H3B-120

Expression of ornithine–urea cycle enzymes in early life stages of air-breathing walking catfish Clarias batrachus and induction of ureogenesis under hyper-ammonia stress

Zaiba Y. Kharbuli a, Shritapa Datta a, Kuheli Biswas a, Debajit Sarma b, Nirmalendu Saha a,*

Abstract

The air-breathing walking catfish Clarias batrachus is a potential ureogenic teleost with having a full complement of ornithine–urea cycle (OUC) enzymes expressed in various tissues. The present study was aimed at determining the pattern of nitrogenous waste excretion in the form of ammonia-N and urea-N along with the changes of tissue ammonia and urea levels, and the expression of OUC enzymes and glutamine synthetase (GSase) in early life stages of this teleost, and further, to study the possible induction of ureogenesis in 15-day old fry under hyper-ammonia stress. The ammonia and urea excretion was visible within 12 h post-fertilization (hpf), which increased several-fold until the yolk was completely absorbed by the embryo. Although all the early developing stages were primarily ammoniotelic, they also excreted significant amount of nitrogen (N) in the form of urea-N (about 35–40% of total N). Tissue levels of ammonia and urea also increased along with subsequent developmental stages at least until the yolk absorption stage. All the OUC enzymes and GSase were expressed within 4–12 hpf showing an increasing trend of activity for all the enzymes until 350 hpf. There was a significant increase of activity of GSase, carbamyl phosphate synthetase III (CPSase III) and argininosuccinate lyase enzymes (ASL), accompanied with significant increase of enzyme protein concentration of at least two enzymes (GSase and CPSase III) in the 15-day old fry following exposure to 10 mM NH4Cl as compared to respective controls kept in water over a period of 72 h. Thus, it appears that the OUC enzymes are expressed in early life stages of walking catfish like other teleosts, but at relatively high levels and remain expressed all through the life stages with a potential of stimulation of ureogenesis throughout the life cycle as a sort of physiological adaptation to survive and breed successfully under hyper-ammonia and various other environmental-related stresses. D 2005 Elsevier Inc. All rights reserved.

Keywords: Ammonia; Ammoniotelic; Urea; Ureogenic; Glutamine synthetase; Carbamyl phosphate synthetase III; Ammonium chloride

1. Introduction

Although teleost fishes excrete predominantly ammonia as a nitrogenous waste, a small amount of urea is also found to be excreted by most teleosts, which is usually around 10–15% of the total nitrogenous wastes (for review, see Saha and Ratha, 1998). The formation of urea in most teleosts is thought to result from the breakdown of dietary arginine and/or uric acid (for reviews, see Mommsen and Walsh, 1991; Wright, 1993; Walsh, 1998), not from the conventional ornithine–urea cycle (OUC), the primary source for urea synthesis in mammals, adult amphibian and elasmobranch fishes. It was believed for many years that the genes of OUC enzymes had been lost from the genome of teleosts because the enzyme activity could not be detected or was found to be very low in the liver tissue of various fish species (Brown and Cohen, 1960). In recent years, however, there has been renewed interest on the studies of OUC in teleosts after the reports of expression of high OUC enzymes accompanied by active ureogenesis in several teleosts species primarily as an adaptation to survive in unique environmental circumstances. Examples include the marine toadfishes Opsanus beta, O. tau (Read, 1971; Mommsen and Walsh, 1989), alkaline lake-adapted tilapia Alcolapia grahami (Randall et al., 1989), two Indian freshwater air-breathing catfishes Heteropneustes fossilis and Clarias batrachus (Saha and Ratha, 1987, 1989; Saha et al., 1999), and the gobiid fish Mugilogobius abei (Iwata et al., 2000). Furthermore, some of these species have been reported to excrete significant amount of urea in response to various adverse environmental conditions such as confinement (stress), severely alkaline water, ammonia loading, and exposure to air or while living inside the mud peat under water restricted conditions (Randall et al., 1989; Walsh et al., 1990, 1994; Walsh and Milligan, 1995; Ratha et al., 1995; Saha and Ratha, 1994; Saha and Das, 1999; Saha et al., 1995, 2001, 2002a, 2003).
More interestingly, ureogenesis via the OUC has been reported in early embryonic and larval stages of rainbow trout (De´peˆche et al., 1979; Wright et al., 1995; Korte et al., 1997), Atlantic cod (Chadwick and Wright, 1999), Atlantic halibut (Terjesen et al., 2000) and African catfish (Terjesen et al., 2001), which is otherwise non-functional in most of these adult teleosts. Recently, the expression of significant levels of OUC enzymes has also been reported during the early life stages in ureogenic marine gulf toadfish with significant increase of activity of some of these enzymes under hyper-ammonia stress (Barimo et al., 2004). Griffith, in his review (Griffith, 1991) reasoned that urea synthesis is essential during early development of teleosts to convert ammonia to less toxic compound, when the opportunity to excrete ammonia is restricted and the rate of protein catabolism is relatively high. Indeed, Wright and Land (1998) showed that in embryos and yolk-sac larvae of rainbow trout, urea excretion increased several-fold upon exposure to ammonia or high pH, factors which likely would inhibit or reduce ammonia excretion. Thus, it is believed that all teleosts have retained the genes for OUC enzymes, but the expression is suppressed in the adult stage except for a few species inhabiting in adverse environmental conditions.
The freshwater amphibious air-breathing walking catfish (C. batrachus) that are predominantly found in the eastern part of India, usually inhabit stagnant, slow flowing swampy water bodies or wet lands, which are often covered with macrovegetation like water hyacinth and are characterized by low dissolved oxygen, high carbon dioxide, methane and ammonia, and are generally not suitable for survival of fishes other than air-breathers (for reviews, see Rao et al., 1994; Saha and Ratha, 1998). Rice fields, which harbor a variety of insects, form another congenial habitat for this air-breathing catfish. During summer, these swamps usually dry up due to prolonged drought and this catfish burrows inside the mudflats, possibly to avoid total dehydration. In nature, this catfish breeds only once during the period of June to August after a heavy rainfall. In ponds and swamps, these fishes make small pits along the margin, congregate there in pairs and spawn with the onset of rain. The female fishes move out of the pits immediately after spawning and males guard the young ones for a few days. The fry start moving around in search of food after the adsorption of yolk-sac (for review, see Rao et al., 1994). In adult walking catfish, the presence of full complement of OUC enzymes along with glutamine synthetase (GSase) activity, a closely linked enzyme in fish ureogenesis, has been reported (Saha and Ratha, 1989; Saha et al., 1999). Further, the pattern of N excretion in the form of ammonia-N and urea-N both in controls as well as in fishes exposed to various environmental stresses under the laboratory conditions has been studied in detail (Saha and Ratha, 1998; Saha and Das, 1999; Saha et al., 2002a,b, 2003). However, no attention has been made to the ontogeny of urea and ammonia excretion along with the expression of OUC enzymes in this catfish, although their excretory needs may vary due to changing water quality in their natural habitat during early life stages. Further, like in adults, the early developmental stages also may face the problem of ammonia toxicity in their natural habitat mainly because of decomposition of macrovegetation and deposition of fertilizers from the cultivable lands through rain water. Therefore, the main objectives of the present study were (i) to assess the pattern of expression of all the OUC enzymes including that of GSase, the pattern of excretion and tissue levels of ammonia and urea in early developmental stages of C. batrachus, and (ii) to look for the possible induction of ureogenesis in 15-day old fry of C. batrachus under hyperammonia stress caused by exposing the fry in 10 mM NH4Cl over a period of 72 h.

