Signal molecule changes in the gills and lungs of the African lungfish Protopterus annectens, during the maintenance and arousal phases of aestivation.

African lungfishes are obligate air breathers, with reduced gills and pulmonary breathing throughout their life. During the dry season they aestivate on land, with the collapse of secondary lamellae of their gills and the establishment of an exclusive aerial ventilation through the vascularization and expansion of their lungs. To date, the mechanisms underlining the respiratory organ remodeling in aestivating lungfishes are unknown. This study aimed to identify key switch components of the stress-induced signal transduction networks implicated in both rapid and medium-long term remodeling of the gills and lungs of the African lungfish Protopterus annectens during aestivation. Through immunofluorescence microscopy and Western blotting, the localization and the expression of nitric oxide synthase (NOS), Akt, Hsp-90 and HIF-1α were evaluated in both gills and lungs exposed to three experimental conditions: freshwater (FW), 6 months of experimentally induced aestivation (6mAe), and 6 days after arousal from 6 months of aestivation (6mAe6d). After 6mAe, the expression of NOS (p-eNOS antibody), Akt (p-Akt antibody), and Hsp-90 decreased in the gills, while NOS and Hsp-90 expression increased with Akt remained unchanged in the lungs. Upon 6mAe6d, NOS, Akt and Hsp-90 expression in the gills returned to the respective FW values. In the lungs of the aroused fish, NOS and Akt decreased to their respective FW levels, while Hsp-90 expression was enhanced with respect to aestivation. In both respiratory organs, the qualitative and quantitative patterns of HIF-1α expression correlated inversely to those of NOS. Overall, our findings suggest that the molecular components of the NOS/NO system changed in a tissue-specific manner in parallel with organ readjustment in the gills and lungs of P. annectens during aestivation and arousal.

Excretory nitrogen metabolism and defence against ammonia toxicity in air-breathing fishes.

With the development of air-breathing capabilities, some fishes can emerge from water, make excursions onto land or even burrow into mud during droughts. Air-breathing fishes have modified gill morphology and morphometry and accessory breathing organs, which would tend to reduce branchial ammonia excretion. As ammonia is toxic, air-breathing fishes, especially amphibious ones, are equipped with various strategies to ameliorate ammonia toxicity during emersion or ammonia exposure. These strategies can be categorized into (1) enhancement of ammonia excretion and reduction of ammonia entry, (2) conversion of ammonia to a less toxic product for accumulation and subsequent excretion, (3) reduction of ammonia production and avoidance of ammonia accumulation and (4) tolerance of ammonia at cellular and tissue levels. Active ammonia excretion, operating in conjunction with lowering of ambient pH and reduction in branchial and cutaneous NH3 permeability, is theoretically the most effective strategy to maintain low internal ammonia concentrations. NH3 volatilization involves the alkalization of certain epithelial surfaces and requires mechanisms to prevent NH3 back flux. Urea synthesis is an energy-intensive process and hence uncommon among air-breathing teleosts. Aestivating African lungfishes detoxify ammonia to urea and the accumulated urea is excreted following arousal. Reduction in ammonia production is achieved in some air-breathing fishes through suppression of amino acid catabolism and proteolysis, or through partial amino acid catabolism leading to alanine formation. Others can slow down ammonia accumulation through increased glutamine synthesis in the liver and muscle. Yet, some others develop high tolerance of ammonia at cellular and tissue levels, including tissues in the brain. In summary, the responses of air-breathing fishes to ameliorate ammonia toxicity are many and varied, determined by the behaviour of the species and the nature of the environment in which it lives.

Nitric oxide synthase-dependent "on/off" switch and apoptosis in freshwater and aestivating lungfish, Protopterus annectens: skeletal muscle versus cardiac muscle.

