The snakehead Channa asiatica accumulates alanine during aerial exposure, but is incapable of sustaining locomotory activities on land through partial amino acid catabolism

Shit F. Chew1,*, Mei Y. Wong1, Wai L. Tam2 and Yuen K. Ip2

1 Natural Sciences, National Institute of Education, Nanyang Technological University, 1 Nanyang Walk, Singapore 637616, Republic of Singapore
2 Department of Biological Sciences, National University of Singapore, 10 Kent Ridge Road, Singapore 117543, Republic of Singapore

* Author for correspondence (e-mail: sfchew{at}nie.edu.sg)

Accepted 11 November 2002


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 Summary
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The freshwater snakehead Channa asiatica is an obligatory air-breather that resides in slow-flowing streams and in crevices near riverbanks in Southern China. In its natural habitat, it may encounter bouts of aerial exposure during the dry seasons. In the laboratory, the ammonia excretion rate of C. asiatica exposed to terrestrial conditions in a 12h:12h dark:light regime was one quarter that of the submerged control. Consequently, the ammonia contents in the muscle, liver and plasma increased significantly, and C. asiatica was able to tolerate quite high levels of ammonia in its tissues. Urea was not the major product of ammonia detoxification in C. asiatica, which apparently did not possess a functioning ornithine urea cycle. Rather, alanine increased fourfold to 12.6 µmolg-1 in the muscle after 48h of aerial exposure. This is the highest level known in adult teleosts exposed to air or an ammonia-loading situation. The accumulated alanine could account for 70% of the deficit in ammonia excretion during this period, indicating that partial amino acid catabolism had occurred. This would allow the utilization of certain amino acids as energy sources and, at the same time, maintain the new steady state levels of ammonia in various tissues, preventing them from rising further. There was a reduction in the aminating activity of glutamate dehydrogenase from the muscle and liver of specimens exposed to terrestrial conditions. Such a phenomenon has not been reported before and could, presumably, facilitate the entry of {alpha}-ketoglutarate into the Krebs cycle instead of its amination to glutamate, as has been suggested elsewhere. However, in contrast to mudskippers, C. asiatica was apparently unable to reduce the rates of proteolysis and amino acid catabolism, because the reduction in nitrogenous excretion during 48 h of aerial exposure was completely balanced by nitrogenous accumulation in the body. Alanine accumulation also occurred in specimens exposed to terrestrial conditions in total darkness, with no change in the total free amino acid content in the muscle. Exercise on land led to a decrease in glycogen content, and an increase in lactate levels, with no significant effect on ammonia and alanine contents in the muscle of C. asiatica. Hence, unlike the mudskipper Periophthalmodon schlosseri, C. asiatica was incapable of increasing the rate of partial amino acid catabolism to sustain locomotory activities on land. Alanine formation therefore appears to be a common strategy adopted by obligatory air-breathing fishes to avoid ammonia toxicity (not a strategy to detoxify ammonia) on land, but not all of them can utilize it to fuel muscular activities.

Key words: aerial exposure, alanine, amino acid, ammonia, Channa asiatica, glutamate dehydrogenase, proteolysis, snakehead


    Introduction
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 Summary
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Some teleostean fishes actively lead an amphibious life (e.g. mudskippers) while others occasionally get trapped in puddles of water (e.g. marble goby) or in the intertidal zone in their natural habitats. Without the buoyancy of water, gills collapse and lamellae coalesce, so branchial ammonia excretion would be inefficient if no external water current were available to irrigate the gills during aerial exposure. Consequently, endogenously produced ammonia would accumulate and these fishes would need to deal with the problem of ammonia intoxication in the absence of water.

Three different major nitrogenous products accumulate in the tissues of some tropical teleostean fishes during aerial exposure (Ip et al., 2001aGo). These are alanine (e.g. mudskipper; Ip et al., 2001bGo), glutamine (e.g. marble goby and sleeper; Jow et al., 1999Go; Ip et al., 2001cGo; Anderson et al., 2001Go, 2002Go) and urea (e.g. Singhi catfish; Saha and Ratha, 1998Go). The weather loach (Chew et al., 2001Go) and the mangrove killifish (Frick and Wright, 2002Go) accumulate both alanine and glutamine, although to a lesser extent than the mudskipper and the sleeper, respectively, when exposed to terrestrial conditions. These two fishes are uniquely capable of excreting ammonia through volatilization of NH3 during aerial exposure (Frick and Wright, 2002Go; Tsui et al., 2002Go).

