NITROGEN EXCRETION AND EXPRESSION OF UREA CYCLE ENZYMES IN THE ATLANTIC COD (GADUS MORHUA L.): A COMPARISON OF EARLY LIFE STAGES WITH ADULTS

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1 The Journal of Experimental Biology 22, (1999) Printed in Great Britain The Company of Biologists Limited 1999 JEB NITROGEN EXCRETION AND EXPRESSION OF UREA CYCLE ENZYMES IN THE ATLANTIC COD (GADUS MORHUA L.): A COMPARISON OF EARLY LIFE STAGES WITH ADULTS TERRY D. CHADWICK AND PATRICIA A. WRIGHT* Department of Zoology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 *Author for correspondence ( patwrigh@oguelph.ca) Accepted 2 July; published on WWW 13 September 1999 For many years, the urea cycle was considered to be relatively unimportant in the life history of most teleost fishes. In previous studies, we were surprised to find that newly hatched freshwater rainbow trout embryos had relatively high activities of the key urea cycle enzyme, carbamoyl phosphate synthetase III (CPSase III), and other enzymes in the pathway, whereas adult trout had much lower or non-detectable activities. The present study tested the hypothesis that urea cycle enzyme expression is unique to early stages of rainbow trout. In marine Atlantic cod (Gadus morhua) embryos, CPSase III, ornithine transcarbamoylase (OTCase), glutamine synthetase (GSase) and arginase activities were all expressed prior to hatching. Urea excretion was detected shortly after fertilization and rates were high relative to those of ammonia excretion (5 1 % of total nitrogen excreted as urea nitrogen; total=ammonia+urea). Urea concentration was relatively constant in embryos, but ammonia concentration increased by about fourfold during Summary embryogenesis. Two populations of cod embryos were studied (from Newfoundland and New Brunswick), and significant differences in enzyme activities and excretion rates were detected between the two populations. In adult cod, CPSase III was not detectable in liver, white muscle, intestine and kidney tissues, but OTCase, GSase and arginase were present. Adult cod excreted about 17 % of nitrogenous waste as urea. Taken together, these data indicate that early urea cycle enzyme expression is not unique to rainbow trout but is also a feature of Atlantic cod development, and possibly other teleosts. The relatively high urea excretion rates underline the importance of urea as the primary nitrogen excretory product in Atlantic cod during early embryogenesis. Key words: urea, ammonia, carbamoyl phosphate synthetase, glutamine synthetase, arginase, ornithine transcarbamoylase, embryo, larvae, Atlantic cod, Gadus morhua. Introduction Almost all teleost fishes excrete predominantly ammonia as a nitrogenous waste, but they also excrete a small amount of urea, usually around 2 % of the total nitrogen excreted (Wood, 1993). The formation of urea in most teleosts is thought to result from the breakdown of dietary arginine and/or uric acid (Goldstein and Forster, 1965; Mommsen and Walsh, 1991; Wright, 1993), but not from the urea cycle, which is the primary pathway for urea synthesis in mammals, adult amphibians and elasmobranch fishes. The enzymology of the urea cycle in fishes differs from the mammalian urea cycle in two ways. First, the enzyme that catalyzes the first step of the urea cycle, carbamoyl phosphate synthetase, is different in fishes (CPSase III) from that in mammals (CPSase I); CPSase III utilizes glutamine as the nitrogen-donating substrate, whereas CPSase I uses ammonia. Second, because CPSase III requires glutamine as a substrate, glutamine synthetase (GSase) is an important accessory enzyme to the fish urea cycle. For many years it was assumed that the genes of urea cycle enzymes had been lost from the genome of teleosts because enzyme activities were low or undetectable in the liver tissue of various species (Brown and Cohen, 196). In recent years, there has been renewed interest in the urea cycle in teleosts because the complete urea cycle has been reported in the liver of a few unusual species: the alkaline lake tilapia Oreochromis alcalicus grahami (Randall et al., 1989), the air-breathing catfish Heteropneustes fossilis (Saha and Ratha, 1987, 1989, 1994) and the gulf toadfish Opsanus beta (Mommsen and Walsh, 1989; Walsh et al., 199, 1994). In the case of O. a. grahami, all nitrogen wastes are excreted as urea; this novel situation appears to be related to the extremely alkaline environment in which this species lives (Lake Magadi, ph 1; Randall et al., 1989). During periods of drought, H. fossilis is thought to detoxify ammonia that accumulates in its tissues by synthesizing urea (Saha and Ratha, 1987). O. beta becomes facultatively ureotelic when exposed to stressful environmental

