas, acid-base and hematological variables that match blood samples taken
from fish via a surgically implanted cannula (Fig. 6).
For valid blood samples it is key that fish are not disturbed prior to anes-
thesia, as any anaerobic muscle use results in a sufficient rise in blood
lactic acid to disturb blood acid-base status. The choice of anesthetic is also
important as some can have a major influence on the water acid-base chem-
istry (e.g., tricaine methanesulfonate; MS-222 or TMS), resulting in rapid ele-
vations in water PCO2 and even more so when water pH is buffered by adding
sodium bicarbonate as is common practice (see Davison et al. in preparation
for a fuller discussion). Rapid transfer of fish (within seconds) to a gill
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Dorsal Aorta via Cannula

8.4
Caudal Puncture with Gill Irrigation
Blood pH
8.2
8.0 A
B
7.8
7.6
B
20
HCO3- (mM)
15
A
10
0
B
8
PCO2 (mmHg)
4 A
2
0
Control 7.5 mmHg
External CO2
Fig. 6. Blood acid-base status of 200 g rainbow trout exposed to control conditions (water equil-
ibrated with atmospheric air) and after 24 h exposure to 7.5 mmHg (1% CO2 10,000 μatm;
N ¼ 6). Fish were cannulated and allowed 48 h to recover in individual chambers of flow-through
aerated water. Under both treatments blood was sampled via the dorsal aorta catheter after 24 h
exposure to CO2 and 0.5 h later the same fish was anesthetized in the chamber with a 60 mg/L dose
of MS222 that had been pH adjusted using NaOH to the exact pH of the water in each chamber.
Once fish had lost the righting reflex and did not respond to a tail pinch, they were held upside in a
custom built sling within the same chamber with the head completely submerged but the ventral
portion of the operculum just touching the water surface so that the gill ventilator flow could be
easily visualized. Blood was then sampled via caudal puncture with the tail portion just raised
above the water surface. Pairwise t-tests were used to compare acid-base variables (pH, PCO2
and ½HCO3 ) between treatments (control v. 7.5 mmHg), where letters that differ indicate statis-
tically significant differences between CO2 treatments. Pairwise t-tests were used to compare acid-
base variables (pH, PCO2 and ½HCO3 ) between blood sampling techniques (dorsal aorta catheter
v. caudal puncture with gill irrigation) but no statistically significantly differences were observed.
irrigation system is also vital to minimize the restriction of branchial gas

exchange due to gill collapse out of water. As far as possible it is also important
to have a natural flow of water over the gills, while allowing easy access to the
blood vessel puncture site. This procedure is very species-specific, and a clear
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understanding of the branchial anatomy and mode of ventilation is important

to ensure that the entire gill basket is submerged during the process, and to
avoid either under- or over-ventilating the gills.
As an example of how this can work well to obtain valid blood gas and
acid-base values in fish, Fig. 6 shows data from rainbow trout that had the
dorsal aorta cannulated under anesthesia, and then following recovery where
blood was sampled under control conditions and then again after 24 h of expo-
sure to increased PCO2 (1% CO2, 7.5 mmHg, 10,000 μatm) exposure.
Under both treatments blood was sampled first via the cannula and then
30 min later via the caudal vessels using the anesthetic/gill irrigation technique
described above. Blood pH, PCO2 and ½HCO3 were indistinguishable in the
samples taken via these two methods (Fig. 6). This therefore raises the possi-
bility of using the same technique in animals that are difficult or impossible to
cannulate, either due to small size, problematic anatomy, or lack of surgical
experience, training, facilities, or ethical approval.
Finally, from a suitably obtained blood sample, pH is best measured using
either glass pH electrodes (See Heisler, 1989) or fiber optic pH sensors (both
thermostatted at the temperature to which the animal is acclimated). Blood
PCO2 is usually calculated from total CO2 of the plasma (measured using a
Corning CO2 analyzer or Cameron chamber; Cameron, 1971) using the mea-
sured blood pH value and a rearrangement of the Henderson-Hasselbalch
equation (Eq. 2; Boutilier et al., 1984). However, there is increasing interest
in the use of portable clinical analyzers for these measurements, such as the
i-STAT system, largely because of their ease of use. Clinical analyzers have
been designed for human blood, which differs markedly from that of fish,
and thus, they must be validated for the species and environmental conditions
of interest. In studies on trout (Harter and Brauner, 2017) and sand bar shark
(Harter et al., 2015), blood pH values measured with i-STAT were accurate
(with appropriate temperature correction), however, parameters related to
blood gases (O2 and CO2) were not. Values obtained for blood PCO2 levels
of control resting fish were either below detection or highly variable. However,
as blood PCO2 values increased (>10 mmHg), error was reduced. Thus, in sal-
monids at high CO2 (i.e., RAS) blood PCO2 values may be accurate, but in
control fish at low CO2 levels it will not be. In a tropical air-breathing catfish
at high temperature with high blood PCO2 (temperature and CO2 levels more
similar to those of humans for which the i-STAT was developed) the reported
values for PCO2 were deemed accurate (Damsgaard et al., 2015). Until clinical
analyzers are carefully validated for the species and condition in question,
reported values in the literature should be interpreted critically. There are
many published examples of blood pH and blood gas values measured
using clinical analyzer systems that are clearly not representative of in vivo
values, likely due to a combination of the sampling method to obtain blood
(as described above) and the clinical analyzer used for measurement.
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5. INTRACELLULAR pH REGULATION
To this point, we have focused predominantly on how elevated environ-

mental CO2 affects blood acid-base status. However, elevated environmental
CO2 will also affect intracellular acid-base status (Cameron, 1980; Powers,
1930) because CO2 moves rapidly across membranes and dissociates as
described in Eq. (1). The magnitude of the intracellular pH (pHi) depression
will be species and tissue-specific, influenced in part by the buffer value of that
tissue, with compensation and eventual recovery of pHi being dependent on a
range of cellular transporters as discussed below.
5.1. Consequences of Intracellular pH Dysregulation
The consequences of pHi dysregulation may be widespread, as virtually

every biochemical process is pH sensitive. Perturbations in pH can affect vari-
ous biological processes at the level of the cell, tissue, and whole animal. Within
the cell, regulation of pH within a physiologically optimal range is important
for biochemical reactions and the optimal function of transporters, receptors,
structural and contractile proteins, ionic conductivity and metabolic activity
of enzymes and synthesis of macromolecules (Madshus, 1988; Occhipinti and
Boron, 2015; Putnam and Roos, 1997; Roos and Boron, 1981). Additionally,
the cytosol bathes cellular organelles (e.g., mitochondria, endoplasmic reticu-
lum, nucleus), each of which has their own optimal internal pH (Roos and
Boron, 1981); thus cytosolic pHi has an indirect role in the function of these
structures. The CO2 induced increase in H+ will firstly be dampened by intra-
cellular buffers; however, if and when the disturbance is sufficiently severe, pHi
will be reduced. Once this occurs, additional H+ will reduce pH and disrupt
charges of intracellular macromolecules altering their structure and function
(e.g., Moody, 1981; Squirrell et al., 2001).
Uncompensated reductions in pHi affect not only cellular function but
also whole animal function. For example, reduction in brain pH is associated
with loss of equilibrium in common carp (Cyprinus carpio; Yoshikawa et al.,
1994) and reduced muscle pHi was hypothesized to be the cause of post-
exercise mortality in rainbow trout (Wood et al., 1983). Additionally, cardiac
function is believed to be impaired due to failure of pHi recovery leading to
mortality during severe elevations in environmental CO2 in marine fish
despite near-complete pHe recovery (Hayashi et al., 2004; Vaughan-Jones
et al., 2009). Given the consequences of dysregulation, cellular pH is regu-
lated and typically occurs to some degree in tandem with the extracellular
compartment, which has been referred to as “coupled pH regulation”
(Shartau et al., 2016a).
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5.2. Cellular Mechanisms of pH Regulation
All cells have some capacity for pHi regulation during an acid-base distur-
bance (see Section 5) which can be achieved using a range of transporters that
translocate acid-base equivalents across the plasma membrane. This process
can be either active or passive, depending on the opposing electrochemical
gradients (Occhipinti and Boron, 2015). As the membrane potential of the cell
is negative relative to the extracellular fluid, acid extrusion requires energy,
whether that is through the use of an active H+ transporter (e.g., VHA) or
indirectly using the energy from electrogenic transporters such as NKA that
then drive passive transporters (e.g., NHE; Occhipinti and Boron, 2015) as
mentioned above. There are many excellent reviews on cellular acid-base
transporters in a range of biological systems, including pHi regulation in
cancer biology (Parks and Pouyssegur, 2017; Swietach, 2019), vertebrate
cardiomyocyte pH regulation (Vaughan-Jones et al., 2009), renal acid-base
regulation (Boron, 2006; Hamm et al., 2015), pH regulation during mamma-
lian development (Baltz, 1993; FitzHarris and Baltz, 2009), modeling of pHi
regulation (Occhipinti and Boron, 2015), and of specific transporters involved
in pHi regulation (e.g., Na+-coupled HCO3 transporters; Parker and Boron,
2013). As such, this section will only briefly review some of the transporters
involved in pHi regulation, with an emphasis on those identified, or likely
to be involved, in fish.
In general, during an acidosis, net cellular acid excretion to the extracellu-
lar space occurs via extrusion of intracellular H+ or uptake of extracellular
HCO 3 ; the net effect being an increase in pHi. Similar to branchial and
renal mechanisms (Section 3), tissues use various isoforms of acid-transporters
(i.e., NHE, VHA), and base transporters (i.e., AE), which can be independent
or dependent on Na+ transport. Although the specifics of pHi regulation
are complex, with several families of transporters, various isoforms, and
numerous arrangements of these transporters in the cell and among species,
Fig. 7 shows a general schematic of a non-specified cell containing the key
transporters used for pHi regulation in fish. As the cell experiences an acidosis
(e.g., from elevated CO2), these key transporters may be involved in restoring
pHi in conjunction with other ion transporters not indicated on the figure
such as NCC, NKA, NKCC, Na+/Ca2+ exchanger (NCX) (Evans et al.,
2005; Hwang et al., 2011; Kumai and Perry, 2012); of course, not all these
transporters will be present or functioning similarly in all cell types. Along
with those classic acid-base transporters (Fig. 7), a lactate-H+ cotransporter
(monocarboxylate transporter; MCT) has been found to function during hyp-
oxia (where metabolic acidosis typically occurs) to remove intracellular lactate
and H+. The transport of H+ via MCT is facilitated by CA to rapidly provide
the MCT with H+ (Parks and Pouyssegur, 2017; Vaughan-Jones et al., 2009).
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Fig. 7. A simplified schematic of a generalized cell containing the transporters involved with active
pHi regulation in fish. The specific arrangements of transporters will vary among cell types, and
species, but this represents the key acid-base transporters identified in fish. Other transporters are
involved in the maintenance of pH homeostasis and ion regulation, for more in depth review see
Hwang et al. (2011) and Occhipinti et al. (2015). Transporters involved in net removal of acid
(transporters indicated in blue) from the cell includes Cl/HCO3 exchanger (CBE also known
as anion exchanger (AE)), Na+/H+ exchanger (NHE), V-type H+ pump (VHA), and electroneutral
Na/HCO3 (NBC). Transporters involved in net removal of base (transporters indicated in red)
from the cell include anion exchanger (AE) and the electrogenic Na/HCO3 exchanger (NBCe).
Many of these transporters are also found in invertebrates such as crabs

(Fehsenfeld and Weihrauch, 2016), marine worms (P€ ortner et al., 1998),
and cephalopods (Gutowska et al., 2010).
During acid-base disturbances of early-stage mammalian embryos (human,
bovine, mouse and hamster) NHE, AE, and MCT are used (Baltz, 1993;
Erdogan et al., 2005; FitzHarris and Baltz, 2006; Gibb et al., 1997; Lane,
1999; Lane and Bavister, 1999; Phillips et al., 2000; Siyanov and Baltz,
2013; Squirrell et al., 2001; Steeves et al., 2001; Thompson et al., 2006;
Zhao and Baltz, 1996;). In cultured chick heart cells, the Na+-dependent AE
regulates the pHi response to acidification and is involved in the steady-state
maintenance of pHi (Liu et al., 1990). NHE, NBC (acid extruders) and AE
and CHE (acid loaders) have been found in mammalian cardiac cells (rat,
rabbit, and guinea pig) (Vaughan-Jones et al., 2009; Yamamoto et al.,
2005). In mouse hippocampus neurons, AE are involved in pHi regulation
(Salameh et al., 2016), mammalian neuronal pHi regulation has been found
to use NHE, NBC, AE, (reviewed in Ruffin et al., 2014). Additionally, even
maladaptive cells in the form of cancerous tumors demonstrate well-developed
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capacity for pHi regulation using NHEs, MCT, NBC, VHA, AE, and CHE to
safeguard pHi despite extremely acidotic external environments with a pH as
low as six (reviewed in Parks and Pouyssegur, 2017; Reshkin et al., 2014;
Swietach, 2019).
Many of the transporters and enzymes indicated above are also found in
fish and used for pHi regulation. Using teleost hepatocytes, several studies
have demonstrated that NHEs are largely responsible for returning pHi to
steady-state conditions following acidification. Although only a few species
have been investigated, the use of NHE appears to be involved in recovery
following acidosis in rainbow trout (Ahmed, 2006; Furimsky et al., 1999,
2000; Tuominen et al., 2003; Walsh, 1986; Walsh et al., 1988), brown bull head
(Ictalurus nebulosus; Furimsky et al., 1999) and toadfish (Walsh, 1989). In
American eel (Furimsky et al., 1999) and goldfish (Krumschnabel et al.,
2001), NHEs were found to be involved in regulating pHi but not during
recovery from an acid load, which instead appeared to involve AE indicating
species-specific differences in mechanisms used. In rainbow trout and carp
thrombocytes, NHE and NBC is used to regulate pHi following acidification
(Nikinmaa et al., 1999). While, in the kidney and intestine, regulation of acid-
base equivalents has been found to involve AE (Gilmour et al., 2007b; Grosell
et al., 2001).
As mentioned above, all cells have some ability for pHi regulation, but
there are limits to this capacity, and in most cases, pHi recovery is to some
degree linked with pHe recovery (Shartau et al., 2016a; “coupled pH regula-
tion”). However, at very high environmental CO2 levels (see Figs. 3 and 4),
pHe recovery may be very limited. It appears that fish that are able to tolerate
these very high CO2 levels possess an exceptional capacity for pHi regulation:
preferential pHi regulation (Shartau et al., 2016a), which is described in the
following section.
5.3. Preferential Intracellular pH Regulation

