Acute toxicity of ammonia and nitrite to Siamese fighting fish (Betta splendens).

The acute toxicity and sublethal effects of ammonia and nitrite on the air-beathing Siamese fighting fish, betta (Betta splendens) was studied for 96 h. The LC50 (50% Lethal Concentration) for 96 h for adult bettas to ammonia-N and nitrite-N was 123.4 mM (1.7 g/L, 95% confidence limits: 114.7-130.0 mM) and 24.6 mM (343.6 mg/L, 95% confidence limits: 22.7-26.4 mM) respectively. Exposure to 90 mM ammonia did not affect ammonia and urea excretion rates in bettas. There was no significant difference in values between control and ammonia-loaded (90 mM ammonia) individuals in either brain or liver activities of glutamine synthase, while plasma ammonia levels slightly increased. It appears unlikely that ammonia was converted to urea or amino acids for detoxification. Sublethal nitrite (24.6 mM nitrite) affected plasma nitrite, methemoglobin and hemoglobin. Plasma nitrite values remained much lower than ambient concentrations. Betta has a labyrinth organ and can breathe air. Bettas may temporarily reduce the entry of ammonia and nitrite into the body by increasing the rate of air respiration and reducing the contribution of the gill epithelium, which is highly permeable to these nitrogenous pollutants.

Distributional shift of urea production site from the extraembryonic yolk sac membrane to the embryonic liver during the development of cloudy catshark (Scyliorhinus torazame).

Urea is an essential osmolyte for marine cartilaginous fishes. Adult elasmobranchs and holocephalans are known to actively produce urea in the liver, muscle and other extrahepatic organs; however, osmoregulatory mechanisms in the developing cartilaginous fish embryo with an undeveloped urea-producing organ are poorly understood. We recently described the contribution of extraembryonic yolk sac membranes (YSM) to embryonic urea synthesis during the early developmental period of the oviparous holocephalan elephant fish (Callorhinchus milii). In the present study, to test whether urea production in the YSM is a general phenomenon among oviparous Chondrichthyes, we investigated gene expression and activities of ornithine urea cycle (OUC) enzymes together with urea concentrations in embryos of the elasmobranch cloudy catshark (Scyliorhinus torazame). The intracapsular fluid, in which the catshark embryo develops, had a similar osmolality to seawater, and embryos maintained a high concentration of urea at levels similar to that of adult plasma throughout development. Relative mRNA expressions and activities of catshark OUC enzymes were significantly higher in YSM than in embryos until stage 32. Concomitant with the development of the embryonic liver, the expression levels and activities of OUC enzymes were markedly increased in the embryo from stage 33, while those of the YSM decreased from stage 32. The present study provides further evidence that the YSM contributes to embryonic urea homeostasis until the liver and other extrahepatic organs become fully functional, and that urea-producing tissue shifts from the YSM to the embryonic liver in the late developmental period of oviparous marine cartilaginous fishes.

Urea-based osmoregulation in the developing embryo of oviparous cartilaginous fish (Callorhinchus milii): contribution of the extraembryonic yolk sac during the early developmental period.

Marine cartilaginous fish retain a high concentration of urea to maintain the plasma slightly hyperosmotic to the surrounding seawater. In adult fish, urea is produced by hepatic and extrahepatic ornithine urea cycles (OUCs). However, little is known about the urea retention mechanism in developing cartilaginous fish embryos. In order to address the question as to the mechanism of urea-based osmoregulation in developing embryos, the present study examined the gene expression profiles of OUC enzymes in oviparous holocephalan elephant fish (Callorhinchus milii) embryos. We found that the yolk sac membrane (YSM) makes an important contribution to the ureosmotic strategy of the early embryonic period. The expression of OUC enzyme genes was detectable in the embryonic body from at least stage 28, and increased markedly during development to hatching, which is most probably due to growth of the liver. During the early developmental period, however, the expression of OUC enzyme genes was not prominent in the embryonic body. Meanwhile, we found that the mRNA expression of OUC enzymes was detected in the extra-embryonic YSM; the mRNA expression of cmcpsIII in the YSM was much higher than that in the embryonic body during stages 28-31. Significant levels of enzyme activity and the existence of mitochondrial-type cmgs1 transcripts in the YSM supported the mRNA findings. We also found that the cmcpsIII transcript is localized in the vascularized inner layer of the YSM. Taken together, our findings demonstrate for the first time that the YSM is involved in urea-based osmoregulation during the early to mid phase of development in oviparous cartilaginous fish.