Urea production does not appear to be a common strategy adopted by teleosts to detoxify endogenous ammonia during aerial exposure, probably because it is energy expensive. 5 mol ATP are required for the formation of 1 mol urea containing 2 nitrogen atoms (Ip et al., 2001aGo). Glutamine formation is also energy dependent and appears to be adopted mainly by fishes that remain completely quiescent on land. If the reaction begins with glutamate and NH4+, 1 mol ATP is utilized for every mol ammonia detoxified. If the starting point is {alpha}-ketoglutarate and NH4+, 4 mol ATP-equivalents (3 from NADH) would be needed to detoxify 2 mol ammonia (Ip et al., 2001). By contrast, alanine formation through partial amino acid catabolism would lead to the production of ATP without releasing ammonia (Ip et al., 2001aGo). Hence, this pathway cannot be regarded as an ammonia-detoxifying mechanism, but rather a way to use certain amino acids as energy sources without leading to ammonia accumulation in the body. When the mudskipper Periophthalmodon schlosseri was forced to exercise on land after 24 h of aerial exposure, muscle alanine levels increased significantly, with no apparent change in glycogen content (Ip et al., 2001bGo), indicating that the apparent increase in energy demand was not met by an increase in the rate of fermentative glycolysis. Hence, Ip et al. (2001bGo) concluded that P. schlosseri was capable of using certain amino acids as metabolic fuels to support locomotory activities on land and avoided ammonia toxicity through partial amino acid catabolism. They further suggested that such a strategy may be widely adopted, especially by obligatory air-breathing fishes, to avoid ammonia intoxication during aerial exposure (Ip et al., 2001bGo).

The aim of this study was to investigate the effects of aerial exposure on the snakehead Channa asiatica, which is an obligatory air-breather, and to test the hypothesis set forth by Ip et al. (2001bGo). Channa asiatica and other snakeheads are valued freshwater food fishes in Southern China and Far East Asia. They command very high prices (S$10-20 kg-1) when fresh, because people there believe that consumption of this fish aids the recovery of injuries (especially for post-surgical patients) (Lim and Ng, 1990Go; Ng and Lim, 1990Go). Channa asiatica is a predaceous fish that resides in slow-flowing streams and in crevices near riverbanks. In its natural habitat, it may encounter bouts of aerial exposure during the dry seasons. While `walking' fishes like Anabas sp. and Clarias sp. (see Smith, 1945Go) have frequently been reported upon, `walking snakeheads' are less well known. Smith (1945Go) and Mohsin and Ambak (1983Go) all noted that snakeheads could move across land. They can move a considerable distance on all kinds of surfaces, squirming and skipping (Ng and Lim, 1990Go). However, unlike mudskippers, C. asiatica is incapable of using its pectoral fins to move actively on land, and does not exhibit any feeding, territorial or courtship behaviour while out of water. When trapped in puddles of water, it tried to struggle back to water with eel-like body movements, which could last several minutes. After struggling for a while without managing to get back to water, C. asiatica turned motionless and remained quiescent, with only occasional eel-like movements, for an extended period. We therefore hypothesized that C. asiatica was capable of undergoing partial amino acid catabolism, leading to the formation of alanine to reduce the rate of ammonia accumulation in its tissues during aerial exposure; however, it might not be able to use amino acids to fuel locomotory activities on land as observed in P. schlosseri (Ip et al., 2001bGo). Experiments were therefore undertaken to examine the effects of aerial exposure or locomotory activities on the nitrogen metabolism and excretion in this fish.


    Materials and methods
 TOP
 Summary
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection and maintenance of Channa asiatica
Channa asiatica L. (40-100 g body mass) were purchased from Hong Kong. They were maintained in trays (43 cmx28 cmx12 cm, LxWxH) containing air-saturated dechlorinated tapwater at 25±2°C, and fed with live guppies ad libitum. No attempt was made to separate the sexes. The water was changed three times weekly.

Exposure of C. asiatica to experimental conditions and collection of samples
Effects of aerial exposure
All experiments performed in this study were done under a 12h:12h dark:light regime unless stated otherwise. 24h before the experiment, a group of specimens was separated individually and immersed completely in water with continuous aeration. Food was withdrawn so that the fish had empty guts. At the end of the 24h period (0h control), some of these specimens were anaesthetized by the introduction of 3-aminobenzoic acid ethyl ester (MS-222) at a final concentration of 0.02% and killed. Others were exposed to aerial conditions individually in plastic aquaria (20.5 cmx14.5 cmx6 cm, LxWxH) containing a thin film of 50 ml dechlorinated tapwater, for a specific period (6 h, 12 h, 24 h or 48 h) at 25°C and 80-90% humidity. Another group of fish was submerged in air-saturated, dechlorinated tapwater at 25°C for 48 h as a control to eliminate the effects of fasting on the fish.

Effects of locomotory activity
Specimens were submerged for 24 h in a 12h:12h dark:light regime. Some of these specimens were then stimulated by mechanical disturbance to swim in water for 3 min. A separate group of specimens was exposed to terrestrial conditions for 24 h in the 12h:12h dark:light regime before being stimulated to move on land for 3 min. This period was chosen because the fish became refractory to stimulation after such a period of locomotory activity on land. No anaesthesia was applied to the exercised fish, simply because recovery would have taken place during the period of anaesthesia treatment (approximately 5-7 min). Specimens were killed by a quick blow to the head followed by pithing.

Effects of constant darkness
Specimens were kept in submerged conditions or exposed to air as stated before but in constant darkness in a cabinet for 48 h, in order to minimize visual stimulation and hence to reduce locomotory activities.