In 1982, Heisler (1982) observed that in the tropical facultative air-breathing
fish, the marbled swamp eel (Synbranchus marmoratus), hypoxia-induced air-
breathing resulted in an increase in blood PCO2 to 26 mmHg in a few hours.
Blood pHe fell by 0.6 units but impressively, pHi of white and heart muscle
were tightly regulated through a rapid elevation of intracellular HCO 3 . In
his paper, he remarked, “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.” Heisler
found a similar ability to tightly regulate tissue pHi despite a large reduction in
pHe in salamanders exposed to elevated aquatic CO2 (Heisler et al., 1982) and
later a similar response was observed in the tropical facultative air-breathing
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catfish (Pterygoplichthys pardalis; Brauner et al., 2004b), where pHi of heart,

brain, liver, and muscle were tightly regulated despite reductions in pHe.
This pattern of complete pHi regulation early during exposure to an acid-base
disturbance, despite a pronounced reduction in pHe has been called
“preferential pHi regulation” and differs from other patterns, where a large
reduction in pHe is accompanied by a reduction in pHi (see Brauner and
Baker, 2009) and recovery of pHi is to some degree coupled with recovery of
pHe (coupled pH regulation; Shartau et al., 2016a).
Preferential pHi regulation is defined as ΔpHi/ΔpHe 0 immediately
following the onset of an acid-base disturbance, whereby for a given reduction
in pHe, pHi does not change, or in some cases even increases during early expo-
sure to elevated environmental CO2 despite the reduction of pHe (Baker et al.,
2009; Shartau et al., 2016a). The strategy of preferential pHi regulation was
considered to be rare among vertebrates by Brauner et al. (2004b) and possibly
associated with air-breathing or living in ion-poor waters, such as those of
the Amazon River basin. Several studies have indicated that the temperate,
exclusively water breather, white sturgeon, also uses preferential pHi regula-
tion which appears to be associated with CO2 tolerance (Baker and Brauner,
2012; Baker et al., 2009, 2011; Huynh et al., 2011; Shartau et al., 2017a).
Recent work now suggests that preferential pHi regulation may be a relatively
common strategy, occurring in at least eight species of fish (Shartau and
Brauner, 2014; Shartau et al., 2016a; Fig. 8) that are phylogenetically diverse,
include both water and air breathers and are found in habitats ranging from
temperate to tropical. Just how widespread preferential pHi regulation is
among fish species is clearly an area worthy of further investigation. While
preferential pHi regulation appears to be absent in most tetrapods studied
to date (Fig. 8), recent studies on embryonic reptiles shows use of preferential
pHi regulation during development but a reduced capacity in yearlings, with
no evidence in adults (Shartau et al., 2016b, 2018). Little work has been con-
ducted on embryonic fish, but early-stage zebrafish embryos do possess the
capacity to recover pHi during sustained respiratory acidosis (Molich and
Heisler, 2005). Thus, it has been proposed that preferential pHi regulation
may be an embryonic trait that is either retained or lost in adult vertebrates,
likely influenced by the need to protect pHi in environments subject to severe
acid-base disturbances (Shartau et al., 2016b, 2018, 2019).
The following sections discuss what is known about the cellular
mechanism(s) involved and the functional significance of this pattern of
acid-base regulation in vertebrates.
5.3.1. PUTATIVE MECHANISMS

As the rate at which pHi regulation occurs in preferential pHi regulation is
extremely rapid, the cellular mechanisms involved relative to coupled pH
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Fig. 8. Evolution of the strategy for pH regulation in adult vertebrates when exposed to an acute
(<48 h) respiratory acidosis of >1 kPa blood PCO2 inferred by stochastic character mapping.
Patterns of acid-base regulation for each species were taken from literature (references indicated
by superscript number). Preferential pHi regulation was defined as ΔpHi/ΔpHe 0 immediately
following onset of an acid-base disturbance. Coupled pH regulation was defined as when reductions
in pHe were associated with qualitatively similar reductions in pHi. Pie charts on internal nodes
and branches are color coded for Bayesian posterior probabilities for the strategy for pH regulation.
1 (Baker et al., 2015), 2 (Wood et al., 1990), 3 (Snyder and Nestler, 1991), 4 (Heisler et al., 1982),
5 (Wasser et al., 1991), 6 (Snyder et al., 1995), 7 (Nattie and Edwards, 1981), 8 (Yaksh and
Anderson, 1987), 9 (Wood and Schaefer, 1978), 10 (Malan et al., 1985), 11 (Gonzalez
and Clancy, 1986), 12 (Litt et al., 1985), 13 (Baker et al., 2009), 14 (Shartau et al., 2016a),
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15 (Wood and LeMoigne, 1991), 16 (Larsen et al., 1997), 17 (Heisler, 1982), 18 (Wright et al., 1988),
19 (Brauner et al., 2004), 20 (Sackville et al., 2018). Phylogenetic relationships are based on Hedges
and Kumar (2009) and branch lengths are taken from various references utilizing fossil and molec-
ular estimates of divergence times (Aschliman et al., 2012; Betancur-R et al., 2013, 2015; Blair, 2005;
Macqueen and Johnston, 2014; Meredith et al., 2011; Zhang et al., 2013); the phylogenetic tree was
created using Mesquite (Maddison and Maddison, 2017).
regulation may involve either different or modified transporters from those

discussed above (see Section 5.2). Preferential pHi regulation cannot simply
be explained by exceptional intracellular buffer capacity as that will only
reduce the magnitude of the pHi reduction during elevated PCO2. Preferential
pHi regulation must involve the net transport of acid equivalents across the
plasma membrane into the extracellular fluid that bathes the tissues. Net base
uptake into (or net acid excretion from) tissues is due to cellular transmem-
brane exchange of acid-base relevant ions that are ultimately associated
with an increase in intracellular HCO3 . The specific transporters involved
in preferential pHi regulation are unknown, but both AE and NHE are strong
candidates (Baker et al., 2009). During elevated PCO2, blood pH falls along
the blood buffer line elevating plasma HCO3 levels, thus Cl/HCO3
exchange may be more favorable energetically (Baker and Brauner, 2012).
As whole-body oxygen uptake in white sturgeon is reduced during exposure
to elevated environmental CO2, this provides some support for this idea;
however, further work needs to be done to link whole-body metabolism to
possible reductions in tissue oxygen consumption during preferential pHi
regulation.
There has yet to be a comprehensive investigation into the cellular mech-
anisms of preferential pHi regulation. The ability to regulate pHi in this fash-
ion likely depends on modifications to existing acid-base relevant ion
regulatory transporters discussed above and is an excellent area for investiga-
tion. However, as most studies investigating transporter mechanism use
in vitro methods, this may be challenging to address the questions associated
with the mechanism of preferential pHi regulation as differences have been
observed between in vivo and in vitro pHi regulation in white sturgeon liver.
White sturgeon hepatocytes exposed to elevated PCO2 in vitro experience a
reduction in pHi that was compensated for over the following 96h (Huynh
et al., 2011)—this represents a vastly different response to that observed
in vivo where no reduction in sturgeon liver pHi is observed, and (in some cases)
is overcompensated. This suggests some influence/effect from the extracellular
compartment on preferential pHi regulation; whether it indicates differences in
transporter mechanisms between in vivo and in vitro preparations is unknown
but should be considered.
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5.3.2. FUNCTIONAL SIGNIFICANCE OF PREFERENTIAL PHI REGULATION

The ability to protect pHi against a CO2 induced acidosis is likely impor-
tant for fish that inhabit high and fluctuating CO2 environments due to puta-
tive limits on pHe compensation (discussed above in Section 3). An extreme
example of the putative benefit of preferential pHi regulation is demonstrated
by tambaqui exposed to a PCO2 of 150 mmHg (20 kPa) which completely
regulated heart, brain and liver pHi despite a nearly 1 pH unit reduction in
pHe. If tambaqui required even partial pHe recovery to regulate pHi, an
increase of plasma HCO–3 of over 150 mM would be required (Fig. 4B); this
is physiologically improbable (Shartau, 2017). Additionally, as some high
CO2 environments are ion-poor and low pH (e.g., Rio Negro), preferential
pHi regulation may allow for pHi to be protected in conditions unfavorable
for pHe compensation (Brauner et al., 2004b; Sackville et al., 2018).
Retention of normal, or near normal, physiological performance may be
achieved by protecting pHi. Of particular importance is cardiomyocyte pHi,
which if reduced pHi would negatively affect cardiac function (Vaughan-
Jones et al., 2009). Thus minimizing pHi disturbance could allow animals
to maintain cardiac performance. Indeed, the armored catfish and white
sturgeon tightly control heart pHi during elevated environmental CO2 despite
severe pHe reduction (Baker et al., 2009; Brauner et al., 2004b), yet largely
maintain cardiac function (Baker et al., 2011; Hanson et al., 2009).
Finally, numerous air-breathing fish use preferential pHi regulation, these
fish may be subjected to respiratory acidosis due to buildup of metabolically
produced CO2 arising from the physiochemical differences between water and
air during air-breathing episodes (Bayley et al., 2019; Graham, 1997; Shartau
and Brauner, 2014), and by inhabiting environments prone to hypoxia and
elevated PCO2 (Ultsch, 1987). While preferential pHi regulation is not exclu-
sively associated with air-breathing fish (Shartau, 2017), it may represent
an exaptation for air-breathing (Brauner and Baker, 2009; Shartau et al.,
2016a). Additionally, preferential pHi regulation has been hypothesized to
play a role in the terrestrialization of early tetrapods. Transitioning from an
aquatic water breather to a terrestrial air-breather involved changes in blood
acid-base status as the former have low PCO2, low plasma ½HCO3 , and high
pHe, while the latter have high PCO2, high plasma ½HCO3 , and low pHe
(Bayley et al., 2019; Randall et al., 1981; Ultsch, 1996). Acid-base regulation
between the two differs in that water breathers rely on physicochemical buff-
ering and net transport of acid-base equivalents, while air breathers depend
mainly on changes in ventilation rate to alter PCO2 and thus pH (Brauner
and Baker, 2009; Heisler, 1986; Truchot, 1987). It is unknown exactly how
early vertebrates made this transition, but preferential pHi regulation is
hypothesized to have been involved to minimize the effects of an air-breathing
induced respiratory acidosis (Brauner and Baker, 2009; Shartau and Brauner,
2014; Shartau et al., 2016a); this remains to be investigated further.
ARTICLE IN PRESS
6. CONCLUSIONS AND FUTURE DIRECTIONS
Over the last half a century, a great deal has been learned about CO2 trans-
port and excretion in fish, and there are clearly similarities and differences
between fish groups. Over this same time frame, a considerable amount has been
learned about how elevated environmental CO2 affects blood acid-base status
in fish and the role of the gill, kidney and gastrointestinal systems in acid-base
regulation and compensation during exposure to elevated CO2. This research
was largely conducted on a relatively few number of model species, and initially,
to understand the basic physiology of fish. This information has formed an
invaluable foundation from which to investigate the more applied aspects of
environmental CO2 on fish; ranging from those due to climate change, to those
associated with intensive rearing of fish at high densities to provide protein for
an ever-growing population, and those to increase understanding of how fish
deal with natural spatial and temporal variation in marine and freshwater
CO2 levels. While a great deal has been learned on these topics in the last decade,
as discussed in this chapter, the following questions represent large knowledge
gaps that are timely and topical for further investigation:
(1) Acid-base regulation is ultimately driven by active ionoregulation,

which has a metabolic cost, but what is the metabolic cost of acid-base
regulation in fish?
(2) What is the relationship between water pH and water ion composition
on the dynamics of acid-base regulation in fish?
(3) How does exercise intensity or fish activity level affect the rate and com-
pleteness of acid-base regulation during elevated environmental CO2?
(4) Most of the studies on kidney tubular HCO3 reabsorption have been
conducted at relatively high CO2 levels. What is the role of the kidney
under climate change relevant CO2 levels?
(5) What is the relative role of gill H+ excretion relative to HCO3 uptake
from the water in fish? And how widespread are the morphological
changes at the gills (reduction in MRC fractional surface area) during
exposure to elevated CO2 in freshwater fish and is this pattern observed
in seawater fish?
(6) What is the effect of climate change relevant CO2 increases in freshwater
fish? While 40% of all fish species are freshwater, very few have been
investigated to date.
(7) What is the effect of oscillating CO2 tensions that naturally occur spa-
tially and temporally in both marine and freshwater systems that fish
inhabit? Depending upon the metabolic cost of acid-base regulation
(point 1 above), oscillations could have far greater physiological effects
than constant CO2 tensions.
ARTICLE IN PRESS
(8) Are low blood PCO2 values in larval and juvenile fish the basis for their
increased sensitivity to OA relative to adult fishes?
(9) How widespread is preferential pHi regulation among fish species? And
does it represent an embryonic trait in vertebrates that is then retained
or lost during development?
(10) What are the cellular mechanisms associated with preferential pHi
regulation?
REFERENCES
Ahmed, K.H., 2006. Signalling pathways involved in hypertonicity- and acidification-induced