Transcriptome responses in the rectal gland of fed and fasted spiny dogfish shark (Squalus acanthias) determined by suppression subtractive hybridization.

Prior studies of the elasmobranch rectal gland have demonstrated that feeding induces profound and rapid up regulation of the gland's ability to secrete concentrated NaCl solutions and the metabolic capacity to support this highly ATP consuming process. We undertook the current study to attempt to determine the degree to which up regulation of mRNA transcription was involved in the gland's activation. cDNA libraries were created from mRNA isolated from rectal glands of fasted (7days post-feeding) and fed (6h and 22h post-feeding) spiny dogfish sharks (Squalus acanthias), and the libraries were subjected to suppression subtractive hybridization (SSH) analysis. Quantitative real time PCR (qPCR) was also used to ascertain the mRNA expression of several genes revealed by the SSH analysis. In total the treatments changed the abundance of 170 transcripts, with 103 up regulated by feeding, and 67 up regulated by fasting. While many of the changes took place in 'expected' Gene Ontology (GO) categories (e.g., metabolism, transport, structural proteins, DNA and RNA turnover, etc.), KEGG analysis revealed a number of categories which identify oxidative stress as a topic of interest for the gland. GO analysis also revealed that branched chain essential amino acids (e.g., valine, leucine, isoleucine) are potential metabolic fuels for the rectal gland. In addition, up regulation of transcripts for many genes in the anticipated GO categories did not agree (i.e., fasting down regulated in feeding treatments) with previously observed increases in their respective proteins/enzyme activities. These results suggest an 'anticipatory' storage of selected mRNAs which presumably supports the rapid translation of proteins upon feeding activation of the gland.

Hepatic and extrahepatic distribution of ornithine urea cycle enzymes in holocephalan elephant fish (Callorhinchus milii).

Cartilaginous fish comprise two subclasses, the Holocephali (chimaeras) and Elasmobranchii (sharks, skates and rays). Little is known about osmoregulatory mechanisms in holocephalan fishes except that they conduct urea-based osmoregulation, as in elasmobranchs. In the present study, we examined the ornithine urea cycle (OUC) enzymes that play a role in urea biosynthesis in the holocephalan elephant fish, Callorhinchus milii (cm). We obtained a single mRNA encoding carbamoyl phosphate synthetase III (cmCPSIII) and ornithine transcarbamylase (cmOTC), and two mRNAs encoding glutamine synthetases (cmGSs) and two arginases (cmARGs), respectively. The two cmGSs were structurally and functionally separated into two types: brain/liver/kidney-type cmGS1 and muscle-type cmGS2. Furthermore, two alternatively spliced transcripts with different sizes were found for cmgs1 gene. The longer transcript has a putative mitochondrial targeting signal (MTS) and was predominantly expressed in the liver and kidney. MTS was not found in the short form of cmGS1 and cmGS2. A high mRNA expression and enzyme activities were found in the liver and muscle. Furthermore, in various tissues examined, mRNA levels of all the enzymes except cmCPSIII were significantly increased after hatching. The data show that the liver is the important organ for urea biosynthesis in elephant fish, but, extrahepatic tissues such as the kidney and muscle may also contribute to the urea production. In addition to the role of the extrahepatic tissues and nitrogen metabolism, the molecular and functional characteristics of multiple isoforms of GSs and ARGs are discussed.

The influence of feeding and fasting on plasma metabolites in the dogfish shark (Squalus acanthias).

