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.     

Balancing the competing requirements of air-breathing and display behaviour during male–male interactions in Siamese fighting fish Betta splendens

Air-breathing fish of the Anabantoidei group meet their metabolic requirements for oxygen through both aerial and aquatic gas exchange. Siamese fighting fish Betta splendens are anabantoids that frequently engage in aggressive male–male interactions which cause significant increases in metabolic rate and oxygen requirements. These interactions involve opercular flaring behaviour that is thought to limit aquatic oxygen uptake, and combines with the increase in metabolic rate to cause an increase in air-breathing behaviour. Air-breathing events interrupt display behaviour and increase risk of predation, raising the question of how Siamese fighting fish manage their oxygen requirements during agonistic encounters. Using open-flow respirometry, we measured rate of oxygen consumption in displaying fish to determine if males increase oxygen uptake per breath to minimise visits to the surface, or increase their reliance on aquatic oxygen uptake. We found that the increased oxygen requirements of Siamese fighting fish during display behaviour were met by increased oxygen uptake from the air with no significant changes in aquatic oxygen uptake. The increased aerial oxygen uptake was achieved almost entirely by an increase in air-breathing frequency. We conclude that limitations imposed by the reduced gill surface area of air-breathing fish restrict the ability of Siamese fighting fish to increase aquatic uptake, and limitations of the air-breathing organ of anabantoids largely restrict their capacity to increase oxygen uptake per breath. The resulting need to increase surfacing frequency during metabolically demanding agonistic encounters has presumably contributed to the evolution of the stereotyped surfacing behaviour seen during male–male interactions, during which one of the fish will lead the other to the surface, and each will take a breath of air.     

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.     

Changes in cardiac output during swimming and aquatic hypoxia in the air-breathing Pacific tarpon

Pacific tarpon (Megalops cyprinoides) use a modified gas bladder as an air-breathing organ (ABO). We examined changes in cardiac output (V(b)) associated with increases in air-breathing that accompany exercise and aquatic hypoxia. Juvenile (0.49 kg) and adult (1.21 kg) tarpon were allowed to recover in a swim flume at 27 degrees C after being instrumented with a Doppler flow probe around the ventral aorta to monitor V(b) and with a fibre-optic oxygen sensor in the ABO to monitor air-breathing frequency. Under normoxic conditions and in both juveniles and adults, routine air-breathing frequency was 0.03 breaths min(-1) and V(b) was about 15 mL min(-1) kg(-1). Normoxic exercise (swimming at about 1.1 body lengths s(-1)) increased air-breathing frequency by 8-fold in both groups (reaching 0.23 breaths min(-1)) and increased V(b) by 3-fold for juveniles and 2-fold for adults. Hypoxic exposure (2 kPa O2) at rest increased air-breathing frequency 19-fold (to around 0.53 breaths min(-1)) in both groups, and while V(b) again increased 3-fold in resting juvenile fish, V(b) was unchanged in resting adult fish. Exercise in hypoxia increased air-breathing frequency 35-fold (to 0.95 breaths min(-1)) in comparison with resting normoxic fish. While juvenile fish increased V(b) nearly 2-fold with exercise in hypoxia, adult fish maintained the same V(b) irrespective of exercise state and became agitated in comparison. These results imply that air-breathing during exercise and hypoxia can benefit oxygen delivery, but to differing degrees in juvenile and adult tarpon. We discuss this difference in the context of myocardial oxygen supply.     

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.     