2 2654 T. D. CHADWICK AND P. A. WRIGHT conditions such as elevated ammonia, confinement, crowding or exposure to air (Walsh et al., 199, 1994; Walsh and Milligan, 1995). Taken together, these findings strongly suggest that all teleosts have retained the genes for urea cycle enzymes, even though the urea cycle may be functional in only a few species. However, it remains unclear why fishes that are not exposed to unique environmental conditions conserve the genes for some or all of the urea cycle enzymes. Griffith (1991) suggested that urea synthesis may play a significant role during the protracted embryonic development of teleosts, when the opportunity to excrete ammonia is restricted and the rate of protein catabolism is relatively high. Recently, Wright et al. (1995) demonstrated that four enzymes that are involved with the urea cycle (including the key enzyme CPSase III) were induced in trout just after hatching, and found that newly hatched trout excreted urea. The presence of CPSase III activity in the early development of trout was corroborated by measurement of CPSase III mrna, which was detected as early as 3 days postfertilization (d.p.f.) and reached maximum levels at 1 14 d.p.f. (Korte et al., 1997). The expression of urea cycle enzymes in trout during early life stages has been clearly established, but not whether the early expression of the urea cycle is universal among teleosts (for reviews, see Anderson, 1995; Korsgaard et al., 1995), nor whether rainbow trout are exceptional rather than representative among teleosts in this regard. To investigate this, we therefore tested the hypothesis that urea cycle enzyme expression is unique to early stages of rainbow trout. We investigated the expression of the urea cycle during the early ontogeny of Atlantic cod (Gadus morhua L.), a marine teleost with a very different early life history from the rainbow trout. Cod embryos float at the surface layers of the ocean (pelagic), whereas trout embryos lie at the bottom of lakes or rivers (demersal) and are typically buried in several inches of gravel (Orcutt et al., 1961), where ammonia diffusion away from the embryo may be restricted. Cod eggs are small compared with trout eggs (1.4 mm and 4. mm diameter, respectively; Russell, 1976; Velsen, 1987) and have lower calorific content (Kato and Kamler, 1983; Finn et al., 1995a); consequently cod hatch at approximately 2 d.p.f. at 4 C (Russell, 1976), which is considerably earlier than trout (at approximately 33 d.p.f. at 1 C; Humpesch, 1985). The first objective of this study was to investigate whether urea is produced and excreted as a nitrogenous end product during the early life stages of the Atlantic cod. Tissue concentrations and excretion rates of urea and ammonia were measured in cod embryos and larvae up to 52 d.p.f. For comparison, urea and ammonia excretion rates were also measured in adult cod. The second objective was to establish whether urea cycle enzymes were induced during early development in cod. Activities of CPSase III, glutamine synthetase (GSase), ornithine transcarbamoylase (OTCase) and arginase were measured in whole embryos and larvae (up to 52 d.p.f.), and in various tissues of adult cod for comparative purposes. Materials and methods Animals and experimental protocol Atlantic cod (Gadus morhua L.) embryos were obtained from St Andrew s Biological Station (St Andrew s, New Brunswick, Canada) and the Ocean Sciences Centre (St John s, Newfoundland, Canada). New Brunswick (NB) broodstock were caught off the coast of St Andrew s and maintained in captivity for at least 1 year prior to spawning in February Newfoundland (NF) broodstock were caught off the coast of St John s and maintained in captivity for 2 years prior to spawning in April For each of the NB and NF batches, eggs from 6 8 females were mixed together and fertilized with the milt from 2 3 males. Embryos were maintained in static trays (1 l) with reconstituted salt water (Instant Ocean TM, 4 C, ph 7.6, 35 salinity) for up to 27 days. Embryos floated in a single layer at the water surface, and water