activation of Na+/H+ exchange in trout hepatocytes. J. Exp. Biol. 209, 3101–3113.
Alderman, S.L., Harter, T.S., Wilson, J.M., Supuran, C.T., Farrell, A.P., Brauner, C.J., 2016.
Evidence for a plasma-accessible carbonic anhydrase in the lumen of salmon heart that
may enhance oxygen delivery to the myocardium. J. Exp. Biol. 219, 719–724.
Alin, S.R., Rasera, M.F.F.L., Salimon, C.I., Richey, J.E., Holtgrieve, G.W., Krusche, A.V.,
Snidvongs, A., 2011. Physical controls on carbon dioxide transfer velocity and flux in
low-gradient river systems and implications for regional carbon budgets. J. Geophys. Res.
Biogeosci. 116, G01009.
Allmon, E.B., Esbaugh, A.J., 2017. Carbon dioxide induced plasticity of branchial acid-base
pathways in an estuarine teleost. Sci. Rep. 7, 45680.
Aranda, V., Martinez, I., Melero, S., Lecanda, J., Banales, J.M., Prieto, J., Medina, J.F., 2004.
Shared apical sorting of anion exchanger isoforms AE2a, AE2b1, and AE2b2 in primary
hepatocytes. Biochem. Biophys. Res. Commun. 319, 1040–1046.
Aschliman, N.C., Nishida, M., Miya, M., Inoue, J.G., 2012. Body plan convergence in the
evolution of skates and rays (Chondrichthyes: Batoidea). Mol. Phylogenet. Evol. 63, 28–42.
Aufdenkampe, A.K., Mayorga, E., Raymond, P.A., Melack, J.M., Doney, S.C., Alin, S.R.,
Aalto, R.E., Yoo, K., 2011. Riverine coupling of biogeochemical cycles between land, oceans,
and atmosphere. Front. Ecol. Environ. 9, 53–60.
Axelsson, M., Fritsche, R., 1994. Cannulation techniques. In: Hochachka, P.W., Mommsen, T.
(Eds.), Biochemistry and Biology of Fishes. Elesvier Science, pp. 17–36.
Baker, D.W., Brauner, C.J., 2012. Metabolic changes associated with acid–base regulation during
hypercarbia in the CO2-tolerant chondrostean, white sturgeon (Acipenser transmontanus).
Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 161, 61–68.
Baker, D.W., Matey, V., Huynh, K.T., Wilson, J.M., Morgan, J.D., Brauner, C.J., 2009.
Complete intracellular pH protection during extracellular pH depression is associated with
hypercarbia tolerance in white sturgeon, Acipenser transmontanus. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 296, R1868–R1880.
Baker, D.W., Hanson, L.M., Farrell, A.P., Brauner, C.J., 2011. Exceptional CO2 tolerance in
white sturgeon (Acipenser transmontanus) is associated with protection of maximum cardiac
performance during hypercapnia in situ. Physiol. Biochem. Zool. 84, 239–248.
Baker, D.W., Sardella, B., Rummer, J.L., Sackville, M., Brauner, C.J., 2015. Hagfish: champions
of CO2 tolerance question the origins of vertebrate gill function. Sci. Rep. 5, 11182.
Baltz, J.M., 1993. Intracellular pH regulation in the early embryo. Bioessays 15, 523–530.
Baumann, H., 2019. Experimental assessments of marine species sensitivities to ocean acidifica-
tion and co-stressors: how far have we come? Can. J. Zool. 97, 399–408.
Baumann, H., Wallace, R.B., Tagliaferri, T., Gobler, C.J., 2015. Large natural pH, CO2 and O2
fluctuations in a temperate tidal salt marsh on diel, seasonal, and interannual time scales.
Estuar. Coasts 38, 220–231.
ARTICLE IN PRESS
Bayaa, M., Vulesevic, B., Esbaugh, A., Braun, M., Ekker, M.E., Grosell, M., Perry, S.F., 2009.
The involvement of SLC26 anion transporters in chloride uptake in zebrafish (Danio rerio)
larvae. J. Exp. Biol. 212, 3283–3295.
Bayley, M., Damsgaard, C., Thomsen, M., Malte, H., Wang, T., 2019. Learning to air-breathe:
the first steps. Physiology 34, 14–29.
Berenbrink, M., Koldkjaer, P., Kepp, O., Cossins, A.R., 2005. Evolution and oxygen secretion in
fishes and the emergence of a complex physiological system. Science 307, 1752–1757.
Betancur-R, R., Broughton, R.E., Wiley, E.O., Carpenter, K., Lopez, J.A., Li, C., Holcroft, N.I.,
Arcila, D., Sanciangco, M., Cureton II, J.C., Zhang, F., Buser, T., Campbell, M.A.,
Ballesteros, J.A., Roa-Varon, A., Willis, S., Borden, W.C., Rowley, T., Reneau, P.C.,
Hough, D.J., Lu, G., Grande, T., Arratia, G., Orti, G., 2013. The tree of life and a new
classification of bony fishes. PLoS Curr. 5.
Betancur-R, R., Orti, G., Pyron, R.A., 2015. Fossil-based comparative analyses reveal ancient
marine ancestry erased by extinction in ray-finned fishes. Ecol. Lett. 18, 441–450.
Blair, J.E., 2005. Molecular phylogeny and divergence times of deuterostome animals. Mol. Biol.
Evol. 22, 2275–2284.
Blancheton, J.P., 2000. Developments in recirculation systems for Mediterranean fish species.
Aquacult. Eng. 22, 17–31.
Blinn, D.W., Sanderson, M.W., 1989. Aquatic insects in Montezuma well, Arizona, USA:
a travertine spring mound with high alkalinity and dissolved carbon dioxide. West. N.
Am. Nat. 49, 85–88.
Boisen, A.M.Z., Amstrup, J., Novak, I., Grosell, M., 2003. Sodium and chloride transport in
soft water and hard water acclimated zebrafish (Danie rerio). Biochim. Biophys. Acta
1618, 207–218.
Boron, W.F., 2006. Acid-base transport by the renal proximal tubule. J. Am. Soc. Nephrol.
17, 2368–2382.
Boutilier, R.G., Heming, T.A., Iwama, G.K., 1984. Appendix: physicochemical parameters
for use in fish respiratory physiology. In: Hoar, W.S., Randall, D.J. (Eds.), Fish Physiology.
In: vol. XA. Academic Press, New York, pp. 403–430.
Brauner, C.J., Baker, D.W., 2009. Patterns of acid–base regulation during exposure to hyper-
carbia in fishes. In: Glass, M.L., Wood, S.C. (Eds.), Cardio-Respiratory Control in Verte-
brates. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 43–63.
Brauner, C.J., Randall, D.J., 1998. The linkage between oxygen and carbon dioxide transport.
In: Perry, S.F., Tufts, B.L. (Eds.), Fish Physiology. Fish Respiration, vol. 17. Academic
Press, Toronto, pp. 283–319.
Brauner, C.J., Thorarensen, H., Gallaugher, P., Farrell, A.P., Randall, D.J., 2000. CO2 transport
and excretion in rainbow trout (Oncorhynchus mykiss) during graded sustained exercise.
Respir. Physiol. 119, 69–82.
Brauner, C.J., Matey, V., Wilson, J.M., Bernier, N.J., Val, A.L., 2004a. Transition in organ
function during the evolution of air-breathing; insights from Arapaima gigas, an obligate
air-breathing teleost from the Amazon. J. Exp. Biol. 207, 1433–1438.
Brauner, C.J., Wang, T., Wang, Y., Richards, J.G., Gonzalez, R.J., Bernier, N.J., Xi, W.,
Patrick, A., Va, A.L., 2004b. Limited extracellular but complete intracellular acid-base
regulation during short-term environmental hypercapnia in the armoured catfish, Liposarcus
pardalis. J. Exp. Biol. 207, 3381–3390.
Brix, K.V., Grosell, M., 2012. Comparative characterization of Na+ transport in Cyprinodon
variegatus variegatus and Cyprinodon variegatus hubbsi: a model species complex for studying
teleost invasion of freshwater. J. Exp. Biol. 215, 1199–1209.
Bury, N.R., Wood, C.M., 1999. Mechanisms of branchial apical silver uptake by rainbow trout is
via the proton coupled Na+ channel. Am. J. Physiol. 277, R1385–R1391.
ARTICLE IN PRESS
Cai, W.-J., Hu, X., Huang, W.-J., Murrell, M.C., Lehrter, J.C., Lohrenz, S.E., Chou, W.-C.,
Zhai, W., Hollibaugh, J.T., Wang, Y., Zhao, P., Xianghui, G., Gundersen, K., Dai, M.,
Gong, G.-C., 2011. Acidification of subsurface coastal waters enhanced by eutrophication.
Nat. Geosci. 4, 766–770.
Caldeira, K., Wickett, M.E., 2003. Anthropogenic carbon and ocean pH. Nature 425, 365.
Cameron, J., 1971. A rapid method for determination of total carbon dioxide in small blood
samples. J. Appl. Physiol. 31, 632–634.
Cameron, J.N., 1978. Regulation of blood pH in teleost fish. Respir. Physiol. 33, 129–144.
Cameron, J.N., 1980. Body fluid pools, kidney function, and acid-base regulation in the freshwater
catfish Ictalurus punctatus. J. Exp. Biol. 86, 171–185.
Cameron, J.N., Iwama, G.K., 1987. Compensation of progressive hypercapnia in channel catfish
and blue crabs. J. Exp. Biol. 133, 183–197.
Cameron, J.N., Polhemus, J.A., 1974. Theory of CO2 exchange in trout gills. J. Exp. Biol.
60, 183–191.
Chen, L.M., Zhao, J., Musa-Aziz, R., Pelletier, M.F., Drummond, I.A., Boron, W.F., 2010.
Cloning and characterization of a zebrafish homologue of human AQP1: a bifunctional water
and gas channel. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, R1163–R1174.
Christianson, J., Douglas, C.G., Haldane, J.S., 1914. The absorption and dissociation of carbon
dioxide by human blood. J. Physiol. 48, 244–277.
Claiborne, J.B., 1998. Acid-base regulation. In: Evans, D.H. (Ed.), The Physiology of Fishes.
Boca Raton, CRC Press, pp. 171–198.
Claiborne, J.B., Heisler, N., 1986. Acid-base regulation and ion transfers in the carp (Cyprinus-
Carpio)—pH compensation during graded long-term and short-term environmental
hypercapnia, and the effect of bicarbonate infusion. J. Exp. Biol. 126, 41–61.
Claiborne, J.B., Edwards, S.L., Morrison-Shetlar, A.I., 2002. Acid-base regulation in fishes:
cellular and molecular mechanisms. J. Exp. Zool. 293, 302–319.
Cloern, J.E., Abreu, P.C., Carstensen, J., Chauvaud, L., Elmgren, R., Grall, J., Greening, H.,
Johansson, J.O.R., Kahru, M., Sherwood, E.T., Xu, J., Yin, K., 2016. Human activities
and climate variability drive fast-paced change across the world’s estuarine–coastal ecosys-
tems. Glob. Change Biol. 22, 513–529.
Cole, J., Caraco, N., 2001. Carbon in catchments: connecting terrestrial carbon losses with aquatic
metabolism. Mar. Freshw. Res. 52, 101–110.
Cooper, G.J., Boron, W.F., 1998. Effect of PCMBS on CO2 permeability of Xenopus oocytes
expressing aquaporin 1 or its C189S mutant. Am. J. Physiol. 275, C1481–C1486.
Cooper, C.A., Whittamore, J.M., Wilson, R.W., 2010. Ca2+-driven intestinal HCO 3 secretion
and CaCO3 precipitation in the European flounder in vivo: influences on acid-base
regulation and blood gas transport. Am. J. Physiol. Regul. Integr. Comp. Physiol. 298,
R870–R876.
Currie, S., Kieffer, J.D., Tufts, B.L., 1995. The effects of blood CO2 reaction rates on CO2 removal
from muscle in exercised trout. Respir. Physiol. 100, 261–269.
Damsgaard, C., Gam, L.T.H., Tuong, D.D., Thinh, P.V., Thanh, D.T.H., Wang, T., Bayley, M.,
2015. High capacity for extracellular acid–base regulation in the air-breathing fish
Pangasianodon hypophthalmus. J. Exp. Biol. 218, 1290–1294.
Davenport, H.W., 1974. The ABC of Acid-Base Chemistry, sixth ed. The University of Chicago
Press, Chicago.
Deigweiher, K., Koschnick, N., Portner, H.O., Lucassen, M., 2008. Acclimation of ion regulatory
capacities in gills of marine fish under environmental hypercapnia. Am. J. Physiol. Regul.
Demars, B.O.L., Tremolières, M., 2009. Aquatic macrophytes as bioindicators of carbon dioxide
in groundwater fed rivers. Sci. Total Environ. 407, 4752–4763.
ARTICLE IN PRESS
Desforges, P.R., Gilmour, K., Perry II, S.F., 2001. The effects of exogenous extracellular carbonic
anhydrase on CO2 excretion in rainbow trout (Oncorhynchus mykiss): role of plasma buffering
capacity. J. Comp. Physiol. B 171, 465–473.
Desforges, P.R., Harman, S.S., Gilmour, K.M., Perry, S.F., 2002. Sensitivity of CO2 excretion to
blood flow changes in trout is determined by carbonic anhydrase availability. Am. J. Physiol.
Regul. Integr. Comp. Physiol. 282, R501–R508.
Doney, S.C., Fabry, V.J., Feely, R.A., Kleypas, J.A., 2009. Ocean acidification: the other CO2
problem. Ann. Rev. Mar. Sci. 1, 169–192.
Dymowska, A.K., Hwang, P.-P., Goss, G.G., 2012. Structure and function of ionocytes in the
freshwater fish gill. Respir. Physiol. Neurobiol. 184, 282–292.
Edwards, S.L., Wall, B.P., Morrison-Shetlar, A., Sligh, S., Weakley, J.C., Claiborne, J.B., 2005.
The effect of environmental hypercapnia and salinity on the expression of NHE-like isoforms
in the gills of a euryhaline fish (Fundulus heteroclitus). J. Exp. Zool. A Comp. Exp. Biol.
303A, 464–475.
Edwards, S.L., Weakley, J.C., Diamanduros, A.W., Claiborne, J.B., 2010. Molecular identifica-
tion of Na+-H+ exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus
heteroclitus. J. Fish Biol. 76, 415–426.
Ellis, R.P., Urbina, M.A., Wilson, R.W., 2017. Lessons from two high CO2 worlds—future oceans
and intensive aquaculture. Glob. Chang. Biol. 23, 2141–2148.
Ellory, J.C., Wolowyk, M.W., Young, J.D., 1987. Hagfish (Eptatretus stouti) erythrocytes show