Dogfish sharks are opportunistic predators, eating large meals at irregular intervals. Here we present a synthesis of data from several previous studies on responses in plasma metabolites after natural feeding and during prolonged fasting (up to 56days), together with new data on changes in plasma concentrations of amino acids and non-esterified fatty acids. Post-prandial and long-term fasting responses were compared to control sharks fasted for 7days, a typical inter-meal interval. A feeding frenzy was created in which dogfish were allowed to feed naturally on dead teleosts at two consumed ration levels, 2.6% and 5.5% of body weight. Most responses were more pronounced at the higher ration level. These included increases in urea and TMAO concentrations at 20h, followed by stability through to 56days of fasting. Ammonia levels were low and exhibited little short-term response to feeding, but declined to very low values during the extended fast. Glucose and beta-hydroxybutyrate both fell after feeding, the latter to a greater and more prolonged extent (up to 60h), whereas acetoacetate did not change. During prolonged fasting, glucose concentrations were well regulated, but beta-hydroxybutyrate increased to 2-3-fold control levels. Total plasma amino acid concentrations increased in a biphasic fashion, with peaks at 6-20h, and 48-60h after the meal, followed by homeostasis during the extended fast. Essential and non-essential amino acids generally followed this same pattern, though some exhibited different trends after feeding: taurine, beta-alanine, and glycine (decreases or stability), alanine and glutamine (modest prolonged increases), and threonine, serine, asparagine, and valine (much larger short-term increases). Plasma non-esterified fatty acid concentrations declined markedly through 48h after the 2.6% meal. These data are interpreted in light of companion studies showing elevations in aerobic metabolic rate, urea production, rectal gland function, metabolic base excretion, and activation of ornithine-urea cycle and aerobic enzymes after the meal, and muscle N-depletion but maintenance of osmolality and urea production during long-term fasting.

Is the alkaline tide a signal to activate metabolic or ionoregulatory enzymes in the dogfish shark (Squalus acanthias)?

Experimental metabolic alkalosis is known to stimulate whole-animal urea production and active ion secretion by the rectal gland in the dogfish shark. Furthermore, recent evidence indicates that a marked alkaline tide (systemic metabolic alkalosis) follows feeding in this species and that the activities of the enzymes of the ornithine-urea cycle (OUC) for urea synthesis in skeletal muscle and liver and of energy metabolism and ion transport in the rectal gland are increased at this time. We therefore evaluated whether alkalosis and/or NaCl/volume loading (which also occurs with feeding) could serve as a signal for activation of these enzymes independent of nutrient loading. Fasted dogfish were infused for 20 h with either 500 mmol L(-1) NaHCO3 (alkalosis + volume expansion) or 500 mmol L(-1) NaCl (volume expansion alone), both isosmotic to dogfish plasma, at a rate of 3 mL kg(-1) h(-1). NaHCO3 infusion progressively raised arterial pH to 8.28 (control = 7.85) and plasma [HCO3-] to 20.8 mmol L(-1) (control = 4.5 mmol L(-1)) at 20 h, with unchanged arterial P(CO2), whereas NaCl/volume loading had no effect on blood acid-base status. Rectal gland Na+,K+-ATPase activity was increased 50% by NaCl loading and more than 100% by NaHCO3 loading, indicating stimulatory effects of both volume expansion and alkalosis. Rectal gland lactate dehydrogenase activity was elevated 25% by both treatments, indicating volume expansion effects only, whereas neither treatment increased the activities of the aerobic enzymes citrate synthase, NADP-isocitrate dehydrogenase, or the ketone body-utilizing enzyme beta-hydroxybutyrate dehydrogenase in the rectal gland or liver. The activity of ornithine-citrulline transcarbamoylase in skeletal muscle was doubled by NaHCO3 infusion, but neither treatment altered the activities of other OUC-related enzymes (glutamine synthetase, carbamoylphosphate synthetase III). We conclude that both the alkaline tide and salt loading/volume expansion act as signals to activate some but not all of the elevated metabolic pathways and ionoregulatory mechanisms needed during processing of a meal.

Intertissue regulation of carnitine palmitoyltransferase I (CPTI): mitochondrial membrane properties and gene expression in rainbow trout (Oncorhynchus mykiss).

Carnitine palmitoyltransferase (CPT) I is regulated by several genetic and non-genetic factors including allosteric inhibition, mitochondrial membrane composition and/or fluidity and transcriptional regulation of enzyme content. To determine the intrinsic differences in these regulating factors that may result in differences between tissues in fatty acid oxidation ability, mitochondria were isolated from red, white and heart muscles and liver tissue from rainbow trout. Maximal activity (V(max)) for beta-oxidation enzymes and citrate synthase per mg tissue protein as well as CPT I in isolated mitochondria followed a pattern across tissues of red muscle>heart>white muscle>liver suggesting both quantitative and qualitative differences in mitochondria. CPT I inhibition showed a similar pattern with the highest malonyl-CoA concentration to inhibit activity by 50% (IC(50)) found in red muscle while liver had the lowest. Tissue malonyl-CoA content was highest in white muscle with no differences between the other tissues. Interestingly, the gene expression profiles did not follow the same pattern as the tissue enzyme activity. CPT I mRNA expression was greatest in heart>red muscle>white muscle>liver. In contrast, PPARalpha mRNA was greatest in the liver>red muscle>heart>white muscle. There were no significant differences in the mRNA expression of PPARbeta between tissues. As well, no significant differences were found in the mitochondrial membrane composition between tissues, however, there was a tendency for red muscle to exhibit higher proportions of PUFAs as well as a decreased PC:PE ratio, both of which would indicate increased membrane fluidity. In fact, there were significant correlations between IC(50) of CPT I for malonyl-CoA and indicators of membrane fluidity across tissues. This supports the notion that sensitivity of CPT I to its allosteric regulator could be modulated by changes in mitochondrial membrane composition and/or fluidity.

