Characterization of carbonic anhydrase and anion exchange in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish

The purpose of this study was to investigate the characteristics of carbonic anhydrase (CA) and the Cl⁻/HCO₃⁻ exchanger (Band 3; AE1) in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish, in order to further understand the strategies of blood CO₂ transport in lower vertebrates and gain insights into the evolution of the vertebrate erythrocyte proteins, CA and Band 3. A significant amount of CA activity was measured in the erythrocytes of bowfin (70 mmol CO₂ min⁻¹ ml⁻¹), although it appeared to be lower than that in the erythrocytes of teleost fish. The turnover number (K_cat) of bowfin erythrocyte CA was intermediate between that of the slow type I CA isozyme in agnathans and elasmobranchs and the fast type II CA in the erythrocytes of the more recent teleost fishes, but the inhibition properties of bowfin erythrocyte CA were similar to the fast mammalian CA isozyme, CA II. In contrast to previous findings, a plasma CA inhibitor was found to be present in the blood of bowfin. Bowfin erythrocytes were also found to possess a high rate of Cl⁻/HCO₃⁻ exchange (6 nmol HCO₃⁻ s⁻¹ cm⁻²) that was sensitive to DIDS. Visualization of erythrocyte membrane proteins by SDS-PAGE revealed a major band in the 100 kDa range for the trout, which would be consistent with the anion exchanger. In contrast, the closest major band for the membranes of bowfin erythrocytes was around the 140 kDa range. Taken together, these results suggest that the strategy for blood CO₂ transport in bowfin is probably similar to that in most other vertebrates despite several unique characteristics of erythrocyte CA and Band 3 in these primitive fish.     

Neuropeptides and nitric oxide synthase in the gill and the air-breathing organs of fishes

Anatomical and histochemical studies have demonstrated that the bulk of autonomic neurotransmission in fish gill is attributed to cholinergic and adrenergic mechanisms (Nilsson. 1984. In: Hoar WS, Randall DJ, editors. Fish physiology, Vol. XA. Orlando: Academic Press. p 185-227; Donald. 1998. In: Evans DH, editor. The physiology of fishes, 2nd edition. Boca Raton: CRC Press. p 407-439). In many tissues, blockade of adrenergic and cholinergic transmission results in residual responses to nerve stimulation, which are termed NonAdrenergic, NonCholinergic (NANC). The discovery of nitric oxide (NO) has provided a basis for explaining many examples of NANC transmissions with accumulated physiological and pharmacological data indicating its function as a primary NANC transmitter.Little is known about the NANC neurotransmission, and studies on neuropeptides and NOS (Nitric Oxide Synthase) are very fragmentary in the gill and the air-breathing organs of fishes. Knowledge of the distribution of nerves and effects of perfusing agonists may help to understand the mechanisms of perfusion regulation in the gill (Olson. 2002. J Exp Zool 293:214-231).Air breathing as a mechanism for acquiring oxygen has evolved independently in several groups of fishes, necessitating modifications of the organs responsible for the exchange of gases. Aquatic hypoxia in freshwaters has been probably the more important selective force in the evolution of air breathing in vertebrates. Fishes respire with gills that are complex structures with many different effectors and potential control systems. Autonomic innervation of the gill has received considerable attention. An excellent review on branchial innervation includes Sundin and Nilsson's (2002. J Exp Zool 293:232-248) with an emphasis on the anatomy and basic functioning of afferent and efferent fibers of the branchial nerves. The chapters by Evans (2002. J Exp Zool 293:336-347) and Olson (2002) provide new challenges about a variety of neurocrine, endocrine, paracrine and autocrine signals that modulate gill perfusion and ionic transport.The development of the immunohistochemical techniques has led to a new phase of experimentation and to information mainly related to gills rather than air-breathing organs of fishes. During the last few years, identification of new molecules as autonomic neurotransmitters, monoamines and NO, and of their multiple roles as cotransmitters, has reshaped our knowledge of the mechanisms of autonomic regulation of various functions in the organs of teleosts (Donald, '98).NO acts as neurotransmitter and is widely distributed in the nerves and the neuroepithelial cells of the gill, the nerves of visceral muscles of the lung of polypterids, the vascular endothelial cells in the air sac of Heteropneustes fossilis and the respiratory epithelium in the swimbladder of the catfish Pangasius hypophthalmus. In addition, 5-HT, enkephalins and some neuropeptides, such as VIP and PACAP, seem to be NANC transmitter candidates in the fish gill and polypterid lung. The origin and function of NANC nerves in the lung of air-breathing fishes await investigation.Several mechanisms have developed in the Vertebrates to control the flow of blood to respiratory organs. These mechanisms include a local production of vasoactive substances, a release of endocrine hormones into the circulation and neuronal mechanisms.Air breathers may be expected to have different control mechanisms compared with fully aquatic fishes. Therefore, we need to know the distribution and function of autonomic nerves in the air-breathing organs of the fishes.     

