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 pO₂ 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.     

The African lungfish, Protopterus dolloi, detoxifies ammonia to urea during environmental ammonia exposure

The African lungfish, Protopterus dolloi, was able to maintain a low level of blood plasma ammonia during exposure to high concentrations of environmental ammonia. After 6 d of exposure to 30 or 100 mM NH(4)Cl, the total ammonia concentrations in the blood plasma were 0.288 and 0.289 mM, respectively, which were only 1.7-fold greater than the control value of 0.163 mM. In addition, accumulation of ammonia occurred only in the muscle, but not in the liver. This was achieved in part through urea synthesis, as reflected by significant increases in urea contents in the muscle, liver, and plasma of the experimental animals. In contrast with plasma ammonia, the plasma urea concentrations of specimens exposed to 30 or 100 mM NH(4)Cl for 6 d increased 15.4-fold and 18.8-fold, respectively. Taken together, these results suggest that P. dolloi upregulated the rate of urea synthesis to detoxify ammonia during environmental ammonia exposure and that the increased rate of urea synthesis was fast enough to compensate for the rate of endogenous ammonia production plus the net influx of exogenous ammonia in these experimental animals. Simultaneously, there were increases in the rates of urea excretion in the experimental animals between day 2 and day 6 of environmental ammonia exposure. Interestingly, the rates of urea excretion in specimens exposed to 100 mM NH(4)Cl were lower than those exposed to 30 mM NH(4)Cl, despite the presumably greater load of ammonia to be detoxified to urea in the former situation. It would appear that P. dolloi was regulating the rate of urea excretion during ammonia exposure to retain urea, which might have some physiological functions under environmental stresses yet to be determined. There were decreases in the contents of glutamate, glutamine, and total free amino acids in the liver of the experimental animals, which indirectly suggest that a reduction in the rate of proteolysis and/or amino acid catabolism would have occurred that might lead to a decrease in ammonia production. Our results suggest that, unlike marine elasmobranchs and coelacanths, which synthesize and retain urea for osmoregulatory purposes, the ureogenic P. dolloi was adapted to synthesizing and excreting urea for the purpose of ammonia detoxification.     

Effects of hypoxia on the energy status and nitrogen metabolism of African lungfish during aestivation in a mucus cocoon

We examined the energy status, nitrogen metabolism and hepatic glutamate dehydrogenase activity in the African lungfish Protopterus annectens during aestivation in normoxia (air) or hypoxia (2% O(2) in N(2)), with tissues sampled on day 3 (aerial exposure with preparation for aestivation), day 6 (entering into aestivation) or day 12 (undergoing aestivation). There was no accumulation of ammonia in tissues of fish exposed to normoxia or hypoxia throughout the 12-day period. Ammonia toxicity was avoided by increased urea synthesis and/or decreased endogenous N production (as ammonia), but the dependency on these two mechanisms differed between the normoxic and the hypoxic fish. The rate of urea synthesis increased 2.4-fold, with only a 12% decrease in the rate of N production in the normoxic fish. By contrast, the rate of N production in the hypoxic fish decreased by 58%, with no increase in the rate of urea synthesis. Using in vivo (31)P NMR spectroscopy, it was demonstrated that hypoxia led to significantly lower ATP concentration on day 12 and significantly lower creatine phosphate concentration on days 1, 6, 9 and 12 in the anterior region of the fish as compared with normoxia. Additionally, the hypoxic fish had lower creatine phosphate concentration in the middle region than the normoxic fish on day 9. Hence, lowering the dependency on increased urea synthesis to detoxify ammonia, which is energy intensive by reducing N production, would conserve cellular energy during aestivation in hypoxia. Indeed, there were significant increases in glutamate concentrations in tissues of fish aestivating in hypoxia, which indicates decreases in its degradation and/or transamination. Furthermore, there were significant increases in the hepatic glutamate dehydrogenase (GDH) amination activity, the amination/deamination ratio and the dependency of the amination activity on ADP activation in fish on days 6 and 12 in hypoxia, but similar changes occurred only in the normoxic fish on day 12. Therefore, our results indicate for the first time that P. annectens exhibited different adaptive responses during aestivation in normoxia and in hypoxia. They also indicate that reduction in nitrogen metabolism, and probably metabolic rate, did not occur simply in association with aestivation (in normoxia) but responded more effectively to a combined effect of aestivation and hypoxia.     