2. Materials and methods

2.1. Animals

To get all the early developmental stages of C. batrachus, a specific breeding place in a suitable natural breeding pond was targeted in June 2003 in Nalbari, situated in the state of Assam. Immediately after fertilization in their natural habitat, a group of fertilized eggs were segregated and kept separately in an area of 9 m2 using a meshed cloth (0.75 mm pore size) in the same pond so that the eggs were restricted to one place in the pond. The development of fertilized eggs and embryos was monitored microscopically at timed intervals. The different stages of development were: embryo formation after 8 h (2.0–2.5 mg), differentiation of head and tail ends after 10–11 h (2.5–3.0 mg), twisting movement stage of embryo after 23 h (2.8–3.5 mg), hatching after 24–26 h (3.5–4.2 mg), and complete yolk absorption stage 90 h post-fertilization (hpf) (16–22 mg) at a temperature ranging between 26 and 29 -C. After 90 hpf, larvae were fed regularly with micro crustaceans until 200 hpf (200–250 mg), followed by feeding with small insects and worms until 350 hpf (400–500 mg). In NH4Cl-treated experiment with 15-day old fry, no food was provided both to treated and control fry during the period of experiment.

2.2. Experimental protocol

For studying the rate of excretion of N in the form of ammonia-N and urea-N, a number of different age group stages (pre-weighed) were kept in known volumes of bacteria-free filtered and properly aerated pond water in five different sets for 30–60 min at 28T2 -C. Immediately after collecting the sample for estimation of ammonia and urea, all the stages were dipped into liquid nitrogen and then stored at 60 -C for analysis of tissue ammonia and urea levels, and enzyme assays. For all studies, whole animals were used as it was difficult to isolate different tissues in the early developmental stages. For NH4Cl exposure, a group of eight to ten pre-weighed 15-day old fry was kept in an individual bucket containing 500 mL of 10 mM NH4Cl solution (pH 7.15T0.15, temperature 28T2 -C) for 72 h. Another set of fry (8–10 individuals) was kept in plastic buckets containing 500 mL of bacteria-free filtered pond water (pH 6.95T0.10), which served as controls. Further, streptopenicillin (a mixture of streptomycin sulphate, penicillin G Na and procain penicillin) was added in each bucket (10 mg L1) to stop microbial growth. Both the NH4Cl solution and water from control buckets were replaced with fresh medium at every 12 h of exposure and the media were aerated regularly at timed intervals; aerial respiration starts in this fish after 10–12 days of hatching (Rao et al., 1994). After 24, 48 and 72 h of exposure, a set of fish fry, both from treated and control buckets, was removed, killed immediately by decapitation, plunged into liquid nitrogen and stored at 60 -C for later analysis of tissue ammonia and urea and enzyme assays. All enzyme assays and analysis were completed within two weeks of preserving the animal.

2.3. Estimation of ammonia and urea

Amounts of excreted ammonia and urea were measured enzymatically (Kun and Kearney, 1974). Concentrations of ammonia and urea in whole bodies were measured by the same enzymatic methods after processing the tissue as described by Saha and Ratha (1989).

