Teleost fishes constitute 95% of extant aquatic vertebrates, and we suggest that this is related in part to their unique mode of tissue oxygenation. We propose the following sequence of events in the evolution of their oxygen delivery system. First, loss of plasma-accessible carbonic anhydrase (CA) in the gill and venous circulations slowed the Jacobs–Stewart cycle and the transfer of acid between the plasma and the red blood cells (RBCs). This ameliorated the effects of a generalised acidosis (associated with an increased capacity for burst swimming) on haemoglobin (Hb)–O 2 binding. Because RBC pH was uncoupled from plasma pH, the importance of Hb as a buffer was reduced. The decrease in buffering was mediated by a reduction in the number of histidine residues on the Hb molecule and resulted in enhanced coupling of O 2 and CO 2 transfer through the RBCs. In the absence of plasma CA, nearly all plasma bicarbonate ultimately dehydrated to CO 2 occurred via the RBCs, and chloride/bicarbonate exchange was the rate-limiting step in CO 2 excretion. This pattern of CO 2 excretion across the gills resulted in disequilibrium states for CO 2 hydration/dehydration reactions and thus elevated arterial and venous plasma bicarbonate levels. Plasma-accessible CA embedded in arterial endothelia was retained, which eliminated the localized bicarbonate disequilibrium forming CO 2 that then moved into the RBCs. Consequently, RBC pH decreased which, in conjunction with pH-sensitive Bohr/Root Hbs, elevated arterial oxygen tensions and thus enhanced tissue oxygenation. Counter-current arrangement of capillaries (retia) at the eye and later the swim bladder evolved along with the gas gland at the swim bladder. Both arrangements enhanced and magnified CO 2 and acid production and, therefore, oxygen secretion to those specialised tissues. The evolution of β-adrenergically stimulated RBC Na + /H + exchange protected gill O 2 uptake during stress and further augmented plasma disequilibrium states for CO 2 hydration/dehydration. Finally, RBC organophosphates (e.g. NTP) could be reduced during hypoxia to further increase Hb–O 2 affinity without compromising tissue O 2 delivery because high-affinity Hbs could still adequately deliver O 2 to the tissues via Bohr/Root shifts. We suggest that the evolution of this unique mode of tissue O 2 transfer evolved in the Triassic/Jurassic Period, when O 2 levels were low, ultimately giving rise to the most extensive adaptive radiation of extant vertebrates, the teleost fishes.

SUMMARY The mechanisms of ammonia excretion at fish gills have been studied for decades but details remain unclear, with continuing debate on the relative importance of non-ionic NH 3 or ionic NH 4 + permeation by various mechanisms. The presence of an apical Na + /NH 4 + exchanger has also been controversial. The present study utilized an in vitro cultured gill epithelium (double seeded insert, DSI) of freshwater rainbow trout as a model to investigate these issues. The relationship between basolateral ammonia concentration and efflux to apical freshwater was curvilinear, indicative of a saturable carrier-mediated component ( K m =66 μmol l –1 ) superimposed on a large diffusive linear component. Pre-exposure to elevated ammonia (2000 μmol l –1 ) and cortisol (1000 ng ml –1 ) had synergistic effects on the ammonia permeability of DSI, with significantly increased Na + influx and positive correlations between ammonia efflux and Na + uptake. This increase in ammonia permeability was bidirectional. It could not be explained by changes in paracellular permeability as measured by[ 3 H]PEG-4000 flux. The mRNA expressions of Rhbg, Rhcg2,H + -ATPase and Na + /H + exchanger-2 (NHE-2) were up-regulated in DSI pre-exposed to ammonia and cortisol, CA-2 mRNA was down-regulated, and transepithelial potential became more negative. Bafilomycin (1 μmol l –1 ), phenamil (10 μmol l –1 ) and 5-( N,N -hexamethylene)amiloride (HMA, 10μmol l –1 ) applied to the apical solution significantly inhibited ammonia efflux, indicating that H + -ATPase, Na + channel and NHE-2 pathways on the apical surface were involved in ammonia excretion. Apical amiloride (100 μmol l –1 ) was similarly effective, while basolateral HMA was ineffective. Pre-treatment with apical freshwater low in [Na + ] caused increases in both Rhcg2 mRNA expression and ammonia efflux without change in paracellular permeability. These data suggest that Rhesus glycoproteins are important for ammonia transport in the freshwater trout gill, and may help to explain in vivo data where plasma ammonia stabilized at 50% below water levels during exposure to high environmental ammonia (∼2300 μmol l –1 ). We propose an apical`Na + /NH 4 + exchange complex' consisting of several membrane transporters, while affirming the importance of non-ionic NH 3 diffusion in ammonia excretion across freshwater fish gills.

