Colin J. Brauner

Patterns of acid-base regulation during exposure to hypercarbia in fishes

Acid–base regulation is one of the most tightly regulated physiological processes among vertebrates, and the specific mechanisms and patterns of acid–base regulation in fish have been investigated for decades, although primarily on a few species of teleosts and elasmobranchs. The most common response observed in fish during short-term (up to 96 h) exposure to hypercarbia is that of blood pH compensation for the induced respiratory acidosis by a net increase in plasma \({\rm HCO}_3^{-}\) in exchange for Cl−, predominantly through processes at the gills. Studies on hagfish indicate that this pattern of pH compensation (i.e. net plasma \({\rm HCO}_3^-/{\rm Cl}^-\) exchange, driving pH recovery) probably represents the ancestral state for fishes. Due to an apparent limit to this net \({\rm HCO}_3^-/{\rm Cl}^-\) exchange, most fishes examined to date exhibit incomplete pH compensation for the acidosis, in both plasma and tissues associated with CO2 tensions greater than 10–16 mmHg; in CO2-sensitive fishes, this may be the basis for mortality during exposure to high CO2. A few fish species, however, are capable of tolerating PaCO2s well above 10–16 mmHg; in some of these species, this tolerance appears to be associated with the ability to completely regulate intracellular pH (preferential pHi regulation) of tissues, such as brain, muscle and liver, despite a large reduction in extracellular pH. We hypothesize that: (a) preferential pHi regulation in fish evolved in the ancestors of the pleisiomorphic freshwater (non-teleost) actinopterygiians, (b) is associated with high CO2 tolerance, and (c)was an exaptation for air-breathing. A great deal of research remains to test these hypotheses, and to elucidate the origin and ubiquity of preferential pHi regulation among fishes and the cellular and molecular mechanisms involved.

The effect of acclimation to hypoxia and sustained exercise on subsequent hypoxia tolerance and swimming performance in goldfish (Carassius auratus)

SUMMARY The objective of this study was to determine whether acclimation to hypoxia and sustained exercise would increase hypoxia tolerance (as indicated by a decrease in critical oxygen tension, Pcrit) and swimming performance in goldfish (Carassius auratus), and to investigate the relationship between changes in performance and gill remodelling and tissue metabolic capacity. Goldfish were acclimated to either hypoxia (48 h at 0.3 mg O2 l–1) or sustained exercise (48 h at 70% of critical swimming speed, Ucrit) and then Pcrit and Ucrit were determined in normoxia (10 mg O2 l–1) and hypoxia (1 mg O2 l–1) and compared with values from control fish. Acclimation to both hypoxia and sustained exercise improved hypoxia tolerance (Pcrit was reduced by 49% and 39%, respectively), which was associated with an increase in lamellar surface area (71% and 43%, respectively) and an increase in blood [Hb] (26% in both groups). Exercise acclimation also resulted in a decrease in routine (). Acclimation to both hypoxia and sustained exercise resulted in a significant increase in Ucrit in hypoxia (18% and 17%, respectively), which was associated with an increase in maximal O2 consumption rate at Ucrit (; 35% and 39%, respectively). While hypoxia acclimation resulted in an increase in Ucrit in normoxia, acclimation to sustained exercise did not improve subsequent swimming performance in normoxia. This lack of improvement was possibly due to depleted oxidizable substrates during exercise acclimation.

Transition in organ function during the evolution of air-breathing; insights from Arapaima gigas, an obligate air-breathing teleost from the Amazon