was replaced every 2 days. Hatching occurred between 19 and 21 d.p.f. The term embryo will be used to refer to the developing fish prior to hatch, and the term larva will refer to all of the other early developmental stages of cod examined in this study. Although larvae at 27 d.p.f. were used to measure excretion rates, insufficient tissue was available for measurement of nitrogen contents or enzyme activities. For this reason, and because exogenously feeding cod larvae are difficult to maintain in non-coastal facilities, larvae (23, 39 and 52 d.p.f., raised at 4 C) were acquired from the Department of Fisheries and Oceans (Mont-Joli, Québec, Canada). Due to the different sources of embryos and larvae, variability of data between embryonic and larval stages may have arisen from population differences as well as ontogenetic differences. Adult cod ( kg) were purchased from the H&H Market and Man Ming Lobsters Inc. (Mississauga, Ontario, Canada) and transported to the University of Guelph in large (75 l), well-oxygenated containers, where they were either killed immediately for tissue analyses or held for 1 2 weeks prior to experimentation. Fishes were maintained in circular tanks (7 l) supplied with recirculating salt water (Instant Ocean TM, flow rate 15 l min 1 ) at the experimental temperature (1 C), salinity (33 ) and ph (7.8). Ammonia levels in the water were kept below 8 µmol l 1 by passing water through a biological filter. Fishes were fed minced krill daily for the first 2 days, but were fasted thereafter in order to eliminate the effect of feeding history on nitrogen metabolism (Wood, 1993). To measure the rates of urea and ammonia excretion, 2 5 embryos or larval cod (.3.8 g) were placed in each of 4 1 open chambers containing 1 ml of aerated salt water (Instant Ocean TM, 4 C, ph 7.6, 35 salinity) for 4 h, except for day 7 NB (N=3) and day 27 NB (N=2). Mortality was not observed during the experiments. Water samples (1 ml) were collected from the bulk water container at h and from the experimental chamber at 4 h, and stored at 2 C for subsequent analysis of urea and ammonia concentrations. Nitrogen (N) excretion rates (µmolng 1 min 1 ) were calculated as the difference in concentration (µmol N l 1 )

3 Urea cycle enzyme expression in Atlantic cod 2655 between and 4 h, multiplied by the volume of the chamber (l), divided by the mass of the tissue (g) and the time period (24 min) (1 µmol urea l 1 =2 µmol N l 1 and 1 µmol ammonia l 1 =1 µmolnl 1 ). At the end of each experiment, animals were counted, blotted, weighed, frozen in liquid nitrogen and then stored at 8 C for subsequent analysis of tissue ammonia and urea contents and enzyme activities. To measure nitrogen excretion rates in adult cod, fishes were placed individually into opaque aquaria (35 l) with flowthrough salt water (flow rate: 5 l min 1 ) and internal aeration. Fishes were acclimated to the chambers for at least 24 h prior to the experiment, and then inflow was stopped (set volume 11 l) for 4 h. Water samples (1 ml) were collected at and 4 h and stored at 2 C for subsequent analysis of urea and ammonia concentrations. Controls with a known amount of ammonia added (no fish present) demonstrated that aeration did not cause ammonia loss during the experiment. At the end of the experiment, fishes were killed by a sharp blow to the head and weighed. Samples of skeletal muscle, intestine, liver and kidney tissues were excised from each fish, frozen in liquid nitrogen, and stored at 8 C for subsequent analysis of enzyme activities. Water and tissue ammonia and urea analysis Water samples were analyzed for ammonia levels using the indophenol blue method, as described by Ivančič and Degobbis (1984). Urea levels in water samples were measured colorimetrically using the diacetyl-monoxime method (Rahmatullah and Boyde, 198). For tissue measurements, frozen embryos or larvae (15 3 individuals;.25.5 g) were ground into a fine powder in liquid nitrogen, immediately deproteinized in 2 volumes of ice-cold perchloric acid (8 %) and centrifuged (14 g, 4 C) for 5 min. The supernatant was removed, neutralized with saturated KHCO 3 and centrifuged (14 g, 4 C) for 5 min. The final supernatant was analyzed for ammonia concentration using a Sigma diagnostic kit (171- C), and analyzed for urea concentration using the method of Rahmatullah and Boyde (198). Wet mass was used to calculate ammonia and urea