SUMMARY The mechanism(s) of ammonia excretion in the presence of elevated external ammonia are not well understood in fish. Recent studies in other organisms have revealed a new class of ammonia transporters, Rhesus glycoprotein genes( Rh genes), which may also play a role in ammonia excretion in fish. The first objective of this study was to clone and characterize Rh genes in a fish species with a relatively high tolerance to environmental ammonia, the mangrove killifish Kryptolebias marmoratus (formerly Rivulus marmoratus ). We obtained full-length cDNAs of three Rh genes in K. marmoratus : RhBG (1736 bp), RhCG1 (1920 bp) and RhCG2 (2021 bp), which are highly homologous with other known Rh gene sequences. Hydropathy analysis revealed that all three Rh genes encode membrane proteins with 10–12 predicted transmembrane domains. RhBG , RhCG1 and RhCG2 are highly expressed in gill tissue, with RhBG also present in skin of K. marmoratus . Exposure to elevated environmental ammonia (2 mmol l –1 NH 4 HCO 3 ) for 5 days resulted in a modest (+37%) increase in whole-body ammonia levels, whereas gill RhCG2 and skin RhCG1 mRNA levels were upregulated by 5.8- and 7.7-fold, respectively. RhBG mRNA levels were also increased in various tissues, with 3- to 7-fold increases in the liver and skeletal muscle. In a separate group of killifish exposed to air for 24 h, RhCG1 and RhCG2 mRNA levels were elevated by 4- to 6-fold in the skin. Thus, the multifold induction of Rh mRNA levels in excretory tissues (gills and skin) and internal tissues in response to conditions that perturb normal ammonia excretion suggests that RhBG , RhCG1 and RhCG2 may be involved in facilitating ammonia transport in this species. Furthermore, the findings support earlier studies demonstrating that the skin is an important site of ammonia excretion in K. marmoratus .

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.

SUMMARY The weather loach Misgurnus anguillicaudatus inhabits rice fields that experience drought in summer and ammonia loading during agricultural fertilisation. Exposure of specimens to ammonia led to the accumulation of ammonia in muscle, liver and blood. The level of ammonia reached in the plasma was the highest reported among fishes. Ammonia was not detoxified to urea, and urea excretion rate was unaffected by ammonia exposure. Fish acidified the water to reduce ammonia loading. Ammonia loading, unlike aerial exposure, did not induce glutamine synthesis, and there was no accumulation of glutamine. This is a unique observation different from those reported for other fishes in the literature. An initial switch to partial amino acid catabolism led to the accumulation of alanine and was probably associated with a decreased rate of ammonia production. Aerial exposure led to decreases in rates of ammonia and urea excretion, as well as the accumulation of tissue ammonia. As the internal ammonia levels increased, M. anguillicaudatus was able to excrete some ammonia in the gaseous form (NH 3 ). The percentage of ammonia excreted as NH 3 increased with time of exposure and with increasing temperature. It appears that air-breathing through the gut is involved, with the anterior portion of the digestive tract playing a central role: it became significantly more alkaline in fish exposed to air or to environmental ammonia. The skin, which also became more alkaline during air exposure, may also be involved in ammonia volatilization in air-exposed fish. This represents the first report of a fish using volatilization of NH 3 as part of a defence against ammonia toxicity. It can be concluded that the main strategy adopted by M. anguillicaudatus confronted with ammonia loading or air exposure is to tolerate high ammonia levels in the tissues. During periods of elevated tissue ammonia levels, some ammonia is lost by volatilization via air-breathing using the gut. In addition, some ammonia may be lost across the skin during air exposure.