Improved heat tolerance in air drives the recurrent evolution of air-breathing

The transition to air-breathing by formerly aquatic species has occurred repeatedly and independently in fish, crabs and other animal phyla, but the proximate drivers of this key innovation remain a long-standing puzzle in evolutionary biology. Most studies attribute the onset of air-breathing to the repeated occurrence of aquatic hypoxia; however, this hypothesis leaves the current geographical distribution of the 300 genera of air-breathing crabs unexplained. Here, we show that their occurrence is mainly related to high environmental temperatures in the tropics. We also demonstrate in an amphibious crab that the reduced cost of oxygen supply in air extends aerobic performance to higher temperatures and thus widens the animal''s thermal niche. These findings suggest that high water temperature as a driver consistently explains the numerous times air-breathing has evolved. The data also indicate a central role for oxygen- and capacity-limited thermal tolerance not only in shaping sensitivity to current climate change but also in underpinning the climate-dependent evolution of animals, in this case the evolution of air-breathing.     

Autonomic nervous control of the circulatory system in the air-breathing fish Channa argus

The control of the cardiovascular system with particular emphasis on the regulation of blood distribution in the gills and air-breathing organ was studied in the air-breathing teleost Channa argus. Perfused head preparations were used in addition to experiments with isolated strip preparations of arteries and heart chambers. The distribution of adrenergic nerves was investigated using Falck-Hillarp fluorescence histochemistry. This preliminary study shows an adrenergic control system composed of chromaffin cells and adrenergic nerves similar to that found in other teleosts investigated, although the systemic arteries (coeliac artery, dorsal aorta and the vasculature of the air-breathing organ) appear to lack an adrenergic innervation. The reactions of isolated artery strip preparations to acetylcholine and adrenaline resemble those seen in other teleosts, and there is a prominent inhibitory effect of L-isoprenaline suggestive of arterial beta-adrenoceptors. The general vascular resistance of the gill apparatus-air-breathing organ increases in response to acetylcholine or adrenaline, and there is a redistribution of perfusion flow from the air-breathing organ circuit (anterior venous outflow from the first and second pair of gills and the air-breathing organ) to the general systemic circuit (dorsal aortic outflow from the third and fourth pair of gills). Stimulation of the vagal branch entering the air-breathing organ mimics the effects of acetylcholine or adrenaline. This innervation is probably non-adrenergic since no adrenergic nerve fibres could be demonstrated in the vasculature of the air-breathing organ using the histochemical technique. An adrenergic control of the vasculature of the air-breathing organ is not likely, since the concentration of adrenaline needed to affect the vasculature is not reached in the plasma even during "stress".     

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.     

Hypoxia induces surfacing behaviour in brown-striped frog (Limnodynastes peronii) larvae

In their natural habitat, brown-striped frog (Limnodynastes peronii) larvae periodically swim rapidly from the bottom of their ponds to the water surface and then immediately dive to the bottom again. This behaviour is presumably related to air-breathing. We examined the behavioural and metabolic responses to aquatic hypoxia in L. pernoii larvae. Gas filled lungs were found in all free-swimming larval stages of L. peronii, but air-breathing occurred infrequently in normoxic water. The frequency of air-breathing at 30°C increased rapidly in hypoxic water when oxygen partial pressure (P_o2) fell below 10 kPa. Only a slight increase was observed at similar oxygen partial pressures at 20°C. The critical oxygen tension at 30°C was about 7kPa, below which, aquatic breathing larvae become metabolic oxygen conformers. In natural habitats where surfacing behaviour was observed, temperatures during summer months frequently exceed 25°C and some ponds become extremely hypoxic (po₂ < 3.0 kPa); therefore air-breathing appears to be the only way in which these larvae can maintain a fully aerobic metabolism.     