Which came first, the lung or the breath?

Lungs are the characteristic air-filled organs (AO) of the Polypteriformes, lungfish and tetrapods, whereas the swimbladder is ancestral in all other bony fish. Lungs are paired ventral derivatives of the pharynx posterior to the gills. Their respiratory blood supply is the sixth branchial artery and the venous outflow enters the heart separately from systemic and portal blood at the sinus venosus (Polypteriformes) or the atrium (lungfish), or is delivered to a separate left atrium (tetrapods). The swimbladder, on the other hand, is unpaired, and arises dorsally from the posterior pharynx. It is employed in breathing in Ginglymodi (gars), Halecomorphi (bowfin) and in basal teleosts. In most cases, its respiratory blood supply is homologous to that of the lung, but the vein drains to the cardinal veins. Separate intercardiac channels for oxygenated and deoxygenated blood are lacking. The question of the homology of lungs and swimbladders and of breathing mechanisms remains open. On the whole, air ventilatory mechanisms in the actinopterygian lineage are similar among different groups, including Polypteriformes, but are distinct from those of lungfish and tetrapods. However, there is extreme variation within this apparent dichotomy. Furthermore, the possible separate origin of air breathing in actinopterygian and 'sarcopterygian' lines is in conflict with the postulated much more ancient origin of vertebrate air-breathing organs. New studies on the isolated brainstem preparation of the gar (Lepisosteus osseus) show a pattern of efferent activity associated with a glottal opening that is remarkably similar to that seen in the in-vitro brainstem preparation of frogs and tadpoles. Given the complete lack of evidence for AO in chondrichthyans, and the isolated position of placoderms for which buoyancy organs of uncertain homology have been demonstrated, it is likely that homologous pharyngeal AO arose in the ancestors of early bony fish, and was pre-dated by behavioral mechanisms for surface (water) breathing. The primitive AO may have been the posterior gill pouches or even the modified gills themselves, served by the sixth branchial artery. Further development of the dorsal part may have led to the respiratory swimbladder, whereas the paired ventral parts evolved into lungs.     

Cadmium toxicity in fishes

Cadmium from industrial effluents pollutes sea and river water and hence may effect the fish population therein. The present investigation shows that resistivity is greater in case of air-breathing fishes as compared to non air-breathing fishes with regards to their duration of survival and growth when exposed to a cadmium containing environment. Under sub-acute levels of cadmium exposure, they respond differently so far as the activities of their liver, gill and intestinal lysosomal enzymes are concerned. Though both species show a definite stress condition on cadmium exposure, the two anatomically different fishes respond differently towards carbohydrate metabolism as observed from liver and muscle glycogen content, liver microsomal glucose-6-phosphatase activity and serum glucose level.     

Aspiratory respiration in Arapaima gigas (Teleostei, Osteoglossomorpha): A reappraisal

Recently it has been hypothesized that the osteoglossoid fish Arapaima gigas is unique amongst air-breathing bony fishes in that it ventilates its respiratory swimbladder by aspiration. Aspiration, it was suggested, is effected by a diaphragm-like septum lying below the swimbladder, and activated through lateral movement of the ribs.
By using both high-speed X-ray and light cinematography this hypothesis can be refuted. Furthermore, dissection shows that the presumed anatomical basis for aspiration stems from a misinterpretation of the usual retroperitoneal position of the swimbladder, and that the ribs are capable of only a very restricted lateral movement. Arapaima gigas , like other bony fishes with a respiratory swimbladder, ventilates that organ by means of a buccopharyngeal pump.
The principal afferent vascular supply to the swimbladder is, however, unusual since it stems from the caudal vein via the renal-portal system. This and other aspects of swimbladder structure and anatomy may throw further light on the interrelationships of osteoglossoid fishes.     