2.4. Enzyme assays

A 10% homogenate (w/v) of the whole animal of early life stages or individual tissues of adult fish was prepared in a homogenizing buffer containing 100 mM Tris–HCl buffer (pH 7.5), 50 mM KCl, 1 mM ethylene diamine tetra acetic acid (EDTA), 1 mM dithiothreitol (DTT) and a protease inhibitor cocktail (Roche), using a motor-driven Potter– Elvehjem glass homogenizer with a Teflon pestle. The homogenate was treated with 0.5% Triton X-100 in a 1:1 ratio for 30 min. The homogenate was then subjected to mild sonication for proper breakage of mitochondria and centrifuged at 10,000 g for 10 min. The supernatant was used for assaying the enzymes. All steps were carried out at 4 -C. The OUC enzymes, ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase (ARG) were assayed following the method of Saha et al. (1995). The OUC-related CPSase activity was assayed separately in the presence of 25 mM glutamine (CPSase III) and 25 mM ammonium chloride (CPSase I-like) as nitrogen donors, and contained 1 mM of uridine-5Vtriphosphate (UTP) to inhibit CPSase II activity (Saha et al., 1997, 1999). The reaction for all the enzymes was stopped by adding 0.5 mL of 10% perchloric acid per 1 mL of reaction mixture, followed by centrifugation to remove precipitated protein. Citrulline (CPSase, OTC, and ASS assays) and urea (ASL and ARG assays) were measured spectrophotometrically in the supernatants (Moore and Kauffman, 1970) and expressed as enzyme activity. All the enzyme assays were carried out at 30 -C. One unit of enzyme activity was defined as the amount of enzyme catalyzing 1 Amol of product formed or substrate used h1 at 30 -C. Glutamine synthetase (GSase) was assayed by the g-glutamyl transferase reaction (Webb and Brown, 1976). One unit of enzyme activity for GSase was expressed as the amount of enzyme catalyzing the formation of 1 Amol of g-glutamyl hydroxamate h1 at 30 -C.

2.5. Western blot analysis

Tissues from both the control and NH4Cl-treated 15-day old fry were homogenized in 20 mM Tris–HCl buffer (pH 7.4) containing 0.33 M sucrose, 1 mM EDTA, 1% Triton X-100 and a protease inhibitor cocktail (Roche) and sonicated for 30 s. The homogenate was centrifuged at 10,000 g for 20 min at 4 -C. Supernatants were mixed with loading buffer containing 150 mM Tris–HCl (pH 8.8), 15 mM EDTA, 60 mM DTT, 1.2 M sucrose, 6% sodium dodecyl sulphate (SDS) and 0.125% bromophenol blue in a ratio of 2:1, followed by incubation at 37 -C for 30 min. In each lane, 75 Ag of protein was applied and electrophoresed in 7.5% and 10% SDS-PAGE in cases of CPSase III and GSase enzymes, respectively. In one lane, 5 AL of Rainbow molecular weight marker (Amersham) was also run. Proteins were electroblotted from gels onto nitrocellulose membrane in a semidry blotter (Hoefer TE 70) for 2 h in presence of a transfer buffer containing 40 mM glycine, 48 mM Tris, 0.03% SDS and 20% methanol. Blots were blocked overnight in 5% bovine serum albumin (BSA) prepared in TBST (20 mM Tris–HCl, pH 7.6, 147 mM NaCl and 0.1% Tween) at 4 -C. Blots were then incubated either with rabbit antibody to GSase conserved oligopeptide conjugated to keyhole limpet hemocyanin (KLH) (AcetylcysteinylCPRSVGQEKKGYFEDRRPS-amide, as produced in Anderson et al., 2002) or with rabbit antibody to marble goby CPSase III peptide sequence (acetyl-KKAWSDSHNLQQELAC-amide; P. M. Anderson, personal communication) (1:2500 dilution) for 2 h, followed by incubation with horseradish peroxidase (HRP) conjugated anti-rabbit IgG (1:5000 dilution) secondary antibody for 2 h with washing steps with TBST in between. Immunodetection was performed with an enhanced chemiluminescence kit (ECL-PLUS, Amersham). Blots were scanned with a computer assisted densitometer (Kodak, ID 3.6) and photographed.

2.6. Measurement of protein concentration

Protein concentrations in individual tissue samples were determined by the dye-binding method (Bradford, 1976) with bovine serum albumin as standard.

2.7. Chemicals

All the enzymes, co-enzymes and substrates were purchased from Sigma-Aldrich Chemicals, USA. The GSase and CPSase III antibodies, raised in rabbit, were generous gifts from Drs. P. J. Walsh and P. M. Anderson, respectively. Anti-rabbit secondary HRP conjugated antibody was obtained from Sigma. Other chemicals were of analytical grade and obtained from local sources. Deionized double-glass distilled water was used in all preparations.