Collection of samples for further analyses
Anaesthetized or exercised fish were killed immediately by pithing. The lateral muscle and the liver were quickly excised. No attempt was made to separate the red and white muscle, but care was taken to sample muscles from the same region of the body. The excised tissues and organs were immediately freeze-clamped in liquid nitrogen with pre-cooled tongs. Frozen samples were kept at -80°C until analysed (within a month). A separate group of fish exposed to similar conditions was used for the collection of blood samples. The caudal peduncle of the anaesthetized fish was severed, and blood exuding from the caudal artery was collected in heparinized capillary tubes. The collected blood was centrifuged at 4000 g at 4°C for 10 min to obtain the plasma. The plasma was deproteinized in an equal volume (v/v) of ice-cold 6% trichloroacetic acid (TCA) and centrifuged at 10 000 g at 4°C for 15 min. The resulting supernatant fluid was kept at -80°C until analysed (within a month).

Ammonia and urea excretion rates in specimens in 12 h: 12 h dark:light regime
Specimens were submerged individually in plastic aquaria tank (25 cmx14 cmx12 cm, LxWxH) containing 3.51 of water at 25°C with aeration. Preliminary experiments on the analysis of ammonia and urea in the water sampled at 6 and 24 h showed that the ammonia and urea excretion rates were linear for at least 24 h. Thus, subsequently, water was sampled for ammonia and urea analysis after 24 h of exposure. The same individuals were then exposed to terrestrial conditions in plastic tanks containing 20 ml of water. After 24 h, the fish were sprayed thoroughly with water. The water collected was used for ammonia and urea analyses. After aerial exposure, the fish were resubmerged in water to study the rates of ammonia and urea excretion upon recovery. Ammonia and urea concentrations were determined as described below.

Analyses of ammonia and urea in tissue samples
The frozen sample was weighed, ground to a powder in liquid nitrogen, and homogenized in 5 volumes (w/v) of 6% TCA for determinations of ammonia and urea levels. For ammonia analysis, the pH of the deproteinized sample was adjusted to between 5.5 and 6.0 with 2 mol l-1 KHCO3. The ammonia content was determined as described previously (Bergmeyer and Beutler, 1985Go). Freshly prepared NH4Cl solution was used as the standard for comparison.

Urea concentration was determined colorimetrically according to the method of Jow et al. (1999Go). The difference in absorbance obtained from the samples with and without urease treatment was used for the estimation of the urea concentration in the sample. Urea standards were processed by the same procedure for comparison. Results were expressed as µmol g-1 wet mass tissue or µmol ml-1 plasma.

Determination of activities of ornithine—urea cycle enzymes
The livers of experimental fishes were excised quickly for the isolation of mitochondrial and cytosolic fractions, using a modification of the procedure of Jow et al. (1999Go). The excised livers were minced and suspended in 10 vol. (w/v) of ice-cold mitochondria extraction buffer (285 mmol l-1 sucrose, 3 mmol l-1 EDTA, 3 mmol l-1 Tris-HCl, pH 7.2), and homogenized using three strokes of a Teflon-glass homogenizer. The homogenized sample was centrifuged at 600 g for 15 min at 4°C to remove any unbroken cells and nuclei. The supernatant obtained was further centrifuged for 15 min at 10 000 g to obtain a mitochondrial pellet. The mitochondrial pellet was washed twice with the extraction buffer, and centrifuged for 15 min at 10 000 g. The final mitochondrial pellet was suspended in 1 ml of suspension buffer (100 mmol l-1 Hepes, pH 7.6, 100 mmol l-1 KCl and 1 mmol l-1 EDTA). It was then sonicated three times for 20 s with a 10 s break between each sonication. The sonicated sample was centrifuged at 4000 g at 4°C for 3 min. After centrifugation, the supernatant was passed through a 10 ml Bio-Rad P-6DG column (Bio-Rad Laboratories; CA, USA) equilibrated with suspension buffer.

To obtain the cytosolic fraction, the freshly excised liver was homogenized with 5 vol. (w/v) of mitochondria extraction buffer and centrifuged at 10 000 g for 15 min. The resulting supernatant was collected and passed through a 10 ml Bio-Rad column equilibrated with suspension buffer. The collected filtrates of the mitochondrial and cytosolic fractions were used for the subsequent enzyme analyses.

Carbamoyl phosphate synthetase (CPS; E.C. 2.7.2.5) activity was determined in the mitochondrial fraction according to the method of Anderson and Walsh (1995Go). Radioactivity was measured using a Wallac 1414 liquid scintillation counter (Bio Laboratories, USA). The CPS activity was expressed as [14C]urea formed (µmol min-1 mg-1 protein).

Ornithine transcarbamoylase (E.C. 2.1.3.3) activity was determined in the mitochondrial fraction by combining the methods of Anderson and Walsh (1995Go) and Xiong and Anderson (1989Go). Absorbance was measured at 466 nm using a Shimadzu UV 160 UV VIS recording spectrophotometer. The ornithine transcarbamoylase activity was expressed as citrulline formed (µmol min-1 mg-1 protein).