Natural feeding influences protein expression in the dogfish shark rectal gland: A proteomic analysis.

The rectal gland is the principal salt-secreting organ in elasmobranchs, yet its functional response to normal physiological variation (e.g., due to feeding, stress) has only recently been examined. To complement studies on acid-base, digestive, and osmoregulatory physiology in response to natural feeding, we investigated protein-level responses in the rectal gland of spiny dogfish (Squalus acanthias) 6 h, 20 h, and 5 days (reference control) after a meal. Our objective was to identify proteins involved in regulation of osmoregulatory and metabolic processes in response to feeding. Proteins were separated by two-dimensional gel electrophoresis, and protein spots that were significantly up- or down-regulated >2 fold (i.e., abundance increased more than 100% or decreased more than 50%) were detected using gel image analysis software. Of 684 proteins analyzed on 2D gels, 16 proteins changed significantly 6 h after feeding vs. 5 day controls (5 decreased; 11 increased), and 12 proteins changed >2 fold 20 h after feeding vs. 5 day controls (2 decreased; 10 increased). Thirteen of these proteins were identified using mass spectrometry and classified into functional pathways using the PANTHER bioinformatics database. Rectal gland proteins that were regulated following feeding fell into three main categories: cytoskeletal/muscular (e.g., tropomyosin alpha chain, transgelin), energy metabolism (e.g., malate dehydrogenase, ATP synthase), and nucleotide metabolism (nucleoside diphosphate kinase). The data also revealed that previously documented increases in the activity of isocitrate dehydrogenase after feeding are at least partially due to increased abundance of a cytosolic, NADP-dependent isoform of this enzyme. One of the primary components of the rectal gland's response to feeding appears to be maintenance of the cellular supply of energy, which would be necessary to fuel increased activities of enzymes involved in salt secretion and oxidative metabolism in the rectal gland following a meal.

Osmoregulation, ionoregulation and acid-base regulation by the gastrointestinal tract after feeding in the elasmobranch (Squalus acanthias).

In order to study the physiological consequences of voluntary feeding in the gastrointestinal tract of a ureotelic marine elasmobranch, dogfish (fasted for 96 h) were sampled at various times up to 360 h after consuming a 5-6% ration of teleost fish (hake) under natural feeding conditions. Digestion and absorption were completed between 120 and 360 h post-feeding. The tissue masses of different segments of the gastrointestinal tract increased and decreased markedly as the chyme moved through, mainly because of fluid engorgement rather than hyperplasia. In fasted dogfish, the cardiac and pyloric stomachs contained only small volumes of highly acidic fluid (pH 1.77+/-1.12, 2.05+/-0.08) similar in composition to seawater. Feeding resulted in gastric pHs of 3.20+/-0.31 and 3.95+/-0.40 at 6 h, followed by slow declines through 60 h. An alkaline tide in the blood also occurred at 6 h. In the face of large changing masses of highly acidic chyme in the stomachs, the pH (6.50+/-0.10), ionic composition and volume of chyme in the intestine (spiral valve) were precisely regulated from 6 to 60 h post-feeding at very different values from those in the stomachs, and intestinal HCO3(-) remained low (5.12+/-0.83 mmol l(-1)). The colon was usually empty and its pH constant at 7.20+/-0.16 at all times. Despite the ingestion of strongly hypo-osmotic teleost tissue, the osmolality of the chyme remained in equilibrium with that of the blood plasma in all segments at all times after feeding. Much of the osmotic equilibration was because of the secretion of urea into the chyme, particularly in the intestine. After feeding, gastric fluid concentrations of Na(+) and Mg(2+) declined, K(+) and Ca(2+) increased, whereas Cl(-) exhibited little change, indicating that additional drinking of seawater was minimal. Na(+), K(+), water and especially Cl(-) were absorbed in the intestine, whereas Mg(2+) and Ca(2+) were largely excluded. Our results illustrate the complex integration of digestive and ionoregulatory function in the elasmobranch digestive tract, and marked differences from the teleost pattern.