minimal chloride transport activity. J. Exp. Biol. 129, 377–383.
Endeward, V., Cartron, J.P., Ripoche, P., Gros, G., 2008. RhAG protein of the Rhesus complex is
a CO2 channel in the human red cell membrane. FASEB J. 22, 64–73.
Erdogan, S., FitzHarris, G., Tartia, A.P., Baltz, J.M., 2005. Mechanisms regulating intracellular
pH are activated during growth of the mouse oocyte coincident with acquisition of meiotic
competence. Dev. Biol. 286, 352–360.
Ern, R., Esbaugh, A.J., 2016. Hyperventilation and blood acid-base balance in hypercapnia
exposed red drum (Sciaenops ocellatus). J. Comp. Physiol. B 186 (4), 447–460.
Esbaugh, A.J., Tufts, B.L., 2004. Evidence for a membrane-bound carbonic anhydrase in the
heart of an ancient vertebrate, the sea lamprey (Petromyzon marinus). J. Comp. Physiol.
B, Biochem. Syst. Environ. Physiol. 174, 399–406.
Esbaugh, A.J., Tufts, B.L., 2006a. The structure and function of carbonic anhydrase isozymes in
the respiratory system of vertebrates. Respir. Physiol. Neurobiol. 154, 185–198.
Esbaugh, A.J., Tufts, B.L., 2006b. Evidence of a high activity carbonic anhydrase isozyme in the
red blood cells of an ancient vertebrate, the sea lamprey (Petromyzon marinus). J. Exp. Biol.
209, 1169–1178.
Esbaugh, A.J., Lund, S.G., Tufts, B.L., 2004. Comparative physiology and molecular analysis of
carbonic anhyrdase from the red blood cells of teleost fish. J. Comp. Physiol. B, Biochem.
Syst. Environ. Physiol. 174, 429–438.
Esbaugh, A.J., Perry, S.F., Bayaa, M., Georgalis, T., Nickerson, J., Tufts, B.L., Gilmour, K.M.,
2005. Cytoplasmic carbonic anhydrase isozymes in rainbow trout Oncorhynchus mykiss:
comparative physiology and molecular evolution. J. Exp. Biol. 208, 1951–1961.
Esbaugh, A.J., Perry, S.F., Gilmour, K.M., 2009a. Hypoxia-inducible carbonic anhydrase IX
expression is insufficient to alleviate intracellular metabolic acidosis in the muscle of
zebrafish, Danio rerio. Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, R150–R160.
Esbaugh, A.J., Gilmour, K.M., Perry, S.F., 2009b. Membrane-associated carbonic anhydrase in
the respiratory system of the Pacific hagfish (Eptatretus stouti). Respir. Physiol. Neurobiol.
166, 107–116.
Esbaugh, A.J., Heuer, R., Grosell, M., 2012. Impacts of ocean acidification on respiratory gas
exchange and acid-base balance in a marine teleost, Opsanus beta. J. Comp. Physiol. B, Bio-
chem. Syst. Environ. Physiol. 182, 921–934.
ARTICLE IN PRESS
Esbaugh, A.J., Ern, R., Nordi, W.M., Johnson, A.S., 2016. Respiratory plasticity is insufficient to
alleviate blood acid-base disturbances after acclimation to ocean acidification in the estuarine
red drum, Sciaenops ocellatus. J. Comp. Physiol. B 186, 97–109.
Evans, D.H., Piermarini, P.M., Choe, K.P., 2005. The multifunctional fish gill: dominant site
of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste.
Physiol. Rev. 85, 97–177.
Farrell, A.P., 2006. Bulk oxygen uptake measured with over 60,000 kg of adult salmon during
live-haul transportation at sea. Aquaculture 254, 646–652.
Feely, R.A., Sabine, C.L., Hernandez-Ayon, J.M., Ianson, D., Hales, B., 2008. Evidence for
upwelling of corrosive “acidified” water onto the continental shelf. Science 320, 1490–1492.
Fehsenfeld, S., Weihrauch, D., 2016. Mechanisms of acid–base regulation in seawater-acclimated
green crabs (Carcinus maenas). Can. J. Zool. 94, 95–107.
Ferguson, R.A., Sehdev, N., Bagatto, B., Tufts, B.L., 1992. In vitro interactions between oxygen
and carbon dioxide transport in the blood of the sea lamprey (Petromyzon marinus). J. Exp.
Biol. 173, 25–41.
FitzHarris, G., Baltz, J.M., 2006. Granulosa cells regulate intracellular pH of the murine growing
oocyte via gap junctions: development of independent homeostasis during oocyte growth.
Development 133, 591–599.
FitzHarris, G., Baltz, J.M., 2009. Regulation of intracellular pH during oocyte growth and
maturation in mammals. Reproduction 138, 619–627.
Fivelstad, S., 2013. Long-term carbon dioxide experiments with salmonids. Aquacult. Eng.
53, 40–48.
Fivelstad, S., Haavik, H., Løvik, G., Olsen, A.B., 1998. Sublethal effects and safe levels of carbon
dioxide in seawater for Atlantic salmon postsmolts (Salmo salar L.): ion regulation and
growth. Aquaculture 160, 305–316.
Fivelstad, S., Olsen, A.B., Kløften, H., Ski, H., Stefansson, S., 1999. Effects of carbon dioxide on
Atlantic salmon (Salmo salar L.) smolts at constant pH in bicarbonate rich freshwater.
Aquaculture 178, 171–187.
Fivelstad, S., Olsen, A.B., Åsgård, T., Baeverfjord, G., Rasmussen, T., Vindheim, T.,
Stefansson, S., 2003a. Long-term sublethal effects of carbon dioxide on Atlantic salmon
smolts (Salmo salar L.): ion regulation, haematology, element composition, nephrocalcinosis
and growth parameters. Aquaculture 215, 301–319.
Fivelstad, S., Waagbø, R., Zeitz, S.F., Hosfeld, A.C.D., Olsen, A.B., Stefansson, S., 2003b.
A major water quality problem in smolt farms: combined effects of carbon dioxide, reduced
pH and aluminium on Atlantic salmon (Salmo salar L.) smolts: physiology and growth.
Aquaculture 215, 339–357.
Fivelstad, S., Kvamme, K., Handeland, S., Fivelstad, M., Olsen, A.B., Hosfeld, C.D., 2015.
Growth and physiological models for Atlantic salmon (Salmo salar L.) parr exposed to
elevated carbon dioxide concentrations at high temperature. Aquaculture 436, 90–94.
Frommel, A.Y., Maneja, R., Lowe, D., Malzahn, A.M., Geffen, A.J., Folkvord, A.,
Piatkowski, U., Reusch, T.B.H., Clemmesen, C., 2012. Severe tissue damage in Atlantic
cod larvae under increasing ocean acidification. Nat. Clim. Chang. 2, 42–46.
Frommel, A.Y., Maneja, R., Lowe, D., Pascoe, C.K., Geffen, A.J., Folkvord, A., Piatkowski, U.,
Clemmesen, C., 2014. Organ damage in Atlantic herring larvae as a result of ocean acidifi-
cation. Ecol. Appl. 24, 1131–1143.
Frommel, A.Y., Margulies, D., Wexler, J.B., Stein, M.S., Scholey, V.P., Williamson, J.E.,
Bromhead, D., Nicol, S., Havenhand, J., 2016. Ocean acidification has lethal and sub-lethal
effects on larval development of yellowfin tuna, Thunnus albacares. J. Exp. Mar. Biol. Ecol.
482, 18–24.
Furch, K., Junk, W.J., 1997. Physicochemical conditions in the floodplains. In: The Central
Amazon Floodplain. Springer, Berlin, Heidelberg, Berlin, Heidelberg, pp. 69–108.
ARTICLE IN PRESS
Furimsky, M., Moon, T.W., Perry, S.F., 1999. Intracellular pH regulation in hepatocytes isolated
from three teleost species. J. Exp. Zool. 284, 361–367.
Furimsky, M., Moon, T.W., Perry, S.F., 2000. Evidence for the role of a Na+/HCO3
cotransporter in trout hepatocyte pHi regulation. J. Exp. Biol. 203, 2201–2208.
Geers, C., Gros, G., 2000. Carbon dioxide transport and carbonic anhydrase in blood and muscle.
Physiol. Rev. 80, 681–715.
Genz, J., Taylor, J.R., Grosell, M., 2008. Effects of salinity on intestinal bicarbonate secretion and
compensatory regulation of acid-base balance in Opsanus beta. J. Exp. Biol. 211, 2327–2335.
Genz, J., McDonald, D.M., Grosell, M., 2011. Concentration of MgSO4 in the intestinal lumen of
Opsanus beta limits osmoregulation in response to acute hypersalinity stress. Am. J. Physiol.
300, R895–R909.
Georgalis, T., Perry, S.F., Gilmour, K.M., 2006a. The role of branchial carbonic anhydrase in
acid-base regulation in rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 209, 518–530.
Georgalis, T., Perry, S.F., Gilmour, K.M., 2006b. Roles of cytosolic and membrane-bound
carbonic anhydrase in renal control of acid-base balance in rainbow trout, Oncorhynchus
mykiss. J. Exp. Biol. 209, 518–530.
Gervais, M.R., Tufts, B.L., 1998. Evidence for membrane-bound carbonic anhydrase in the
air bladder of bowfin (Amia calva), a primitive air-breathing fish. J. Exp. Biol.
201, 2205–2212.
Geyer, R.R., Parker, M.D., Toye, A.M., Boron, W.F., Musa-Aziz, R., 2013. Relative CO2/NH3
permeabilities of human RhAG, RhBG and RhCG. J. Membr. Biol. 246, 915–926.
Gibb, C.A., Poronnik, P., Day, M.L., Cook, D.I., 1997. Control of cytosolic pH in two-cell mouse
embryos: roles of H+-lactate cotransport and Na+/H+ exchange. Am. J. Physiol. Cell Physiol.
273, C404–C419.
Gilmour, K.M., 2001. The CO2/pH ventilatory drive in fish. Comp. Biochem. Physiol. A
130, 219–240.
Gilmour, K.M., Perry, S.F., 2004. Branchial membrane-associated carbonic anhydrase activity
maintains CO2 excretion in severely anemic dogfish. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 286, R1138–R1148.
Gilmour, K.M., Perry, S.F., 2009a. Carbonic anhydrase and acid-base regulation in fish. J. Exp.
Biol. 212, 1647–1661.
Gilmour, K.M., Perry, S.F., 2009b. Gas transfer in dogfish: a unique model of CO2 excretion.
Comp. Biochem. Physiol. A Mol. Integr. Physiol. 155, 476–485.
Gilmour, K.M., Perry, S.F., Bernier, N.J., Henry, R.P., Wood, C.M., 2001. Extracellular
carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion. Physiol. Bio-
chem. Zool. 74, 477–492.
Gilmour, K.M., Shah, B., Szebedinszky, C., 2002. An investigation of carbonic anhydrase activity
in the gills and blood plasma of brown bullhead (Ameiurus nebulosus), longnose skate
(Raja rhina), and spotted raffish (Hydrolagus colliei). J. Comp. Physiol. B. Biochem. Syst.
Environ. Physiol. 172, 77–86.
Gilmour, K.M., Bayaa, M., Kenney, L., McNeill, B., Perry, S.F., 2007a. Type IV carbonic
anhydrase is present in the gills of spiny dogfish (Squalus acanthias). Am. J. Physiol. Regul.
Gilmour, K.M., Euverman, R.M., Esbaugh, A.J., Kenney, L., Chew, S.F., Ip, Y.K., Perry, S.F.,
2007b. Mechanisms of acid-base regulation in the African lungfish Protopterus annectens.
J. Exp. Biol. 210, 1944–1959.
Golub, M., 2016. Controls on Temporal Variation in Ecosystem-Atmosphere CO2 Exchange in
Lakes and Reservoirs. PhD thesis. University of Wisconsin-Madison.
Gonzalez, N.C., Clancy, R.L., 1986. Intracellular pH regulation during prolonged hypoxia in rats.
Respir. Physiol. 65, 331–339.
ARTICLE IN PRESS
Good, C., Davidson, J., Welsh, C., Snekvik, K., Summerfelt, S., 2010. The effects of carbon
dioxide on performance and histopathology of rainbow trout Oncorhynchus mykiss in water
recirculation aquaculture systems. Aquacult. Eng. 42, 51–56.
Good, C., Davidson, J., Terjesen, B.F., Takle, H., Kolarevic, J., Bæverfjord, G., Summerfelt, S.,
2018. The effects of long-term 20 mg/L carbon dioxide exposure on the health and perfor-
mance of Atlantic salmon Salmo salar post-smolts in water recirculation aquaculture systems.
Aquacult. Eng. 81, 1–9.
Goss, G.G., Perry, S.F., 1993. Physiological and morphological regulation of acid-base status
during hypercapnia in rainbow trout (Oncorhynchus mykiss). Can. J. Zool. 71, 1673–1680.
Goss, G.G., Laurent, P., Perry, S.F., 1992. Evidence for a morphological component in acid-base
regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus).
Cell Tissue Res. 268, 539–552.
Graham, J.B., 1997. Air Breathing Fishes: Evolution, Diversity, and Adaptation. Academic press,
San Diego, CA.
Green, L., Jutfelt, F., 2014. Elevated carbon dioxide alters the plasma composition and behaviour
of a shark. Biol. Lett. 10 (9), pii: 20140538.
Grosell, M., 2006. Intestinal anion exchange in marine fish osmoregulation. J. Exp. Biol.
209, 2813–2827.
Grosell, M., 2011. The role of the gastrointestinal tract in salt and water balance. Fish Physiol.
30, 135–164.
Grosell, M., 2019. CO2 and calcification processes in fish. In: Grosell, M., Munday, P.L.,
Farrell, A.P., Brauner, C.J. (Eds.), Carbon Dioxide. Fish Physiology, vol. 37. Academic
Press, San Diego.
Grosell, M., De Boeck, G., Johannsson, O., Wood, C.M. The effects of silver on intestinal ion and
acid-base regulation in the marine teleost fish, Parophrys vetulus. Comparative Biochemistry
and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 124, 1999, 259–270.
Grosell, M., Laliberte, C.N., Wood, S., Jensen, F.B., Wood, C.M., 2001. Intestinal HCO3
secretion in marine teleost fish: evidence for an apical rather than a basolateral
Cl/HCO3 exchanger. Fish Physiol. Biochem. 2, 81–95.
Grosell, M., Mager, E.M., Williams, C., Taylor, J.R., 2009. High rates of HCO 3 secretion and cl

