Lympho-granulocytic tissue associated with the wall of the spiral valve in the African lungfish Protopterus annectens.

We describe the structure of the lympho-granulocytic tissue associated with the wall of the spiral valve of the African lungfish Protopterus annectens. The study was performed under freshwater conditions and after 6 months of aestivation. The lympho-granulocytic tissue consists of nodes surrounded by reticular tissue. The nodes are formed by an outer and an inner component separated by a thin collagenous layer. The outer component is a reticular-like tissue that contains two types of granulocytes, developing and mature plasma cells and melanomacrophage centres (MMCs). The inner component, the parenchyma, contains a meshwork of trabeculae and vascular sinusoids and shows dark and pale areas. The dark areas contain diffuse lymphoid tissue, with a large number of mitoses and plasma cell clusters. The pale areas contain a small number of macrophages and lymphocytes. Macrophages and sinus endothelial cells are filled with haemosiderin granules and appear to form part of the reticuloendothelial system of the lungfish. The reticular tissue houses granulocytes, plasma cells and MMCs and might serve for the housing and maturation of cells of the white series. After aestivation, the nodes undergo lymphocyte depletion, the suppression of mitosis, granulocyte invasion and the occurrence of cell death. By contrast, few histological changes occur in the reticular tissue. Whereas the nodes appear to be involved in lymphocyte proliferation and plasma cell maturation, the function of the reticular tissue remains obscure.

The Chinese soft-shelled turtle, Pelodiscus sinensis, excretes urea mainly through the mouth instead of the kidney.

The Chinese soft-shelled turtle, Pelodiscus sinensis, is well adapted to aquatic environments, including brackish swamps and marshes. It is ureotelic, and occasionally submerges its head into puddles of water during emersion, presumably for buccopharyngeal respiration. This study was undertaken to test the hypothesis that the buccophyaryngeal cavity constitutes an important excretory route for urea in P. sinensis. Results indicate that a major portion of urea was excreted through the mouth instead of the kidney during immersion. When restrained on land, P. sinensis occasionally submerged their head into water (20-100 min), during which urea excretion and oxygen extraction occurred simultaneously. These results indicate for the first time that buccopharyngeal villiform processes (BVP) and rhythmic pharyngeal movements were involved in urea excretion in P. sinensis. Urea excretion through the mouth was sensitive to phloretin inhibition, indicating the involvement of urea transporters (UTs). In addition, saliva samples collected from the buccopharyngeal surfaces of P. sinensis injected intraperitoneally with saline contained ~36 mmol N l(-1) urea, significantly higher than that (~2.4 mmol N l(-1)) in the plasma. After intraperitoneal injection with 20 µmol urea g(-1) turtle, the concentration of urea in the saliva collected from the BVP increased to an extraordinarily high level of ~614 µmol N ml(-1), but the urea concentration (~45 µmol N ml(-1)) in the plasma was much lower, indicating that the buccopharyngeal epithelium of P. sinensis was capable of active urea transport. Subsequently, we obtained from the buccopharyngeal epithelium of P. sinensis the full cDNA sequence of a putative UT, whose deduced amino acid sequence had ~70% similarity with human and mouse UT-A2. This UT was not expressed in the kidney, corroborating the proposition that the kidney had only a minor role in urea excretion in P. sinensis. As UT-A2 is known to be a facilitative urea transporter, it is logical to deduce that it was localized in the basolateral membrane of the buccopharyngeal epithelium, and that another type of primary or secondary active urea transporter yet to be identified was present in the apical membrane. The ability to excrete urea through the mouth instead of the kidney might have facilitated the ability of P. sinensis and other soft-shelled turtles to successfully invade the brackish and/or marine environment.

The spleen of the African lungfish Protopterus annectens: freshwater and aestivation.

We describe the structure of the spleen of the African lungfish Protopterus annectens in freshwater conditions, and after 6 months of aestivation. The spleen is formed by cortical tissue that surrounds the splenic parenchyma. The cortex is a reticulum that contains two types of granulocytes, developing and mature plasma cells, and melanomacrophage centres (MMCs). The parenchyma is divided into lobules that show a subcapsular sinus and areas of red pulp and white pulp. Red pulp contains vascular sinuses and atypical cords formed by delicate trabeculae. White pulp also contains vascular sinuses and cords. Structural data indicate that red pulp is involved in erythropoiesis, destruction of effete erythrocytes, and plasma cell differentiation. White pulp appears to be involved in the production of immune responses. Macrophages and sinus endothelial cells constitute the reticulo-endothelial system of the spleen. After aestivation, the number of MMCs increases, and spleen tissue is infiltrated by lymphocytes, granulocytes, and monocytes. Also, white pulp is reduced, and sinus endothelial cells undergo vacuolar degeneration. Lungfish spleen shares structural characteristics with secondary lymphoid organs of both ectothermic and endothermic vertebrates, but appears to have evolved in unique ways.