2.8. Statistical analysis

The data collected from different replicates were statistically analyzed and presented as meanTS.E.M. (n) where n equals the number of animals in the sample. One-way ANOVA was done between the controls and the results of NH4Cl-treated experiment, and P <0.05 was taken as statistically significant. 3. Results 3.1. Excretion of ammonia and urea by early life history stages The pattern of N excretion in the form of ammonia-N and urea-N by early life history stages of C. batrachus, starting from 4 to 348 hpf, is shown in Fig. 1. The rate of excretion of ammonia sharply increased from 0.025T0.005 Amol g1 h1 at 12 hpf to 0.256T0.029 Amol g1 h1 at 84 hpf. Thereafter followed a decreasing trend until 204 h and then again increased gradually at later stages of development. The excretion of urea also increased gradually from 0.010T0.002 Amol g1 h1 at 12 hpf to 0.167T0.013 Amol g1 h1 at 84 hpf. Thereafter followed a decreasing trend until 240 h and then gradually increased at later stages of development. Although the excretion of nitrogenous wastes in the form of ammonia-N dominated over the urea-N excretion by the early life history stages of C. batrachus, as evidenced by the fact that the ratio of ammonia-N:urea-N excretion varied between 1.9 to 2.5 and 348 hpf, urea-N also contributed a significant part of nitrogenous waste (about 35–40% of the total N excretion as ammonia-N and urea-N) at least from 156 hpf. 3.2. Tissue ammonia and urea levels at early life history stages As shown in Fig. 2, the concentration of ammonia in the whole body increased gradually from 1.01T0.11 Amol g1 body mass at 4 hpf to 4.35T0.41 Amol g1 body mass at 84 hpf, followed by a slight decrease at later stages of development. Likewise, the concentration of urea also increased gradually from 0.21T0.05 Amol g1 body mass at 4 hpf to 2.86T0.27 Amol g1 body mass at 84 hpf, followed by a slight decrease at later stages. 3.3. Expression of OUC and related enzymes at early life history stages The pattern of expression of GSase, the enzyme related to urea synthesis in fish, and all the OUC enzymes at early life history stages of C. batrachus up to 350 hpf are presented in Fig. 3. All the OUC enzymes including the GSase were seen to express between 4 and 12 hpf. In case of GSase, the enzyme activity was recorded to be 1.56T0.25 U g1 wet mass at 4 h with a sharp increase of activity to 37.81T3.75 U g1 wet mass at 60 hpf, followed by a slight decrease between 60 and 84 hpf and then again gradual increase at later stages of development. The urea cycle-related CPSase was assayed separately in the presence of glutamine (CPSase III) and in the presence of ammonia (CPSase I-like; Saha et al., 1999). In both cases, the CPSase activity increased sharply until 108 hpf (2.85T0.35 U g1 wet mass (CPSase III) and 3.57T0.44 U g1 wet mass (CPSase I-like) with slight or no increase of activity at later stages of development. The changing pattern of OTC activity at early life stages was similar to that of CPSase III with a peak of activity at 156 hpf (85.25T3.57 U g1 wet mass), followed by a gradual decrease of activity until 252 hpf and an increase at later stages. Activity of ASS increased with increasing age of the embryo showing at least two peaks of activity, one at 36 hpf (15.64T1.56 U g1 wet mass) and one at 156 hpf (32.51T2.48 U g1 wet mass). The changing pattern of ASL activity at early developmental stages also followed the similar pattern as that of ASS. The enzyme activity increased gradually with increasing age of the embryo showing again at least two peaks of activity, one at 156 hpf (19.22T1.51 U g1 wet mass) and one at 252 hpf (25.45T2.74 U g1 wet mass). ARG activity increased gradually with increasing age of early life history stages, initially with a sharp rise until 108 hpf, followed by a slower increase at later stages. All OUC enzymes including GSase remained expressed at relatively high levels in adult tissues of C. batrachus with maximum activity in liver, followed by kidney and muscle (Table 1). 3.4. Changes in tissue concentrations of ammonia and urea in the NH4Cl-treated fry The concentration of ammonia in the NH4Cl-treated fry almost doubled from the control value of 3.15T0.35 Amol g1 body mass to 6.29T0.56 Amol g1 body mass (P<0.001) after mass (about 1.8-fold, P <0.01) in the fry after 24 h of exposure to NH4Cl, which increased further to 3.86T0.41 Amol g1 body mass (about 2.3-fold; P <0.001) after 48 h with no further changes at later stages (Fig. 4). 3.5. Changes in the activity of GSase and OUC enzymes in NH4Cl-treated fry As evidenced by the western blot analysis (Fig. 6), the increase of activities of GSase and CPSase III enzymes in the NH4Cl-treated fry was accompanied by significant increase of GSase enzyme protein concentration by 95% (P<0.01) and CPSase III enzyme protein concentration by 55% (P<0.01) after 72 h of exposure compared to respective controls. 