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.     

The morphology of the lung of the African lungfish,Protopterus aethiopicus

Summary The lung of the African lungfish (Protopterus aethiopicus) is paired, long and cylindrical. It is situated on the dorsal aspect of the coelomic cavity ventral to the ribs. Much of the gas exchange tissue is found in the proximal aspect of the lung with the caudal part largely taken up by a centrally situated air-duct with a few large peripherally located alveoli. Interalveolar septa, arranged at differing hierarchical levels from the air-duct, subdivide the lung into alveoli, the gas exchange compartments. The alveolar surface is covered by some cells characterized by microvilli on their free surface, while others are devoid of such structures. The general organization of the lung of Protopterus aethiopicus is similar to that of the other genera of Dipnoi, Neoceratodus and Lepidosiren, with the interalveolar septa increasing the surface area for gas exchange through pulmonary compartmentation. The abundant septal smooth muscle fibres and elastic tissue may contribute to the physiomechanical compliance of the lung. The undifferentiated alveolar pneumocytes and the double capillary system, observed in Protopterus, in general appear to characterize the very primitive lungs of the lower air-breathing vertebrates.     

Ornithine-urea cycle and urea synthesis in African lungfishes, Protopterus aethiopicus and Protopterus annectens, exposed to terrestrial conditions for six days

The objectives of this study were (1) to determine the type of carbamoyl phosphate synthetase (CPS) present, and the compartmentalization of arginase, in the livers of the African lungfishes, Protopterus aethiopicus and Protopterus annectens, and (2) to elucidate if these two lungfishes were capable of increasing the rates of urea synthesis and capacities of the ornithine-urea cycle (OUC) during 6 days of aerial exposure without undergoing aestivation. Like another African lungfish, Protopterus dolloi, reported elsewhere, the CPS activities from the livers of P. aethiopicus and P. annectens had properties similar to that of the marine ray (Taeniura lymma), but dissimilar to that of the mouse (Mus musculus). Hence, they possessed CPS III, and not CPS I as reported previously. CPS III was present exclusively in the liver mitochondria of both lungfishes, but the majority of the arginase activities were present in the cytosolic fractions of their livers. Glutamine synthetase (GS) activity was also detected in the hepatic mitochondria of both specimens. Therefore, our results suggest that the evolution of CPS III to CPS I might not have occurred before the evolution of extant lungfishes as suggested previously, prompting an examination of the current view on the evolution of CPS and OUC in vertebrates. Aerial exposure led to significant decreases in rates of ammonia excretion in P. aethiopicus and P. annectens, but there were no accumulations of ammonia in their tissues. However, urea contents in their tissues increased significantly after 6 days of aerial exposure. The estimated rates of urea synthesis in P. aethiopicus and P. annectens increased 1.2- and 1.47-fold, respectively, which were smaller than that in P. dolloi (8.6-fold) reported elsewhere. In addition, unlike P. dolloi, 6 days of aerial exposure had no significant effects on the hepatic CPS III activities of P. aethiopicus and P. annectens. In contrast, aerial exposure induced relatively greater degrees of reductions in ammonia production in P. aethiopicus (34%) and P. annectens (37%) than P. dolloi (28%) as previously reported. Thus, our results suggest that various species of African lungfishes respond to aerial exposure differently with respect to nitrogen metabolism and excretion, and it can be concluded that P. aethiopicus and P. annectens depended more on reductions in ammonia production than on increases in urea synthesis to ameliorate ammonia toxicity when exposed to terrestrial conditions.     