African lungfishes (Protopterus spp.) are obligate air breathers which enter in a prolonged torpor (aestivation) in association with metabolic depression, and biochemical and morpho-functional readjustments during the dry season. During aestivation, the lungfish heart continues to pump, while the skeletal muscle stops to function but can immediately contract during arousal. Currently, nothing is known regarding the orchestration of the multilevel rearrangements occurring in myotomal and myocardial muscles during aestivation and arousal. Because of its universal role in cardio-circulatory and muscle homeostasis, nitric oxide (NO) could be involved in coordinating these stress-induced adaptations. Western blotting and immunofluorescence microscopy on cardiac and skeletal muscles of Protopterus annectens (freshwater, 6months of aestivation and 6days after arousal) showed that expression, localization and activity of the endothelial-like nitric oxide synthase (eNOS) isoform and its partners Akt and Hsp-90 are tissue-specifically modulated. During aestivation, phospho-eNOS/eNOS and phospho-Akt/Akt ratios increased in the heart but decreased in the skeletal muscle. By contrast, Hsp-90 increased in both muscle types during aestivation. TUNEL assay revealed that increased apoptosis occurred in the skeletal muscle of aestivating lungfish, but the myocardial apoptotic rate of the aestivating lungfish remained unchanged as compared with the freshwater control. Consistent with the preserved cardiac activity during aestivation, the expression of apoptosis repressor (ARC) also remained unchanged in the heart of aestivating and aroused fish as compared with the freshwater control. Contrarily, ARC expression was strongly reduced in the skeletal muscle of aestivating lungfish. On the whole, our data indicate that changes in the eNOS/NO system and cell turnover are implicated in the morpho-functional readjustments occurring in lungfish cardiac and skeletal muscle during the switch from freshwater to aestivation, and between the maintenance and arousal phases of aestivation.

Nitrogen metabolism and excretion during aestivation.

In this chapter, up-to-date information on nitrogen metabolism and excretion in various aestivators is presented. Although aestivation involves long-term fasting and corporal torpor, adaptive responses with regard to excretory nitrogen metabolism exhibited by aestivators during aestivation differ from those exhibited by nonaestivators undergoing fasting or immobilization. Special efforts were made to address current issues pertaining to excretory nitrogen metabolism and related phenomena in aestivators. Adaptations exhibited by aestivators were discussed in relation to the induction, maintenance, and arousal phases of aestivation. For the induction phase, we included topics like urea as an internal induction signal for aestivation, alteration in the permeability of the skin to ammonia, and changes in rate of ammonia production and urea synthesis. For the maintenance phase, the emphasis was on protein synthesis and degradation, ammonia production, and urea synthesis and accumulation. For the arousal phase, the focus was on rehydration, urea excretion, and phenomena related to feeding. Adaptations exhibited by aestivators specifically to each of these three phases of aestivation are essential to the understanding of the overall aestivation process, but, at present, only limited information is available on excretory nitrogen metabolism in animals during the induction or arousal phases of aestivation. Therefore, future efforts should be made to identify adaptive responses particular to each of the three phases of aestivation in various aestivators.

Cytochrome c oxidase is regulated by modulations in protein expression and mitochondrial membrane phospholipid composition in estivating African lungfish.

We examined some of the potential mechanisms lungfish (Protopterus dolloi) use to regulate cytochrome c oxidase (CCO), during metabolic depression. CCO activity was reduced by 67% in isolated liver mitochondria of estivating fish. This was likely accomplished, in part, by the 46% reduction in CCO subunit I protein expression in the liver. No change in the mRNA expression levels of CCO subunits I, II, III, and IV were found in the liver, suggesting CCO is under translational regulation; however, in the kidney, messenger limitation may be a factor as the expression of subunits I and II were depressed ( approximately 10-fold) during estivation, suggesting tissue-specific mechanisms of regulation. CCO is influenced by mitochondrial membrane phospholipids, particularly cardiolipin (CL). In P. dolloi, the phospholipid composition of the liver mitochondrial membrane changed during estivation, with a approximately 2.3-fold reduction in the amount of CL. Significant positive correlations were found between CCO activity and the amount of CL and phosphatidylethanolamine within the mitochondrial membrane. It appears CCO activity is regulated through multiple mechanisms in P. dolloi, and individual subunits of CCO are regulated independently, and in a tissue-specific manner. It is proposed that altering the amount of CL within the mitochondrial membrane may be a means of regulating CCO activity during metabolical depression in the African lungfish, P. dolloi.