Argininosuccinate synthetase (E.C. 6.3.4.5) and lyase (E.C. 4.3.2.1) activities were determined together in the cytosolic fraction, assuming that both were present, by measuring the formation of [14C]fumarate from [14C]aspartate using the method of Cao et al. (1991Go). Radioactivity was measured using a Wallac 1414 liquid scintillation counter. Argininosuccinate synthetase and lyase activity was expressed as [14C]fumarate formed (µmol min-1 mg-1 protein).

Arginase (E.C. 3.5.3.1) was assayed in both mitochondrial and cytosolic fractions as described by Felskie et al. (1998Go). Urea was determined as described above. Arginase activity was expressed as urea formed (µmol min-1 mg-1 protein).

Analysis of free amino acids
The frozen muscle and liver samples were weighed, ground to a powder in liquid nitrogen, and homogenized in 5 vol. (w/v) of 6% TCA three times (20 s each at 10 s intervals) using an Ultra-Turrax homogenizer at 24 000 revs min-1. The homogenates were centrifuged at 10 000 g at 4°C for 15 min to obtain the supernatant fluid for free amino acid (FAA) analyses. The supernatant obtained or the deprotenized plasma was adjusted to pH 2.2 with 4 mol l-1 lithium hydroxide and diluted appropriately with 0.2 mol l-1 lithium citrate buffer, pH 2.2. Free amino acids were analyzed using a Shimadzu LC6A amino acid analysis system (Kyoto, Japan) with a Shimpack ISC-07/S1504 Li-type column. The results of FAA analyses are expressed as µmol g-1 wet mass tissue or µmol ml-1 plasma.

Determination of activities associated with alanine or glutamine formation
Alanine aminotransferase (ALT; E.C. 2.6.1.2) activities in both the cytosolic and mitochondrial fractions in the direction of alanine degradation were assayed according to Peng et al. (1994Go) with some modifications. The reaction mixture of 3.1 ml contained 50 mmol l-1 imidazole-HCl, pH 7.4, 10 mmol l-1 {alpha}-ketoglutarate ({alpha}-KG), 0.17 mmol l-1 NADH, 0.025 mmol l-1 pyridoxal phosphate, 6.2 i.u. lactate dehydrogenase (LDH) (Sigma Chemical Co., USA) and 0.2 ml of sample. The reaction was initiated with 0.4 ml of alanine at a final concentration of 200 mmol l-1. ALT activity was expressed as NADH utilized (µmol min-1 mg-1 protein).

The glutamate dehydrogenase (GDH; E.C. 1.4.1.3) activity in the mitochondrial fraction was assayed in the aminating and deaminating directions according to Ip et al. (1993Go), with some modifications. For the amination reaction, the reaction mixture of 3 ml contained 200 mmol l-1 imidazole-HCl, pH 7.8, 10 mmol l-1 {alpha}-KG, 1 mmol l-1 ADP, 0.17 mmol l-1 NADH and 0.2 ml of sample. The reaction was initiated with 0.4 ml of ammonium acetate at a final concentration of 250 mmol l-1. GDH activity was expressed in NADH utilized (µmol min-1 mg-1 protein). For the deamination reaction, the reaction mixture of 2.8 ml contained 200 mmol l-1 glycine-NaOH, pH 9, 1 mmol l-1 ADP, 0.1 mmol l-1 nicotinamide adenine dinucleotide (NAD), 0.09 mmol l-1 iodonitrotetrazolium chloride (INT), 0.28 i.u. diaphorase (Sigma Chemical Co., USA) and 0.2 ml of sample. The reaction was initiated with 0.2 ml of glutamate at a final concentration of 100 mmol l-1. The change in absorbance was monitored at 492 nm and 25°C. The specific activity was expressed as formazan formed (µmol min-1 mg-1 protein).

The activity of malic enzyme (E.C.1.1.1.40) was determined according to Peng et al. (1994Go). The reaction mixture of 1.45 ml contained 67 mmol l-1 triethanolamine-HCl (TEA), pH 7.4, 4 mmol l-1 MgCl2, 2 mmol l-1 NAD and 0.15 ml of sample. The reaction was initiated by the addition of malate at a final concentration of 25 mmol l-1. The change in absorbance was monitored at 340 nm at 25°C. The activity of malic enzyme was expressed as NADH formed (µmol min-1 mg-1 protein).

Analyses of glycogen, ammonia, urea, alanine, lactate, ATP, ADP and AMP in samples from the exercised specimens
For the determination of glycogen, frozen samples (0.5-1.0 g) were ground to a powder and digested in 2 ml of 30% (w/v) KOH in a boiling water bath for 10 min. The glycogen was extracted according to the method of Lim and Ip (1989Go), and was determined by combining the methods of Bergmeyer et al. (1974Go) and Roehrig and Allred (1974Go). The glycogen content was expressed as glycosyl units (µmol g-1 wet mass tissue).

For the other metabolites, samples were homogenized as stated above except in 5 vol. of 6% HClO4. After centrifugation at 10 000 g for 15 min, the supernatant was decanted and the pH adjusted to 5.5-6.0 with 2 mol l-1 K2CO3. The ammonia and urea contents were determined as stated before. The alanine content was determined according to the method of Williamson (1974Go). Lactate was determined using the method of Gutmann and Wahlefeld (1974Go) while ATP, ADP and AMP were determined spectrophotometrically by the procedures of Scheibel et al. (1968Go). Results were expressed as µmol g-1 wet mass tissue or µmol ml-1 plasma.