absorption against adverse gradients in the marine teleost intestine: the involvement of an
electrogenic anion exchanger and H+-pump metabolon? J. Exp. Biol. 212, 1684–1696.
Guffey, S.C., Fliegel, L., Goss, G.G., 2015. Cloning and characterization of Na+/H+ exchanger
isoforms NHE2 and NHE3 from the gill of Pacific dogfish Squalus suckleyi. Comp. Biochem.
Physiol. B Biochem. Mol. Biol. 188, 46–53.
Gutowska, M., Melzner, F., Langenbuch, M., Bock, C., Claireaux, G., P€ ortner, H., 2010.
Acid–base regulatory ability of the cephalopod (Sepia officinalis) in response to environmen-
tal hypercapnia. J. Comp. Physiol. B, Biochem. Syst. Environ. Physiol. 180, 323–335.
Hamm, L.L., Nakhoul, N., Hering-Smith, K.S., 2015. 3. Clin. J. Am. Soc. Nephrol. 10, 2232–2242.
Hanson, L.M., Baker, D.W., Kuchel, L.J., Farrell, A.P., Val, A.L., Brauner, C.J., 2009. Intrinsic
mechanical properties of the perfused armoured catfish heart with special reference to the effects
of hypercapnic acidosis on maximum cardiac performance. J. Exp. Biol. 212, 1270–1276.
Harter T.S., Brauner C.J., 2017. The O2 and CO2 transport system in teleosts and the specialized mech-
anisms to enhance Hb-O2 unloading to tissues, In: Gamperl A.K., Gillis T.E., Farrell A.P., Braune
C.J., (Volume Editors), Fish Physiology Volume 36B “Phenotypic and Physiological Responses”,
Farrell A.P., Brauner C.J., (Series Editors), Elsevier, New York, pp. 128–219.
Harter, T.S., Morrison, P.R., Mandelman, J.W., Rummer, J.L., Farrell, A.P., Brill, R.W.,
Brauner, C.J., 2015. Validation of the i-STAT system for the analysis of blood gases and
acid-base status in juvenile sandbar shark (Carcharhinus plumbeus). Conserv. Physiol. 3.
https://doi.org/10.1093/conphys/cov002.
ARTICLE IN PRESS
Harter, T.S., Sackville, M., Wilson, J.M., Metzger, D.C.H., Egginton, S., Esbaugh, A.J.,
Farrell, A.P., Brauner, C.J., 2018. A solution to Nature’s haemoglobin knockout: a
plasma-accessible carbonic anhydrase catalyses CO2 excretion in Antarctic icefish gills.
J. Exp. Biol. 221. https://doi.org/10.1242/jeb
Hasler, C.T., Butman, D., Jeffrey, J.D., Suski, C.D., 2016. Freshwater biota and rising pCO2?
Ecol. Lett. 19, 98–108.
Hayashi, M., Kita, J., Ishimatsu, A., 2004. Acid-base responses to lethal aquatic hypercapnia in
three marine fishes. Mar. Biol. 144, 153–160.
Hedges, S.B., Kumar, S., 2009. The Timetree of Life. Oxford University Press.
Heisler, N., 1982. Intracellular and extracellular acid-base regulation in the tropical fresh-water
teleost fish Synbranchus marmoratus in response to the transition from water breathing to
air breathing. J. Exp. Biol. 99, 9–28.
Heisler, N., 1984. Acid-base regulation in fishes. Fish Physiol. 10, 315–401.
Heisler, N., 1986. Acid-Base Regulation in Animals. Elsevier, Amsterdam, p. 492.
Heisler, N., 1988. Acid-base regulation. In: Shuttleworth, T.J. (Ed.), The Physiology of Elasmo-
branch Fishes. Berlin, Springer, pp. 215–252.
Heisler, N., 1989. Parameters and methods in acid-base physiology. In: Bridges, C.R., Butler, P.J.
(Eds.), Techniques in Comparative Respiratory Physiology—An Experimental Approach.
Cambridge University Press, Cambridge, pp. 305–332.
Heisler, N., 1993. Acid-base regulation. In: Evans, D.H. (Ed.), The Physiology of Fishes. Boca
Raton, CRC Press, pp. 343–377.
Heisler, N., Forcht, G., Ultsch, G.R., Anderson, J.F., 1982. Acid-base regulation in response to
environmental hypercapnia in two aquatic salamanders, Siren lacertina and Amphiuma
means. Respir. Physiol. 49, 141–158.
Heming, T.A., Randall, D.J., Boutilier, R.G., Iwama, G.K., Primmett, D., 1986. Ionic equilibria
in red blood cells of rainbow trout (Salmo gairdneri): cl, HCO +
3 and H . Respir. Physiol.
65, 223–234.
Henriksson, P., Mandic, M., Richards, J.G., 2008. The osmorespiratory compromise in sculpins:
impaired gas exchange is associated with freshwater tolerance. Physiol. Biochem. Zool.
81, 310–319.
Henry, R.P., Heming, T.A., 1998. Carbonic anhydrase and respiratory gas exchange.
In: Perry, S.F., Tufts, B.L. (Eds.), Fish Physiology. In: Fish Respiration, vol. 17.
Academic Press, Toronto, pp. 75–111.
Henry, R.P., Swenson, E.R., 2000. The distribution and physiological significance of carbonic
anhydrase in vertebrate gas exchange organs. Respir. Physiol. 121, 1–12.
Henry, R.P., Tufts, B.L., Boutilier, R.G., 1993. The distribution of carbonic anhydrase type I and
II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate
exchange. J. Comp. Physiol. B. Biochem. Syst. Environ. Physiol. 163, 380–388.
Henry, R.P., Wang, Y., Wood, C.M., 1997a. Carbonic anhydrase facilitates CO2 and NH3 trans-
port across the sarcolemma of trout white muscle. Am. J. Physiol. 272, R1754–R1761.
Henry, R.P., Gilmour, K.M., Wood, C.M., Perry, S.F., 1997b. Extracellular carbonic anhydrase
activity and carbonic anhydrase inhibitors in the circulatory system of fish. Physiol. Zool.
70, 650–659.
Heuer, R.M., Grosell, M., 2014. Physiological impacts of elevated carbon dioxide and ocean acid-
ification on fish. Am. J. Physiol. Regul. Integr. Comp. Physiol. 307, R1061–R1084.
Heuer, R.M., Grosell, M., 2016. Elevated CO2 increases energetic cost and ion movement in the
marine fish intestine. Sci. Rep. 6, 34480.
Heuer, R.M., Esbaugh, A.J., Grosell, M., 2012. Ocean acidification leads to counterproductive
intestinal base loss in the Gulf toadfish (Opsanus beta). Physiol. Biochem. Zool.
85, 450–459.
ARTICLE IN PRESS
Heuer, R.M., Welch, M.J., Rummer, J.L., Munday, P.L., Grosell, M., 2016. Altered brain ion
gradients following compensation for elevated CO2 are linked to behavioural alterations in
a coral reef fish. Sci. Rep. 6, 33216.
Heuer, R.M., Hamilton, T.J., Nilsson, G.E., 2019. The physiology of behavioral impacts of high
CO2. In: Grosell, M., Munday, P.L., Farrell, A.P., Brauner, C.J. (Eds.), Carbon Dioxide.
Fish Physiology, vol. 37. Academic Press, San Diego.
Hoffert, J.R., Fromm, P.O., 1973. Effect of acetazolamide on some hematological parameters and
ocular oxygen concentration in rainbow-trout. Comp. Biochem. Physiol. 45, 371–378.
Hoppe, C.J.M., Langer, G., Rokitta, S.D., Wolf-Gladrow, D.A., Rost, B., 2012. Implications of
observed inconsistencies in carbonate chemistry measurements for ocean acidification studies.
Biogeosciences 9, 2401–2405.
Horng, J.L., Lin, L.Y., Huang, C.J., Katoh, F., Kaneko, T., Hwang, P.P., 2007. Knockdown
of V-ATPase subunit a (atp6v1a) impairs acid secretion and ion balance in zebrafish
(Danio rerio). Am. J. Physiol. Regul. Integr. Comp. Physiol. 292, R2068–R2076.
Horng, J.L., Lin, L.Y., Hwang, P.P., 2009. Functional regulation of H+-ATPase-rich cells in
zebrafish embryos acclimated to an acidic environment. Am. J. Physiol. Cell Physiol.
296, C682–C692.
Hsu, H.-H., Lin, L.-Y., Tseng, Y.-C., Horng, J.-L., Hwang, P.-P., 2014. A new model for fish ion
regulation: identification of ionocytes in freshwater- and seawater-acclimated medaka
(Oryzias latipes). Cell Tissue Res. 357, 225–243.
Hulikova, A., Swietach, P., 2014. Rapid CO2 permeation across biological membranes: implica-
tions for CO2 venting from tissue. FASEB J. 28, 2762–2774.
Huynh, K.T., Baker, D.W., Harris, R., Church, J., Brauner, C.J., 2011. Capacity for intracellular
pH compensation during hypercapnia in white sturgeon primary liver cells. J. Comp. Physiol.
B. Biochem. Syst. Environ. Physiol. 181, 893–904.
Hwang, P.-P., Lee, T.-H., Lin, L.-Y., 2011. Ion regulation in fish gills: recent progress in the
cellular and molecular mechanisms. Am. J. Physiol. Regul. Integr. Comp. Physiol. 301,
R28–R47.
Ivanis, G., Esbaugh, A.J., Perry, S.F., 2008. Branchial expression and localization of SLC9A2
and SLC9A3 sodium/hydrogen exchangers and their possible role in acid–base regulation
in freshwater rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 211, 2467–2477.
Iwama, G.K., Ishimatsu, A., 1994. Cannulation of blood vessels. In: Stolen, J.S., Fletcher, T.C.,
Rowley, J.T., Zeliko, J.T., Kaattari, S.A., Smith, S.A. (Eds.), Techniques in Fish Immunol-
ogy. In: vol. 3. SOS Publications, Jefferson City, MO, USA, pp. 1–16.
Jarrold, M.D., Humphrey, C., McCormick, M.I., Munday, P.L., 2017. Diel CO2 cycles reduce
severity of behavioural abnormalities in coral reef fish under ocean acidification. Sci. Rep.
7, 10153.
Katoh, F., Hyodo, S., Kaneko, T., 2003. Vacuolar-type proton pump in the basolateral plasma
membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus
heteroclitus, adapted to a low ion environment. J. Exp. Biol. 206, 793–803.
Khan, J.R., Johansen, D., Skov, P.V., 2018. The effects of acute and long-term exposure to CO2
on the respiratory physiology and production performance of Atlantic salmon (Salmo salar)
in freshwater. Aquaculture 491, 20–27.
Krumschnabel, G., Manzl, C., Schwarzbaum, P.J., 2001. Regulation of intracellular pH in
anoxia-tolerant and anoxia-intolerant teleost hepatocytes. J. Exp. Biol. 204, 3943–3951.
Kumai, Y., Perry, S.F., 2011. Ammonia excretion via Rhcg1 facilitates Na+ uptake in larval
zebrafish, Danio rerio, in acidic water. Am. J. Physiol. Regul. Integr. Comp. Physiol.
301, R1517–R1528.
Kumai, Y., Perry, S.F., 2012. Mechanisms and regulation of Na+ uptake by freshwater fish. Res-
pir. Physiol. Neurobiol. 184, 249–256.
ARTICLE IN PRESS
Kurita, Y., Nakada, T., Kato, A., Doi, H., Mistry, A.C., Chang, M.H., Romero, M.F., Hirose, S.,
2008. Identification of intestinal bicarbonate transporters involved in formation of carbonate
precipitates to stimulate water absorption in marine teleost fish. Am. J. Physiol. Regul. Integr.
Comp. Physiol. 294, R1402–R1412.
Kustu, S., Inwood, W., 2006. Biological gas channels for NH3 and CO2: evidence that Rh (Rhesus)
proteins are CO2 channels. Transfus. Clin. Biol. 13, 103–110.
Kwong, R.W.M., Kumai, Y., Perry, S.F., 2014. The physiology of fish at low pH: the zebrafish as
a model system. J. Exp. Biol. 217, 651–662.
Lane, M., 1999. Bicarbonate/chloride exchange regulates intracellular pH of embryos but not
oocytes of the hamster. Biol. Reprod. 61, 452–457.
Lane, M., Bavister, B.D., 1999. Regulation of intracellular pH in bovine oocytes and cleavage
stage embryos. Mol. Reprod. Dev. 54, 396–401.
Larsen, B.K., Jensen, F.B., 1997. Influence of ionic composition on acid-base regulation in
rainbow trout (Oncorhynchus mykiss) exposed to environmental hypercapnia. Fish Physiol.
Biochem. 16, 157–170.
Larsen, B.K., Portner, H.O., Jensen, F.B., 1997. Extra- and intracellular acid-base balance and
ionic regulation in cod (Gadus morhua) during combined and isolated exposures to hypercap-
nia and copper. Mar. Biol. 128, 337–346.
Larsen, E.H., Deaton, L.E., Onken, H., O’Donnell, M., Grosell, M., Dantzler, W.H.,
Weihrauch, D., 2014. Osmoregulation and excretion. Compr. Physiol. 4, 405–573.
Lee, Y.C., Yan, J.J., Cruz, S.A., Horng, J.L., Hwang, P.P., 2011. Anion exchanger 1b, but not
sodium-bicarbonate cotransporter 1b, plays a role in transport functions of zebrafish
H+-ATPase-rich cells. Am. J. Physiol. 300, C295–C307.
Lefevre, S., 2019. Effects of high CO2 on oxygen consumption rates, aerobic scope and swimming
performance. In: Grosell, M., Munday, P.L., Farrell, A.P., Brauner, C.J. (Eds.), Carbon
Dioxide. Fish Physiology, vol. 37. Academic Press, San Diego.
Lin, H., Randall, D., 1995. 9 proton pumps in fish gills. Fish Physiol. 14, 229–255.
Lin, T.Y., Liao, B.K., Horng, J.L., Yan, J.J., Hsiao, C.D., Hwang, P.P., 2008. Carbonic
anhydrase 2-like a and 15a are involved in acid-base regulation and Na+ uptake in zebrafish
H+-ATPase-rich cells. Am. J. Physiol. Cell Physiol. 294, C1250–C1260.
Litt, L., González-Mendez, R., Severinghaus, J.W., Hamilton, W.K., Shuleshko, J., Murphy-
Boesch, J., James, T.L., 1985. Cerebral intracellular changes during supercarbia: an in vivo
31P nuclear magnetic resonance study in rats. J. Cereb. Blood Flow Metab. 5, 537–544.
Liu, S., Piwnica-Worms, D., Lieberman, M., 1990. Intracellular pH regulation in cultured embry-
onic chick heart cells. Na+-dependent Cl/HCO 3 exchange. J. Gen. Physiol. 96, 1247–1269.
Maberly, S.C., 1996. Diel, episodic and seasonal changes in pH and concentrations of inorganic
carbon in a productive lake. Freshwater Biol. 35, 579–598.
Macqueen, D.J., Johnston, I.A., 2014. A well-constrained estimate for the timing of the salmonid
whole genome duplication reveals major decoupling from species diversification. Proc. R.
Soc. B Biol. Sci. 281, 20132881.
Maddison, W.P., Maddison, D., 2017. Mesquite: A Modular System for Evolutionary Analysis.
Version 3.6. https://www.mesquiteproject.org/.
Madshus, I.H., 1988. Regulation of intracellular pH in eukaryotic cells. Biochem. J. 250, 1–8.
Malan, A., Rodeau, J.L., Daull, F., 1985. Intracellular pH in hibernation and respiratory
acidosis in the European hamster. J. Comp. Physiol. B. Biochem. Syst. Environ. Physiol.
156, 251–258.
Maren, T.H., Friedland, B.R., Rittmaster, R.S., 1980. Kinetic properties of primitive vertebrate
carbonic anhydrases. Comp. Biochem. Physiol. 67B, 69–74.
Marshall, W.S., Grosell, M., 2006. Ion transport, osmoregulation and acid-base balance.
In: Evans, D.H., Claiborne, J.B. (Eds.), Physiology of Fishes. Boca Raton, CRC Press,
pp. 179–214.
ARTICLE IN PRESS
Martens, L.G., Witten, P.E., Fivelstad, S., Huysseune, A., Sævareid, B., Vikeså, V., Obach, A.,
2006. Impact of high water carbon dioxide levels on Atlantic salmon smolts (Salmo salar L.):
effects on fish performance, vertebrae composition and structure. Aquaculture 261, 80–88.
Matey, V., Richards, J.G., Wang, Y., Wood, C.M., Rogers, J., Davies, R., Murray, B.W.,
Chen, X.-Q., Du, J., Brauner, C.J., 2008. The effect of hypoxia on gill morphology and
ionoregulatory status in the Lake Qinghai scaleless carp, Gymnocypris przewalskii. J. Exp.
Biol. 211, 1063–1074.
McKenzie, D.J., Piccolella, M., Valle, A.Z.D., Taylor, E.W., Bolis, C.L., Steffensen, J.F., 2003.
Tolerance of chronic hypercapnia by the European eel Anguilla anguilla. J. Exp. Biol.
206, 1717–1726.
McMillan, O.J.L., Dichiera, A.M., Harter, T.S., Wilson, J.M., Esbaugh, A.J., Brauner, C.J.,