The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water.

This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food.

Ionoregulatory physiology of two species of African lungfishes Protopterus dolloi and Protopterus annectens.

Basic ionoregulatory physiology was characterized in two species of African lungfish, slender African lungfish Protopterus dolloi and West African lungfish Protopterus annectens, largely under aquatic conditions. There were no substantive differences between the two species. Plasma [Na], [Cl] and [Ca] were only 60-80% of those typical of freshwater teleosts, and plasma Ca activity was particularly low. Unidirectional Na and Cl influx rates from water were also very low, only c. 10% of teleost values, whereas unidirectional Ca influx rates were comparable with teleost rates. Protopterus spp. were fed a 3% ration of bloodworms every 48 h. The bloodworm diet provided similar amounts of Na and Ca as uptake from water, but almost no Cl. Efflux rates of Na and Cl through the urine were greater than via the faeces, whereas the opposite was true for Ca. Net ion flux measurements and ionic balance sheet calculations indicated that (1) both water and dietary uptake routes are important for Na and Ca acquisition; (2) the waterborne route predominates for Cl uptake; (3) unidirectional ion effluxes across the body surface (gills and skin) rather than urine and faeces are the major routes of loss for Na, Cl and Ca. Tissues (muscle, liver, lung, kidney, intestine and heart) and plasma ions were also examined in P. dolloi'terrestrialized' in air for up to 5 months, during which plasma ion concentrations (Na, Cl, Ca and Mg) did not change and there were only a few alterations in tissue ions, that is, increased [Na] in intestine, decreased [Cl] in kidney and increased [Ca] in liver and kidney.

Control of breathing in African lungfish (Protopterus dolloi): a comparison of aquatic and cocooned (terrestrialized) animals.

African lungfish, Protopterus dolloi exhibited constant rates of O(2) consumption before (0.95+/-0.07 mmol kg(-1) h(-1)), during (1.21+/-0.32 mmol kg(-1) h(-1)) and after (1.14+/-0.14 mmol kg(-1) h(-1)) extended periods (1-2 months) of terrestrialization while cocooned. Although a breathing event in terrestrialized fish consisted of multiple bouts of inspiration and expiration in rapid succession, the mean frequency of pulmonary breathing events was unaltered in the terrestrialized fish (16.7+/-1.4 h(-1)versus 20.1+/-4.9 h(-1) in the aquatic and terrestrialized fish, respectively). Hypoxia (approximately 20 mmHg) increased the frequency of breathing events by 16 and 23 h(-1) in the aquatic and terrestrialized fish, respectively. Hyperoxia (approximately 550 mmHg) decreased breathing event frequency by 10 and 15 h(-1) in the aquatic and terrestrialized animals. Aquatic hypercapnia (approximately 37.5 mmHg) increased pulmonary breathing frequency (from 15.3+/-2.3 to 28.7+/-5.4 h(-1)) in free swimming lungfish, whereas aerial hypercapnia was without effect in aquatic or terrestrialized fish.

Mechanisms of and defense against acute ammonia toxicity in the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis.