Statistical analysis
Results are presented as means ± the standard error of the mean (S.E.M.). Student's t-test or analysis of variance (ANOVA) followed by multiple comparisons of means by Duncan's procedure was used to evaluate differences between means in groups where appropriate. Differences where P<0.05 were regarded as statistically significant.


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 Introduction
 Materials and methods
 Results
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Effects of aerial exposure in a 12 h: 12 h dark:light regime
Aerial exposure significantly decreased the rates of ammonia excretion by C. asiatica in comparison with the submerged control (Fig. 1A). Upon resubmergence after aerial exposure, the rates returned to the initial control values. Similar observations were made on the rates of urea excretion (Fig. 1B).



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Fig. 1. The effects of terrestrial exposure for 2 days followed by resubmersion on the rates of (A) ammonia and (B) urea excretion by C. asiatica. Values are means ± S.E.M. (N=4). aSignificantly different from Day 1 value, P<0.05; bSignificantly different from Day 2 value, P<0.05; csignificantly different from Day 3 value, P<0.05. S, submerged; T, terrestrial.

 

There were significant increases in concentrations of ammonia in the muscle, liver and plasma after exposure to terrestrial conditions for 48 h (Fig. 2A). The urea level increased slightly after 12 h of aerial exposure and returned to the normal value thereafter (Fig. 2B). Aerial exposure had no effect on the urea content in the liver (Fig. 2B). There was a significant increase in the urea concentration in the plasma of specimens exposed to 24 or 48 h of terrestrial conditions (Fig. 2B), but the change was minor compared to that for ammonia (Fig. 2A).



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Fig. 2. The effects of terrestrial exposure for up to 48 h on the concentrations of (A) ammonia and (B) urea in the muscle (open bars), liver (shaded bars) and plasma (hatched bars) of C. asiatica. Values are means ± S.E.M. (N=4). aSignificantly different from the value of 0 h, S, P<0.05; bsignificantly different from the value of 6 h, T, P<0.05; csignificantly different from the value of 12 h, T, P<0.05; dsignificantly different from the value of 24 h, T, P<0.05; esignificantly different from the value of 48 h, T, P<0.05. S, submerged; T, terrestrial; 48 h, S, fasted control.

 

In the absence or presence of glutamine and N-acetylglutamate in the assay medium, no carbamoylphosphate synthetase-III activity could be detected from liver mitochondria isolated from specimens kept under control conditions or exposed to terrestrial conditions for 48 h. In addition, no argininosuccinate synthetase + lyase activity could be detected in the cytosol of the liver of these specimens. Ornithine transcarbamoylase and arginase activities were present in the liver mitochondria (0.0022±0.0004 and 0.093±0.023 µmol min-1 mg-1 protein, respectively, N=3) from control specimens. The former was unaffected by aerial exposure, but the latter increased significantly to 0.39±0.04 µmol min-1 mg-1 protein (N=3) after exposure to terrestrial conditions for 48 h. Arginase activity could also be detected in the cytosol, though at a much lower level (0.0072±0.0036 µmol min-1 mg-1 protein), and was unaffected by 48 h of aerial exposure.

Levels of alanine, asparagine, glutamine, histidine, serine and tyrosine in the muscle of C. asiatica increased significantly after 48 h of aerial exposure (Table 1). The alanine level (3.7 µmol g-1) rose to 6.4 and 12.6 µmol g-1 after 24 h and 48 h of exposure to terrestrial conditions, respectively. The TFAA level in the muscle of specimen exposed to 48 h of aerial exposure was significantly higher than that of the 0 h submerged control, but was comparable to the submerged control fasted for 48 h (Table 1). There were significant changes to some of the FAAs in the liver of specimens exposed to terrestrial conditions for 48 h, as compared to the 0 h submerged control (Table 2). However, the majority of these changes were also detected in the specimens kept in the submerged conditions and fasted for the same period (Table 2). Aerial exposure induced only minor changes to some of the amino acids in the plasma of C. asiatica (Table 3).


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Table 1. Effects of various periods of terrestrial exposure on the concentrations of various free amino acids (FAA) and total FAA (TFAA) values in the muscle of C. asiatica

 

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Table 2. Effects of various periods of terrestrial exposure on the concentrations of various free amino acids (FAA) and total FAA (TFAA) values in the liver of C. asiatica

 

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Table 3. Effects of various periods of terrestrial exposure on the concentrations of various free amino acids (FAA) and total FAA (TFAA) values in the plasma of C. asiatica

 

Activities of ALT in the cytosolic and mitochondrial fractions of the muscle and liver were unaffected by aerial exposure (Table 4). However, there were significant decreases in the aminating activity of GDH from the muscle and liver of specimens exposed to terrestrial conditions, leading to significant decreases in the amination:deamination ratio (Table 5). Malic enzyme is present in the muscle of C. asiatica (0.0030±0.0007 µmol min-1 mg-1, N=3) and was unaffected by aerial exposure (0.0042±0.0007).