Autonomic control of the heart in the Asian swamp eel (Monopterus albus)

The Asian swamp eel (Monopterus albus) is an air-breathing teleost with very reduced gills that uses the buccal cavity for air-breathing. Here we characterise the cardiovascular changes associated with the intermittent breathing pattern in M. albus and we study the autonomic control of the heart during water- and air-breathing. The shift from water- to air-breathing was associated with a rise in heart rate from 27.7 ± 1.6 to 41.4 ± 2.6 min(-1) and an increase in cardiac output from 23.1 ± 3.0 to 58.7 ± 6.5 mLmin(-1)kg(-1), while mean systemic blood pressure did not change (39.0 ± 3.5 and 46.4 ± 1.3 cmH(2)O). The autonomic control of the heart during water- and air-breathing was revealed by infusion of the β-adrenergic antagonist propranolol and muscarinic antagonist atropine (3 mgkg(-1)) in eels instrumented with an arterial catheter. Inhibition of the sympathetic and parasympathetic innervations of the heart revealed a strong vagal tone on the heart of water-breathing eels and that the tachycardia during air-breathing is primarily mediated by withdrawal of cholinergic tone.     

Ontogenetic changes and developmental adjustments in lactate dehydrogenase isozymes of an obligate air-breathing fish Channa punctatus during deprivation of air access

In air-breathing snakehead Channa punctatus, Ldh-B is expressed at all ontogenetic and developmental stages, while Ldh-A is expressed temporally in pre-hatchlings 12-13 days ahead of bimodal respiration marked by air-breathing. Remarkable differences are observed in the LDH isozyme expression among various ontogenetic and developmental stages upon denying air access. When denied air access, water-breathing larvae show two distinct characteristics: (i) they survive longer than transitory air-breathers due to independence from air-breathing and (ii) there is more transient induction of Ldh-B than Ldh-A. Transition to bimodal breathing, which occurred post-hatching in 15-day old larvae, is coincidental with inducibility of Ldh-A and concomitant down-regulation of Ldh-B. Heart tissue from air-breathing adults denied air access shows a preferential expression of LDH-A subunit and slight down-regulation of LDH-B. Heterotetramers of A and B subunits participate in adjusting LDH levels among those stages which either precede air-breathing switchover, or are subsequent to this transition. The contribution of heterotetramers depends on the stage-specific levels of LDH homotetramers A(4) or B(4). Scaling of muscle mass during growth, tolerance to extended deprivation of air access and induction of Ldh-A are correlated. Response to restoring air contact indicated that advanced air-breathing stages of C. punctatus possess an inherent capacity to sense surface air. In kinetic properties, LDH isozymes of C. punctatus are teleost-like but species specificity is displayed in oxidative potential by cardiac muscle and in L-lactate reduction by skeletal muscle.     

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.     

Respiratory vasculatures of the intertidal air-breathing eel goby, <Emphasis Type="Italic">Odontamblyopus lacepedii</Emphasis> (Gobiidae: Amblyopinae)

Lacking a propensity to emerge over the mud surface, the eel goby, Odontamblyopus lacepedii, survives low tide periods by continuously breathing air in burrows filled with hypoxic water. As with most marine air-breathing fishes, O. lacepedii does not possess an accessory air-breathing organ, but holds air in the buccal–opercular cavity. The present study aimed to clarify how the respiratory vasculature has been modified in this facultative air-breathing fish. Results showed that the gills apparently lacked structural modifications for air breathing, whereas the inner epithelia of the opercula were richly vascularized. Comparison with two sympatric gobies revealed that the density of blood capillaries within 10μm from the inner opercular epithelial surface in O. lacepedii (14.5 ± 3.0 capillaries mm⁻¹; mean ± s.d., n = 3) was significantly higher than in the aquatic non-air-breathing Acanthogobius hasta (0.0 ± 0.0) but significantly lower than in the amphibious air-breathing mudskipper, Periophthalmus modestus (59.1 ± 8.5). The opercular capillary bed was supplied predominantly by the 1st efferent branchial arteries (EBA1) and drained by the opercular veins, which open into the anterior cardinal vein. Deep invaginations at the distal end of the EBA1 and the junction with EBA2 are suggestive of blood flow regulatory sites during breath-holding and apnoeic periods. It remains to be investigated how blood flow through the gills is maintained during breath holding when the buccal–opercular cavity is filled with air.     

Oxygen uptake capacity of gills and skin in relation to body weight of the air-breathing siluroid fish, Clarias batrachus (Linn.).