The objective of this study was to elucidate the mechanisms of acute ammonia toxicity in the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis, and to examine how this turtle defended against a sublethal dose of NH(4)Cl injected into its peritoneal cavity. The ammonia and glutamine contents in the brains of turtles that succumbed within 3h to an intraperitoneal injection with a lethal dose (12.5 micromolg(-1) turtle) of NH(4)Cl were 21 and 4.4 micromolg(-1), respectively. Since the brain glutamine content increased to 8 micromolg(-1) at hour 6 and recovered thereafter in turtles injected with a sub-lethal dose of NH(4)Cl (7.5 micromolg(-1) turtle), it can be concluded that increased glutamine synthesis and accumulation was not the major cause of acute ammonia toxicity in P. sinensis. Indeed, the administration of l-methionine S-sulfoximine (MSO; 82 microgg(-1) turtle), a glutamine synthetase (GS) inhibitor, prior to the injection of a lethal dose of NH(4)Cl had no significant effect on the mortality rate. Although the prior administration of MSO led to an extension of the time to death, it was apparently a result of its effects on glutamate dehydrogenase and glutamate formation, instead of glutamine synthesis and accumulation, in the brain. By contrast, a prior injection with MK801 (1.6 microgg(-1) turtle), a NMDA receptor antagonist, reduced the 24h mortality of turtles injected with a lethal dose of NH(4)Cl by 50%. Thus, acute ammonia toxicity in P. sinensis was probably a result of glutamate dysfunction and the activation of NMDA receptors. NMDA receptor activation could also be exacerbated through membrane depolarization caused by the extraordinarily high level of ammonia (21 micromolg(-1) brain) in the brain of turtles that succumbed to a lethal dose of NH(4)Cl. One hour after the injection with a sub-lethal dose of NH(4)Cl, the brain of P. sinensis exhibited an extraordinarily high tolerance of ammonia (16 micromolg(-1) brain). The transient nature of ammonia accumulation indicates that P. sinensis could ameliorate ammonia toxicity through the suppression of endogenous ammonia production and/or the excretion of exogenous ammonia. Despite being ureogenic and ureotelic, only a small fraction of the exogenous ammonia was detoxified to urea. A major portion of ammonia was excreted unchanged, resulting in an apparent ammonotely in the experimental turtles. Since there were increases in total essential free amino acid contents in the brain, liver and muscle, it can be deduced that a suppression of amino acid catabolism had occurred, reducing the production of endogenous ammonia and hence alleviating the possibility of ammonia intoxication.

Defense against environmental ammonia toxicity in the African lungfish, Protopterus aethiopicus: Bimodal breathing, skin ammonia permeability and urea synthesis.

This study aimed to determine how the African lungfish Protopterus aethiopicus defended against ammonia toxicity when confronted with high concentrations (30 or 100 mmoll(-1)) of environmental ammonia. Exposure to 100 mmoll(-1) of NH(4)Cl for 1 or 6 days had no significant effect on the rate of O(2) uptake from water or from air, and the rate of total O(2) consumption. Using an Ussing-like apparatus, we report for the first time that the skin of P. aethiopicus had low permeability (1.26 x 10(-4) micromol min(-1)cm(-1)) to NH(3)in vitro. Indeed, the influx of exogenous ammonia into fish exposed to 30 mmoll(-1) NH(4)Cl was low (0.117 micromol min(-1) 100g(-1) fish). As a result, P. aethiopicus could afford to maintain relatively low ammonia contents in plasma, muscle, liver and brain even after 6 days of exposure to 100 mmoll(-1) NH(4)Cl. Surprisingly, fish exposed to 30 or 100 mmoll(-1) NH(4)Cl had comparable ammonia contents in the muscle and the brain in spite of the big difference (70 mmoll(-1)) in environmental ammonia concentrations. Significant increases in urea contents occurred in various tissues of fish exposed to 30 mmoll(-1) NH(4)Cl for 6 days, but there were no significant differences in tissue urea contents between fish exposed to 30 mmoll(-1) and 100 mmoll(-1) NH(4)Cl. Between days 3 and 6, the rate of urea excretion in fish exposed to 30 mmoll(-1) NH(4)Cl was significantly greater than that of the control. By contrast, there was no significant difference in urea excretion rates between fish exposed to 100 mmoll(-1) NH(4)Cl and control fish throughout the 6-day period, and such a phenomenon has not been reported before for other lungfish species. Thus, our results suggest that P. aethiopicus was capable of decreasing the NH(3) permeability of its body surface when exposed to high concentrations of environmental ammonia. Indeed, after 6 days of exposure to 100 mmoll(-1) NH(4)Cl, the NH(3) permeability constant of the skin (0.55 x 10(-4) micromol min(-1)cm(-1)) decreased to half of that of the control. A decrease in the already low cutaneous NH(3) permeability and an increased urea synthesis, working in combination, allowed P. aethiopicus to effectively defend against environmental ammonia toxicity without elevating the plasma ammonia level. Therefore, unlike other fishes, glutamine and alanine contents did not increase in the muscle and liver, and there was no accumulation of glutamine in the brain, even when the fish was immersed in water containing 100 mmoll(-1) NH(4)Cl.