Hepatic carbamoyl phosphate synthetase (CPS) I and urea contents in the hylid tree frog, Litoria caerulea: transition from CPS III to CPS I.

The complete cDNA sequence of CPS I obtained from the liver of the hylid tree frog, Litoria caerulea, consisted of 4,485 bp which coded for 1,495 amino acids with an estimated molecular mass of 163.7 kDa. The deduced CPS I consisted of a mitochondrial targeting sequence of 33 amino acid residues, a glutaminase amidotransferase component spanning from tyrosine 95 to leucine 425, and a methylglyoxal synthetase-like component spanning from valine 441 to lysine 1566. It also comprised two cysteine residues (cysteine 1360 and cysteine 1370) that are characteristic of N-acetyl-L-glutamate dependency. Similar to the CPS I of Rana catesbeiana and Cps III of lungfishes and teleosts, it contained the Cys-His-Glu catalytic triad (cysteine 304, histidine 388 and glutamate 390). All Cps III contain methionine 305 and glutamine 308, which are essential for the Cys-His-Glu triad to react with glutamine, but the CPS I of R. catesbeiana contains lysine 305 and glutamate 308, and therefore cannot effectively utilize glutamine as a substrate. However, the CPS I of L. caerulea, unlike that of R. catesbeiana, contained besides glutamate 308, methionine 305 instead of lysine 305, and thus represented a transitional form between Cps III and CPS I. Indeed, CPS I of L. caerulea could utilize glutamine or NH4⁺ as a substrate in vitro, but the activity obtained with glutamine + NH4⁺ reflected that obtained with NH4⁺ alone. Furthermore, only <5 % of the glutamine synthetase activity was present in the hepatic mitochondria, indicating that CPS I of L. caerulea did not have an effective supply of glutamine in vivo. Hence, our results confirmed that the evolution of CPS I from Cps III occurred in amphibians. Since L. caerulea contained high levels of urea in its muscle and liver, which increased significantly in response to desiccation, its CPS I had the dual functions of detoxifying ammonia to urea and producing urea to reduce evaporative water loss.

Roles of three branchial Na(+)-K(+)-ATPase α-subunit isoforms in freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus.

Three Na(+)-K(+)-ATPase (nka) α-subunit isoforms, nka α1a, nka α1b, and nka α1c, were identified from gills of the freshwater climbing perch Anabas testudineus. The cDNA sequences of nka α1a and nka α1b consisted of 3,069 bp, coding for 1,023 amino acids, whereas nka α1c was shorter by 22 nucleotides at the 5' end. In freshwater, the quantity of nka α1c mRNA transcripts present in the gills was the highest followed by nka α1a and nka α1b that was almost undetectable. The mRNA expression of nka α1a was downregulated in the gills of fish acclimated to seawater, indicating that it could be involved in branchial Na(+) absorption in a hypoosmotic environment. By contrast, seawater acclimation led to an upregulation of the mRNA expression of nka α1b and to a lesser extent nka α1c, indicating that they could be essential for ion secretion in a hyperosmotic environment. More importantly, ammonia exposure led to a significant upregulation of the mRNA expression of nka α1c, which might be involved in active ammonia excretion. Both seawater acclimation and ammonia exposure led to significant increases in the protein abundance and changes in the kinetic properties of branchial Na(+)-K(+)-ATPase (Nka), but they involved two different types of Nka-immunoreactive cells. Since there was a decrease in the effectiveness of NH(4)(+) to substitute for K(+) to activate branchial Nka from fish exposed to ammonia, Nka probably functioned to remove excess Na(+) and to transport K(+) instead of NH(4)(+) into the cell to maintain intracellular Na(+) and K(+) homeostasis during active ammonia excretion.

Cystic fibrosis transmembrane conductance regulator in the gills of the climbing perch, Anabas testudineus, is involved in both hypoosmotic regulation during seawater acclimation and active ammonia excretion during ammonia exposure.