Oxygen consumption through gills and skin in relation to body weight was estimated in the air-breathing catfish, Clarias batrachus, under two experimental conditions, viz., (i) when access to air was allowed and (ii) when air-breathing was prevented. There was a positive correlation between VO2 (ml/hr) and body weight in both experimental conditions. Oxygen consumption (ml/hr) increased by a power of 0.869 when access to air was allowed whereas the power was slightly less (b = 0.841) when air-breathing was prevented. As the values for exponent (b) were less than 1.0, the weight specific VO2 (ml/kg/hr) decreased with increasing body weight. The decrease was more marked (b = - 0.180) in fishes which were not allowed air than in those where access to air was allowed (b = - 0.148). Under normal conditions of water and air-breathing the rate of VO2 (ml/kg/hr) via gills and skin from water ranged from 39.7 +/- 3.21 to 76.7 +/- 9.01 and this increased to 42.17 +/- 6.2 to 105.9 +/- 8.33 when air-breathing was prevented. The increase in the rate of VO2 was perhaps associated with the increase in the volume of water irrigating the gills per unit time.     

Oxidation of 5-methyltetrahydrofolate in cobalamin-inactivated rats.

Rats were exposed to nitrous oxide, which inactivates cob(I)alamin (Cbl). As in air-breathing rats methionine administration led to the conversion of hepatic 5-methyltetrahydrofolate (MeH4 folate) into formyltetrahydrofolate. The recovery of MeH4 folate levels in liver after its oxidation initiated by methionine was noted and the rate compared with that for air-breathing rats. Oxidation of MeH4 folate was less complete and occurred more slowly in Cbl-inactivated rats as compared with controls. However, recovery of MeH4 folate levels was more rapid in Cbl inactivation. S-Adenosylmethionine did not produce a significant change in MeH4 folate levels in Cbl-inactivated rats, whereas it did so in air-breathing animals.     

Air-breathing rhythm in Clarias batrachus (Linn.): modulatory role of eyes, pineal and exogenous melatonin

Effects of enucleation followed by pinealectomy and administration of exogenous melatonin on air-breathing activity rhythm in a fresh water catfish, C. batrachus maintained at LD 12:12 and laboratary temperature during its prepratory phase, were examined. Results of cosinor analysis clearly reveal that most of the intact individuals exhibited circadian rhythm in their air-breathing activity and such rhythm persists even after enucleation followed by pinealectomy and then melatonin administration. However, the period (tau) of the activity obtained by power spectrum analysis was prominent 24 hr in most of the intact individuals, but it was increased (tau > 24 hr) after enucleation in most of the individuals. In most of the enucleated + pinealectomized individuals tau was less than 24 hr, and after receiving melatonin treatment tau was shifted to prominent 24 hr in most of the individuals. In addition, visual analysis of the actograms depicted that in intact individuals air-breathing activity is entrained with the timings of lights on/off with elevation of activity during dark period and decreased activity during light hours. However, enucleated and enucleated + pinealectomized individuals showed free run in their activity rhythm. The treatment of melatonin reestablished the entrainment of activity at least with the timing of lights off, in most of the studied individuals. Further, daily mean of the air-breathing activity was decreased in enucleated + pinealectomized individuals as compared with other studied groups (intact, enucleated, enucleated + pinealectomized + melatonin receiving). It could be speculated that there may be existence of extraretinal and extrapineal photoreceptors in C. batrachus. However, eyes play an important role in regulating air-breathing activity rhythm in such species. In addition, exogenous melatonin may also have some modulatory effect on such rhythm.     

Circulatory Anatomy in Bimodally Breathing Fish

SYNOPSIS. The development of air-breathing organs in bimodallybreathing fish has necessitated a degree of vascular remodellingin order to enhance gas exchange and support other homeostaticactivities. Macrocirculatory changes include several plumbingschemes that allow perfusion of the gills, air-breathing organ,and systemic circulations in a variety of in-parallel and in-seriesarrangements. The incorporation of structural adaptations designedto minimize admixture of oxygenated and deoxygenated blood intransit through the heart as well as vascular shunts furtherincreases the efficiency of the gas exchange process. A numberof anatomical modifications in capillary architecture and endothelialcell structure are found in air-breathing fish and appear tobe unique to these vertebrates. The physiological significanceof the microcirculatory adaptations remains, to a large extent,speculative.     