SUMMARY The transition from aquatic to aerial respiration is associated with dramatic physiological changes in relation to gas exchange, ion regulation, acid–base balance and nitrogenous waste excretion. Arapaima gigas is one of the most obligate extant air-breathing fishes, representing a remarkable model system to investigate (1) how the transition from aquatic to aerial respiration affects gill design and (2) the relocation of physiological processes from the gills to the kidney during the evolution of air-breathing. Arapaima gigas undergoes a transition from water- to air-breathing during development, resulting in striking changes in gill morphology. In small fish (10 g), the gills are qualitatively similar in appearance to another closely related water-breathing fish (Osteoglossum bicirrhosum); however, as fish grow (100–1000 g), the inter-lamellar spaces become filled with cells, including mitochondria-rich (MR) cells, leaving only column-shaped filaments. At this stage, there is a high density of MR cells and strong immunolocalization of Na+/K+-ATPase along the outer cell layer of the gill filament. Despite the greatly reduced overall gill surface area, which is typical of obligate air-breathing fish, the gills may remain an important site for ionoregulation and acid–base regulation. The kidney is greatly enlarged in A. gigas relative to that in O. bicirrhosum and may comprise a significant pathway for nitrogenous waste excretion. Quantification of the physiological role of the gill and the kidney in A. gigas during development and in adults will yield important insights into developmental physiology and the evolution of air-breathing.

Root Effect Hemoglobin May Have Evolved to Enhance General Tissue Oxygen Delivery

Holding Your Breath Hemoglobin and myoglobin are widely responsible for oxygen transport and storage (see the Perspective by Rezende). The ability of diving mammals to obtain enough oxygen to support extended dives and foraging is largely dependent on muscle myoglobin (Mb) content. Mirceta et al. (p. 1303) found that in mammalian lineages with an aquatic or semiaquatic lifestyle, Mb net charge increases, which may represent an adaptation to inhibit self-association of Mb at high intracellular concentrations. Epistasis results from nonadditive genetic interactions and can affect phenotypic evolution. Natarajan et al. (p. 1324) found that epistatic interactions were able to explain the increased hemoglobin oxygen-binding affinity observed in deer mice populations at high altitude. In mammals, the offloading of oxygen from hemoglobin is facilitated by a reduction in the bloods pH, driven by metabolically produced CO2. However, in fish, a reduction in blood pH reduces oxygen carrying capacity of hemoglobin. Rummer et al. (p. 1327) implanted fiber optic oxygen sensors within the muscles of rainbow trout and found that elevated CO2 levels in the water led to acidosis and elevated oxygen tensions. The evolutionary origin of the unloading of oxygen at low pH is traced back to teleosts. [Also see Perspective by Rezende] The Root effect is a pH-dependent reduction in hemoglobin-O2 carrying capacity. Specific to ray-finned fishes, the Root effect has been ascribed specialized roles in retinal oxygenation and swimbladder inflation. We report that when rainbow trout are exposed to elevated water carbon dioxide (CO2), red muscle partial pressure of oxygen (PO2) increases by 65%—evidence that Root hemoglobins enhance general tissue O2 delivery during acidotic stress. Inhibiting carbonic anhydrase (CA) in the plasma abolished this effect. We argue that CA activity in muscle capillaries short-circuits red blood cell (RBC) pH regulation. This acidifies RBCs, unloads O2 from hemoglobin, and elevates tissue PO2, which could double O2 delivery with no change in perfusion. This previously undescribed mechanism to enhance O2 delivery during stress may represent the incipient function of Root hemoglobins in fishes.

Limited extracellular but complete intracellular acid-base regulation during short-term environmental hypercapnia in the armoured catfish, Liposarcus pardalis.