4. Discussion The excretion of N as ammonia-N and urea-N became prominent within 12 hpf of eggs in C. batrachus, with a sharp increase of excretion of both ammonia-N and urea-N until 84 hpf (Fig. 1). Although ammonia was found to be the predominant nitrogen excretory product during early life stages, urea also contributed a significant amount of total N (as ammonia-N+urea-N) excreted especially from 156 hpf (about 35–40% of the total). Yolk absorption in C. batrachus embryo normally takes place between 70 and 90 hpf (Rao et al., 1994), when the catabolism of yolk protein and amino acids is believed to take place at a higher rate. This could be the reason of obtaining the peak of excretion of ammonia and urea between 84 and 108 hpf, as observed in the present study. In line with our findings, significant levels of urea excretion during embryogenesis have also been reported in Atlantic cod Gadus morhua (Chadwick and Wright, 1999), rainbow trout Oncorhynchus mykiss (Wright et al., 1995; Wright and Land, 1998), common carp Cyprinus carpio (Kaushik et al., 1982), whitefish Coregonus lavaretus (Dabrowski et al., 1984) and African catfish Clarias gariepinus (Terjesen et al., 1997), constituting about 10–35% of total N excretion. Urea excretion in early developmental stages of ureogenic gulf toadfish (O. beta), however, was found to dominate over the ammonia excretion under natural conditions, constituting about 60% of the total N (ammonia-N+urea-N), and was suggested to be mainly due to high expression of all the OUC enzymes in fertilized eggs, larvae and juvenile stages of this fish (Barimo et al., 2004). During early development of viviparous eelpout Zoarces viviparous is also reported to excrete predominantly urea (about 65% of total N; Korsgaard, 1994). Relatively high accumulation of both ammonia and urea in the whole body tissue with a peak of accumulation between 84 and 108 hpf in C. batrachus, as observed in the present study (Fig. 2), was possibly due to high catabolic activity of the yolk protein at early life stages. Steady rise of tissue ammonia and urea levels was also seen in rainbow trout (De´peˆche et al., 1979; Wright et al., 1995), in African catfish (Terjesen et al., 1997), and also in some marine fish species (Fyhn and Serigstad, 1987; Finn et al., 1996; Chadwick and Wright, 1999) during embryogenesis, where the expression of OUC enzymes in early life stages has been confirmed. Accumulation of toxic ammonia was suspected to be one of the triggers for hatching in certain fish species (for review, see Wright and Fyhn, 2001). Later, however, it was shown at least in rainbow trout that higher accumulation of ammonia does not have any effect on hatching (Steele et al., 2001). This will possibly also be true in C. batrachus. However, it needs to be studied in detail before drawing any definite conclusion. Although the activities of all the OUC enzymes and GSase were seen to express within 4–12 hpf, the peaks of activities of most of the enzymes were obtained after 84 hpf, time when complete yolk absorption took place. Further, in the present study, the enzyme activities were measured from the whole animal preparation without separating out the yolk from the larval body, which constituted the major part of the body until the yolk was absorbed during the early part of development. It is likely that all the OUC and GSase enzymes are present in the larval body with very little or no expression in the yolk-sac, as shown in rainbow trout (Chadwick and Wright, 1999), thus causing a dilution effect of enzyme activities. This, possibly, explains the fact that the activities of most of the OUC and GSase enzymes had risen sharply immediately after complete absorption of yolk in early life stages. Furthermore, from 252 h onwards the levels of activity of GSase, CPSase (both glutamine- and ammonia-dependent), ASS and ASL were almost comparable to the activities reported in adult liver and kidney, and at higher levels compared to the levels in muscle tissue (Fig. 3; Table 1). Like in adults (Saha et al., 1999), detection of OUC-related CPSase activity in the presence of glutamine (CPSase III) as well as in the presence of ammonia (CPSase I-like) almost at the same levels at the early life history stages is of special significance. Whether or not these two activities of CPSase are due to the presence of two different enzymes as products of two different genes, or these represent an adapted form of CPSase III with a separate ammonia and glutamine binding sites, as suggested in adults (Saha et al., 1999), nevertheless, it is clear that this uniqueness with relation to the CPSase activity in this catfish occurs from the early life history stages. Both GSase and ARG enzymes showed an increased trend of activity throughout the developmental period, at least until 350 h, thus suggesting that the activities of these two enzymes remain more expressed in adults than in early life stages. In general, other than the role of ureogenesis, these two enzymes are known to have other multiple functions (Wood, 1993; Anderson, 2001), thus justifying the over expression of enzymic activities in adults. Hence, the high rate of urea excretion and also accumulation of urea in body tissues, which has been observed in yolk-sac and juvenile stages of this catfish, are mainly attributable to relatively high levels of activity of OUC enzymes in early life history stages. It is, therefore, evident from these results that the OUC enzymes are expressed in C. batrachus from the early life history stages and remain expressed at higher levels in adults. This is in contrast to some other teleost species such as common carp C. carpio (Kaushik et al., 1982), coregonid Coregonus schinzi palea (Dabrowski and Kaushik, 1984), whitefish C. lavaretus (Dabrowski et al., 1984) and Atlantic halibut H. hippoglossus (Terjesen et al., 2000), where the OUC enzymes are expressed (but at low levels) only in the early embryonic stages, but are undetectable in the adult fish. Thus, majority of fish appear to have genes for the OUC enzymes, even though expression of all enzymes at measurable levels has been