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Table 4. Effects of 48 h terrestrial exposure on the specific activities of alanine transaminase from the mitochondrial and cytosolic fractions of the muscle and liver of C. asiatica

 

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Table 5. Effects of 48 h terrestrial exposure on the specific activities of glutamate dehydrogenase in the amination and deamination reactions, and their ratios, in the muscle and liver of C. asiatica

 

Effects of short burst of locomotory activity
Exercise in water for 3 min after 24 h of submergence had no significant effect on the energy charge and content of ATP, ADP, AMP, alanine, glutamate, glutamine, ammonia and glycogen in the muscle of C. asiatica (Table 6). However, there was a significant increase in lactate levels in the muscle of these exercised specimens (Table 6). Similar results were obtained for fish that were exercised on land after 24 h of aerial exposure, except that the increase in lactate in the muscle was accompanied by a significant decrease in the glycogen content (Table 6).


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Table 6. Effects of exercise (3 min) on the concentrations of various metabolites and energy charge in the muscle of C. asiatica after terrestrial or submerged conditions

 

Effects of constant darkness
The contents of ammonia, urea, alanine and total free amino acids (TFAA) in the muscle of specimens exposed to terrestrial conditions in constant darkness and those of specimens in a 12 h:12 h dark:light regime were comparable (Fig. 3).



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Fig. 3. Effects of 24 h terrestrial exposure under a 12 h:12 h L:D regime or in constant darkness on urea (open bars), ammonia (hatched bars), alanine (shaded bars) and TFAAs (black bars) levels in the muscle of C. asiatica. Values are means ± S.E.M. (N=4).

 


    Discussion
 TOP
 Summary
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of aerial exposure in a 12 h:12 h dark:light regime
The most important excretory products of nitrogen metabolism in teleostean fishes are ammonia and urea, with ammonia usually the largest component (Mommsen and Walsh, 1992Go). In freshwater teleosts, over 99% of the ammonia is excreted via the gills, kidney and skin contributing the remainder (Mommsen and Walsh, 1992Go). However, amphibious teleostean fishes with limited gill ventilation during aerial exposure may face problems in excreting the endogenous ammonia. Indeed, the ammonia excretion rate of C. asiatica exposed to terrestrial conditions was one quarter that of the submerged control. Consequently, ammonia contents in the muscle, liver and plasma increased significantly. The first strategy adopted by this fish to survive aerial exposure is therefore to tolerate the build-up of ammonia levels in its body. However, in order to prevent ammonia from building up to an intolerable level, other strategies must subsequently be involved to maintain the new steady state level of internal ammonia. In other fishes, accumulating ammonia is detoxified to less toxic substances, e.g. urea or glutamine, depending on the species (Ip et al., 2001aGo). There may also be a reduction in ammonia production through suppression of proteolysis and/or amino acid catabolism, or through partial amino acid catabolism leading to the formation of alanine (Ip et al., 2001aGo,bGo; Lim et al., 2001Go).

There was no change in the rate of urea excretion in C. asiatica exposed to terrestrial conditions compared to submerged controls. In addition, there was no major change in the urea content of muscle, liver and plasma after 48 h of aerial exposure. C. asiatica did not therefore detoxify ammonia to urea when out of water, contrary to the belief that there is a tendency towards predominance of ureotelism in amphibious species (Wright, 1995Go; Mommsen and Walsh, 1992Go; Walsh, 1997Go; Wright and Land, 1998Go), which are found mainly in the tropics. Furthermore, carbamoylphosphate synthetase and argininosuccinate synthetase + lyase activities remained below the detection limits of the methods adopted in this study, although ornithine transcarbamoylase and arginase activities were detected in the hepatic mitochondria, so it is unlikely that C. asiatica possesses a `functional' ornithine—urea cycle due to the low activities of some of the enzymes.

Ramaswamy and Reddy (1983Go) concluded that another snakehead Channa gachua shifted toward ureotelism during aerial exposure. This has been cited to support the hypothesis of urea formation as a means of detoxifying ammonia in air-breathing teleosts exposed to land (Anderson, 1995Go; Graham, 1997Go; Saha and Ratha, 1998Go; Frick and Wright, 2002Go). However, we wish to point out that there were apparent discrepancies in results reported by Ramaswamy and Reddy (1983Go). Taking the normal ammonia excretion rate over 5 h in water from their results as 7.13 mg N h-1 kg-1, and assuming that no ammonia excretion took place when C. gachua was exposed to terrestrial conditions for 10 h, the total amount of ammonia excreted in the subsequent 5 h (a total of 15 h) of resubmergence should have been 7.13x15=106.95 mg N kg-1. Since the reported result obtained for ammonia excretion during this subsequent 5 h period was only 14.03x5= 70.15 mg N kg-1, there was a deficit of 106.95-70.15= 36.8 mg ammonia-N kg-1. However, Ramaswamy and Reddy (1983Go) reported the urea excretion rate in this subsequent 5 h period, during which the urea and ammonia levels in the blood and liver returned back to normal, to be 50.14 mg N h-1 kg-1. This represents a total of 50.14x5 or 250.7 mg N kg-1, or a surplus of 250.7-(9.91x15) or 102.05 mg urea-N kg-1, which was much greater than the deficit of 36.8 mg ammonia-N kg-1 in ammonia excretion. Actually, the only possible explanation for such results is that increases in proteolysis and amino acid catabolism had taken place, but such an adaptation is physiologically unfitting during aerial exposure and was unlikely to have occurred. Alternatively, the discrepancy might have arisen from the high variability and low sensitivity of the method of ammonia and urea assay (urease treatment plus Nesslerization) adopted in their study. There may be physiological differences between two species of fishes, even if they are grouped under one single genus (in this case, Channa). However, Ramaswamy and Reddy (1983Go) did not examine urea and ammonia contents in the muscle, which constitutes the bulk of the fish, nor assay for ornithine—urea cycle enzymes in C. gachua. Since our results on C. asiatica do not support urea formation as the major strategy for detoxifying ammonia during aerial exposure, whether C. gachua would turn ureotelic on land is an issue that needs to be re-examined.