2019. Blood and gill carbonic anhydrase in the context of a chondrichthyan model of CO2
excretion. Physiol. Biochem. Zool. (In Press).
McNeil, B., Matsumoto, K., 2019. The changing ocean and freshwater CO2 system.
In: Grosell, M., Munday, P.L., Farrell, A.P., Brauner, C.J. (Eds.), Carbon Dioxide. Fish
Physiology, vol. 37. Academic Press, San Diego.
McNeil, B.I., Sasse, T.P., 2016. Future ocean hypercapnia driven by anthropogenic amplification
of the natural CO2 cycle. Nature 529, 383–386.
Meinshausen, M., Smith, S.J., Calvin, K., Daniel, J.S., Kainuma, M.L.T., Lamarque, J.-F.,
Matsumoto, K., Montzka, S.A., Raper, S.C.B., Riahi, K., Thomson, A., Velders, G.J.M.,
van Vuuren, D.P.P., 2011. The RCP greenhouse gas concentrations and their extensions from
1765 to 2300. Clim. Change 109, 213–241.
Melzner, F., Gobel, S., Langenbuch, M., Gutowska, M.A., Portner, H.O., Lucassen, M., 2009.
Swimming performance in Atlantic cod (Gadus morhua) following long-term (4–12 months)
acclimation to elevated seawater pCO2. Aquat. Toxicol. 92, 30–37.
Meredith, R.W., Janecka, J.E., Gatesy, J., Ryder, O.A., Fisher, C.A., Teeling, E.C., Goodbla, A.,
Eizirik, E., Simao, T.L.L., Stadler, T., et al., 2011. Impacts of the cretaceous terrestrial
revolution and KPg extinction on mammal diversification. Science 334, 521–524.
Mitrovic, D., Dymowska, A., Nilsson, G.E., Perry, S.F., 2009. Physiological consequences of
gill remodeling in goldfish (Carassius auratus) during exposure to long-term hypoxia. Am.
J. Physiol. Regul. Integr. Comp. Physiol. 297, R224–R234.
Molich, A., Heisler, N., 2005. Determination of pH by microfluorometry: intracellular and
interstitial pH regulation in developing early-stage fish embryos (Danio rerio). J. Exp. Biol.
208, 4137–4149.
Moody Jr., W.J., 1981. The ionic mechanism of intracellular pH regulation in crayfish neurones.
J. Physiol. 316, 293–308.
Morrison, P.R., Gilmour, K.M., Brauner, C.J., 2016. Oxygen and carbon dioxide transport
in elasmobranchs. In: Shadwick, R., Farrell, A.P., Brauner, C.J. (Eds.), Fish Physiology.
In: Farrell, A.P., Brauner, C.J. (Eds.), Physiology of Elasmobranch Fishes: Internal
Processes, vol. 34B. Elsevier, New York, pp. 128–219. Series Editors.
Mota, V.C., Nilsen, T.O., Gerwins, J., Gallo, M., Ytteborg, E., Bæverfjord, G., Kolarevic, J.,
Summerfelt, S.T., Terjesen, B.F., 2019. The effects of carbon dioxide on growth performance,
welfare, and health of Atlantic salmon post-smolt (Salmo salar) in recirculating aquaculture
systems. Aquaculture 498, 578–586.
Munday, P.L., Jarrold, M.D., Nagelkerken, I., 2019a. Ecological effects of elevated CO2 on
marine and freshwater fishes: from individual to community effects. In: Grosell, M.,
Munday, P.L., Farrell, A.P., Brauner, C.J. (Eds.), Carbon Dioxide. Fish Physiology,
vol. 37. Academic Press, San Diego.
Munday, P.L., Rummer, J.L., Baumann, H., 2019b. Adaptation and evolutionary responses
to high CO2. In: Grosell, M., Munday, P.L., Farrell, A.P., Brauner, C.J. (Eds.), Carbon
ARTICLE IN PRESS
Musa-Aziz, R., Chen, L.M., Pelletier, M.F., Boron, W.F., 2009. Relative CO2/NH3
selectivities of AQP1, AQP4, AQP5, AmtB, and RhAG. Proc. Natl. Acad. Sci. U.S.A.
106, 5406–5411.
Nakhoul, N.L., Davis, B.A., Romero, M.F., Boron, W.F., 1998. Effect of expressing the water
channel aquaporin-1 on the CO2 permeability of Xenopus oocytes. Am. J. Physiol.
274, C543–C548.
Nattie, E.E., Edwards, W.H., 1981. CSF acid-base regulation and ventilation during acute hyper-
capnia in the newborn dog. J. Appl. Physiol. 50, 566–574.
Nikinmaa, M., 1986. Red cell pH of lamprey (Lampetra fluviatilis) is actively regulated. J. Comp.
Physiol. B, Biochem. Syst. Environ. Physiol. 5, 747–750.
Nikinmaa, M., Mattsoff, L., 1992. Effects of oxygen saturation on the CO2 transport properties of
Lampetra red cells. Respir. Physiol. 87, 219–230.
Nikinmaa, M., Railo, E., 1987. Anion movements across lamprey (Lampetra fluviatilis) red cell
membrane. Biochim. Biophys. Acta 899, 134–136.
Nikinmaa, M., Kunnamo-Ojala, T., Railo, E., 1986. Mechanisms of pH regulation in lamprey
(Lampetra fluviatilis) red blood cells. J. Exp. Biol. 122, 355–367.
Nikinmaa, M., Tufts, B.L., Boutilier, R.G., 1993. Volume and pH regulation in agnthan
erythrocytes—comparsions between the hagfish, Myxine glutinosa, and the lampreys, Per-
tromyzon marinus and Lampetra fluviatilis. J. Comp. Physiol. B, Biochem. Syst. Environ. Phy-
siol. 163, 608–613.
Nikinmaa, M., Bogdanova, A., Virkki, L.V., 1999. Intracellular pH regulation of rainbow trout
and carp thrombocytes. Fish Physiol. Biochem. 21, 269–275.
Nikinmaa, M.M., Berenbrink, M., Brauner, C.J., 2019. Regulation of erythrocyte function:
multiple evolutionary solutions for respiratory gas transport and its regulation in fish. Acta
Physiol. e13299, in press. doi.org/10.1111/apha.13299.
Nilsson, G.E., Lefevre, S., 2016. Physiological challenges to fishes in a warmer and acidified
future. Phys. Ther. 31, 409–417.
Nilsson, G.E., Dixson, D.L., Domenici, P., McCormick, M.I., Sørensen, C., Watson, S.-A.,
Munday, P.L., 2012. Near-future carbon dioxide levels alter fish behaviour by interfering with
neurotransmitter function. Nat. Clim. Chang. 2, 201–204.
Noble, C., Kankainen, M., Set€al€a, J., Berrill, I.K., Ruohonen, K., Damsgård, B., Toften, H.,
2012. The bio-economic costs and benefits of improving productivity and fish welfare in
aquaculture: utilizing CO2 stripping technology in Norwegian Atlantic salmon production.
Aquac. Econ. Manag. 16, 414–428.
Obaid, A.L., Critz, A.M., Crandall, E.D., 1979. Kinetics of bicarbonate/chloride exchange in dog-
fish erythrocytes. Am. J. Physiol. 237, R132–R138.
Occhipinti, R., Boron, W.F., 2015. Mathematical modeling of acid-base physiology. Prog.
Biophys. Mol. Biol. 117, 557–573.
Orellana, J., Wecker, B., 2014. Degassing to reduce carbon dioxide during live transport of adult
salmon in wellboats: a model approach. J. Appl. Ichthyol. 30, 411–414.
Ou, M., Hamilton, T.J., Eom, J., Lyall, E.M., Gallup, J., Jiang, A., Lee, J., Close, D.A.,
Yun, S.-S., Brauner, C.J., 2015. Responses of pink salmon to CO2-induced aquatic acidifica-
tion. Nat. Clim. Chang. 5, 950–955.
Parker, M.D., Boron, W.F., 2013. The divergence, actions, roles, and relatives of sodium-coupled
bicarbonate transporters. Physiol. Rev. 93, 803–959.
Parks, S.K., Pouyssegur, J., 2017. Targeting pH regulating proteins for cancer therapy-progress
and limitations. Semin. Cancer Biol. 43, 66–73.
Perry, S.F., 1986. Carbon dioxide excretion in fishes. Can. J. Zool. 64, 565–572.
Perry, S.F., Abdallah, S., 2012. Mechanisms and consequences of carbon dioxide sensing in fish.
Respir. Physiol. Neurobiol. 184 (3), 309–315.
ARTICLE IN PRESS
Perry, S.F., Gilmour, K.M., 1993. An evaluation of factors limiting carbon dioxide excretion by
trout red blood cells in vitro. J. Exp. Biol. 180, 39–54.
Perry, S.F., Gilmour, K.M., 2006. Acid-base balance and CO2 excretion in fish: unanswered
questions and emerging models. Respir. Physiol. Neurobiol. 154, 199–215.
Perry, S.F., Goss, G.G., Laurent, P., 1992. The interrelationships between gill chloride cell
morphology and ionic uptake in four freshwater teleosts. Can. J. Zool. 70 (9), 1775–1786.
Perry, S.F., Wood, C.M., Walsh, P.J., Thomas, S., 1996. Fish red blood cell carbon dioxide excre-
tion in vitro: a comparative study. Comp. Biochem. Physiol. A 113, 121–130.
Perry, S.F., Brauner, C.J., Tufts, B., Gilmour, K.M., 1997. Acid-base disequilibrium in the venous
blood of rainbow trout (Oncorhynchus mykiss). Exp. Biol. Online 2, 1.
Perry, S.F., Furimsky, M., Bayaa, M., Georgalis, T., Nickerson, J.G., Moon, T.W., 2003. Inter-
grated involvement of Na+/HCO 3 cotransporters and V-type ATPases in branchial and renal
acid-base regulation in freshwater fishes. Biochim. Biophys. Acta 1618, 175–184.
Perry, S.F., Esbaugh, A., Braun, M., Gilmour, K.M., 2009. Gas transport and gill function in
water-breathing fish. In: Glass, M., Woods, S. (Eds.), Cardio-Respiratory Control in Verte-
brates. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 5–42.
Perry, S.F., Braun, M.H., Noland, M., Dawdy, J., Walsh, P.J., 2010a. Do zebrafish Rh proteins
act as dual ammonia-CO2 channels? J. Exp. Zool. A Ecol. Genet. Physiol. 313, 618–621.
Perry, S.F., Braun, M.H., Genz, J., Vulesevic, B., Taylor, J.R., Grosell, M., Gilmour, K.M.,
2010b. Acid-base regulation in the plainfin midshipman (Porichthys notatus), an aglomerular
marine teleost. J. Comp. Physiol. 180, 1213–1225.
Petochi, T., Di Marco, P., Priori, A., Finoia, M.G., Mercatali, I., Marino, G., 2011. Coping
strategy and stress response of European sea bass Dicentrarchus labrax to acute and chronic
environmental hypercapnia under hyperoxic conditions. Aquaculture 315, 312–320.
Phillips, K.P., Leveille, M.-C., Claman, P., Baltz, J.M., 2000. Intracellular pH regulation in
human preimplantation embryos. Hum. Reprod. 15, 896–904.
Phuong, L.M., Huong, D.T.T., Nyengaard, J.R., Bayley, M., 2017. Gill remodelling and growth
rate of striped catfish Pangasianodon hypophthalmus under impacts of hypoxia and temper-
ature. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 203, 288–296.
Piermarini, P.M., Verlander, J.W., Royaux, I.E., Evans, D.H., 2002. Pendrin immunoreactivity in
the gill epithelium of a euryhaline elasmobranch. Am. J. Physiol. Regul. Integr. Comp. Phy-
siol. 283, R983–R992.
P€
ortner, H.O., Boutilier, R.G., Tang, Y., Toews, D.P., 1990. Determination of intracellular pH
and pCO2 after metabolic inhibition by fluoride and nitrilotriacetic acid. Respir. Physiol.
81, 255–273.
P€
ortner, H.O., Reipschlager, A., Heisler, N., 1998. Acid-base regulation, metabolism and energet-
ics in Sipunculus nudus as a function of ambient carbon dioxide level. J. Exp. Biol. 201, 43–55.
Powers, E.B., 1930. The relation between pH and aquatic animals. Am. Nat. 64, 342–366.
Putnam, R.W., Roos, A., 1997. Intracellular pH. In: Hoffman, J., Jamieson, J. (Eds.), Handbook
of Physiology. In: Cell Physiology, vol 14. Oxford University Press, Oxford, pp. 389–440.
Randall, D.J., Burggren, W.W., Farrell, A.P., Haswell, M.S., 1981. The Evolution of Air Breath-
ing in Vertebrates. Cambridge University press, Cambridge.
Randall, D.J., Rummer, J., Wilson, J., Wang, W., Brauner, C.J., 2014. A unique mode of tissue
oxygenation and the success of teleost fish. J. Exp. Biol. 217, 1205–1214.
Raymond, P.A., Hartmann, J., Lauerwald, R., Sobek, S., McDonald, C., Hoover, M.,
Butman, D., Striegl, R., Mayorga, E., Humborg, C., Kortelainen, P., D€ urr, H.,
Meybeck, M., Ciais, P., Guth, P., 2013. Global carbon dioxide emissions from inland waters.
Nature 503, 355–359.
Reshkin, S.J., Greco, M.R., Cardone, R.A., 2014. Role of pHi, and proton transporters in
oncogene-driven neoplastic transformation. Phil. Trans. R. Soc. B 369, 20130100.
ARTICLE IN PRESS
Richey, J.E., Melack, J., Audfenkampe, A., Ballester, M.V.R., Hess, L., 2002. Outgassing from
Amazonian rivers and wetlands as a large tropical source of atmospheric CO2. Nature
416, 617–620.
Rimoldi, S., Terova, G., Brambilla, F., Bernardini, G., Gornati, R., Saroglia, M., 2009. Molecular
characterization and expression analysis of Na+/H+ exchanger (NHE)-1 and c-Fos genes in
sea bass (Dicentrarchus labrax, L) exposed to acute and chronic hypercapnia. J. Exp. Mar.
Biol. Ecol. 375, 32–40.
Romero, M.F., Fulton, C.M., Boron, W.F., 2004. The SLC4 family of HCO 3 transporters.
Pfl€
ugers Arch. 447, 495–509.
Roos, A., Boron, W.F., 1981. Intracellular pH. Physiol. Rev. 61, 296–434.
Roughton, F.J.W., 1935. Recent work on carbon dioxide transport by the blood. Physiol. Rev.
15, 241–296.
Ruffin, V.A., Salameh, A.I., Boron, W.F., Parker, M.D., 2014. Intracellular pH regulation by
acid-base transporters in mammalian neurons. Front. Physiol. 5, 43.
Rummer, J.L., Brauner, C.J., 2011. Plasma-accessible carbonic anhydrase at the tissue of a teleost
fish may greatly enhane oxygen delivery: in vitro evidence in rainbow trout (Oncorhynchus
mykiss). J. Exp. Biol. 214, 2319–2328.
Rummer, J.L., McKenzie, D.J., Innocenti, A., Supuran, C.T., Brauner, C.J., 2013. Root effect
hemoglobin may have evolved to enhance general tissue oxygen delivery. Science
340, 1327–1329.
Sackville, M., Shartau, R.B., Damsgaard, C., Hvas, M., Phuong, L.M., Wang, T., Bayley, M.,
Huong, D.T.T., Phuong, N.T., Brauner, C.J., 2018. Water pH limits extracellular but not
intracellular pH compensation in the CO2 tolerant freshwater fish, Pangasianodon hypo-
phthalmus. J. Exp. Biol. 221, 1–5.
Sahu, B.C., Adhikari, S., Mahapatra, A.S., Dey, L., 2013. Carbon, nitrogen, and posphorous
budget in scampi (Macrobrachium rosenbergii) culture ponds. Environ. Monit. Assess.
185, 10157–10166.
Saksena, D.N., Gaidhane, D.M., Singh, H., 2006. Limnology of Kharland (saline) ponds of
Ratnagiri, Maharashtra in relation to prawn culture potential. J. Environ. Biol. 27, 49–53.
Salameh, A.I., H€ ubner, C.A., Boron, W.F., 2016. Role of Cl—HCO 3 exchanger AE3 in intracel-
lular pH homeostasis in cultured murine hippocampal neurons, and in crosstalk to adjacent
astrocytes. J. Physiol. 595, 93–124.
Sattin, G., Mager, E.M., Beltramini, M., Grosell, M., 2010. Cytosolic carbonic anhydrase in the
gulf toadfish is important for tolerance to hypersalinity. Comp. Biochem. Physiol. A Mol.
Integr. Physiol. 156, 169–175.
Seidelin, M., Brauner, C.J., Jensen, F.B., Madsen, S.S., 2001. Vacuolar-type H+-ATPase
and Na, K-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated
and combined exposure to hyperoxia and hypercapnia in fresh water. Zoolog. Sci.
18, 1199–1205.
Shartau, R.B., 2017. Vertebrate Preferential Intracellular pH Regulation During Severe Acute
Hypercarbia. University of British Columbia.
Shartau, R.B., Brauner, C.J., 2014. Acid-base and ion balance in fishes with bimodal respiration.
J. Fish Biol. 84, 682–704.
Shartau, R.B., Baker, D.W., Crossley, D.A., Brauner, C.J., 2016a. Preferential intracellular pH
regulation: hypotheses and perspectives. J. Exp. Biol. 219, 2235–2244.