demonstrated in only a few adult species (Saha and Ratha, 1987, 1989; Mommsen and Walsh, 1989; Randall et al., 1989; Iwata et al., 2000); expression of urea cycle enzymes at significant levels in adult teleosts appear to be correlated with adaptations to unique environmental circumstances (for reviews, see Anderson, 2001; Saha and Ratha, 1998). However, it remains unclear why fishes that are not exposed to unique environmental circumstances conserve the genes of the OUC enzymes. Expression of urea cycle in early life stages of teleost has been suggested to assist in detoxification of ammonia accumulating from amino acid catabolism, which may be a particular problem for species with large embryos (Griffith, 1991; Wright et al., 1995; Korsgaard et al., 1995). High levels of expression of OUC enzymes, which has been observed from the early embryonic stages of development in C. batrachus, may be helping them to challenge the hyperammonia stress and other environmental-related problems even during early life history stages. Significant increase in the concentration of total body ammonia in the 15-day old fry of C. batrachus during exposure to 10 mM NH4Cl might have resulted due to partial inhibition of ammonia excretion generated endogenously, accompanied with passive ammonia transport into the body through the branchial tissues and possibly through the cutaneous surface from the external medium in favour of concentration gradient, similar to the situation observed in adults (Saha et al., 2003). Furthermore, significant upregulation of activities of GSase, CPSase III and also ASL enzymes in the NH4Cl-exposed fry, observed between 24 and 72 h (Fig. 5), was similar to the situation noticed in adults under hyper-ammonia stress (Saha et al., 2003) and also other environmental-related problems like alkaline pH (Saha et al., 2002b). This initial result could suggest that the upregulation of GSase and CPSase III works together as a control point which starts from the early life stages and continues until adulthood and is associated with a shift to ureotelism under hyper-ammonia stress, and possibly under other environmental-related stresses in this air-breathing catfish. This might have led to a higher accumulation of tissue urea level during exposure to NH4Cl. Significant increase of excretion and accumulation of urea was reported in rainbow trout embryo after both acute and chronic NH4Cl exposure, but without altering the activities of any of the OUC and related enzymes (Steele et al., 2001). In juvenile toadfish, all the OUC enzymes including the GSase, except OTC (whose activity increased by 3-fold), were not affected with 1 mM NH4Cl exposure (Barimo et al., 2004) although the upregulation of GSase activity in adult toadfish liver with ammonia exposure and other factors have been reported (Wang and Walsh, 2000; Wood, 1993). Significant increase of enzyme protein concentrations of both GSase and CPSase III, as shown by western blot analysis (Fig. 6), in the fish fry following exposure to NH4Cl is clearly an indication that ammonia stress resulted to upregulation of these enzymes either at transcriptional or at translational level. Similar upregulation has also been observed in adult fish (Saha, Datta and Bhattacharjee, unpublished results). The mechanism(s) of such upregulation of ureogenesis and related enzymes may be difficult to explain at this moment with the available data, but the possible reasons could be: (i) stress-induced related release of cortisol, which is known to cause alterations of protein and amino acid metabolism in adult teleosts (Mommsen et al., 1999), and/or (ii) the accumulation of ammonia itself, as suggested in adult air-breathing catfishes (Saha and Ratha, 1994; Saha and Das, 1999; Saha et al., 1995, 2003). In addition to upregulation of ureogenesis, there could be other possible means of handling the ammonia toxicity by this fish even from the early life history stages such as: (i) decrease in amino acid catabolism rate as suggested in adult mudskippers (Lim et al., 2001) and loach (Chew et al., 2001) during aerial exposure mainly to avoid ammonia toxicity, and/or (ii) excretion of nitrogenous wastes in some other forms other than as ammonia-N and urea-N such as amino acid-N and protein-N as shown in juvenile rainbow trout (Kajimura et al., 2004). Furthermore, possible conversion of some part of accumulated ammonia to non-essential free amino acids (FAAs) in body tissues (Saha et al., 2002a) and more efflux of non-essential FAAs from the perfused liver (Saha et al., 2000) under hyperammonia stress are shown recently in adult walking catfish. In summary, all the OUC enzymes including that of GSase are expressed at relatively high levels at early embryo and juvenile stages in walking catfish, which remain expressed all through the life history stages. Like adults, early developmental stages are also ammoniotelic in natural habitat but excrete a significant amount of nitrogenous wastes as urea-N synthesized possibly via the functional OUC. Accumulation of ammonia inside the body in 15-day old fry, occurred during exposure to 10 mM NH4Cl over a period of 72 h, was likely due to inhibition of ammonia excretion accompanied with a more uptake of ammonia from the external medium, and hence stimulation of ureogenesis is caused possibly by enhancing the protein concentration of certain key enzymes related to ureogenesis such as GSase and CPSase III and/or by some other allosteric regulatory mechanism(s). These physiological adaptive strategies probably help them to tolerate high external ammonia throughout their life cycle, which they face in their natural habitat along with other environmental-related issues. References Anderson, P.M., 2001. Urea and glutamine synthesis: environmental influences on nitrogen excretion. Fish Physiol. 