This study aimed to clone and sequence the cystic fibrosis transmembrane conductance regulator (cftr) from, and to determine the effects of seawater acclimation or exposure to 100 mmol l⁻¹ NH4Cl in freshwater on its mRNA and protein expressions in, the gills of Anabas testudineus. There were 4,530 bp coding for 1,510 amino acids in the cftr cDNA sequence from A. testudineus. The branchial mRNA expression of cftr in fish kept in freshwater was low (<50 copies of transcript per ng cDNA), but significant increases were observed in fish acclimated to seawater for 1 day (92-fold) or 6 days (219-fold). Branchial Cftr expression was detected in fish acclimated to seawater but not in the freshwater control, indicating that Cl⁻ excretion through the apical Cftr of the branchial epithelium was essential to seawater acclimation. More importantly, fish exposed to ammonia also exhibited a significant increase (12-fold) in branchial mRNA expression of cftr, with Cftr being expressed in a type of Na⁺/K⁺-ATPase-immunoreactive cells that was apparently different from the type involved in seawater acclimation. It is probable that Cl⁻ excretion through Cftr generated a favorable electrical potential across the apical membrane to drive the excretion of NH4⁺ against a concentration gradient through a yet to be determined transporter, but it led to a slight loss of endogenous Cl⁻. Since ammonia exposure also resulted in significant decreases in blood pH, [HCO3⁻] and [total CO2] in A. testudineus, it can be deduced that active NH4⁺ excretion could also be driven by the exit of HCO3⁻ through the apical Cftr. Furthermore, A. testudineus uniquely responded to ammonia exposure by increasing the ambient pH and decreasing the branchial bafilomycin-sensitive V-type H⁺-ATPase activity, which suggests that its gills might have low NH3 permeability.

The alimentary canal of the African lungfish Protopterus annectens during aestivation and after arousal.

We describe the structural modifications that occur in the alimentary canal of the African lungfish Protopterus annectens during aestivation and after arousal. With fasting, all gut segments undergo structural modifications. The epithelium covering the intestinal vestibule undergoes bursts of activation at 4 months of aestivation, adopting a more quiescent appearance at 6 months. The ridge area of the spiral intestine shows, at 4 months of aestivation, epithelial disintegration, cell desquamation, cell death, and loss of the freshwater phenotype. Surprisingly, the epithelium adopts a stratified appearance at 6 months of aestivation. Except for epithelial disintegration, the smooth portion of the spiral intestine follows a similar pattern of modifications than the ridge area. The entire epithelium of spiral intestine appears to be renewed during aestivation. The presence of intraepithelial mast cells suggests that inflammation is part of the cellular response to aestivation. After arousal, cell phenotypes are restored in about 6 days, but full structural recovery is not attained during the experimental period (15 days post-aestivation). Several aspects of the cellular response to fasting are shared by a wide range of animal groups. This commonality agrees with the presence of a character that allows to adjust the structural and functional properties of the gut to food availability and food quality, and to the characteristics of the fasting episodes.

Both seawater acclimation and environmental ammonia exposure lead to increases in mRNA expression and protein abundance of Na⁺:K⁺:2Cl⁻ cotransporter in the gills of the climbing perch, Anabas testudineus.

The freshwater climbing perch, Anabas testudineus, is an obligatory air-breathing teleost which can acclimate to seawater, survive long period of emersion, and actively excrete ammonia against high concentrations of environmental ammonia. This study aimed to clone and sequence the Na⁺:K⁺:2Cl⁻ cotransporter (nkcc) from the gills of A. testudineus, and to determine the effects of seawater acclimation or exposure to 100 mmol l⁻¹ NH4Cl in freshwater on its branchial mRNA expression. The complete coding cDNA sequence of nkcc from the gills of A. testudineus consisted of 3,495 bp, which was translated into a protein with 1,165 amino acid residues and an estimated molecular mass of 127.4 kDa. A phylogenetic analysis revealed that the translated Nkcc of A. testudineus was closer to fish Nkcc1a than to fish Nkcc1b or Nkcc2. After a progressive increase in salinity, there were significant increases in the mRNA expression and protein abundance of nkcc1a in the gills of fish acclimated to seawater as compared with that of the freshwater control. Hence, it can be concluded that similar to marine teleosts, Cl⁻ excretion through basolateral Nkcc1 of mitochondrion-rich cells (MRCs) was essential to seawater acclimation in A. testudineus. Exposure of A. testudineus to 100 mmol l⁻¹ NH4Cl for 1 or 6 days also resulted in significant increases in the mRNA expression of nkcc1a in the gills, indicating a functional role of Nkcc1a in active ammonia excretion. It is probable that NH4⁺ enter MRCs through basolateral Nkcc1a before being actively transported across the apical membrane. Since the operation of Nkcc1a would lead to an increase in the intracellular Na⁺ concentration, it can be deduced that an upregulation of basolateral Na⁺/K⁺-ATPase (Nka) activity would be necessary to compensate for the increased influx of Na⁺ into MRCs during active NH4⁺ excretion. This would imply that the main function of Nka in active NH4⁺ excretion is to maintain intracellular Na⁺ and K⁺ homeostasis instead of transporting NH4⁺ directly into MRCs as proposed previously. In conclusion, active salt secretion during seawater acclimation and active NH4⁺ excretion during exposure to ammonia in freshwater could involve similar transport mechanisms in the gills of A. testudineus.