Rapid regulation of Na+ fluxes and ammonia excretion in response to acute environmental hypoxia in the Amazonian oscar, Astronotus ocellatus.

The Amazonian oscar is extremely resistant to hypoxia, and tolerance scales with size. Overall, ionoregulatory responses of small ( approximately 15 g) and large oscars ( approximately 200 g) to hypoxia were qualitatively similar, but the latter were more effective. Large oscars exhibited a rapid reduction in unidirectional Na(+) uptake rate at the gills during acute hypoxia (Po(2) approximately 10 mmHg), which intensified with time (7 or 8 h); Na(+) efflux rates were also reduced, so net balance was little affected. The inhibitions were virtually immediate (1st h) and preceded a later 60% reduction (at 3 h) in gill Na(+)-K(+)-ATPase activity, reflected in a 60% reduction in maximum Na(+) uptake capacity without change in affinity (Km) for Na(+). Upon acute restoration of normoxia, recovery of Na(+) uptake was delayed for 1 h. These data suggest that dual mechanisms may be involved (e.g., immediate effects of O(2) availability on transporters, channels, or permeability, slower effects of Na(+)-K(+)-ATPase regulation). Ammonia excretion appeared to be linked indirectly to Na(+) uptake, exhibiting a Michaelis-Menten relationship with external [Na(+)], but the Km was less than for Na(+) uptake. During hypoxia, ammonia excretion fell in a similar manner to Na(+) fluxes, with a delayed recovery upon normoxia restoration, but the relationship with [Na(+)] was blocked. Reductions in ammonia excretion were greater than in urea excretion. Plasma ammonia rose moderately over 3 h hypoxia, suggesting that inhibition of excretion was greater than inhibition of ammonia production. Overall, the oscar maintains excellent homeostasis of ionoregulation and N-balance during severe hypoxia.

The African lungfish (Protopterus dolloi): ionoregulation and osmoregulation in a fish out of water.

Although urea production and metabolism in lungfish have been thoroughly studied, we have little knowledge of how internal osmotic and electrolyte balance are controlled during estivation or in water. We tested the hypothesis that, compared with the body surface of teleosts, the slender African lungfish (Protopterus dolloi) body surface was relatively impermeable to water, Na(+), and Cl(-) due to its greatly reduced gills. Accordingly, we measured the tritiated water ((3)H-H(2)O) flux in P. dolloi in water and during air exposure. In water, (3)H-H(2)O efflux was comparable with the lowest measurements reported in freshwater teleosts, with a rate constant (K) of 17.6% body water h(-1). Unidirectional ion fluxes, measured using (22)Na(+) and (36)Cl(-), indicated that Na(+) and Cl(-) influx was more than 90% lower than values reported in most freshwater teleosts. During air exposure, a cocoon formed within 1 wk that completely covered the dorsolateral body surface. However, there were no disturbances to blood osmotic or ion (Na(+), Cl(-)) balance, despite seven- to eightfold increases in plasma urea after 20 wk. Up to 13-fold increases in muscle urea (on a dry-weight basis) were the likely explanation for the 56% increase in muscle water content observed after 20 wk of air exposure. The possibility that muscle acted as a "water reservoir" during air exposure was supported by the 20% decline in body mass observed during subsequent reimmersion in water. This decline in body mass was equivalent to 28 mL water in a 100-g animal and was very close to the calculated net water gain (approximately 32 mL) observed during the 20-wk period of air exposure. Tritiated water and unidirectional ion fluxes on air-exposed lungfish revealed that the majority of water and ion exchange was via the ventral body surface at rates that were initially similar to aquatic rates. The (3)H-H(2)O flux declined over time but increased upon reimmersion. We conclude that the slender lungfish body surface, including the gills, has relatively low permeability to water and ions but that the ventral surface is an important site of osmoregulation and ionoregulation. We further propose that an amphibian-like combination of ventral skin water and ion permeability, plus internal urea accumulation during air exposure, allows P. dolloi to extract water from its surroundings and to store water in the muscle when the water supply becomes limited.