Nitrogen metabolism and excretion in the swamp eel, Monopterus albus, during 6 or 40 days of estivation in mud.

Monopterus albus inhabits muddy ponds, swamps, canals, and rice fields, where it can burrow into the moist earth, and it survives for long periods during the dry summer season. However, it had been reported previously that mortality increased when M. albus was exposed to air for 8 d or more. Thus, the objective of this study was to elucidate the strategies adopted by M. albus to defend against ammonia toxicity during 6 or 40 d of estivation in mud and to evaluate whether these strategies were different from those adopted by fish to survive 6 d of aerial exposure. Ammonia and glutamine accumulations occurred in the muscle and liver of fish exposed to air (normoxia) for 6 d, indicating that ammonia was detoxified to glutamine under such conditions. In contrast, ammonia accumulation occurred only in the muscle, with no increases in glutamine or glutamate contents in all tissues, of fish estivated in mud for 6 d. Similar results were obtained from fish estivated in mud for 40 d. While estivating in mud prevented excessive water loss through evaporation, M. albus was exposed to hypoxia, as indicated by significant decreases in blood P(O(2)), muscle energy charge, and ATP content in fish estivated in mud for 6 d. Glutamine synthesis is energy intensive, and that could be the reason why M. albus did not depend on glutamine synthesis to defend against ammonia toxicity when a decrease in ATP supply occurred. Instead, suppression of endogenous ammonia production was adopted as the major strategy to ameliorate ammonia toxicity when M. albus estivated in mud. Our results suggest that a decrease in O(2) level in the mud could be a more effective signal than an increase in internal ammonia level during aerial exposure to induce a suppression of ammonia production in M. albus. This might explain why M. albus is able to estivate in mud for long periods (40 d) but can survive in air for only <10 d.

Circulating catecholamines and cardiorespiratory responses in hypoxic lungfish (Protopterus dolloi): a comparison of aquatic and aerial hypoxia.

Circulating catecholamine levels and a variety of cardiorespiratory variables were monitored in cannulated bimodally breathing African lungfish (Protopterus dolloi) exposed to aquatic or aerial hypoxia. Owing to the purported absence of external branchial chemoreceptors in lungfish and the minor role played by the gill in O2 uptake, it was hypothesized that plasma catecholamine levels would increase only during exposure of fish to aerial hypoxia. The rapid induction of aquatic hypoxia (final PWo2 = 25.9+/-1.6 mmHg) did not affect the levels of adrenaline (A) or noradrenaline (NA) within the plasma. Similarly, none of the measured cardiorespiratory variables--including heart rate (fH), blood pressure, air-breathing frequency (fV), O2 consumption (Mo2), CO2 excretion (Mco2), or blood gases--were influenced by acute aquatic hypoxia. In contrast, however, the rapid induction of aerial hypoxia (inspired Po2=46.6+/-3.3 mmHg) caused a marked increase in the circulating levels of A (from 7.9+/-2.0 to 18.8+/-6.1 nmol L(-1)) and NA (from 7.7+/-2.2 to 19.7+/-6.3 nmol L(-1)) that was accompanied by significant decreases in Mo2, arterial Po2 (Pao2), and arterial O2 concentration (Cao2). Air-breathing frequency was increased (by approximately five breaths per hour) during aerial hypoxia and presumably contributed to the observed doubling of pulmonary Mco2 (from 0.25+/-0.04 to 0.49+/-0.07 mmol kg(-1) h(-1)); fH and blood pressure were unaffected by aerial hypoxia. An in situ perfused heart preparation was used to test the possibility that catecholamine secretion from cardiac chromaffin cells was being activated by a direct localized effect of hypoxia. Catecholamine secretion from the chromaffin cells of the heart, while clearly responsive to a depolarizing concentration of KCl (60 mmol L(-1)), was unaffected by the O2 status of the perfusion fluid. The results of this study demonstrate that P. dolloi is able to mobilize stored catecholamines and increase f(V) during exposure to aerial hypoxia while remaining unresponsive to aquatic hypoxia. Thus, unlike in exclusively water-breathing teleosts, P. dolloi would appear to rely solely on internal/airway O2 chemoreceptors for initiating catecholamine secretion and cardiorespiratory responses.