The gut of the juvenile African lungfish Protopterus annectens: a light and scanning electron microscope study.

We describe the microstructure of the alimentary canal of the juvenile lungfish Protopterus annectens. Following the oesophagus, the gut is formed by a long segment that extends down to the pyloric valve. This segment, classically named stomach, is lined by a transitional epithelium but lacks all characteristics of the vertebrate stomach. It has been defined here as the intestinal vestibule. The spiral valve is divided into a first large chamber, which contains mucosal ridges, and a second smooth portion. The entire spiral valve is lined with a pseudostratified columnar epithelium that contains approximately six cell types: enterocytes, goblet cells, ciliated cells, leukocytes, dark pigment cells, and vascular cells. Enterocytes and goblet cells show a high number of cytoplasmic vacuoles. The number and size of the vacuoles, and the number of ciliated cells, decreases from the anterior toward the posterior end, suggesting that most of the digestive processes take place in the anterior part of the spiral valve. The epithelium overlies a lamina propria in the first large chamber and a vascular plexus in the smooth portion. The cloaca has a thick muscular wall covered by a transitional epithelium. An extensive lymphatic system formed by capillaries and lymphatic micropumps is present along the entire wall of the alimentary canal.

The anatomy of the gastrointestinal tract of the African lungfish, Protopterus annectens.

The gastrointestinal tract of the African lungfish Protopterus annectens is a composite, which includes the gut, the spleen, and the pancreas. The gut is formed by a short oesophagus, a longitudinal stomach, a pyloric valve, a spiraling intestine, and a cloaca. Coiling of the intestine begins dorsally below the pylorus, winding down to form six complete turns before ending into the cloaca. A reticular tissue of undisclosed nature accompanies the winding of the intestinal mucosa. The spleen is located along the right side of the stomach, overlapping the cranial end of the pancreas. The pancreas occupies the shallow area, which indicates on the gut dorsal side the beginning of the intestine coiling. In addition, up to 25 lymphatic-like nodes accompany the inner border of the spiral valve. The mesenteric artery forms a long axis for the intestine. All the components of the gastrointestinal tract are attached to each other by connective sheaths, and are wrapped by connective tissue, and by the serosa externally. We believe that several previous observations have been misinterpreted and that the anatomy of the lungfish gut is more similar among all the three lungfish genera than previously thought. Curiously, the gross anatomical organization is not modified during aestivation. We hypothesize that the absence of function is accompanied by structural modifications of the epithelium, and are currently investigating this possibility.

Glutamine accumulation and up-regulation of glutamine synthetase activity in the swamp eel, Monopterus albus (Zuiew), exposed to brackish water.

The swamp eel, Monopterus albus, is an air-breathing teleost which typically lives in freshwater but can also be found in estuaries, where it has to deal with ambient salinity fluctuations. Unlike other teleosts, its gills are highly degenerate. Hence, it may have uncommon osmoregulatory adaptations, but no information is available on its osmoregulatory capacity and mechanisms at present. In this study M. albus was exposed to a 5 day progressive increase in salinity from freshwater (1 per thousand) to brackish water (25 per thousand) and subsequently kept in 25 per thousand water for a total of 4 days. The results indicate that M. albus switched from hyperosmotic hyperionic regulation in freshwater to a combination of osmoconforming and hypoosmotic hypoionic regulation in 25 per thousand water. Exposure to 25 per thousand water resulted in relatively large increases in plasma osmolality, [Na(+)] and [Cl(-)]. Consequently, fish exposed to 25 per thousand water had to undergo cell volume regulation through accumulation of organic osmolytes and inorganic ions. Increases in tissue free amino acid content were apparently the result of increased protein degradation, decreased amino acid catabolism, and increased synthesis of certain non-essential amino acids. Here we report for the first time that glutamine is the major organic osmolyte in M. albus. Glutamine content increased to a phenomenal level of > 12 micromol g(-1) and > 30 micromol g(-1) in the muscle and liver, respectively, of fish exposed to 25 per thousand water. There were significant increases in glutamine synthetase (GS) activity in muscle and liver of these fish. In addition, exposure to 25 per thousand water for 4 days led to significant increases in GS protein abundance in both muscle and liver, indicating that increases in the expression of GS mRNA could have occurred.

Increased urea synthesis and/or suppressed ammonia production in the African lungfish, Protopterus annectens, during aestivation in air or mud.