Respiratory allocation and standard rate of metabolism in the African lungfish, Protopterus aethiopicus

This paper quantifies the relationship between respiratory allocation (air vs. water) and the standard rate of metabolism (SMR) in the primitive air-breathing lungfish, Protopterus aethiopicus. Simultaneous measurements of oxygen consumed from both air and water were made to determine the SMR at ecologically relevant aquatic oxygen levels for juveniles 2 to 221 g. Total metabolic rate was positively correlated with body mass with a scaling exponent of 0.78. Aerial oxygen consumption averaged 98% (range=94% to 100%) of total respiratory allocation under low aquatic oxygen levels. Measurements of oxygen consumption made across a gradient of dissolved oxygen from normoxia to anoxia showed that P. aethiopicus maintains its SMR despite a change in respiratory allocation between water and air.     

Ultrastructural study on nuclear-cytoplasmic relationships in oocytes of the African lungfish,Protopterus aethiopicus

Summary Growing oocytes of Protopterus, like those of some amphibians and teleosts, show an impressive development of the nucleolar apparatus. Numerous nucleolus-like bodies establish close spatial relationships with the nuclear envelope by extending pedicels and streams of finely dispersed material towards the inner membrane.At such contact points, gaps in the perinuclear cistern are more frequent than elsewhere along the nuclear boundary. Expansion of the outer nuclear membrane gives rise to blebs, with or without visible content, and these become pinched off to form small vesicles in the perinuclear cytoplasm.Small, electron dense aggregates, indistinguishable from nucleolar material occur on both sides of the nuclear envelope opposite to each other, some being connected by a slender portion of the same material within a nuclear pore. Such accumulations are interpreted as detached parts of nucleolar bodies in transit to cytoplasmic sites where they presumably participate in the biogenesis of ribosomes. At the height of nucleolar emission, nucleoplasm and perinuclear cytoplasm are so rich in small electron dense particles that they are almost indistinguishable from each other.At this stage of massive transport, the route provided by the nuclear pores seems to be insufficient and another, more spacious, gateway may be in operation. The latter involves direct passage of material across the nuclear membranes preferentially where these form blebs.This view is supported not only by the overt spatial relationships between nucleolar pedicels and blebs, but by the occurrence within perinuclear lacunae and blebs of particles that seem to be derived from nucleolar bodies. Furthermore, frequent interruptions in the nuclear membranes preferentially located where they expand into outpocketings suggest that at these sites temporary gateways may exist in the living cell that permit easy access of intranuclear components to the cytoplasm.Supported by grants AM-3984, NB-00840, and NB-05219 from the U.S.P.H.S.     

Lipid, ketone body and oxidative metabolism in the African lungfish, Protopterus dolloi following 60 days of fasting and aestivation

The potential importance of lipids and ketone bodies as fuels in the African lungfish, Protopterus dolloi, and the role of oxidative metabolism, were examined under control, fasted and aestivated conditions. In aestivating but not fasting lungfish, the activities of citrate synthase (CS) and cytochrome c oxidase (CCO) (enzymes of oxidative metabolism) showed tissue-specific changes. Significant reductions in CS activity occurred in the kidney, heart, gill and muscle, and in CCO in the liver and kidney tissues. Aestivation, but not fasting, also had a tissue-specific effect on mitochondrial state 3 respiration rates (using succinate as a substrate), with a >50% reduction in the liver, yet no change within muscle mitochondria. There is no indication that enzymes involved in lipid catabolism are up-regulated during periods of fasting or aestivation; however, both 3-hydroxyacyl CoA dehydrogenase (HOAD) and carnitine palmitoyl CoA transferase (CPT) activities were sustained in the liver despite the approximately 42% reduction in CCO activity, potentially indicating lipid metabolism is of importance during aestivation. Lungfish are able to utilize both the d- and l-stereoisomers of the ketone body beta-hydroxybutyrate (beta-HB); however, beta-HB does not appear to be an important fuel source during aestivation or fasting as no changes were observed in beta-HB tissue levels. This study demonstrates that an important aspect of metabolic depression during aestivation in lungfish is the tissue-specific down regulation of enzymes of aerobic metabolism while maintaining the activities of enzymes in pathways that supply substrates for aerobic metabolism.     