20, 239–277. Anderson, P.M., Broderius, M.A., Fong, K.C., Tsui, N.T., Chew, S.F., Ip, Y.K., 2002. Glutamine synthetase expression in liver, muscle, stomach and intestine of Botrichthys sinensis in response to exposure to a high exogenous ammonia concentration. J. Exp. Biol. 205, 2053–2065. Barimo, J.F., Steele, S.L., Wright, P.A., Walsh, P.J., 2004. Dogmas and controversies in the handling of nitrogenous wastes: ureotely and ammonia tolerance in early life stages of the gulf toadfish, Opsanus beta. J. Exp. Biol. 207, 2011–2020. Bradford, P.M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of the protein-dye binding. Anal. Biochem. 72, 248–254. Brown Jr., G.W., Cohen, P.P., 1960. Comparative biochemistry of urea synthesis-III. Activities of urea-cycle enzymes in various higher and lower vertebrates. Biochem. J. 75, 82–91. Chadwick, T.D., Wright, P.A., 1999. Nitrogen excretion and expression of urea cycle enzymes in the Atlantic cod (Gadus morhua L.): a comparison of early life stages with adults. J. Exp. Biol. 202, 2653–2662. Chew, S.F., Jun, Y., Ip, Y.K., 2001. The loach Misgurnus anguillicaudatus reduces amino acid catabolism and accumulates alanine and glutamine during aerial exposure. Physiol. Biochem. Zool. 74, 226–237. Dabrowski, K., Kaushik, S.J., 1984. Rearing of coregonid (Coregonus schinzi palea Cuv. Et Val.) larvae using dry and live food. II. Oxygen consumption and nitrogen excretion. Aquaculture 41, 333–344. Dabrowski, K., Kaushik, S.J., Luquet, P., 1984. Metabolic utilization of body stores during the early life of whitefish Coregonus laveretus L. J. Fish Biol. 24, 721–729. De´peˆche, J., Gilles, R., Daufresne, S., Chapello, H., 1979. Urea content and urea production via the ornithine–urea cycle pathway during the ontogenic development of two teleost fishes. Comp. Biochem. Physiol., A 63, 51–56. Finn, R.N., Fyhn, H.J., Henderson, R.J., Evjen, M.S., 1996. The sequence of catabolic substrate oxidation and enthalpy balance of developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus L.). Comp. Biochem. Physiol., A 115, 133–151. Fyhn, H.J., Serigstad, B., 1987. Free amino acids as energy substrate in developing eggs and larvae of the cod Gadus morhua. Mar. Biol. 96, 335– 341. Griffith, R.W., 1991. Guppies, toadfish, lungfish, coelacanths and frogs: a scenario for the evolution of urea retention in fishes. Environ. Biol. Fishes 32, 199–218. Iwata, K., Kajimura, M., Sakamoto, T., 2000. Functional ureogenesis in the gobiid fish, Mugilogobius abei. J. Exp. Biol. 203, 3703–3715. Kajimura, M., Croke, S., Glover, C.N., Wood, C.M., 2004. 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. J. Exp. Biol. 207, 1993–2002. Kaushik, S.J., Dabrowski, K., Luquet, P., 1982. Patterns of nitrogen excretion and oxygen consumption during ontogenesis of common carp (Cyprinus carpio). Can. J. Fish. Aquat. Sci. 39, 1095–1105. Korsgaard, B., 1994. Nitrogen distribution and excretion during embryonic postyolk sac development in Zoarces viviparous. J. Comp. Physiol. 164B, 42–46. Korsgaard, B., Mommsen, T.P., Wright, P.A., 1995. Nitrogen excretion in teleostean fish: adaptive relationship to environment, ontogenesis, and viviparity. In: Walsh, P.J., Wright, P.A. (Eds.), Nitrogen Metabolism and Excretion. CRC Press, Boca Raton, pp. 259–287. Korte, J.J., Salo, W.L., Cabrera, V.M., Wright, P.A., Felskie, A.K., Anderson, P.M., 1997. Expression of carbamyl phosphate synthetase III mRNA during the early stages of development and in muscle of adult rainbow trout (Oncorhynchus mykiss). J. Biol. Chem. 272, 6270–6277. Kun, E., Kearney, E.B., 1974. Ammonia. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis vol. IV. Academic Press, New York, pp. 1802–1806. Lim, C.B., Chew, S.F., Anderson, P.M., Ip, Y.K., 2001. Reduction in the rates of protein and amino acid catabolism to slow down the accumulation of endogenous ammonia: a strategy potentially adopted by mudskippers (Periophthalmodon schlosseri and Boleophthalmus boddaerti) during aerial exposure in constant darkness. J. Exp. Biol. 204, 1605–1614. Mommsen, T.P., Walsh, P.J., 1989. Evolution of urea synthesis in vertebrates: the piscine connection. Science 243, 72–75. Mommsen, T.P., Walsh, P.J., 1991. Urea synthesis in fishes: evolutionary and biochemical perspectives. In: Hochachka, P.W., Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology of Fishes. Elsevier, Amsterdam, pp. 137–163. Mommsen, T.P., Vijayan, M.M., Moon, T.P., 1999. Cortisol in teleosts: dynamics, mechanisms of action and metabolic regulation. Rev. Fish Biol. Fish. 9, 211–268. Moore, R.B., Kauffman, N.G., 1970. Simultaneous determination of citrulline and urea using diacetyl monoxime. Anal. Biochem. 33, 263–272. Randall, D.J., Wood, C.M., Perry, S.F., Bergman, H., Maloiy, G.M.O., Mommsen, T.P., Wright, P.A., 1989. Urea excretion as a strategy for survival in a fish living in a very alkaline environment. Nature 337, 165–166. Rao, G.R., Tripathi, S.D., Sahu, A.K., 1994. Breeding and seed production of the Asian catfish Clarias batrachus (Linnaeus). Manual Series vol. 3. Central Institute of Freshwater Aquaculture, Bhubaneswar, India, pp. 1–47. Ratha, B.K., Saha, N., Rana, R.K., Choudhury, B., 1995. Evolutionary significance of metabolic detoxification of ammonia to urea in an ammoniotelic freshwater teleost, Heteropneustes fossilis during temporary water deprivation. Evol. Biol. 8 & 9, 107–117. Read, L.J., 1971. The presence of high ornithine–urea cycle enzyme activities in the teleost Opsanus tau. Comp. Biochem. Physiol., B 39, 409–413. Saha, N., Das, L., 1999. Stimulation of ureogenesis in the perfused liver of an Indian air-breathing catfish, Clarias batrachus, infused with different concentrations of ammonium chloride. Fish Physiol. Biochem. 