The objective of this study was to elucidate how the African lungfish, Protopterus annectens, ameliorated ammonia toxicity during 12 or 46 days of aestivation in air or in mud. Twelve days of aestivation in air led to significant increases in contents of urea, but not ammonia, in tissues of P. annectens. The estimated rate of urea synthesis increased 2.7-fold despite the lack of changes in the activities of hepatic ornithine-urea cycle enzymes, but there was only a minor change in the estimated rate of ammonia production. After 46 days of aestivation in air, the ammonia content in the liver decreased significantly and contents of urea in all tissues studied increased significantly, indicating that the fish shifted to a combination of increased urea synthesis (1.4-fold of the day 0 value) and decreased ammonia production (56% of the day 0 value) to defend against ammonia toxicity. By contrast, 12 days of aestivation in mud produced only minor increases in tissue urea contents, with ammonia contents remained unchanged. This was apparently achieved through decreases in urea synthesis and ammonia production (40 and 15%, respectively, of the corresponding day 0 value). Surprisingly, 46 days of aestivation in mud resulted in no changes in tissue urea contents, indicating that profound suppressions of urea synthesis and ammonia production (2.6 and 1.2%, respectively, of the corresponding day 0 value) had occurred. This is the first report on such a phenomenon, and the reduction in ammonia production was so profound that it could be the greatest reduction known among animals. Since fish aestivated in mud had relatively low blood pO(2) and muscle ATP content, they could have been exposed to hypoxia, which induced reductions in metabolic rate and ammonia production. Consequently, fish aestivating in mud had a lower dependency on increased urea synthesis to detoxify ammonia, which is energy intensive, than fish aestivating in air.

Postprandial increases in nitrogenous excretion and urea synthesis in the Chinese soft-shelled turtle, Pelodiscus sinensis.

The objective of this study was to determine the effects of feeding on the excretory nitrogen (N) metabolism of the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis, with a special emphasis on the role of urea synthesis in ammonia detoxification. P. sinensis is ureogenic and possesses a full complement of ornithine-urea cycle enzymes in its liver. It is primarily ureotelic in water, and the estimated rate of urea synthesis in unfed animals was equivalent to only 1.5% of the maximal capacity of carbamoyl phosphate synthetase I (CPS I) in its liver. Approximately 72 h was required for P. sinensis to completely digest a meal of prawn meat. During this period, there were significant increases in ammonia contents in the stomach at hour 24 and in the intestine between hours 12 and 36, which could be a result of bacterial activities in the intestinal tract. However, ammonia contents in the liver, muscle, brain and plasma remained unchanged throughout the 72-h post-feeding. In contrast, at hour 24, urea contents in the stomach, intestine, liver, muscle, brain and plasma increased significantly by 2.9-, 3.5-, 2.6-, 2.9-, 3.4 and 3.0-fold, respectively. In addition, there was a 3.3- to 8.0-fold increase in the urea excretion rate between hours 0 and 36 post-feeding, which preceded the increase in ammonia excretion between hours 12 and 48. By hour 48, 68% of the assimilated N from the feed was excreted, 54% of which was excreted as urea-N. The rate of urea synthesis apparently increased sevenfold during the initial 24 h after feeding, which demanded only 10% of the maximal CPS I capacity in P. sinensis. The postprandial detoxification of ammonia to urea in P. sinensis effectively prevented postprandial surges in ammonia contents in the plasma and other tissues, as observed in other animals, during the 72-h period post-feeding. In addition, postprandial ammonia toxicity was ameliorated by increased transamination and synthesis of certain amino acids in the liver and muscle of P. sinensis. After feeding, a slight but significant increase in the glutamine content occurred in the brain at hour 24, indicating that the brain might experience a transient increase in ammonia and ammonia was detoxified to glutamine.

Active ammonia transport and excretory nitrogen metabolism in the climbing perch, Anabas testudineus, during 4 days of emersion or 10 minutes of forced exercise on land.