The evolution of Root effect hemoglobins in the absence of intracellular pH protection of the red blood cell: insights from primitive fishes

The Root effect, a reduction in blood oxygen (O₂) carrying capacity at low pH, is used by many fish species to maximize O₂ delivery to the eye and swimbladder. It is believed to have evolved in the basal actinopterygian lineage of fishes, species that lack the intracellular pH (pH_i) protection mechanism of more derived species’ red blood cells (i.e., adrenergically activated Na⁺/H⁺ exchangers; βNHE). These basal actinopterygians may consequently experience a reduction in blood O₂ carrying capacity, and thus O₂ uptake at the gills, during hypoxia- and exercise-induced generalized blood acidoses. We analyzed the hemoglobins (Hbs) of seven species within this group [American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), mooneye (Hiodon tergisus), and pirarucu (Arapaima gigas)] for their Root effect characteristics so as to test the hypothesis of the Root effect onset pH value being lower than those pH values expected during a generalized acidosis in vivo. Analysis of the haemolysates revealed that, although each of the seven species displayed Root effects (ranging from 7.3 to 40.5% desaturation of Hb with O₂, i.e., Hb O₂ desaturation), the Root effect onset pH values of all species are considerably lower (ranging from pH 5.94 to 7.04) than the maximum blood acidoses that would be expected following hypoxia or exercise (pH_i 7.15–7.3). Thus, although these primitive fishes possess Hbs with large Root effects and lack any significant red blood cell βNHE activity, it is unlikely that the possession of a Root effect would impair O₂ uptake at the gills following a generalized acidosis of the blood. As well, it was shown that both maximal Root effect and Root effect onset pH values increased significantly in bowfin over those of the more basal species, toward values of similar magnitude to those of most of the more derived teleosts studied to date. This is paralleled by the initial appearance of the choroid rete in bowfin, as well as a significant decrease in Hb buffer value and an increase in Bohr/Haldane effects, together suggesting bowfin as the most basal species capable of utilizing its Root effect to maximize O₂ delivery to the eye.     

Biochemical responses of fish exposed to a harmful dinoflagellate Cochlodinium polykrikoides

To elucidate the ichthyotoxic mechanisms of a harmful dinoflagellate Cochlodinium polykrikoides, biochemical responses of fish exposed to blooms were investigated. Particularly, based on our finding that oxidative damages of gill were associated with fish mortality (J. Plankton Res. 21 (1999) 2105-2115), dysfunction of ion-transporting enzymes and secretion of gill mucus of fish exposed to this bloom species were examined. The susceptibilities of several fishes to C. polykrikoides were different; the active pelagic fishes such as black scraper Thamnaconus septentrionalis, red sea bream Pagrus major, beakperch Oplegnathus fasciatus and seaperch Malakichthys wakiyae, were more vulnerable than the benthic fishes, flounder Paralichthys olivaceus and rockfish Sebastes inermis. In addition, the higher the algal cell density, the higher the fish mortality. When the test fishes were exposed to C. polykrikoides of 5000 cells ml(-1), the transport-related enzymes, carbonic anhydrase and Na(+)/K(+)-ATPase activities were significantly decreased. The activity of carbonic anhydrase was decreased with increasing algal cell density and exposure time. The quantity of total polysaccharide in gill mucus is higher in the fish exposed to C. polykrikoides than in the control fish; the magnitudes were higher in the pelagic fishes than that of benthic fishes. Moreover, a drop of blood pH and oxygen partial pressure (pO(2)) was also observed in red sea bream and flounder subjected to C. polykrikoides. These results suggest that the inactivation of gill transport-related enzymes activities, the fall in blood pO(2) and abnormal secretion of gill mucus by the C. polykrikoides may be one of the principal causes of fish kill.     

Pulmonary carbonic anhydrase in vertebrate gas exchange organs

Carbonic anhydrase (CA) catalyzes the interconversion of CO(2) and HCO(3)(-). Intracellular (extravascular) and intravascular (extracellular) CA has been identified and localized in the lungs of reptiles and mammals. Less information is known, however, on the presence of intravascular CA in the lungs of amphibians and avians. In the present study, perfusion studies were used to compare the catalytic activity of pulmonary intravascular CA in reptiles and mammals. In addition, SDS-resistant CA activity was examined in microsomal fractions prepared from gill/lung tissue from representative animals in each vertebrate class. Finally, the CNO(-) sensitivity of the microsomal CA activity was compared. No SDS-resistant CA activity was found in gill microsomal fractions of several fish species. In contrast, the data suggest that SDS-resistant, intravascular pulmonary CA activity is present in air-breathing vertebrates with vastly differing lung morphologies and that the kinetics of inhibition is remarkably comparable between the vertebrate classes.     