SUMMARY Environmental hypercapnia induces a respiratory acidosis that is usually compensated within 24-96 h in freshwater fish. Water ionic composition has a large influence on both the rate and degree of pH recovery during hypercapnia. Waters of the Amazon are characteristically dilute in ions, which may have consequences for acid-base regulation during environmental hypercapnia in endemic fishes. The armoured catfish Liposarcus pardalis, from the Amazon, was exposed to a water PCO2 of 7, 14 or 42 mmHg in soft water (in μmol l-1: Na+, 15, Cl-, 16, K+, 9, Ca2+, 9, Mg2+, 2). Blood pH fell within 2 h from a normocapnic value of 7.90±0.03 to 7.56±0.04, 7.34±0.05 and 6.99±0.02, respectively. Only minor extracellular pH (pHe) recovery was observed in the subsequent 24-96 h. Despite the pronounced extracellular acidosis, intracellular pH (pHi) of the heart, liver and white muscle was tightly regulated within 6 h (the earliest time at which these parameters were measured) via a rapid accumulation of intracellular HCO3-. While most fish regulate pHi during exposure to environmental hypercapnia, the time course for this is usually similar to that for pHe regulation. The degree of extracellular acidosis tolerated by L. pardalis, and the ability to regulate pHi in the face of an extracellular acidosis, are the greatest reported to date in a teleost fish. The preferential regulation of pHi in the face of a largely uncompensated extracellular acidosis in L. pardalis is rare among vertebrates, and it is not known whether this is associated with the ability to air-breathe and tolerate aerial exposure, or living in water dilute in counter ions, or with other environmental or evolutionary selective pressures. The ubiquity of this strategy among Amazonian fishes and the mechanisms employed by L. pardalis are clearly worthy of further study.

Complete intracellular pH protection during extracellular pH depression is associated with hypercarbia tolerance in white sturgeon, Acipenser transmontanus

Sturgeons are among the most CO2 tolerant of fishes investigated to date. However, the basis of this exceptional CO2 tolerance is unknown. Here, white sturgeon, Acipenser transmontanus, were exposed to elevated CO2 to investigate the mechanisms associated with short-term hypercarbia tolerance. During exposure to 1.5 kPa Pco2, transient blood pH [extracellular pH (pHe)] depression was compensated within 24 h and associated with net plasma HCO3- accumulation and equimolar Cl- loss, and changes in gill morphology, such as a decrease in apical surface area of mitochondrial-rich cells. These findings indicate that pHe recovery at this level of hypercarbia is accomplished in a manner similar to most freshwater teleost species studied to date, although branchial mechanisms involved may differ. White sturgeon exposed to more severe hypercarbia (3 and 6 kPa Pco2) for 48 h exhibited incomplete pH compensation in blood and red blood cells. Despite pHe depression, intracellular pH (pHi) of white muscle, heart, brain, and liver did not decrease during a transient (6 h of 1.5 kPa Pco2) or prolonged (48 h at 3 and 6 kPa Pco2 blood acidosis. This pHi protection was not due to high intrinsic buffering in tissues. Such tight active cellular regulation of pHi in the absence of pHe compensation represents a unique pattern for non-air-breathing fishes, and we hypothesize that it is the basis for the exceptional CO2 tolerance of white sturgeon and, likely, other CO2 tolerant fishes. Further research to elucidate the specific mechanisms responsible for this tremendous pH regulatory capacity in tissues of white sturgeon is warranted.

Ions first: Na+ uptake shifts from the skin to the gills before O2 uptake in developing rainbow trout, Oncorhynchus mykiss

This is the first direct physiological evidence in support of the ionoregulatory hypothesis, challenging the long-held assumption that teleost gills develop initially for gas exchange. Resting unidirectional sodium (Na+) uptake and oxygen (O2) uptake across the skin and gills were measured simultaneously in larval rainbow trout, Oncorhynchus mykiss, during development. In soft and hard water, Na+ uptake shifted to the gills by 15 and 16 days post-hatch (dph) while O2 uptake took 50–80% longer and shifted by 23 and 28 dph, respectively. This suggests that gills are required for ionoregulation prior to gas exchange in developing rainbow trout. The age of transition for Na+ uptake, gill Na+, K+-ATPase (NKA) α-subunit protein expression and gill NKA enzyme activity were not significantly different between soft and hard water-reared groups, which suggests a lack of plasticity in gill ionoregulatory development. In rainbow trout, the gills assume a dominant role in ionoregulation before gas exchange, suggesting that ionoregulation may be the initial driving force for gill development. Further investigation is required to determine whether this pattern is consistent with other teleosts and more basal fishes during early development to gain insight into the role of ionoregulation in vertebrate gill evolution.