The African sharptooth catfish Clarias gariepinus can tolerate high levels of ammonia in its tissues and organs during four days of aerial exposure.

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and can survive on land during drought. The objective of this study was to elucidate how C. gariepinus defends against ammonia toxicity when exposed to terrestrial conditions. During 4 d of aerial exposure, there was no accumulation of urea in its tissues, and the rate of urea excretion remained low. Thus, exposure to terrestrial conditions for 4 d did not induce ureogenesis or ureotely in C. gariepinus. Volatilization of NH(3) was not involved in excreting ammonia during aerial exposure. In addition, there were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus; nor were there any changes in the glutamine levels in these tissues. However, there were extraordinarily high levels of ammonia in the muscle (14 micromol g(-1)), liver (18 micromol g(-1)), and brain (11 micromol g(-1)) of fish exposed to terrestrial conditions for 4 d. This is the first report on a fish adopting high tolerance of ammonia in cells and tissues as the single major strategy to defend against ammonia toxicity during aerial exposure. At present, it is uncertain how C. gariepinus tolerates such high levels of ammonia, especially in its brain, but it can be concluded that, contrary to previous reports on two air-breathing catfishes (Clarias batrachus and Heteropneustes fossilis) from India, C. gariepinus does not detoxify ammonia to urea or free amino acids on land.

Cyanide exposure affects the production and excretion of ammonia by the mudskipper Boleophthalmus boddaerti.

The concentrations of ammonia in the plasma of the mudskipper Boleophthalmus boddaerti exposed to cyanide for 1-6 days were significantly greater than the respective values of the controls. This was due to an increase in the production of NH3 in the muscle and an increase in the retention of NH3 and/or NH4+ in the blood of the cyanide-exposed fish when compared to controls. Cyanide exposure significantly increased the specific activity of muscle AMP deaminase. Since adenylosuccinate synthetase and lyase were also present in the muscle, exposure to cyanide might increase the production of NH3 from the catabolism of purine nucleotides. B. boddaerti exposed to cyanide excreted significantly less ammonia than the control fish. Results indicate changes in the permeability of the epithelial surfaces of the cyanide-exposed fish to NH3 and/or NH4+. Since the tissues and organs needed time to activate the inducible cyanide detoxification mechanisms, the increase in the production of NH3 might be an important defensive mechanism for B. boddaerti during the early phase of cyanide exposure.

The loach Misgurnus anguillicaudatus reduces amino acid catabolism and accumulates alanine and glutamine during aerial exposure.

The loach Misgurnus anguillicaudatus inhabits rice fields in Southern China. It encounters drought during summer and ammonia loading during agricultural fertilization. In the laboratory, aerial exposure led to decreases in its ammonia and urea excretion. Ammonia accumulated to very high levels in the muscle and liver. Urea synthesis through the ornithine-urea cycle was not involved in ammonia detoxification in M. anguillicaudatus. However, M. anguillicaudatus was capable of partial amino acid catabolism leading to the accumulation of alanine in the first 24 h of aerial exposure. This was apparently coupled to a possible decrease in protein/amino acid catabolism. These are not detoxification mechanisms but mechanisms that avoid internal fouling by ammonia. Misgurnus anguillicaudatus was also capable of detoxifying internally produced ammonia in part to glutamine, which appears to be an important adaptation after 24 h of aerial exposure. However, unlike the case of the marble goby (Oxyeleotris marmoratus), there was no alteration to the kinetic properties of the hepatic glutamine synthetase. During dry seasons, M. anguillicaudatus moves actively on land until it encounters soft mud in which it can bury itself through several strong wriggling actions of the body. Hence, it is possible that M. anguillicaudatus uses partial amino acid catabolism to fuel its short period of activities on land and switches to the formation of glutamine to detoxify internally produced ammonia when it remains relatively inactive in the mud.