tissue contents, which were expressed as µmolng 1. Preparation of extracts for enzyme analysis All preparative procedures were carried out at 4 C. Tissue (.3.5 g) was minced immediately following retrieval from 8 C and then homogenized (Euro Turrax T2b homogenizer) in 4.5 volumes of extract buffer [5 mmol l 1 4- (2-hydroxyethyl)-1-piperazine ethanesulfonic acid (Hepes), ph 7.5, 5 mmol l 1 KCl, 1 mmol l 1 dithiothreitol and.5 mmol l 1 ethylenedinitrilo-tetraacetic acid (EDTA)]. The resulting homogenate was sonicated (Vibracell CV ) for 32 s and then centrifuged (14 g, 4 C) for 1 min. Next, the supernatant was added to a Sephadex G-25 column (6 ml) equilibrated with extract buffer to remove low molecular mass (<5 1 3 M r ) substrates and effectors. Fractions containing most of the protein were pooled. The protein concentration of extract was measured before and after filtration to establish the dilution of the extract. Enzyme activities were corrected for this dilution and expressed per gram of wet mass (µmol g 1 min 1 ). Enzyme assays were performed immediately following elution of the extract. Enzyme assays CPSase, OTCase, arginase and GSase activities were measured at 26 C, essentially as previously described (see below). CPSase III and OTCase appear to function only in arginine or urea synthesis via the urea cycle, so the absence of either of these enzymes suggests that urea synthesis does not occur through this pathway (Felskie et al., 1998). Activities of argininosuccinate synthetase and argininosuccinate lyase were not measured because the levels of activities in fishes are typically very low (even in ureogenic teleosts) and difficult to quantify (Felskie et al., 1998). Reaction mixtures for the CPSase assay contained 5 mmol l 1 Hepes (ph 7.5), 5 mmol l 1 KCl, 25 mmol l 1 MgCl 2, 22 mmol l 1 phospho(enol)pyruvate, 2 i.u. pyruvate kinase, 2 mmol l 1 adenosine 5 -triphosphate (ATP), 5 mmol l 1 NaHCO 3,.75 mmol l 1 [ 14 C]NaHCO 3 (approximately cts min 1 ),.5 mmol l 1 dithiothreitol,.5 mmol l 1 EDTA,.1 ml of tissue extract and, where indicated, 15 mmol l 1 glutamine,.5 mmol l 1 N-acetyl-L-glutamate (AGA) and/or 2.5 mmol l 1 uridine triphosphate (UTP), in a final volume of.3 ml. [ 14 C]carbamoyl phosphate formed after 6 min was measured as described by Anderson et al. (197). Mitochondrial CPSase III (a urea cycle enzyme) and cytosolic CPSase II (involved in pyrimidine biosynthesis) can both be present in fish tissues. CPSase III requires glutamine as a substrate and AGA as a positive effector. CPSase II can use either ammonia or glutamine as a substrate, does not require AGA, and is inhibited by uridine triphosphate (UTP). To differentiate between the two types, the effects of glutamine, AGA and UTP on CPSase activity were measured. Total CPSase (II+III) activity was defined as activity in the presence of glutamine and AGA, and CPSase II activity was defined as activity in the presence of glutamine alone. CPSase III activity was calculated by subtracting CPSase II activity from total CPSase activity. In teleost fishes, CPSase III activity is typically low relative to CPSase II activity (Randall et al., 1989; Felskie et al., 1998), so variability of CPSase II activity can have large effects on estimates of CPSase III activity (P. M. Anderson, personal communication). Previous studies employing the assay of Anderson et al. (197) have used the ratio of total CPSase activity to CPSase II activity in order to qualify the presence of CPSase III (Anderson, 198; Anderson and Walsh, 1995; Korte et al., 1997; Felskie et al., 1998; Kong et al., 1998). In the present study, apparent levels of product formation by CPSase III were well above the estimated limits of detection for the CPSase assay, so absolute CPSase III activity was reported. OTCase activity was measured by monitoring the production of citrulline after and 4 min, using the reaction mixtures of Wright et al. (1995) and the colorimetric method described by Xiong and Anderson (1989). Reaction mixtures