Hypoxia tolerance and air-breathing ability correlate with habitat preference in coral-dwelling fishes

Hypoxia tolerance and air-breathing occur in a range of freshwater, estuarine and intertidal fishes. Here it is shown for the first time that coral reef fishes from the genera Gobiodon, Paragobiodon and Caracanthus, which all have an obligate association with living coral, also exhibit hypoxia tolerance and a well-developed air-breathing capacity. All nine species maintained adequate respiration in water at oxygen concentrations down to 15–25% air saturation. This hypoxia tolerance is probably needed when the oxygen levels in the coral habitat drops sharply at night. Air-breathing abilities of the species correlated with habitat association, being greatest (equaling oxygen uptake in water) in species that occupy corals extending into shallow water, where they may become air exposed during extreme low tides. Air-breathing was less well-developed or absent in species inhabiting corals from deeper waters. Loss of scales and a network of subcutaneous capillaries appear to be key adaptations allowing cutaneous respiration in air. While hypoxia tolerance may be an ancestral trait in these fishes, air-breathing is likely to be a more recent adaptation exemplifying convergent evolution in the unrelated genera Gobiodon and Caracanthus in response to coral-dwelling lifestyles.     

Air-breathing adaptation in a marine Devonian lungfish

Recent discoveries of tetrapod trackways in 395 Myr old tidal zone deposits of Poland (Niedźwiedzki et al. 2010 Nature 463, 43–48 (doi:10.1038/nature.08623)) indicate that vertebrates had already ventured out of the water and might already have developed some air-breathing capacity by the Middle Devonian. Air-breathing in lungfishes is not considered to be a shared specialization with tetrapods, but evolved independently. Air-breathing in lungfishes has been postulated as starting in Middle Devonian times (ca 385 Ma) in freshwater habitats, based on a set of skeletal characters involved in air-breathing in extant lungfishes. New discoveries described herein of the lungfish Rhinodipterus from marine limestones of Australia identifies the node in dipnoan phylogeny where air-breathing begins, and confirms that lungfishes living in marine habitats had also developed specializations to breathe air by the start of the Late Devonian (ca 375 Ma). While invasion of freshwater habitats from the marine realm was previously suggested to be the prime cause of aerial respiration developing in lungfishes, we believe that global decline in oxygen levels during the Middle Devonian combined with higher metabolic costs is a more likely driver of air-breathing ability, which developed in both marine and freshwater lungfishes and tetrapodomorph fishes such as Gogonasus.     

Microanatomy and cytochemistry of the gastro-respiratory tract of an air-breathing cobitidid fish,Lepidocephalichthys guntea

Gastro-respiratory tract of the loach,Lepidocephalichthys guntea has been studied with special reference to the nature of its mucus secreting epithelia. The mucous cells are strongly PAS-positive and their number per unit area (mm²) in the mucosal layers of oesophagus, intestinal bulb, intestine and rectum are 733, 531, 223 and 540, respectively. The air-breathing segment of the gut is completely devoid of neutral mucosubstances, and there is a predominance of acidic mucosubstances over the neutral ones throughout the digestive tube. The air-blood pathway of the accessory respiratory organ is about 2.6 μm which is higher than the values of air-breathing organs of other fishes.     

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.     

Partitioning of oxygen uptake and cost of surfacing during swimming in the air-breathing catfish Pangasianodon hypophthalmus

Though air-breathing has probably evolved mainly as a response to hypoxia, it may provide an important oxygen supplement when metabolism is elevated, as for example during swimming. Due to the increased travelling distance involved when an air-breathing fish swims to and from the surface, and the increased drag when the surface is breached, it can be proposed that air-breathing results in a rise in the apparent cost of transport. In order to investigate this hypothesis, it is necessary to use a fish that is able to swim equally well with and without access to air. The striped catfish Pangasianodon hypophthalmus has been shown to have a sufficiently high capacity for aquatic oxygen uptake in normoxia, to allow for such a comparison. Here, we measured the partitioning of oxygen uptake ( $ \dot{M}{\text{O}}_{2} $ ) during swimming and recovery, and calculated the apparent cost of transport with and without access to air, under normoxic conditions. Aerial $ \dot{M}{\text{O}}_{2} $ constituted 25–40 % of the total $ \dot{M}{\text{O}}_{2} $ during swimming and less than 15 % during recovery. The net cost of transport was 25 % lower in fish that did not air-breathe compared to fish that did, showing that the cost of surfacing can be substantial. This is the first study to measure partitioning in an air-breathing fish during swimming at velocities close to the critical swimming speed.