The climbing perch, Anabas testudineus, inhabits large rivers, canals, stagnant water bodies, swamps and estuaries, where it can be confronted with aerial exposure during the dry season. This study aimed to examine nitrogen excretion and metabolism in this fish during 4 days of emersion. Contrary to previous reports, A. testudineus does not possess a functional hepatic ornithineurea cycle because no carbamoyl phosphate synthetase I or III activity was detected in its liver. It was ammonotelic in water, and did not detoxify ammonia through increased urea synthesis during the 4 days of emersion. Unlike many air-breathing fishes reported elsewhere, A. testudineus could uniquely excrete ammonia during emersion at a rate similar to or higher than that of the immersed control. In spite of the fact that emersion had no significant effect on the daily ammonia excretion rate, tissue ammonia content increased significantly in the experimental fish. Thus, it can be concluded that 4 days of emersion caused an increase in ammonia production in A. testudineus, and probably because of this, a transient increase in the glutamine content in the brain occurred. Because there was a significant increase in the total essential free amino acid in the experimental fish after 2 days of emersion, it can be deduced that increased ammonia production during emersion was a result of increased amino acid catabolism and protein degradation. Our results provide evidence for the first time that A. testudineus was able to continually excrete ammonia in water containing 12 mmol l(-1) NH4Cl. During emersion, active ammonia excretion apparently occurred across the branchial and cutaneous surfaces, and ammonia concentrations in water samples collected from these surfaces increased to 20 mmol l(-1). It is probable that the capacities of air-breathing and active ammonia excretion facilitated the utilization of amino acids by A. testudineus as an energy source to support locomotor activity during emersion. As a result, it is capable of wandering long distance on land from one water body to another as reported in the literature.

Light induces an increase in the pH of and a decrease in the ammonia concentration in the extrapallial fluid of the giant clam Tridacna squamosa.

The objective of this study was to examine whether 12 h of light exposure would lead to an increase in the pH of and a decrease in the concentration of total ammonia in the extrapallial fluid of the giant clam Tridacna squamosa. We also aimed to elucidate indirectly whether movements of ammonia and/or protons (H(+)) occurred between the extrapallial fluid and the outer mantle epithelium. The pH of the extrapallial fluid of T. squamosa exposed to 12 h of light was significantly higher than that of clams exposed to 12 h of darkness. Conversely, the total ammonia concentration in the extrapallial fluid of the former was significantly lower than that of the latter. In addition, the glutamine content in the mantle adjacent to the extrapallial fluid of clams exposed to 12 h of light was significantly greater than that of clams exposed to 12 h of darkness. These results suggest that in the extrapallial fluid of T. squamosa exposed to light, NH(3) combined with H(+) as NH(+)(4) and that NH(+)(4) was transported into the mantle and used as a substrate for glutamine formation. Injection of NH(4)Cl into the extrapallial fluid led to an instantaneous increase in the total ammonia concentration therein, but the total ammonia concentration decreased subsequently and returned to the control value within 1 h. This is in support of the proposition that NH(+)(4) could be transported from the extrapallial fluid to the mantle. Injection of HCl into the extrapallial fluid led to an instantaneous decrease in the pH of the extrapallial fluid. However, there was a significant increase in pH within 1 h in light or darkness, achieving a partial recovery toward the control pH value. The increase in pH within this 1-h period in light or darkness was accompanied by a significant decrease in the total ammonia concentration in the extrapallial fluid, which supports the proposition that H(+) could be transported in combination with NH(3) as NH(+)(4). Therefore, our results prompt a reexamination of the previous proposition that the removal of H(+) by NH(3) can facilitate calcification in molluscs in general and an investigation of the relationship between H(+) removal through NH(+)(4) transport and light-enhanced calcification in T. squamosa.

Exposure to air, but not seawater, increases the glutamine content and the glutamine synthetase activity in the marsh clam Polymesoda expansa.

Polymesoda expansa spends a considerable portion of its life exposed to air in mangrove swamps where salinity fluctuates greatly. Thus, the aim of this study was to evaluate the effects of aerial exposure (transfer from 10 per thousand brackish water directly to air) or salinity changes (transfer from 10 per thousand brackish water directly to 30 per thousand seawater) on nitrogen metabolism in P. expansa. We concluded that P. expansa is non-ureogenic because carbamoyl phosphate (CPS) III activity was undetectable in the adductor muscle, foot muscle, hepatopancreas and mantle when exposed to brackish water (control), seawater or air for 17 days. It is ammonotelic as it excretes nitrogenous wastes mainly as ammonia in brackish water or seawater. After transfer to seawater for 17 days, the contents of total free amino acids (TFAA) in the adductor muscle, foot muscle, hepatopancreas and mantle increased significantly. This could be related to an increase in protein degradation because exposure to seawater led to a greater rate of ammonia excretion on days 15 and 17, despite unchanged tissue ammonia contents. Alanine was the major free amino acid (FAA) in P. expansa. The contribution of alanine to the TFAA pool in various tissues increased from 43-48% in brackish water to 62-73% in seawater. In contrast, in clams exposed to air for 17 days there were no changes in alanine content in any of the tissues studied. Thus, the functional role of alanine in P. expansa is mainly connected with intracellular osmoregulation. Although 8.5-16.1% of the TFAA pool of P. expansa was attributable to glutamine, the glutamine contents in the adductor muscle, foot muscle, hepatopancreas and mantle were unaffected by 17 days of exposure to seawater. However, after exposure to air for 17 days, there were significant increases in ammonia content in all these tissues in P. expansa, accompanied by significant increases in glutamine content (2.9-, 2.5-, 4.5- and 3.4-fold, respectively). Simultaneously, there were significant increases in glutamine synthetase activities in the adductor muscle (1.56-fold) and hepatopancreas (3.8-fold). This is the first report on the accumulation of glutamine associated with an upregulation of glutamine synthetase in a bivalve species in response to aerial exposure, and these results reveal that the evolution of glutamine synthesis as a means for detoxification of ammonia first occurred among invertebrates.