Shartau, R.B., Crossley, D.A., Kohl, Z.F., Brauner, C.J., 2016b. Embryonic common snapping
turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base
challenges. J. Exp. Biol. 219, 1994–2002.
Shartau, R.B., Baker, D.W., Brauner, C.J., 2017a. White sturgeon (Acipenser transmontanus)
acid–base regulation differs in response to different types of acidoses. J. Comp. Physiol. B,
Biochem. Syst. Environ. Physiol. 187, 985–994.
ARTICLE IN PRESS
Shartau, R.B., Brix, K.V., Brauner, C.J., 2017b. Characterization of Na+ transport to gain
insight into the mechanism of acid-base and ion regulation in white sturgeon (Acipenser trans-
montanus). Comp. Biochem. Phys. A 204, 197–204.
Shartau, R.B., Crossley, D.A.I., Kohl, Z.F., Elsey, R., Brauner, C.J., 2018. American alligator
(Alligator mississippiensis) embryos tightly regulate intracellular pH during a severe acidosis.
Can. J. Zool. 96, 723–727.
Shartau, R.B., Damsgaard, C., Brauner, C.J., 2019. Acid-base regulation during elevated
environmental CO2 in fish. Comp. Biochem. Phys. A. 236.
Siyanov, V., Baltz, J.M., 2013. NHE1 is the sodium-hydrogen exchanger active in acute intracel-
lular pH regulation in preimplantation mouse embryos. Biol. Reprod. 88, 157.
Skov, P.V., 2019. CO2 in aquaculture. In: Grosell, M., Munday, P.L., Farrell, A.P., Brauner, C.J.
(Eds.), Carbon Dioxide. Fish Physiology, vol. 37. Academic Press, San Diego.
Smith, L.S., Bell, G.R., 1964. A technique for prolonged blood sampling in free-swimming
salmon. J. Fish. Res. Board Can. 21, 711–717.
Snyder, G.K., Nestler, J.R., 1991. Intracellular pH in the toad Bufo marinus following hypercap-
nia. J. Exp. Biol. 161, 415–422.
Snyder, G.K., Nestler, J.R., Shapiro, J.I., Huntley, J., 1995. Intracellular pH in lizards after
hypercapnia. Am. J. Physiol. 268, R889–R895.
Soivio, A., Nynolm, K., Westman, K., 1975. A technique for repeated sampling of the blood of
individual resting fish. J. Exp. Biol. 63, 207–217.
Sollid, J., De Angelis, P., Gundersen, K., Nilsson, G., 2003. Hypoxia induced adaptive and revers-
ible gross morphological changes in crucian carp gills. J. Exp. Biol. 206, 3667–3673.
Squirrell, J.M., Lane, M., Bavister, B.D., 2001. Altering intracellular pH disrupts development
and cellular organization in preimplantation hamster embryos. Biol. Reprod. 64, 1845–1854.
Steeves, C.L., Lane, M., Bavister, B.D., 2001. Differences in intracellular pH regulation by
Na+/H+ antiporter among two-cell mouse embryos derived from females of different strains.
Biol. Reprod. 65, 14–22.
Strobel, A., Bennecke, S., Leo, E., Mintenbeck, K., Portner, H.O., Mark, F.C., 2012. Metabolic
shifts in the Antarctic fish Notothenia rossii in response to rising temperature and pCO2.
Front. Zool. 9, 28.
Summerfelt, S.T., Vinci, B.J., Piedrahita, R.H., 2000. Oxygenation and carbon dioxide control in
water reuse systems. Aquacult. Eng. 22, 87–108.
Swenson, E.R., 2000. Respiratory and renal roles of carbonic anhydrase in gas exchange and acid
base regulation. In: Chegwidden, W.R., Carter, N.D., Edwards, Y.H. (Eds.), The Carboic
Anhydrases: New Horizons. Birkhauser Verlag, Boston, pp. 281–342.
Swenson, E.R., Maren, T.H., 1978. A quantitative analysis of CO2 transport at rest and during
maximal exercise. Respir. Physiol. 35, 129–159.
Swenson, E.R., Maren, T.H., 1987. Roles of gill and red cell carbonic anhydrase in elasmobranch
HCO 3 and CO2 excretion. Am. J. Physiol. 253, R450–R458.
Swietach, P., 2019. What is pH regulation, and why do cancer cells need it? Cancer and Metastasis
Rev 38, 5–15. doi.org/10.1007/s10555-018-09778-x.
Talbot, K., Kwong, R.W., Gilmour, K.M., Perry, S.F., 2015. The water channel aquaporin-1a1
facilitates movement of CO2 and ammonia in zebrafish (Danio rerio) larvae. J. Exp. Biol.
218, 3931–3940.
Tang, S., Thorarensen, H., Brauner, C.J., Wood, C.M., Farrell, A.P., 2009. Modeling the
accumulation of CO2 during high density, re-circulating transport of adult Atlantic salmon,
Salmo salar, from observations aboard a sea-going commercial live-haul vessel. Aquaculture
296, 102–109.
Taylor, J., Grosell, M., 2008. Basolateral NBC is the hinge of a mechanism serving both osmo-
regulation and acid-base balance in the marine teleost intestine. Comp. Biochem. Physiol.
A Mol. Integr. Physiol. 150, S57–S58.
ARTICLE IN PRESS
Taylor, J.R., Mager, E.M., Grosell, M., 2010. Basolateral NBCe1 plays a rate-limiting role in
transepithelial intestinal HCO 3 secretion, contributing to marine fish osmoregulation.
J. Exp. Biol. 213, 459–468.
Thinh, P.V., Huong, D.T.T., Gam, L.T.H., Damsgaard, C., Phuong, N.T., Bayley, M., Wang, T.,
2019. Renal acid excretion contributes to acid-base regulation during hypercapnia in air-
exposed swamp eel Monopterus albus. J. Exp. Biol. 222, jeb.198259.
Thompson, J., Lane, M., Robertson, S., 2006. Adaptive responses of early embryos to their micro-
environment and consequences for post-implantation development. In: Wintour, E.M.,
Owens, J. (Eds.), Advances in Experimental Medicine and Biology. Springer US, pp. 58–69.
Toews, D.P., Holeton, G.F., Heisler, N., 1983. Regulation of the acid-base status during environ-
mental hypercapnia in the marine teleost fish Conger conger. J. Exp. Biol. 107, 9–20.
Tovey, K.J., Brauner, C.J., 2018. Effects of water ionic composition on acid–base regulation in
rainbow trout, during hypercarbia at rest and during sustained exercise. J. Comp. Physiol.
B 188, 295–304.
Tresguerres, M., Parks, S.K., Katoh, F., Goss, G.G., 2006. Microtubule-dependent relocation of
branchial V-H+-ATPase to the basolateral membrane in the Pacific spiny dogfish (Squalus
acanthias): a role in base secretion. J. Exp. Biol. 209, 599–609.
Tresguerres, M., Parks, S.K., Goss, G.G., 2007. Recovery from blood alkalosis in the Pacific hag-
fish (Eptatretus stoutii): involvement of gill V–H+–ATPase and Na+/K+–ATPase. Comp. Bio-
chem. Phys. A 148, 133–141.
Truchot, J.P., 1987. Comparative Aspects of Extracellular Acid-Base Balance, Zoophysiology.
Springer, Berlin, p. 248.
Tseng, Y.C., Hu, M.Y., Stumpp, M., Lin, L.Y., Melzner, F., Hwang, P.P., 2013. CO2-driven
seawater acidification differentially affects development and molecular plasticity along
life history of fish (Oryzias latipes). Comp. Biochem. Physiol. A Mol. Integr. Physiol.
165, 119–130.
Tufts, B.L., 1992. In vitro evidence for sodium-dependent pH regulation in sea lamprey
(Petromyzon marinus) red blood cells. Can. J. Zool. 70, 411–416.
Tufts, B.L., Boutilier, R.G., 1989. The absence of rapid chloride/bicarbonate exchange in lamprey
erythrocytes: implications for CO2 transport and ion distributions between plasma and eryth-
rocytes in the blood of Petromyson marinus. J. Exp. Biol. 144, 565–576.
Tufts, B.L., Perry, S.F., 1998. Carbon dioxide transport and excretion. In: Perry, S.F., Tufts, B.L.
(Eds.), Fish Physiology. Fish Respiration, vol. 17. Academic Press, San Diego, pp. 229–282.
Tufts, B.L., Bagatto, B., Cameron, B., 1992. In vivo analysis of gas transport in arterial and venous
blood of the sea lamprey, Petromyzon marinus. J. Exp. Biol. 169, 105–119.
Tufts, B.L., Vincent, C.J., Currie, S., 1998. Different red blood cell characteristics in a primitive
agnathan (M. glutinosa) and a more recent teleost (O. mykiss) influence their strategies for
blood CO2 transport. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 119, 533–541.
Tuominen, A., Rissanen, E., Bogdanova, A., Nikinmaa, M., 2003. Intracellular pH regulation in
rainbow trout (Oncorhynchus mykiss) hepatocytes: the activity of sodium/proton exchange is
oxygen-dependent. J. Comp. Physiol. B, Biochem. Syst. Environ. Physiol. 173, 301–308.
Turnbull, J., Bell, A., Adams, C., Bron, J., Huntingford, F., 2005. Stocking density and welfare of
cage farmed Atlantic salmon: application of a multivariate analysis. Aquaculture
243, 121–132.
Tzaneva, V., Gilmour, K.M., Perry, S.F., 2011. Respiratory responses to hypoxia or hypercapnia
in goldfish (Carassius auratus) experiencing gill remodelling. Respir. Physiol. Neurobiol.
175, 112–120.
Ultsch, G.R., 1987. The potential role of hypercarbia in the transition from water-breathing to air-
breathing in vertebrates. Evolution 41, 442–445. https://doi.org/10.2307/2409152.
ARTICLE IN PRESS
Ultsch, G.R., 1996. Gas exchange, hypercarbia and acid-base balance, paleoecology, and the evo-
lutionary transition from water-breathing to air-breathing among vertebrates. Palaeogeogr.
Palaeoclimatol. Palaeoecol. 123, 1–27.
Vaughan-Jones, R.D., Spitzer, K.W., Swietach, P., 2009. Intracellular pH regulation in heart.
J. Mol. Cell. Cardiol. 46, 318–331.
Waisbren, S.J., Geibel, J.P., Modlin, I.M., Boron, W.F., 1994. Unusual permeability properties of
gastric gland cells. Nature 368, 332–335.
Wallace, R.B., Baumann, H., Grear, J.S., Aller, R.C., Gobler, C.J., 2014. Coastal ocean acidifi-
cation: the other eutrophication problem. Estuar. Coast. Shelf Sci. 148, 1–13.
Walsh, P.J., 1986. Ionic requirements for intracellular pH regulation in rainbow trout hepatocytes.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 250, R24–R29.
Walsh, P.J., 1989. Regulation of intracellular pH by toadfish (Opsanus beta) hepatocytes. J. Exp.
Biol. 147, 407.
Walsh, P.J., Mommsen, T.P., Moon, T.W., Perry, S.F., 1988. Effects of acid-base variables on
invitro hepatic-metabolism in rainbow-trout. J. Exp. Biol. 135, 231–241.
Wang, Z., Cai, W.-J., 2004. Carbon dioxide degassing and inorganic carbon export from a marsh-
dominated estuary (the Duplin River): a marsh CO2 pump. Limnol. Oceanogr. 49, 341–354.
Wang, Y., Henry, R.P., Wright, P.M., Heigenhauser, G.J., Wood, C.M., 1998. Respiratory and
metabolic functions of carbonic anhydrase in exercised white muscle of trout. Am. J. Physiol.
275, R1766–R1779.
Wasser, J.S., Warburton, S.J., Jackson, D.C., 1991. Extracellular and intracellular acid-base
effects of submergence anoxia and nitrogen breathing in turtles. Respir. Physiol. 83, 239–252.
Weakley, J.C., Claiborne, J.B., Hyndman, K.A., Edwards, S.L., 2012. The effect of environmental
salinity on H+ efflux in the euryhaline barramundi (Lates calcarifer). Aquaculture
338–341, 190–196.
Weiss, L.C., P€ otter, L., Steiger, A., Kruppert, S., Frost, U., Tollrian, R., 2018. Rising pCO2 in
freshwater ecosystems has the potential to negatively affect predator-induced defenses in
Daphnia. Curr. Biol. 28, 327–332.
Wetzel, R.G., 2001. Limnology: Lake and River Ecosystems, third ed. Academic Press.
Wheatly, M.G., Hobe, H., Wood, C.M., 1984. The mechanisms of acid-base and ionoregulation
in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia.
II. The role of the kidney. Respir. Physiol. 55, 155–173.
Whittamore, J.M., Cooper, C.A., Wilson, R.W., 2010. HCO 3 secretion and CaCO3 precipitation
play major roles in intestinal water absorption in marine teleost fish in vivo. Am. J. Physiol.
Regul. Integr. Comp. Physiol. 298, R877–R886.
Wilson, R.W., Gilmour, K., Henry, R., Wood, C., 1996. Intestinal base excretion in the seawater-
adapted rainbow trout: a role in acid-base balance? J. Exp. Biol. 199, 2331–2343.
Wilson, R.W., Wilson, J.M., Grosell, M., 2002. Intestinal bicarbonate secretion by marine teleost
fish-why and how? Biochim. Biophys. Acta 1566, 182–193.
Wood, C.M., 2019. Internal spatial and temporal CO2 dynamics: fasting, feeding, drinking, and
the alkaline tide. In: Grosell, M., Munday, P.L., Farrell, A.P., Brauner, C.J. (Eds.), Carbon
Wood, C.M., LeMoigne, J., 1991. Intracellular acid-base responses to environmental hyperoxia
and normoxic recovery in rainbow trout. Respir. Physiol. 86, 91–113.
Wood, C.M., Munger, R.S., 1994. Carbonic anhydrase injection provides evidence for the role of
blood acid-base status in stimulating ventilation after exhaustive exercise in rainbow trout.
J. Exp. Biol. 194, 225–253.
Wood, S.C., Schaefer, K.E., 1978. Regulation of intracellular pH in lungs and other tissues during
hypercapnia. J. Appl. Physiol. 45, 115–118.
ARTICLE IN PRESS
Wood, C.M., Turner, J.D., Graham, M.S., 1983. Why do fish die after severe exercise? J. Fish
Biol. 22, 189–201.
Wood, C.M., Wheatly, M.G., Hobe, H., 1984. The mechanisms of acid-base and ionoregulation
in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia.
III. Branchial exchanges. Respir. Physiol. 55, 175–192.
Wood, C.M., Turner, J.D., Munger, R.S., Graham, M.S., 1990. Control of ventilation in the
hypercapnic skate Raja ocellata: II. Cerebrospinal fluid and intracellular pH in the brain
and other tissues. Respir. Physiol. 80, 279–298.
Wright, P.A., Randall, D.J., Wood, C.M., 1988. The distribution of ammonia and H+ between
tissue compartments in lemon sole (Parophrys vetulus) at rest, during hypercapnia and follow-
ing exercise. J. Exp. Biol. 136, 149–175.
Xu, Y.J., Xu, Z., Yang, R., 2019. Rapid daily change in surface water pCO2 and CO2 evasion:
a case study in a subtropical eutrophic lake in Southern USA. J. Hydrol. 570, 486–494.
Yaksh, T.L., Anderson, R.E., 1987. In vivo studies on intracellular pH, focal flow, and vessel
diameter in the cat cerebral cortex: effects of altered CO2 and electrical stimulation.
J. Cereb. Blood Flow Metab. 7, 332–341.
Yamamoto, T., Swietach, P., Rossini, A., Loh, S.-H., Vaughan-Jones, R.D., Spitzer, K.W., 2005.
Functional diversity of electrogenic Na+-HCO 3 cotransport in ventricular myocytes from
rat, rabbit and guinea pig. J. Physiol. 562, 455–475.
Yoshikawa, H., Fumio, K., Masao, K., 1994. The relationship between the EEG and brain pH in
carp, Cyprinus carpio, subjected to environmental hypercapnia at an anesthetic level. Comp.
Biochem. Physiol. A 107, 307–312.
Zhang, P., Liang, D., Mao, R.-L., Hillis, D.M., Wake, D.B., Cannatella, D.C., 2013. Efficient
sequencing of anuran mtDNAs and a mitogenomic exploration of the phylogeny and evolu-
tion of frogs. Mol. Biol. Evol. 30, 1899–1915.
Zhao, Y., Baltz, J.M., 1996. Bicarbonate/chloride exchange and intracellular pH throughout
preimplantation mouse embryo development. Am. J. Physiol. 271, C1512–C1520.