Ip et al. (2001aGo,bGo) suggested that fishes such as the mudskipper P. schlosseri, which are active on land with impeded excretion of ammonia, would partially catabolize certain amino acids to alanine. This would allow amino acids to be used as an energy source without polluting the internal environment. Partial amino acid catabolism coupled with a reduction in amino acid catabolism in general constitutes the most cost-effective way to minimize endogenous ammonia build-up. Alanine may be more suitable for accumulation because it has fewer effects than other amino acids on the kinetics of many enzymes (e.g. versus arginine; Bowlus and Somero, 1979Go). Indeed, alanine levels increased fourfold, from 3.7 to 12.6 µmol g-1, in the muscle of C. asiatica after 48 h of aerial exposure. The accumulated alanine accounted for 70% of the deficit in ammonia excretion during that period. This would allow the utilization of certain amino acids as energy sources and, at the same time, minimize ammonia accumulation. However, in contrast to mudskippers (Ip et al., 2001bGo), the reduction in nitrogenous excretion during 48 h of aerial exposure was completely balanced by nitrogenous accumulation in the tissues (Table 7). Hence, it is unlikely that the rates of proteolysis and amino acid catabolism were reduced during aerial exposure, as in the case of mudskippers (Ip et al., 2001bGo), which implies that C. asiatica does not have the mudskippers' capability of surviving on land. Actually, the ability to reduce rates of amino acid catabolism during aerial exposure may not be a common phenomenon among teleostean fishes. For example, when the marble goby Oxyeleotris marmoratus, which remains inactive on land, is exposed to terrestrial conditions for 72 h, it does not undergo a reduction in amino acid catabolism. Instead, it appears that protein and/or amino acid catabolism may increase during aerial exposure because glutamine accumulates to levels far in excess of those needed to detoxify the ammonia produced during that period (Jow et al., 1999Go).


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Table 7. A balance sheet of nitrogenous accumulation and excretion in a 40 g C. asiatica exposed to submerged or terrestrial conditions for 48 h

 

The presence of malic enzyme and ALT in the tissues of C. asiatica supports the proposition that this fish is capable of undergoing partial amino acid catabolism. In this pathway, malate is channelled out of the Krebs cycle through malic enzyme to form pyruvate. Pyruvate then undergoes transamination through the ALT reaction to form alanine. For this mechanism to work, {alpha}-KG produced must be channelled into the Krebs cycle to maintain a supply of pyruvate for transamination so that no endogenous ammonia is released in the process (Fig. 4). However, {alpha}-KG also acts as a substrate for the GDH reaction, in the aminating direction, which is also occurring in the mitochondria. If {alpha}-KG were converted into glutamate, partial amino acid catabolism would not proceed, ATP would not be produced, and endogenous ammonia would start to accumulate in the terrestrial conditions at a higher rate (Fig. 4). Indeed, aerial exposure significantly decreased the aminating activities of GDH from the muscle and liver of C. asiatica. This can be seen as an important adaptation to facilitate the formation of alanine through partial amino acid catabolism in order to reduce the rate of endogenous ammonia accumulation in adverse conditions. However, suppression of the GDH aminating activity would not be a good strategy for handling exogenous ammonia during ammonia-loading, when ammonia has to be detoxified through glutamate and glutamine formation (Fig. 4) (Ip et al., 2001aGo). At this moment, it is uncertain if C. asiatica can upregulate the aminating activity of GDH in response to exogenous ammonia, as in the mudskipper (Peng et al., 1998Go).



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Fig. 4. Proposed pathway of partial catabolism of certain amino acids, producing alanine without releasing ammonia, in C. asiatica when it is active on land. GDH, glutamate dehydrogenase; GS, glutamine synthetase; OAA, oxaloacetate.