Devonian climate change, breathing, and the origin of the tetrapod stem group

The diversification of the tetrapod stem group occurred duringthe late Middle through the Late Devonian, that is from theGivetian to Famennian stages about 385–365 million yearsago. The relationships between the known taxa representing thisradiation have currently reached a reasonable consensus so thatinterpretations of the order of appearance of tetrapod charactersis possible. The immediate fish relatives of the earliest limbedtetrapods show what is interpreted as a progressive increasein the spiracular chamber and its opening to the outside. Here,this is inferred to be associated with an increased capacityfor air-breathing. Lungs are thought to have been present inmost early bony fishes, and were most likely ventilated by air-gulping.This could have brought about a facultative capacity for air-breathing,which the tetrapod stem group exploited to the greatest degree.These adaptations are shown not only in freshwater forms butalso in estuarine and marginal marine forms. Estimates of oxygenlevels during this period suggest that they were unprecedentedlylow during the Givetian and Frasnian periods. At the same time,plant diversification was at its most rapid, changing the characterof the landscape and contributing, via soils, soluble nutrients,and decaying plant matter, to anoxia in all water systems. Theco-occurrence of these global events may explain the evolutionof air-breathing adaptations in at least two lobe-finned groups,contributing directly to the rise of the tetrapod stem group.In contrast to recent studies, low atmospheric oxygen is notconsidered to be a causal factor in the lack of fossils documentingthe evolution of Early Carboniferous tetrapods.     

Functional differentiation in the anterior gills of the aquatic air-breathing fish, <Emphasis Type="Italic">Trichogaster leeri</Emphasis>

The fish gill is a multifunctional organ responsible for gas exchange and ionic regulation. It is hypothesized that both morphological and functional differentiation can be found in the gills of the aquatic air-breathing fish, Trichogaster leeri. To test this, we used the air-breathing fish, Trichogaster leeri, to investigate various morphological/functional parameters. First, we evaluated the importance of performing the aquatic surface respiration behavior in T. leeri. A reduced survival rate was observed when fish were kept in the restrained cages in hypoxic conditions. On the gross anatomy of gills, we found evidence of both morphological and functional modification in the first and the second gills and are responsible for ionic regulation. There were large-bore arterioarterial shunts in the fourth gill arch. It is specialized for the transport of oxygenated blood and is less responsive to environmental stress. In addition, the anterior and the posterior gills differed in the Na⁺, K⁺-ATPase activity upon ionic stresses. That is, only the Na⁺, K⁺-ATPase activity of the anterior two gills was up-regulated significantly in the deionized water. Lastly, we found that the number of mitochondria-rich cells in the first and the second gills increased following ionic stress and no difference was found in the third and the fourth gills following such an exposure. These results supported the hypothesis that there are morphological and functional differences between anterior and posterior gill arches within the air-breathing Trichogaster leeri. In contrast, no significant difference was found among gills in gross anatomy, filament density and Na⁺, K⁺-ATPase activity in the non-air-breather, Barbodes schwanenfeldi.     

The evolution of the terrestrial vertebrates: environmental and physiological considerations

Physiological evidence has long been used to suggest that the gnathostomous vertebrates (those possessing jaws) were primitively fresh water. The same was also the case for the Osteichthyes (bony fish) and the Tetrapoda (Amphibia, Reptilia, Aves, Mammalia). However, the geological evidence favours a marine origin for the vertebrates as a whole, and, for the gnathostomes and the osteichthyans in particular. Some of the earliest amphibian remains may be associated with tidally influenced sediments. Furthermore, during the early part of the Devonian, fresh water chemistry may well have been different from that of today, lessening the divide between marine and non-marine environments. Urea formation via the ornithine cycle, and urea retention in the body fluids, are useful adaptations for terrestrial life. They prevent excessive water loss associated with the elimination of nitrogenous waste. These abilities may have been primitive for the gnathostomes, and were developed in the marine environment to reduce osmotic dehydration. In the aqueous medium, gaseous exchange is effected by the gills. These organs are, on the whole, useless in air. For vertebrates, air-breathing is effected by an inflatable sac, with moist linings, and an internal location. Some form of air-breathing sac was primitive for the osteichthyans, and may have been primitive for the gnathostomes. Again, this adaptation for terrestrial life developed in response to conditions experienced in the marine, aquatic environment. A new model of tetrapod evolution is proposed in the light of the basic marine origin and character of the ancestors of the tetrapods.     