African sharptooth catfish Clarias gariepinus does not detoxify ammonia to urea or amino acids but actively excretes ammonia during exposure to environmental ammonia.

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and exhibits high tolerance of environmental ammonia. This study aimed at elucidating the strategies adopted by C. gariepinus to defend against ammonia toxicity during ammonia exposure. No carbamoyl phosphate synthetase (CPS) I or III activities were detected in the liver or muscle of the adult C. gariepinus. In addition, activities of other ornithine-urea cycle (OUC) enzymes, especially ornithine transcarbamylase, were low in the liver, indicating that adult C. gariepinus does not have a "functional" hepatic OUC. After being exposed to 50 or 100 mM NH4Cl for 5 d, there was no induction of hepatic OUC enzymes and no accumulation of urea in tissues of the experimental animals. In addition, the rate of urea excretion remained low and unchanged. Hence, ammonia exposure did not induce ureogenesis or ureotely in C. gariepinus as suggested elsewhere for another obligatory air-breathing catfish of the same genus, Clarias batrachus, from India. Surprisingly, the local C. batrachus did not possess any detectable CPS I or III activities in the liver or muscle as had been reported for the Indian counterpart. There were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus exposed to 50 or 100 mM NH4Cl for 5 d; neither were there any changes in the glutamine levels in these tissues. Yet even after being exposed to 100 mM NH4Cl for 5 d, there was no significant increase in the level of ammonia in the muscle, which constitutes the bulk of the specimen. In addition, the level of ammonia accumulated in the plasma was relatively low compared to other tropical air-breathing fishes. More importantly, for all NH4Cl concentrations tested (10, 50, or 100 mM), the plasma ammonia level was maintained relatively constant (2.2-2.4 mM). These results suggest that C. gariepinus was able to excrete endogenous ammonia and infiltrated exogenous ammonia against a very steep ammonia gradient. When exposed to freshwater (pH 7.0) with or without 10 mM NH4Cl, C. gariepinus was able to excrete ammonia continuously to the external medium for at least 72 h. This was achieved while the plasma NH4+ and NH3 concentrations were significantly lower than those of the external medium. Diffusion trapping of NH3 through boundary layer acidification can be eliminated as the pH of the external medium became more alkaline instead. These results represent the first report on a freshwater fish (C. gariepinus) adopting active excretion of ammonia (probably NH4+) as a major strategy to defend against ammonia toxicity when exposed to environmental ammonia.

Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land.

This study aimed to elucidate the strategies adopted by the African slender lungfish, Protopterus dolloi, to ameliorate the toxicity of ammonia during short (6 days) or long (40 days) periods of aestivation in a layer of dried mucus in open air in the laboratory. Despite decreases in rates of ammonia and urea excretion, the ammonia content in the muscle, liver, brain and gut of P. dolloi remained unchanged after 6 days of aestivation compared with the control fasted for 6 days. For specimens aestivated for 40 days, the ammonia contents in the muscle, liver and gut were significantly lower than those of the control fasted for 40 days, which suggests a decrease in the rate of ammonia production. In addition, there were significant increases in contents of alanine, aspartate and glutamate in the muscle, which suggests decreases in their catabolism. During the first 6 days and the last 34 days of aestivation, the rate of ammonia production was reduced to 26% and 28%, respectively, of the control rate (6.83 micromol day(-1) g(-1) on day 0). During the first 6 days and the next 34 days of aestivation, the averaged urea synthesis rate was 2.39-fold and 3.8-fold, respectively, greater than the value of 0.25 micromol day(-1) g(-1) for the day 0 control kept in water. No induction of activities of the ornithine-urea cycle (OUC) enzymes was observed in specimens aestivated for 6 days, because the suppression of ammonia production led to a light demand on the OUC capacity. For specimens aestivated for 40 days, the activities of carbamoyl phosphate synthetase, ornithine transcarbamylase and argininosuccinate synthetase + lyase were significantly greater than those of the control fasted for 40 days. This is in agreement with the observation that the rate of urea synthesis in the last 34 days was greater than that in the first 6 days of aestivation. P. dolloi aestivated in a thin layer of dried mucus in open air with high O(2) tension throughout the 40 days of aestivation, which could be the reason why it was able to sustain a high rate of urea synthesis despite this being an energy-intensive process. Our results indicate that a reduction in ammonia production and decreases in hepatic arginine and cranial tryptophan contents are important facets of aestivation in P. dolloi.