Chloride inhibition of nitrite uptake for non-teleost Actinopterygiian fishes

Fish that transport environmental chloride with a gill uptake mechanism (gill epithelial Cl(-)/HCO(3)(-)cotransport exchange system), also transport nitrite into plasma through the same mechanism. Because of the relationship between nitrite uptake and the gill chloride uptake mechanism, nitrite uptake can provide insight regarding the method of chloride uptake for fish. This study was designed to determine if non-teleost fishes concentrate nitrite in their plasma, and to determine if chloride inhibits nitrite uptake in non-teleost fish. To determine if bowfin Amia calva, spotted gar Lepisosteus oculatus, alligator gar Atractosteus spatula, and paddlefish Polyodon spathula concentrate environmental nitrite in their plasma, individuals were exposed to concentrations of 0, 1, 10, or 100 mg/L nitrite-N. After exposure, all species had plasma nitrite-N concentrations greater than environmental levels. To determine if chloride inhibits nitrite uptake for spotted gar, alligator gar, and paddlefish, fish were exposed to 1 mg/L nitrite-N and 20 mg/L chloride as calcium chloride, or to 1 mg/L nitrite-N only. Chloride effectively prevented nitrite from being concentrated in the plasma of all species. It appears that non-teleost fish concentrate nitrite in their plasma via their chloride uptake mechanism and that this is an ancestral characteristic for teleost.     

Swimming behavior in relation to buoyancy in an open swimbladder fish, the Chinese sturgeon

The swimbladder of fishes is readily compressed by hydrostatic pressure with depth, causing changes in buoyancy. While modern fishes can regulate buoyancy by secreting gases from the blood into the swimbladder, primitive fishes, such as sturgeons, lack this secretion mechanism and rely entirely on air gulped at the surface to inflate the swimbladder. Therefore, sturgeons may experience changes in buoyancy that will affect their behavior at different depths. To test this prediction, we attached data loggers to seven free-ranging Chinese sturgeons Acipenser sinensis in the Yangtze River, China, to monitor their depth utilization, tail-beating activity, swim speed and body inclination. Two distinct, individual-specific, behavioral patterns were observed. Four fish swam at shallow depths (7–31 m), at speeds of 0.5–0.6 m s⁻¹, with ascending and descending movements of 1.0–2.4 m in amplitude. They beat their tails continuously, indicating that their buoyancy was close to neutral with their inflated swimbladders. In addition, their occasional visits to the surface suggest that they gulped air to inflate their swimbladders. The other three fish spent most of their time (88–94%) on the river bottom at a depth of 106–122 m with minimum activity. They occasionally swam upwards at speeds of 0.6–0.8 m s⁻¹ with intense tailbeats before gliding back passively to the bottom, in a manner similar to fishes that lack a swimbladder. Their bladders were probably collapsed by hydrostatic pressure, resulting in negative buoyancy. We conclude that Chinese sturgeons behave according to their buoyancy, which varies with depth due to hydrostatic compression of the swimbladder.     

Effect of air breathing on acid-base and ion regulation after exhaustive exercise and during low pH exposure in the bowfin, Amia calva.

To explore a potential conflict between air breathing and acid-base regulation in the bowfin (Amia calva), we examined how individuals with access to air differed from fish without air access in their response to acidosis. After exhaustive exercise, bowfin with access to air recovered significantly more slowly from the acidosis than fish without air access. While arterial blood pH (pH(a)) of fish without air access recovered to resting levels by 8 h, pH(a) was still significantly depressed in fish having access to air. In addition, Pco(2) was slightly more elevated in fish having air access than those without it. Fish with access to air still had a significant metabolic acid load after 8-h recovery, while those without air access completely cleared the load within 4 h. These results suggest that bowfin with access to air were breathing air and, consequently, were less able to excrete CO(2) and H(+) and experienced a delayed recovery. In contrast, during exposure to low pH, air breathing seemed to have a protective effect on acid-base status in bowfin. During exposure to low pH water, bowfin with access to air developed a much milder acidosis than bowfin without air access. The more severe acidosis in fish without air access was caused by an increased rate of lactic acid production. It appears that enhanced O(2) delivery allowed air-breathing bowfin to avoid acidosis-induced anaerobic metabolism and lactic acid production. In addition, during low pH exposure, plasma Na(+) and Cl(-) concentrations of fish without air access fell slightly more rapidly than those in fish with air access, indicating that the branchial ventilatory changes associated with air breathing limited, to some degree, ion losses associated with low pH exposure.     