Metabolic organization and effects of feeding on enzyme activities of the dogfish shark (Squalus acanthias) rectal gland.

In order to investigate the metabolic poise of the elasmobranch rectal gland, we conducted two lines of experimentation. First, we examined the effects of feeding on plasma metabolites and enzyme activities from several metabolic pathways in several tissues of the dogfish shark, Squalus acanthias, after starvation and at 6, 20, 30 and 48 h post-feeding. We found a rapid and sustained ten-fold decrease in plasma beta-hydroxybutyrate at 6 h and beyond compared with starved dogfish, suggesting an upregulation in the use of this substrate, a decrease in production, or both. Plasma acetoacetate levels remain unchanged, whereas there was a slight and transient decrease in plasma glucose levels at 6 h. Several enzymes showed a large increase in activity post-feeding, including beta-hydroxybutyrate dehydrogenase in rectal gland and liver, and in rectal gland, isocitrate dehydrogenase, citrate synthase, lactate dehydrogenase, aspartate amino transferase, alanine amino transferase, glutamine synthetase and Na(+)/K(+) ATPase. Also notable in these enzyme measurements was the overall high level of activity in the rectal gland in general. For example, activity of the Krebs' TCA cycle enzyme citrate synthase (over 30 U g(-1)) was similar to activities in muscle from other species of highly active fish. Surprisingly, lactate dehydrogenase activity in the gland was also high (over 150 U g(-1)), suggesting either an ability to produce lactate anaerobically or use lactate as an aerobic fuel. Given these interesting observations, in the second aspect of the study we examined the ability of several metabolic substrates (alone and in combination) to support chloride secretion by the rectal gland. Among the substrates tested at physiological concentrations (glucose, beta-hydroxybutyrate, lactate, alanine, acetoacetate, and glutamate), only glucose could consistently maintain a viable preparation. Whereas beta-hydroxybutyrate could enhance gland activity when presented in combination with glucose, surprisingly it could not sustain chloride secretion when used as a lone substrate. Our results are discussed in the context of the in vivo role of the gland and mechanisms of possible upregulation of enzyme activities.

The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.

Tribute to R. G. Boutilier: the effect of size on the physiological and behavioural responses of oscar, Astronotus ocellatus, to hypoxia.

The physiological and behavioural responses of two size groups of oscar (Astronotus ocellatus) to hypoxia were studied. The physiological responses were tested by measuring M(O(2)) during decreasing environmental oxygen tensions. Larger oscars were better able to maintain oxygen consumption during a decrease in P(O(2)), regulating routine M(O(2)) to a significantly lower P(O(2)) threshold (50 mmHg) than smaller oscars (70 mmHg). Previous studies have also demonstrated a longer survival time of large oscars exposed to extreme hypoxia, coupled with a greater anaerobic enzymatic capability. Large oscars began aquatic surface respiration (ASR) at the oxygen tension at which the first significant decrease in M(O(2)) was seen (50 mmHg). Interestingly, smaller oscars postponed ASR to around 22 mmHg, well beyond the P(O(2)) at which they switched from oxyregulation to oxyconformation. Additionally, when given the choice between an hypoxic environment containing aquatic macrophyte shelter and an open normoxic environment, small fish showed a greater preference for the hypoxic environment. Thus shelter from predators appears particularly important for juveniles, who may accept a greater physiological compromise in exchange for safety. In response to hypoxia without available shelter, larger fish reduced their level of activity (with the exception of aggressive encounters) to aid metabolic suppression whereas smaller oscars increased their activity, with the potential benefit of finding oxygen-rich areas.

Alkaline tide and nitrogen conservation after feeding in an elasmobranch (Squalus acanthias).