Five tropical air-breathing fishes, six different strategies to defend against ammonia toxicity on land.

Most tropical fishes are ammonotelic, producing ammonia and excreting it as NH3 by diffusion across the branchial epithelia. Hence, those air-breathing tropical fishes that survive on land briefly or for an extended period would have difficulties in excreting ammonia when out of water. Ammonia is toxic, but some of these air-breathing fishes adopt special biochemical adaptations to ameliorate the toxicity of endogenous ammonia accumulating in the body. The amphibious mudskipper Periophthalmodon schlosseri, which is very active on land, reduces ammonia production by suppressing amino acid catabolism (strategy 1) during aerial exposure. It can also undergo partial amino acid catabolism, leading to the accumulation of alanine (strategy 2) to support locomotory activities on land. In this case, alanine formation is not an ammonia detoxification process but reduces the production of endogenous ammonia. The snakehead Channa asiatica, which exhibits moderate activities on land although not truly amphibious, accumulates both alanine and glutamine in the muscle, with alanine accounting for 80% of the deficit in reduction in ammonia excretion during air exposure. Unlike P. schlosseri, C. asiatica apparently cannot reduce the rates of protein and amino acid catabolism and is incapable of utilizing partial amino acid catabolism to support locomotory activities on land. Unlike alanine formation, glutamine synthesis (strategy 3) represents an ammonia detoxification mechanism that, in effect, removes the accumulating ammonia. The four-eyed sleeper Bostrichyths sinensis, which remains motionless during aerial exposure, detoxifies endogenous ammonia to glutamine for storage. The slender African lungfish Protopterus dolloi, which can aestivate on land on a mucus cocoon, has an active ornithine-urea cycle and converts endogenous ammonia to urea (strategy 4) for both storage and subsequent excretion. Production of urea and glutamine are energetically expensive and appear to be adopted by fishes that remain relatively inactive on land. The Oriental weatherloach Misgurnus anguillicaudatus, which actively burrows into soft mud during drought, manipulates the pH of the body surface to facilitate NH3 volatilization (strategy 5) and develops high ammonia tolerance at the cellular and subcellular levels (strategy 6) during aerial exposure. Hence, with regard to excretory nitrogen metabolism, modern tropical air-breathing fishes exhibit a variety of strategies to survive on land, and they represent a spectrum of specimens through which we may examine various biochemical adaptations that would have facilitated the invasion of the terrestrial habitat by fishes during evolution.

Air breathing and ammonia excretion in the giant mudskipper, Periophthalmodon schlosseri.

The giant mudskipper, Periophthalmodon schlosseri, is an amphibious, obligate, air-breathing teleost fish. It uses its buccal cavity for air breathing and for taking and holding large gulps of air. These fish live in mud burrows at the top of the intertidal zone of mangrove mudflats; the burrow water may be hypoxic and hypercapnic and have high ammonia levels. The buccal epithelium is highly vascularized, with small diffusion distances between air and blood. The gill epithelium is densely packed with mitochondria-rich cells. Periophthalmodon schlosseri can maintain tissue ammonia levels in the face of high ammonia concentrations in the water. This is probably achieved by active ammonium ion transport across the mitochondria-rich cells via an apical Na/H+(NH4+) exchanger and a basolateral Na/K+(NH4+) ATPase. When exposed to air, the animal reduces ammonia production, but there is some increase in tissue ammonia levels after 24 h. There is no detoxification by increased production of glutamine or urea, but there is partial amino acid catabolism, leading to the accumulation of alanine. CO2 production and proton excretion cause acidification of the burrow water to reduce ammonia toxicity. The skin has high levels of cholesterol and saturated fatty acids decreasing membrane fluidity and gas, and therefore ammonia, permeability. Exposure to elevated environmental ammonia further decreases membrane permeability. Acidification of the environment and having a skin with a low NH3 permeability reduces ammonia influx, so that the fish can maintain tissue ammonia levels by active ammonium ion excretion, even in water containing high levels of ammonia.