 

For P. schlosseri exposed to 24 h of terrestrial conditions, alanine accumulates in the body (3-4 µmol g-1 muscle; Ip et al., 1993Go, 2001bGo), but the aminating GDH activity in the muscle is unchanged (Ip et al., 1993Go). The activity (both aminating and deaminating) in the liver decreases, although there is an apparent increase in the amination:deamination ratio (Ip et al., 1993Go). This may represent a compromise between adaptations to aerial exposure (to decrease endogenous ammonia production) during an excursion on land at low tides and adaptations to ammonia loading (to detoxify exogenous ammonia) during a stay in the burrow at high tide or during the breeding seasons.

On the contrary, Iwata et al. (1981Go) reported that the aminating GDH activities in the muscle and liver of the mudskipper Periophthalmus modestus (as P. cantonensis) increased significantly after aerial exposure. Iwata et al. interpreted the results as ammonia (endogenous) being detoxified to amino acids through the amination of {alpha}-KG. However, it would be futile if GDH acts, on the one hand, to release ammonia through transdeamination (Mommsen and Walsh, 1992Go), and on the other hand, to detoxify the released ammonia back to glutamate simultaneously, as suggested by Iwata et al. (1981Go). If indeed alanine is formed through partial amino acid catabolism, the amino groups of various amino acids (e.g. glutamate, valine and leucine) are not physically released, and are reconverted into amino acids (Ip et al., 2001aGo,bGo). Their amino groups are channelled to glutamate, which acts as a substrate for the transamination of pyruvate. Thus, the glutamate involved in this reaction does not arise from the GDH aminating reaction (Fig. 4).

Effects of constant darkness
To slow down the build-up of ammonia internally, it would be necessary to decrease the rate of ammonia production through amino acid catabolism. The steady-state concentration of amino acids in the tissues depends on the rates of their degradation and production. Lim et al. (2001Go) observed decreases in TFAA contents in some of the tissues of two mudskipper species exposed to terrestrial conditions in constant darkness, and there was no accumulation of alanine at all. Lim et al. (2001Go) suggested that simultaneous decreases in the rates of proteolysis and amino acid catabolism would have occurred, and the decrease in proteolytic rate was greater than the decrease in the rate of amino acid catabolism. This would lead to decreases in the steady-state concentrations of various FAAs and, consequently, lowering the TFAA concentrations. This is an effective strategy for slowing down the internal build-up of endogenous ammonia. However, such a phenomenon was not observed in C. asiatica exposed to terrestrial conditions in constant darkness. There was no change in the TFAA contents in all the tissues and organs examined. In addition, alanine accumulated to the same extent in the muscle of these specimens compared to those exposed to terrestrial conditions in a dark:light regime. This would imply that, with respect to nitrogen metabolism, the adaptation of C. asiatica to aerial exposure is less effective than that of the mudskippers.

Effects of short burst of locomotory activity
Exercise in water led to a decrease in glycogen content, and increases in the levels of lactate in the muscle of C. asiatica. However, it had no significant effect on the ammonia and alanine contents in the muscle. It can be deduced that glycogen was mobilized in this situation, and there was an increase in glycolytic rate to supply ATP for locomotory activities, maintaining a relatively constant energy charge in this tissue. Similar results were obtained when specimens were forced to exercise on land after 24 h exposure to terrestrial conditions. Hence, unlike P. schlosseri (Ip et al., 2001bGo), C. asiatica is incapable of increasing the rate of partial amino acid catabolism to sustain locomotory activities on land, although it can undergo partial amino acid catabolism to reduce ammonia production during aerial exposure. In the case of P. schlosseri, exercise on land leads to increases in muscle ammonia and alanine levels with the glycogen content unaffected (Ip et al., 2001bGo). This difference may explain, at least partially, why P. schlosseri can maintain long periods of locomotory activity on land, but under similar conditions C. asiatica can only afford short bursts of muscular activities to try to get back to water.

A comparative perspective
On land, C. asiatica exhibits activity that is intermediate between that of the marble goby (totally quiescent) and the mudskipper (very active). In relation to this, it is important to note that C. asiatica accumulates both alanine and glutamine in the muscle, with glutamine accounting for only 20% of the deficit in reduction in ammonia excretion. In contrast to alanine formation, glutamine synthesis represents an ammonia detoxification mechanism that, in effect, removes the accumulating ammonia. Production of glutamine is energy expensive (Ip et al., 2001aGo), and appears to be adopted by fishes that remain relatively inactive on land such as the marble goby (Jow et al., 1999Go) and sleeper Bostrichthys sinensis (Ip et al., 2001cGo; Anderson et al., 2001Go). As for the amphibious mudskippers Boleophthalmus boddaerti and P. schlosseri, they do not accumulate glutamine during aerial exposure (Lim et al., 2001Go; Ip et al., 2001bGo). Instead, glutamine formation is only used as a means of detoxifying exogenous (and endogenous) ammonia when confronted with sublethal concentrations of external ammonia (Peng et al., 1998Go). Hence, it can be concluded that teleostean fishes exhibit a variety of strategies to survive aerial exposure, and they represent a range of specimens available for us to examine various biochemical adaptations that would have facilitated their invasion of the terrestrial habitat during evolution.


    Acknowledgments
 
This research was supported by the Academic Research Fund RP 5/00 CSF from the Nanyang Technological University, National Institute of Education.


    References
 TOP
 Summary
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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