We investigated the consequences of feeding for acid-base balance, nitrogen excretion, blood metabolites and osmoregulation in the Pacific spiny dogfish. Sharks that had been starved for 7 days were surgically fitted with indwelling stomach tubes for gastric feeding and blood catheters for repetitive blood sampling and were confined in chambers, allowing measurement of ammonia-N and urea-N fluxes. The experimental meal infused via the stomach tube consisted of flatfish muscle (2% of body mass) suspended in saline (4% of body mass total volume). Control animals received only saline (4% of body mass). Feeding resulted in a marked rise in both arterial and venous pH and HCO3- concentrations at 3-9 h after the meal, with attenuation by 17 h. Venous P(O2) also fell. As there were negligible changes in P(CO2), the response was interpreted as an alkaline tide without respiratory compensation, associated with elevated gastric acid secretion. Urea-N excretion, which comprised >90% of the total, was unaffected, while ammonia-N excretion was very slightly elevated, amounting to <3% of the total-N in the meal over 45 h. Plasma ammonia-N rose slightly. Plasma urea-N, TMAO-N and glucose concentrations remained unchanged, while free amino acid and beta-hydroxybutyrate levels exhibited modest declines. Plasma osmolality was persistently elevated after the meal relative to controls, partially explained by a significant rise in plasma Cl-. This marked post-prandial conservation of nitrogen is interpreted as reflecting the needs for urea synthesis for osmoregulation and protein growth in animals that are severely N-limited due to their sporadic and opportunistic feeding lifestyle in nature.

Dogmas and controversies in the handling of nitrogenous wastes: the effect of feeding and fasting on the excretion of ammonia, urea and other nitrogenous waste products in rainbow trout.

Ammonia and urea are the primary forms of nitrogen excretion in teleost fish. There exists, however, a discrepancy between the sum of ammonia plus urea nitrogen and total nitrogen, indicating that 'unknown' nitrogen end products may play an important role in nitrogen metabolism. The current study analysed a wide range of nitrogen end products in both fed and fasted juvenile rainbow trout. Ammonia-N (53-68%) and urea-N (6-10%) were confirmed as the most important forms of nitrogenous waste, but an interesting finding was the considerable excretion of nitrogen as amino acids (4-10%), via the gills, and as protein (3-11%), probably via the body mucus. Use of anal sutures delineated an important role for the gastrointestinal tract in the production of ammonia-N and urea-N in fed fish, but amino acid-N and protein-N output by this route were both negligible. Alternative nitrogen products - trimethylamine, trimethylamine oxide, uric acid, and nitrite + nitrate - were not excreted in detectable quantities. Creatine-N and creatinine-N outputs were detected but contributed only a small fraction to total nitrogen excretion (<1.4%). Despite the wide scope of nitrogenous end products investigated, a considerable proportion (12-20%) of nitrogen excretion remains unknown. Possible alternative end products and methodological considerations are proposed to explain this phenomenon. The findings described above were used to recalculate the nitrogen quotient (NQ=(N)/(O(2))) on trout that had been either fasted or fed various daily rations (1%, 3% or 5% dry food per unit wet body mass per day). Feeding increased oxygen consumption ((O(2))) and total-N excretion ((N)). The NQ is often used as a measure of protein utilisation in aerobic metabolism and assumes that all protein (and amino acid) fuels are converted by oxidation to nitrogenous waste products that are excreted. However, the results showed that calculation of the NQ based on total nitrogen excretion may overestimate protein utilisation in aerobic metabolism because of significant excretion of N in the form of proteins and amino acids, whereas the use of summed ammonia-N and urea-N excretion probably underestimates the contribution of protein towards aerobic metabolism. These errors increase as ration increases, because the discrepancy between total-N excretion and ammonia-N + urea-N excretion increases.

Diurnal nitrogen excretion rhythm of the functionally ureogenic gobiid fish Mugilogobius abei.

This study was performed to determine the daily periodicity of urea excretion in the ureogenic gobiid fish Mugilogobius abei. In 20% seawater, urea excretion of all the fish examined showed daily periodic changes under a 12-h light-dark cycle, and some showed a free-running rhythm under constant darkness. This is the first report of a circadian rhythm in urea excretion in fishes. Daily variations in urea excretion under light-dark cycles were also observed under various conditions, i.e. exposure to water ammonia, confinement/non-confinement and solitary/group. Due to the daily variations in urea excretion, urea contents in tissues changed periodically, whereas enzyme activities related to urea synthesis did not change significantly. The index of urea permeability as determined by changes in body urea contents after 2-h immersion of 25 mM urea solution was high during the peak of daily variation in urea excretion. Locomotor activity and urea excretion showed clear daily variations under light-dark cycles, both of which were diurnal. Furthermore, daily variations in urea excretion were maintained even when the diurnal pattern in the locomotor activity was disturbed. These results suggest that periodic urea excretion was mediated by periodic enhancement of permeability for urea at excretion sites.