Environmental influences on the development of the cardiac system in fish and amphibians

In poikilothermic animals body temperature varies with environmental temperature, and this results in a change in metabolic activity (Q10 of enzymatic reactions typically is around 2-3). Temperature changes also modify gas transport in body fluids. While the diffusion coefficient increases with increasing temperatures, physical solubility and also hemoglobin oxygen affinity decrease. Therefore, an increase in temperature typically requires adjustments in cardiac activity because ventilatory and convectional transport of respiratory gases usually are tightly coupled in adults in order to meet the oxygen demand of body tissues. Hypoxic conditions also provoke adaptations in the central circulatory system, like the hypoxic bradycardia, which has been described for many adult lower vertebrates, combined with an increase in stroke volume and peripheral resistance. In embryos and larvae the situation is much more complicated, because nervous control of the heart is established only late during development, and because the site of gas exchange changes from mainly cutaneous gas exchange during early development to mainly pulmonary or branchial gas exchange in late stages. In addition, recent studies in amphibian and fish embryos and larvae reveal, that at least in very early stages convectional gas transport of the hemoglobin is not essential, which means that in these early stages ventilatory and convectional gas transport are not yet coupled. Accordingly, in early stages of fish and amphibians the central cardiac system often does not respond to hypoxia, although in some species behavioral adaptations indicate that oxygen sensors are functional. If a depression of cardiac activity is observed, it most likely is a direct effect of oxygen deficiency on the cardiac myocytes. Regulated cardiovascular responses to hypoxia appear only in late stages and are similar to those found in adult species.     

The skin of fish as a transport epithelium: a review

The primary function of fish skin is to act as a barrier. It provides protection against physical damage and assists with the maintenance of homoeostasis by minimising exchange between the animal and the environment. However in some fish, the skin may play a more active physiological role. This is particularly true in species that inhabit specialised environmental niches (e.g. amphibious and air-breathing fish such as the lungfish), those with physiological characteristics that may subvert the need for the integument as a barrier (e.g. the osmoconforming hagfish), and/or fish with anatomical modifications of the epidermis (e.g. reduced epithelial thickness). Using examples from different fish groups (e.g. hagfishes, elasmobranchs and teleosts), the importance of fish skin as a transport epithelium for gases, ions, nitrogenous waste products, and nutrients was reviewed. The role of the skin in larval fish was also examined, with early life stages often utilising the skin as a surrogate gill, prior to the development of a functional branchial epithelium.     

Fine structure of the gill epithelium of the terrestrial mudskipper, Periophthalmodon schlosseri

In this paper, we describe the fine structure of the branchial epithelium of the amphibious, air-breathing mudskipper Periophthalmodon schlosseri, and relate the observed structure to functions in gas exchange, and to the elimination of sodium chloride and ammonia. Also, we describe the fine structure of the opercular epithelimicrom. The gill lamellar epithelium is thickened by the presence of large mitochondria-rich (MR) cells. These MR cells are further characterized by an extensive tubular system that is continuous with the basolateral plasma membrane and by a deep apical crypt often lined with microvilli. There are very few specialized MR accessory cells, which are associated with NaCl excretion in marine teleosts. Instead, MR cells are commonly isolated from each other laterally by flattened cells rich in intermediate filaments. These filament-rich (FR) cells are interconnected by desmosomes and have unusual canaliculi. These branchial FR cells are unique to P. schlosseri and may have a structural role. Electron-dense pavement cells rich in vesicles and large vacuous mitochondria compose the superficial layer of the epithelium. The unusual morphology of P. schlosseri's gill lamellae may be related to the animal's ability to effectively eliminate ammonia during air exposure. The inner opercular lining and parts of the leading edge of the filament have intraepithelial capillaries, which provide a more suitable gas exchange surface than the thickened lamellae with its restricted interlamellar water spaces. The arrangement of respiratory and ion exchange epithelia is opposite to that found in all other fish in which the lamellae typically function in gas exchange and the gill filament in ion regulation.     