Alkaline environmental pH has no effect on ammonia excretion in the mudskipper Periophthalmodon schlosseri but inhibits ammonia excretion in the related species Boleophthalmus boddaerti.

Experiments were performed to evaluate the effects of alkaline environmental pH on urea and ammonia excretion rates and on tissue urea, ammonia, and free amino acid concentrations in two mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti. Periophthalomodon schlosseri is known to be capable of actively excreting ammonia. The rate of ammonia excretion in B. boddaerti exposed to 50% seawater (brackish water, BW) at pH 9 decreased significantly during the first 2 d of exposure when compared with that of specimens exposed to pH 7 or 8. This suggested that B. boddaerti was dependent on NH(3) diffusion for ammonia excretion, as in most fishes. It was incapable of detoxifying the accumulating endogenous ammonia to urea but could store and tolerate high concentrations of ammonia in the muscle, liver, and plasma. It did not undergo reductions in proteolysis and/or amino acid catabolism in alkaline water, probably because the buildup of endogenous ammonia was essential for the recovery of the normal rate of ammonia excretion by the third day of exposure to a pH 9 medium. Unlike B. boddaerti, P. schlosseri did not accumulate ammonia in the body at an alkaline pH (i.e., pH 9) because it was capable of actively excreting ammonia. Periophthalmodon schlosseri did not undergo partial amino acid catabolism (no accumulation of alanine) either, although there might be a slight reduction in amino acid catabolism in general. The significant decrease in blood pCO(2) in B. boddaerti at pH 9 might lead to respiratory alkalosis in the blood. In contrast, P. schlosseri was able to maintain its blood pH in BW at pH 9 despite a decrease in pCO(2) in the blood. With 8 mM NH(4)Cl in BW at pH 7, both mudskippers could actively excrete ammonia, although not to the same extent. Only P. schlosseri could sustain ammonia excretion against 8 mM NH(4)Cl in BW at pH 8. In BW containing 8 mM NH(4)Cl at pH 9, both mudskippers died within a short period of time. Boleophthalmus boddaerti consistently died faster than did P. schlosseri. This indicates that the body surfaces of these mudskippers were permeable to NH(3), but the skin of P. schlosseri might be less permeable to NH(3) than that of B. boddaerti. Both mudskippers excreted acid (H(+)) to alter the pH of the alkaline external medium. Such a capability, together with modifications in gill morphology and morphometry as in P. schlosseri, might be essential to the development of an effective mechanism for the active excretion of NH+4.

Quantitative determination of inositol in Hymenolepis diminuta (Cestoda).

Myoinositol and scylloinositol have been identified qualitatively and quantitatively by gas-liquid chromatography in Hymenolepis diminuta. No myoinosose-2 was detected. Myoinositol was unevenly distributed throughout the worm. The scolex and neck regions contained more free- and phosphatidyl-bound inositol. This region also contained more lipid-bound phosphorus, but less total lipid and water.

Membrane transport of inositol by Hymenolepis diminuta (Cestoda).

The absorption of myoinositol by Hymenolepis diminuta involved diffusion that occurred at all substrate concentrations tested; at low substrate concentrations the mediated component predominated. The mediated process exhibited saturation kinetics with a Vmax and Kt of 0.011 mumoles/g of the ethanol-extracted dry wt/4 min and 0.0067 mM, respectively. Myoinositol transport was sensitive to changes in temperature, pH, and sodium ion concentration. D-Glucose was a noncompetitive inhibitor of myoinositol transport, but myoinositol had no effect on D-glucose absorption. Phlorizin interacted competitively with the myoinositol transport system. Various sugar alcohols and amino acids had no effect on myoinositol transport. These results indicate that a separate transport system exists in H. diminuta for myoinositol.