The origin and evolution of the surfactant system in fish: Insights into the evolution of lungs and swim bladders

Several times throughout their radiation fish have evolved either lungs or swim bladders as gas‐holding structures. Lungs and swim bladders have different ontogenetic origins and can be used either for buoyancy or as an accessory respiratory organ. Therefore, the presence of air‐filled bladders or lungs in different groups of fishes is an example of convergent evolution. We propose that air breathing could not occur without the presence of a surfactant system and suggest that this system may have originated in epithelial cells lining the pharynx. Here we present new data on the surfactant system in swim bladders of three teleost fish (the air‐breathing pirarucu Arapaima gigas and tarpon Megalops cyprinoides and the non‐air‐breathing New Zealand snapper Pagrus auratus). We determined the presence of surfactant using biochemical, biophysical, and morphological analyses and determined homology using immunohistochemical analysis of the surfactant proteins (SPs). We relate the presence and structure of the surfactant system to those previously described in the swim bladders of another teleost, the goldfish, and those of the air‐breathing organs of the other members of the Osteichthyes, the more primitive air‐breathing Actinopterygii and the Sarcopterygii. Snapper and tarpon swim bladders are lined with squamous and cuboidal epithelial cells, respectively, containing membrane‐bound lamellar bodies. Phosphatidylcholine dominates the phospholipid (PL) profile of lavage material from all fish analyzed to date. The presence of the characteristic surfactant lipids in pirarucu and tarpon, lamellar bodies in tarpon and snapper, SP‐B in tarpon and pirarucu lavage, and SPs (A, B, and D) in swim bladder tissue of the tarpon provide strong evidence that the surfactant system of teleosts is homologous with that of other fish and of tetrapods. This study is the first demonstration of the presence of SP‐D in the air‐breathing organs of nonmammalian species and SP‐B in actinopterygian fishes. The extremely high cholesterol/disaturated PL and cholesterol/PL ratios of surfactant extracted from tarpon and pirarucu bladders and the poor surface activity of tarpon surfactant are characteristics of the surfactant system in other fishes. Despite the paraphyletic phylogeny of the Osteichthyes, their surfactant is uniform in composition and may represent the vertebrate protosurfactant.

Relationship between toxicant transfer kinetic processes and fish oxygen consumption.

Three organic compounds of different hydrophobicity, 1,2,4,5-tetrachlorobenzene (TeCB), 3,4,5,6-tetrachloroguaiacol (TeCG) and 4,6-dichlorobenzenediol (DBD), were chosen as the test chemicals to carry out a series of investigations to look at the relationship between toxicant transfer and fish metabolic rate. A significant correlation was found between the toxicant uptake rate constant (k(1)) and fish oxygen consumption, regardless of fish size and species. This correlation was improved when fish toxicant body load was expressed on a percent body lipid basis. Similarly, there also existed a significant relationship between the toxicant depuration rate constant (k(2)) and fish oxygen uptake for a range of chemicals with different octanol/water partition coefficients (K(ow)).

Interactions Between Ion and Gas Transfer in Freshwater Teleost Fish

Carbonic anhydrase and proton ATPase are co-distributed, being restricted to the apical regions of the gill epithelium of freshwater teleosts. Carbonic anhydrase supplies protons to the apical proton ATPase. Carbonic anhydrase is absent from the basal regions of the gill epithelium. Plasma flowing through the gills has no available carbonic anhydrase activity and plasma CO2/bicarbonate reactions are uncatalyzed. Thus, bicarbonate dehydration in plasma is negligible, and catalyzed bicarbonate dehydration occurs in erythrocytes in blood flowing through the gills. This results in tight coupling of carbon dioxide excretion to oxygen uptake and the evolution of hemoglobins with large Haldane effects but low buffering capacities, typical of many freshwater teleosts. Tight coupling of carbon dioxide and oxygen transfer in these fish also ensures that the Root shift does not impair oxygen uptake at the gills. Under these conditions, there is a selective advantage for hemoglobins with a Root shift. The presence of a Root shift augments oxygen transfer to the tissues in general and the eye and swimbladder in particular.