Comparative model analysis of central shunts in vertebrate cardiovascular systems

The incomplete double circulation of air-breathing fishes and lungfishes, amphibians, reptiles and embryonic birds and mammals has been analyzed using a simplified mode comprising the intra- and extracardiac shunts and compartments for the gas exchange (gills, lungs, skin, etc.) as well as systemic tissue gas exchange. The intracardiac shunting is defined and given common symbols for all species of animals analyzed. Two types of equations, fluid-flow and mass-flow equations, are derived for each model, which are solved to give shunting rate as a function of blood O2 content of the principal cardiac compartments and vessels. The model analysis not only offers possibility for an overall average evaluation of central shunts, but also suggests which blood samples must be determined for evaluation of the shunt patterns.     

Body size and the air-breathing organ of the Atlantic tarpon Megalops atlanticus

The air-breathing organ (ABO) of the Atlantic tarpon is formed by four parallel ridges of alveolar-like respiratory tissue that extend along the length of the physostomous gas bladder. The large and complex surface of each ridge is formed by a cartilage matrix that is completely infiltrated by a thin respiratory epithelium. Comparison of a size series of specimens demonstrates isometric growth of the ABO, and histological and SEM studies show comparable levels of tissue complexity. These findings suggest that air-breathing capacity, which is required for the survival of juvenile fish in their hypoxic nursery habitat, is retained in older tarpon inhabiting more oxygenated marine coastal habitats. The retention of air breathing in adult tarpon may be related to their occasional occurrence in hypoxic waters and their high rates of aerobic metabolism.     

The venous circulation: a piscine perspective.

Vascular capacitance describes the pressure-volume relationship of the circulatory system. The venous vasculature, which is the main capacitive region in the circulation, is actively controlled by various neurohumoral systems. In terrestrial animals, vascular capacitance control is crucial to prevent orthostatic blood pooling in dependent limbs, while in aquatic animals like fish, the effects of gravity are cancelled out by hydrostatic forces making orthostatic blood pooling an unlikely concern for these animals. Nevertheless, changes in venous capacitance have important implications on cardiovascular homeostasis in fish since it affects venous return and cardiac filling pressure (i.e. central venous blood pressure), which in turn may affect cardiac output. The mean circulatory filling pressure is used to estimate vascular capacitance. In unanaesthetized animals, it is measured as the central venous plateau pressure during a transient stoppage of cardiac output. So far, most studies of venous function in fish have addressed the situation in teleosts (notably the rainbow trout, Oncorhynchus mykiss), while any information on elasmobranchs, cyclostomes and air-breathing fishes is more limited. This review describes venous haemodynamic concepts and neurohumoral control systems in fish. Particular emphasis is placed on venous responses to natural cardiovascular challenges such as exercise, environmental hypoxia and temperature changes.     

Free fatty acid metabolism in the air-breathing African catfish (Clarias gariepinus) during asphyxia

In several waterbreathing fish species, hypoxia induces a decrease in plasma free fatty acid (FFA) levels as opposed to an increase in air-breathing mammals. We hypothesised that this change is coupled to the mode of breathing. Therefore, we followed the metabolic response of cannulated air-breathing African catfish to an 8-h asphyxia period. The hematocrit and hemoglobin increased significantly upon asphyxia. However, no change was observed in the mean cellular hemoglobin concentration, indicating that more erythrocytes were brought into circulation. A continuous increase in plasma lactate concentration during asphyxia showed permanent activation of anaerobic glycolysis, pointing to a persistent oxygen shortage. Plasma glucose levels did not change, but FFA levels decreased significantly upon asphyxia with a concomitant increase in plasma noradrenaline levels. Thus, these results suggest that in the air-breathing African catfish noradrenaline mediated a decrease in plasma FFA levels similar to that in waterbreathing fish species.     

Endogenic upregulations of HIF/VEGF signaling pathway genes promote air breathing organ angiogenesis in bimodal respiration fish

Functional & Integrative Genomics - Air-breathing has evolved independently serval times with a variety of air-breathing organs (ABOs) in fish. The physiology of the air-breathing in bimodal...     