21, 303–311. Saha, N., Ratha, B.K., 1987. Active ureogenesis in a freshwater air-breathing teleost, Heteropneustes fossilis. J. Exp. Zool. 241, 137–141. Saha, N., Ratha, B.K., 1989. Comparative studies of ureogenesis in freshwater, air-breathing teleosts. J. Exp. Zool. 252, 1–8. Saha, N., Ratha, B.K., 1994. Induction of ornithine–urea cycle in a freshwater teleost, Heteropneustes fossilis, exposed to high concentrations of ammonium chloride. Comp. Biochem. Physiol., B 108, 315–325. Saha, N., Ratha, B.K., 1998. Ureogenesis in Indian air-breathing teleosts: adaptation to environmental constraints. Comp. Biochem. Physiol., A 120, 195–208. Saha, N., Dkhar, J., Ratha, B.K., 1995. Induction of ureogenesis in perfused liver of a freshwater teleost, Heteropneustes fossilis, infused with different concentrations of ammonium chloride. Comp. Biochem. Physiol., B 112, 733–741. Saha, N., Dkhar, J., Anderson, P.M., Ratha, B.K., 1997. Carbamyl phosphate synthetase in an air-breathing teleost, Heteropneustes fossilis. Comp. Biochem. Physiol., B 116, 57–63. Saha, N., Das, L., Dutta, S., 1999. Types of carbamyl phosphate synthetase and subcellular localization of urea cycle and related enzymes in air-breathing walking catfish, Clarias batrachus. J. Exp. Zool. 283, 121–130. Saha, N., Dutta, S., Ha¨ussinger, D., 2000. Changes in free amino acid synthesis in the perfused liver of an air-breathing walking catfish, Clarias batrachus infused with ammonium chloride: a strategy to adapt under hyperammonia stress. J. Exp. Zool. 286, 13–23. Saha, N., Das, L., Dutta, S., Goswami, U.C., 2001. Role of ureogenesis in the mud-dwelled singhi catfish (Heteropneustes fossilis) under condition of water shortage. Comp. Biochem. Physiol., A 128, 137–146. Saha, N., Dutta, S., Bhattacharjee, A., 2002a. Role of amino acid metabolism in an air-breathing catfish, Clarias batrachus in response to exposure to a high concentration of exogenous ammonia. Comp. Biochem. Physiol., B 133, 235–250. Saha, N., Kharbuli, Z.Y., Bhattacharjee, A., Goswami, C., Ha¨ussinger, D., 2002b. Effect of alkalinity (pH 10) on ureogenesis in the air-breathing walking catfish, Clarias batrachus. Comp. Biochem. Physiol., A 132, 353–364. Saha, N., Datta, S., Biswas, K., Kharbuli, Z.Y., 2003. Role of ureogenesis in tackling problems of ammonia toxicity during exposure to higher ambient ammonia in the air-breathing walking catfish Clarias batrachus. J. Biosci. 28, 733–742. Steele, S.L., Chadwick, T.D., Wright, P.A., 2001. Ammonia detoxification and localization of urea cycle enzyme activity in embryos of the rainbow trout (Oncorhynchus mykiss) in relation to early tolerance to high environmental ammonia levels. J. Exp. Biol. 204, 2145–2154. Terjesen, B.F., Verreth, J., Fyhn, H.J., 1997. Urea and ammonia excretion by embryos and larvae of the African catfish, Clarias gariepinus (Burchell 1822). Fish Physiol. Biochem. 16, 311–321. Terjesen, B.F., Rønnestad, I., Norberg, B., Anderson, P.M., 2000. Detection and basic properties of carbamyl phosphate synthetase III during teleost ontogeny: a case study in the Atlantic halibut (Hippoglossus hippoglossus L.). Comp. Biochem. Physiol., B 126, 521–535. Terjesen, B.F., Chadwick, T.D., Verreth, J.A.J., Rønnestad, I., Wright, P.A., 2001. Pathways of H3B-120 urea production during early life of an air-breathing teleost, the African catfish, Clarias gariepinus (Burchell 1822). J. Exp. Biol. 204, 2155–2165.
Walsh, P.J., 1998. Nitrogen excretion and metabolism. In: Evans, D.H. (Ed.), The Physiology of Fishes. CRC Press, Boca Raton, pp. 199–214.
Walsh, P.J., Milligan, C.L., 1995. Effects of feeding and confinement on nitrogen metabolism and excretion in the gulf toadfish Opsanus beta. J. Exp. Biol. 198, 1559–1566.
Walsh, P.J., Danulat, E., Mommsen, T.P., 1990. Variation in urea excretion in the gulf toadfish, Opsanus beta. Mar. Biol. 106, 323–328.
Walsh, P.J., Tucker, B.C., Hopkins, T.E., 1994. Effects of confinement/crowding on ureogenesis in the gulf toadfish Opsanus beta. J. Exp. Biol. 191, 195–206.
Wang, Y.X., Walsh, P.J., 2000. High ammonia tolerance in fishes of the family Batrachoididae (toadfish and midshipmen). Aquat. Toxicol. 50, 205–219.
Webb, J.T., Brown, G.W., 1976. Some properties and occurrence of glutamine synthetase in fish. Comp. Biochem. Physiol., B 54, 171–175.
Wood, C.M., 1993. Ammonia and urea metabolism and excretion. In: Evans, D.H. (Ed.), The Physiology of Fishes. CRC Press, Boca Raton, pp. 379–425.
Wright, P.A., 1993. Nitrogen excretion and enzyme pathways for ureogenesis in freshwater tilapia (Oreochromis niloticus). Physiol. Zool. 66, 881–901.
Wright, P.A., Fyhn, H.J., 2001. Ontogeny of nitrogen metabolism and excretion. Fish Physiol. 20, 149–200.
Wright, P.A., Land, M.D., 1998. Urea production and transport in teleost fish. Comp. Biochem. Physiol., A 119, 47–54.
Wright, P.A., Felskie, A., Anderson, P.M., 1995. Induction of ornithine–urea cycle enzymes and nitrogen metabolism and excretion in rainbow trout (Oncorhynchus mykiss) during early life stages. J. Exp. Biol. 198, 127–135.