The metabolic organization of a primitive air-breathing fish, the Florida gar (Lepisosteus platyrhincus)

The metabolic organization of the air-breathing Florida gar, Lepisosteus platyrhincus, was assessed by measuring the maximal activities of key enzymes in several metabolic pathways in selected tissues, concentrations of plasma metabolites including nonesterified fatty acids (NEFA), free amino acids (FAA) and glucose as well as tissue FAA levels. In general, L. platyrhincus has an enhanced capacity for carbohydrate metabolism as indicated by elevated plasma glucose levels and high activities of gluconeogenic and glycolytic enzymes. Based upon these properties, glucose appears to function as the major fuel source in the Florida gar. The capacity for lipid metabolism in L. platyrhincus appears limited as plasma NEFA levels and the activities of enzymes involved in lipid oxidation are low relative to many other fish species. L. platyrhincus is capable of oxidizing both D- and L-beta-hydroxybutyrate, with tissue-specific preferences for each stereoisomer, yet the capacity for ketone body metabolism is low compared with other primitive fishes. Based on enzyme activities, the metabolism of the air-breathing organ more closely resembles that of the mammalian lung than a fish swim bladder. The Florida gar sits phylogenetically and metabolically in an intermediate position between the "primitive" elasmobranchs and the "advanced" teleosts. The apparently unique metabolic organization of the gar may have evolved in the context of a bimodal air-breathing environmental adaptation.     

The source of lamellar mitochondria-rich cells in the air-breathing fish, Trichogaster leeri

Pavement cells and the mitochondria-rich cells (MRCs) are two of the main types of cells in fish gill epithelia. The pavement cells are generally responsible for gas exchange and MRCs for ion regulation. MRCs are found especially in the trailing edge and the interlamellar region of gill filament. In some species, MRCs are also observed in the gill lamellae. A previous study reported the likelihood of having lamellar MRCs in air-breathing fishes. Nevertheless, the source of lamellar MRCs is unclear. We used the air-breathing fish, Trichogaster leeri, to investigate the source of proliferated cells on the lamellae when 5-bromo-2-deoxyuridine (BrdU) was injected at different times before fish were sampled from deionized water. There were two major findings in this study. First, undifferentiated cells were found in the lamellae, as well as in the filaments. And, within 12-24 hr, a proliferated cell, identified as BrdU cell, could differentiate to an MRC in the gill lamellae. Second, the filaments and the lamellae in T. leeri responded to ionic stress differently but the proportion of the proliferated MRCs to the BrdU cells remained constant. Our results suggested that the lamellar MRCs were mainly differentiated from the cells that proliferated earlier from the lamellae.     

Extracellular carbonic anhydrase in the dogfish, Squalus acanthias: a role in CO2 excretion.

In Pacific spiny dogfish (Squalus acanthias), plasma CO(2) reactions have access to plasma carbonic anhydrase (CA) and gill membrane-associated CA. The objectives of this study were to characterise the gill membrane-bound CA and investigate whether extracellular CA contributes significantly to CO(2) excretion in dogfish. A subcellular fraction containing membrane-associated CA activity was isolated from dogfish gills and incubated with phosphatidylinositol-specific phospholipase C. This treatment caused significant release of CA activity from its membrane association, a result consistent with identification of the dogfish gill membrane-bound CA as a type IV isozyme. Inhibition constants (K(i)) against acetazolamide and benzolamide were 4.2 and 3.5 nmol L(-1), respectively. Use of a low dose (1.3 mg kg(-1) or 13 micromol L(-1)) of benzolamide to selectively inhibit extracellular CA in vivo caused a significant 30%-60% reduction in the arterial-venous total CO(2) concentration difference, a significant increase in Pco(2) and an acidosis, without affecting blood flow or ventilation. No effect of benzolamide on any measure of CO(2) excretion was detected in rainbow trout (Oncorhynchus mykiss). These results indicate that extracellular CA contributes substantially to CO(2) excretion in the dogfish, an elasmobranch, and confirm that CA is not available to plasma CO(2) reactions in rainbow trout, a teleost.     

Changes in carbonic anhydrase activity in coho salmon smolts resulting from physical training and transfer into seawater

Carbonic anhydrase (CA) activity was measured in blood and in gill tissue of coho salmon smolts during chronic exercise and subsequent transfer into seawater. The mean level of CA activity was higher in blood than gill tissue in both freshwater and seawater. CA activity in gill tissue increased significantly after the smolts had adapted to seawater. CA activity in blood decreased significantly in the group of fish given the highest exercise level after they had adapted to seawater. There were no significant differences in CA activity in gill tissue between control and exercise groups in either freshwater or seawater.     