Decline of the African lungfish (Protopterus aethiopicus) in Lake Victoria (East Africa)

Catch and effort data for the period 1973–1990 demonstrate a dramatic decline of lungfish in the Tanzanian waters of Lake Victoria. Bottom trawl catches in the Mwanza Gulf showed a decline in catch rates from 67.5 kg h⁻¹ in 1973 to 5.5 kg h⁻¹ in 1986. Trawling of commercial vessels in the Speke Gulf revealed a decline in lungfish catches from 1.3 kg h⁻¹ in 1986 to 0.07 kg h⁻¹ in 1990. The development of anoxia in the deeper waters of Lake Victoria, the algal blooms, and the decline of water transparency, all associated with eutrophication, are not likely to have contributed to the decreased catch rate. However, the lungfish decline may reflect the interaction of overexploitation by the fishery and a low level of Nile perch predation that restricts lungfish to wetland refugia. We suggest that this may have been reinforced over the past few decades by large-scale conversion of wetlands to agricultural land and harvesting of nest-guarding male lungfish leading to decreased recruitment of young.     

Reproductive biology of the East African lungfish (Protopterus aethiopicus) in Mwanza Gulf, Lake Victoria

The population of lungfish (Protopterus aethiopicus (Heckel)) was sampled by bottom trawling for 12 months from June 1984 to May 1985. A total of 576 lungfish were investigated. The sex ratio in the population was 1 male : 1 -98 females. Average size at maturity was found to be 960cm total length (TL) but the smallest matured male measured 72-9 cm and the female 840 cm TL. It was not possible to establish spawning times and reproductive activity trends either from the variation in relative condition factor ‘kn’ or from gonadosomatic index (GSI). Whereas male lungfish seem to show no further increase in GSI after gonadal maturity stage III, females exhibit GSI values that vary directly with oocyte maturation and ovarian weights. Fat deposited along the gonads was found to be a better basis for assessing maturation and spawning in lungfish. The potential spawning period was found to be from September to May. Absence of lungfish with spent gonads over the 12-month period and the presence of different egg sizes in ovaries, suggest that the fish is a multiple spawner. Fecundity was found to range from 705 eggs (in 84-cm TL fish) to 14,922 eggs (in 130-cm TL Fish).     

A radiological study of the central circulation in the lungfish, Protopterus aethiopicus

The dipnoan heart is only in part structurally developed to support a separated circulation in pulmonary and systemic circuits. In the present investigation biplane angiocardiography has been used to describe the extent of such a double circulation and the factors which may modify it in the African lungfish, Protopterus aethiopicus. Contrast injections in the pulmonary vein revealed a clear tendency for aerated blood returing from the lungs to be selectively dispatched to the anterior branchial arteries giving rise to the major systemic circulation. Contrast injections in the vena cava delineated the sinus venosus as a large receiving chamber for systemic venous blood. Contraction of the sinus venosus discharged blood into the right, posterior part of the partially divided atrial space. Contrast injection in the pulmonary vein showed that vessel to pass obliquely from right to left such that blood was emptied distinctly into the left side of the atrium. During contraction the atrial space tended to retain a residual volume in its anterior undivided part which minized mixing. Ventricular filling occurred through separate right and left atrio-ventricular connections. Right-left separation in most of the ventricle was maintained by the partial ventricular septum, the trabeculated, spongelike myocardium and the mode of inflow from the atria. Mixing in the anterior undivided portion of the ventricle during the ejection phase was slight due to a streamlined ejection pattern. The outflow through the bulbus cordis occurred in discrete streams which in part were structurally separated by well developed spiral folds. In the anterior bulbus segment the spiral folds are fused and make completely separate dorsal and ventral outflow tracts. The ventral bulbus channel provides blood to the three anterior branchial arteries. The second and third branchial arteries are large and represent direct shunts to the dorsal aorta. The fourth and fifth branchial arteries are gill bearing and receive blood form the dorsal bulbus channel. The most posterior epibranchial vessels give rise to the pulmonary arteries.     