Urea synthesis in the African lungfish Protopterus dolloi--hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure.

Like the marine ray Taeniura lymma, the African lungfish Protopterus dolloi possesses carbamoyl phosphate III (CPS III) in the liver and not carbamoyl phosphate I (CPS I), as in the mouse Mus musculus or as in other African lungfish reported elsewhere. However, similar to other African lungfish and tetrapods, hepatic arginase of P. dolloi is present mainly in the cytosol. Glutamine synthetase activity is present in both the mitochondrial and cytosolic fractions of the liver of P. dolloi. Therefore, we conclude that P. dolloi is a more primitive extant lungfish, which is intermediate between aquatic fish and terrestrial tetrapods, and represents a link in the fish-tetrapod continuum. During 6 days of aerial exposure, the ammonia excretion rate in P. dolloi decreased significantly to 8-16% of the submerged control. However, there were no significant increases in ammonia contents in the muscle, liver or plasma of specimens exposed to air for 6 days. These results suggest that (1). endogenous ammonia production was drastically reduced and (2). endogenous ammonia was detoxified effectively into urea. Indeed, there were significant decreases in glutamate, glutamine and lysine levels in the livers of fish exposed to air, which led to a decrease in the total free amino acid content. This indirectly confirms that the specimen had reduced its rates of proteolysis and/or amino acid catabolism to suppress endogenous ammonia production. Simultaneously, there were significant increases in urea levels in the muscle (8-fold), liver (10.5-fold) and plasma (12.6-fold) of specimens exposed to air for 6 days. Furthermore, there was an increase in the hepatic ornithine-urea cycle (OUC) capacity, with significant increases in the activities of CPS III (3.8-fold), argininosuccinate synthetase + lyase (1.8-fold) and, more importantly, glutamine synthetase (2.2-fold). This is the first report on the upregulation of OUC capacity and urea synthesis rate in an African lungfish exposed to air. Upon re-immersion, the urea excretion rate increased 22-fold compared with that of the control specimen, which is the greatest increase among fish during emersion-immersion transitions and suggests that P. dolloi possesses transporters that facilitate the excretion of urea in water.

A comparison of the effects of environmental ammonia exposure on the Asian freshwater stingray Himantura signifer and the Amazonian freshwater stingray Potamotrygon motoro.

The white-edge whip tail ray Himantura signifer inhabits a freshwater environment but has retained the capability to synthesize urea de novo through the arginine-ornithine-urea cycle (OUC). The present study aimed to elucidate whether the capacity of urea synthesis in H. signifer could be upregulated in response to environmental ammonia exposure. When H. signifer was exposed to environmental ammonia, fairly high concentrations of ammonia were accumulated in the plasma and other tissues. This would subsequently reduce the net influx of exogenous ammonia by reducing the NH(3) partial pressure gradient across the branchial and body surfaces. There was also an increase in the OUC capacity in the liver. Since the ammonia produced endogenously could not be excreted effectively in the presence of environmental ammonia, it was detoxified into urea through the OUC. In comparison, the South American freshwater stingray Potamotrygon motoro, which has lost the capability to synthesize urea de novo, was unable to detoxify ammonia to urea during ammonia loading. No increase in glutamine was observed in the various tissues of H. signifer exposed to environmental ammonia despite a significant increase in the hepatic glutamine synthetase activity. These results indicate that the excess glutamine formed was channelled completely into urea formation through carbamoyl phosphate synthetase III. It has been reported elsewhere that both urea synthesis and urea retention were upregulated in H. signifer exposed to 20 per thousand water for osmoregulatory purposes. By contrast, for H. signifer exposed to environmental ammonia in freshwater, the excess urea formed was excreted to the external medium instead. This suggests that the effectiveness of urea synthesis de novo as a strategy to detoxify ammonia is determined not simply by an increase in the capacity of urea synthesis but, more importantly, by the ability of the animal to control the direction (i.e. absorption or excretion) and rate of urea transport. Our results suggest that such a strategy began to develop in those elasmobranchs, e.g. H. signifer, that migrate into a freshwater environment from the sea but not in those permanently adapted to a freshwater environment.