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ARTICLE IN PRESS

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS:

2 COLIN J. BRAUNER ET AL.

Acid-base balance is one of the most tightly regulated physiological processes.

Acid-base regulation in vertebrates is tightly controlled and crucial for sur-

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 3

Due to the low solubility of O2 in water, gill ventilation volume in fish is

4 COLIN J. BRAUNER ET AL.

2. CO2 TRANSPORT AND HOMEOSTASIS

2.1. CO2 Transport in the Blood

0.0 0.5 1.0 1.5 2.0 2.5

0 1000 2000 3000

0 0.1 0.2 0.3

0 0.1 0.2 0.3

0 20000 40000 60000 80000

6 COLIN J. BRAUNER ET AL.

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 7

8 COLIN J. BRAUNER ET AL.

2.2. CO2 Transfer Across the Gills

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 9

10 COLIN J. BRAUNER ET AL.

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 11

(Waisbren et al., 1994). It was subsequently demonstrated that members of the

2.3. Non-Teleost Models of CO2 Excretion

12 COLIN J. BRAUNER ET AL.

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 13

2.4. Air-Breathing Fish

The model of teleost CO2 transport described above is complicated some-

14 COLIN J. BRAUNER ET AL.

For example, the obligate air-breathing African lungfish (Protopterus

3. ELEVATED ENVIRONMENTAL CO2 AND EXTRACELLULAR

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 15

elaboration upon these conditions. The physiological disturbances associated

16 COLIN J. BRAUNER ET AL.

1.34 (−0.05) 3.0 mmHg

6.75 7.00 7.25 7.50 7.75

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 17

18 COLIN J. BRAUNER ET AL.

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 19

3.1. Effects of Ambient Ion Composition and Salinity

20 COLIN J. BRAUNER ET AL.

3.2. Organ Systems Used in Acid-Base Regulation and Compensation

3.2.1. BRANCHIAL MECHANISMS

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 21

3.2.1.1. Carbonic Anhydrase. The branchial epithelium CAc (Esbaugh

22 COLIN J. BRAUNER ET AL.

3.2.1.2. Bicarbonate Transport. Based on the limited number of studies con-

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 23

3.2.1.4. Na+/K+-ATPase. Although not an acid-base transporter per se,

24 COLIN J. BRAUNER ET AL.

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 25

to elevate plasma ½HCO3 and limit pHe compensation during exposure to

3.2.3. ROLE OF THE GASTROINTESTINAL TRACT

26 COLIN J. BRAUNER ET AL.

Drinking/ Rectal base

Rainbow Drinking: 11 32 ppt 114  15 Wilson et al.

excretion by the gills, although a transient blood acidosis was observed

ACID-BASE PHYSIOLOGY AND CO2 HOMEOSTASIS 27

Rainbow Drinking: 11 32 ppt 114 15 Wilson et al.