X-ray analysis coupled with scanning and transmission electron microscopic observations of spermatozoa of the African lungfish, Protopterus aethiopicus

Spermatozoa from the reproductive tubules and epididymis of the African lungfish, Protopterus aethiopicus , were examined by scanning, transmission and scanning-transmission electron microscopy. The biflagellate nature of lungfish spermatozoa was confirmed as a general rule by examining large numbers of specimens in the scanning mode. All have two flagella with attached undulating membranes, a short middle piece and a large sperm head without condensation of the nucleus. The various portions of the sperm have been subjected to analysis of the X-rays emitted upon electron bombardment, and the relative concentrations of several elements have been determined.     

Cardiovascular actions of lungfish bradykinin in the unanaesthetised African lungfish, Protopterus annectens

Bradykinin (BK) isolated from plasma of the African lungfish, Protopterus annectens, contains four amino acid substitutions compared with BK from mammals (Arg(1)-->Tyr, Pro(2)-->Gly, Pro(7)-->Ala, Phe(8)-->Pro). Bolus intra-arterial injections of synthetic lungfish BK (1-1000 pmol/kg body wt.) into unanaesthetised, juvenile lungfish (n=5) produced a dose-dependent increase in arterial blood pressure and pulse pressure. The maximum pressor response occurred 2-3 min after injection and persisted for up to 15 min. The threshold dose producing a significant (P<0.01) rise in pressure was 50 pmol/kg and the maximum increase, following injection of 300 pmol/kg, was 9.3 +/- 2.3 mmHg. Injection of the higher doses of lungfish BK produced a significant (P<0.05) increase in heart rate (2.8 +/- 0.8 beats/min at 100 pmol/kg). In contrast, bolus intra-arterial injections of mammalian BK, in doses up to 1000 pmol/kg, produced no significant cardiovascular effects in the lungfish. The data support the existence of a functioning kallikrein-kinin system in the lungfish and demonstrate that the ligand-binding properties of the receptor(s) mediating the cardiovascular actions of lungfish BK are appreciably different from mammalian B1 and B2 receptors.     

Changes in salinity and ionic compositions can act as environmental signals to induce a reduction in ammonia production in the African lungfish Protopterus dolloi

The slender African lungfish, Protopterus dolloi, does not aestivate in a subterranean mud cocoon, but is capable of aestivating inside a layer of dried mucus on land during drought. In this study, we aimed to elucidate if a slight increase in salinity in association with changes in the ionic composition could act as signals for P. dolloi to decrease endogenous ammonia production, in preparation for aestivation when the external medium dries up. Specimens of P. dolloi exposed to 3 per thousand water for 6 days exhibited consistently lower daily urea excretion rate than the freshwater control. This led to significant decreases in the cumulative total nitrogenous wastes excreted on days 3, 5 and 6. On day 6, there were decreases in urea contents in various tissues and organs. Taken together, these results suggest that there was a decrease in the rate of urea synthesis, the magnitude of which was greater than the decrease in the rate of urea excretion, and therefore resulted in decreases in internal urea contents. A decrease in the rate of urea synthesis should result in a decrease in the rate of glutamine utilization, and subsequently led to the accumulations of glutamine and/or ammonia. However, there were no changes in contents of glutamine and ammonia in various tissues and organs in the experimental animals. A logical explanation for this is that there must be a simultaneous reduction in ammonia production; if not, ammonia would accumulate due to the decrease in rate of urea synthesis. Since fish were unfed during the experiment, endogenous ammonia must be derived mainly from amino acid catabolism. Therefore, these results suggest that a suppression of amino acid catabolism occurred in specimens exposed to 3 per thousand for 6 days. The differences in effects of freshwater and 3 per thousand water on endogenous ammonia production could not be due to food deprivation because both groups of fish were fasted for the same period. Because control and experimental fish were kept in water and because there were no changes in the wet mass of the fish and blood osmolality before and after the experiment, dehydration did not occur. Furthermore, both groups of fish have comparable blood pH, pO2 and pCO2 on day 6 as they had free access to air, and therefore CO2 retention could be eliminated as the initiating factor of suppressed endogenous ammonia production. In conclusion, our results suggest that P. dolloi could respond to increases in salinity and changes in ionic composition in the external medium by suppressing ammonia production in preparation for aestivation when the water dries up.