The distribution of mitochondria-rich cells in the gills of air-breathing fishes

Respiration and ion regulation are the two principal functions of teleostean gills. Mainly found in the gill filaments of fish, mitochondria-rich cells (MRCs) proliferate to increase the ionoregulatory capacity of the gill in response to osmotic challenges. Gill lamellae consist mostly of pavement cells, which are the major site of gas exchange. Although lamellar MRCs have been reported in some fish species, there has been little discussion of which fish species are likely to have lamellar MRCs. In this study, we first compared the number of filament and lamellar MRCs in air-breathing and non-air-breathing fish species acclimated to freshwater and 5 g NaCl L(-1) conditions. An increase in filament MRCs was found in both air-breathing and non-air-breathing fish acclimated to freshwater. Lamellar MRCs were found only in air-breathing species, but the number of lamellar MRCs did not change significantly with water conditions, except in Periophthalmus cantonensis. Next, we surveyed the distribution of MRCs in the gills of 66 fish species (including 29 species from the previous literature) from 12 orders, 28 families, and 56 genera. Our hypothesis that lamellar MRCs are more likely to be found in air-breathing fishes was supported by a significant association between the presence of lamellar MRCs and the mode of breathing at three levels of systematic categories (species, genus, and family). Based on this integrative view of the multiple functions of fish gills, we should reexamine the role of MRCs in freshwater fish.     

Carbonic anhydrase activity in the blood and the gills of rainbow trout during long-term hypercapnia in hard,bicarbonate-rich freshwater

Summary Carbonic anhydrase (CA) activities in gills and venous blood, acid-base balance, and haematological variables were studied during environmental hypercapnia in rainbow trout (Salmo gairdneri). Batches of 8–10 fish were exposed to about 3 or 13 mmHg in flow-through tests of various duration from 4 h to 80 days.After initial acidosis, blood pH rose above pre-experimental values. At 3 mmHg it became normal again within 21 days, while at 13 mmHg the overshoot lasted for 80 days. In fish acclimated for 3 weeks or more to 13 mmHg , blood HCO ₃ ^– increased four to five times while plasma Cl^– levels were lower and K⁺ higher. Na⁺ levels did not show any consistent trend associated with exposure to hypercapnia. After an initial acidaemia, Hct, Hb, and RBC remained relatively constant.Patterns of change in CA activity differed between gills and erythrocytes. Initially, blood CA decreased at both levels. It then began rising after about 3 weeks and tended to reach pre-experimental values by 80 day's hypercapnia. At 13 mmHg , gill CA increased to twice the pre-experimental level. Compared with blood CA, gill CA appeared to be more specifically involved in fish acclimation to hypercapnia, which demands an increase in blood bicarbonate to provide a sufficient buffering capacity. Increased CA indicates that the gill enzyme may play a more important role than blood CA in acid-base regulation in fish during hypercapnia.Abbreviations CA carbonic anhydrase - Hb haemoglobin - Hct haematocrit value - RBC red blood cells     

Morphological and biochemical variations in the gills of 12 aquatic air-breathing anabantoid fish

All fish species in the Anabantoidei suborder are aquatic air-breathing fish. These species have an accessory air-breathing organ, called the labyrinth organ, in the branchial cavity and can engulf air at the surface of the water to assist in gas exchange. It is therefore necessary to examine the extent of gill modification among anabantoid fish species and the potential trade-offs in their function. The experimental hypothesis that we aimed to test is whether anabantoid fishes have both morphological and functional variations in the gills among different species. We examined the gills of 12 species from three families and nine genera of Anabantoidei. Though the sizes of the fourth gill arch in three species of Trichogaster were reduced significantly, not all anabantoid species had morphological and functional variations in the gills. In these three species, the specific enzyme activity and relative protein abundance of Na(+)/K(+)-ATPase were significantly higher in the anterior gills as compared with the posterior gills and the labyrinth organ. The relative abundance of cytosolic carbonic anhydrase, an indicator of gas exchange, was found to be highest in the labyrinth organ. The phylogenetic distribution of the fourth gill's morphological differentiation suggests that these variations are lineage specific, which may imply a phylogenetic influence on gill morphology in anabantoid species.     

The importance of a single amino acid substitution in reduced red blood cell carbonic anhydrase function of early-diverging fish

In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO₂) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO₂ excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin’s capacity to bind with oxygen (O₂)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.