Defences against ammonia toxicity in tropical air-breathing fishes exposed to high concentrations of environmental ammonia: a review

In the tropics, air-breathing fishes can be exposed to environmental ammonia when stranded in puddles of water during the dry season, during a stay inside a burrow, or after agricultural fertilization. At low concentrations of environmental ammonia, NH₃ excretion is impeded, as in aerial exposure, leading to the accumulation of endogenous ammonia. At high concentrations of environmental ammonia, which results in a reversed NH₃ partial pressure gradient (P_NH3), there is retention of endogenous ammonia and uptake of exogenous ammonia. In this review, several tropical air-breathing fishes (giant mudskipper, African catfish, oriental weatherloach, swamp eel, four-eyed sleeper, abehaze and slender African lungfish), which can tolerate high environmental ammonia exposure, are used as examples to demonstrate how eight different adaptations can be involved in defence against ammonia toxicity. Four of these adaptations deal with ammonia toxicity at branchial and/or epithelial surfaces: (1) active excretion of NH₄⁺; (2) lowering of environmental pH; (3) low NH₃ permeability of epithelial surfaces; and (4) volatilization of NH₃, while another four adaptations ameliorate ammonia toxicity at the cellular and subcellular levels: (5) high tolerance of ammonia at the cellular and subcellular levels; (6) reduction in ammonia production; (7) glutamine synthesis; and (8) urea synthesis. The responses of tropical air-breathing fishes to high environmental ammonia are determined apparently by behavioural adaptations and the nature of their natural environments.Communicated by I.D. Hume     

Hypoxia tolerance and partitioning of bimodal respiration in the striped catfish (Pangasianodon hypophthalmus)

Air-breathing fish are common in the tropics, and their importance in Asian aquaculture is increasing, but the respiratory physiology of some of the key species such as the striped catfish, Pangasianodon hypophthalmus Sauvage 1878 is unstudied. P. hypophthalmus is an interesting species as it appears to possess both well-developed gills and a modified swim bladder that functions as an air-breathing organ indicating a high capacity for both aquatic and aerial respiration. Using newly developed bimodal intermittent-closed respirometry, the partitioning of oxygen consumption in normoxia and hypoxia was investigated in P. hypophthalmus. In addition the capacity for aquatic breathing was studied through measurements of oxygen consumption when access to air was denied, both in normoxia and hypoxia, and the critical oxygen tension, Pcrit, was also determined during these experiments. Finally, gill ventilation and air-breathing frequency were measured in a separate experiment with pressure measurements from the buccal cavity. The data showed that P. hypophthalmus is able to maintain standard metabolic rate (SMR) through aquatic breathing alone in normoxia, but that air-breathing is important during hypoxia. Gill ventilation was reduced during air-breathing, which occurred at oxygen levels below 8 kPa, coinciding with the measured Pcrit of 7.7 kPa. The findings in this study indicate that the introduction of aeration into the aquaculture of P. hypophthalmus could potentially reduce the need to air-breathe. The possibility of reducing air-breathing frequency may be energetically beneficial for the fish, leaving more of the aerobic scope for growth and other activities, due to the proposed energetic costs of surfacing behavior.     

The venous circulation: a piscine perspective

Vascular capacitance describes the pressure–volume relationship of the circulatory system. The venous vasculature, which is the main capacitive region in the circulation, is actively controlled by various neurohumoral systems. In terrestrial animals, vascular capacitance control is crucial to prevent orthostatic blood pooling in dependent limbs, while in aquatic animals like fish, the effects of gravity are cancelled out by hydrostatic forces making orthostatic blood pooling an unlikely concern for these animals. Nevertheless, changes in venous capacitance have important implications on cardiovascular homeostasis in fish since it affects venous return and cardiac filling pressure (i.e. central venous blood pressure), which in turn may affect cardiac output. The mean circulatory filling pressure is used to estimate vascular capacitance. In unanaesthetized animals, it is measured as the central venous plateau pressure during a transient stoppage of cardiac output. So far, most studies of venous function in fish have addressed the situation in teleosts (notably the rainbow trout, Oncorhynchus mykiss), while any information on elasmobranchs, cyclostomes and air-breathing fishes is more limited. This review describes venous haemodynamic concepts and neurohumoral control systems in fish. Particular emphasis is placed on venous responses to natural cardiovascular challenges such as exercise, environmental hypoxia and temperature changes.