4 2656 T. D. CHADWICK AND P. A. WRIGHT for arginase were as described previously (Felskie et al., 1998); the reaction was terminated after and 4 min and urea was measured using the diacetyl-monoxime method (Rahmatullah and Boyde, 198). Reaction mixtures for measuring GSase activity were as described by Shankar and Anderson (1985); -glutamyl hydroxamate formation in the transferase reaction was measured after reaction for and 4 min. The GSase transferase reaction is about 2-fold higher than the biosynthesis rate. The estimated limits of detection (in µmol g 1 min 1 ) for the enzyme assays were.4 (CPSase),.1 (arginase),.8 (GSase) and.1 (OTCase). Protein measurement Protein concentrations of extracts were measured using the dye-binding method described by Bradford (1976), with reagents from Bio-Rad Laboratories and bovine serum albumin as a standard. Statistics All data are presented as means ± standard error (S.E.M.). Two-way ANOVAs were performed (with embryo population and days post-fertilization as factors) using a General Linear Models procedure with the statistical software SAS (SAS Institute Inc., Cary, NC, USA). Differences between populations of embryos were assessed at P.5 using the Tukey Kramer test for unequal means (Kramer, 1956). Statistical testing for differences between NB and NF embryos was performed by analyzing the data for the entire period of embryogenesis, because the purpose of comparing populations was to observe whether trends were different rather than to observe whether values differed at specific developmental stages. Results Total mass Total mass was relatively constant throughout embryonic development, but decreased by 81 % over the hatching period (Fig. 1). Total mass was not significantly different between the NF and NB embryo populations. Urea and ammonia excretion rates Ammonia excretion was detected shortly after fertilization (2 d.p.f.; Fig. 2) and increased continuously after 7 d.p.f. throughout embryogenesis. The pattern of urea excretion did not parallel that of ammonia; it was initially (5 d.p.f.) relatively high and then diminished to a minimum rate (1 15 d.p.f.) before increasing after hatching. The percentage of total nitrogenous waste (ammonia+urea) excreted as urea by embryos was initially relatively high (5 1 %; Fig. 3), decreased several-fold before hatching (to about 2 % of total), and then increased slightly after hatching (25 35 %). NB embryos excreted ammonia at significantly lower rates, and urea at significantly higher rates, than NF embryos (P.5). Also, the proportion of total nitrogenous waste excreted as urea by NB embryos was significantly higher than by NF embryos Total mass (mg individual -1 ) Hatching Newfoundland, NF New Brunswick, NB Fig. 1. Total mass of embryos (circles) and larvae (filled squares) of Atlantic cod up to 52 days post-fertilization. Values are means ± S.E.M. of 4 1 measurements. Total mass of New Brunswick (NB) embryos was not significantly different from that of Newfoundland (NF) embryos. Larvae used for post-hatching analysis were acquired from Mont-Joli, Québec. See Materials and methods for details. Nitrogen excretion rate (µmol N g -1 min -1 ) Newfoundland, NF New Brunswick, NB Hatching Ammonia Urea Adults Fig. 2. Ammonia-N and urea-n excretion rates (per gram of wet mass) for embryos and larvae (up to 27 days post-fertilization, circles) and adults (open square) of Atlantic cod. Values are means ± S.E.M. of 4 1 measurements, except for day 7 NB (N=3) and day 27 NB (N=2). Ammonia-N and urea-n excretion rates of NB embryos were significantly different from those of NF embryos (P.5; see Materials and methods).

5 Urea cycle enzyme expression in Atlantic cod 2657 % Urea excretion Hatching Newfoundland, NF New Brunswick, NB Adults Fig. 3. Percentage of total nitrogen excretion (ammonia+urea) excreted as urea by embryos and larvae (up to 27 days postfertilization, circles) and adults (open square) of Atlantic cod. Values were calculated from data in Fig. 2. NB embryos excreted significantly higher proportions of urea than NF embryos (P.5; see Materials and methods). (P.5). Ammonia excretion rates of adult cod were fivefold lower than the rates for larvae at 27 d.p.f. and urea excretion rates of adults were an order of magnitude lower than those for larvae at 27 d.p.f. (Fig. 2). Adults excreted 17 % of their total nitrogenous wastes as urea, which is lower than the proportion observed for most embryos (Fig. 3). Urea and ammonia tissue levels Ammonia content increased several-fold throughout embryogenesis, peaking at 15 d.p.f. (about 19 mmol N g 1 wet mass; Fig. 4). Upon hatching, ammonia content decreased by about 53 %, and decreased again by about 81 % from d.p.f. Urea content decreased in NF embryos and increased in NB embryos throughout embryogenesis. The pattern of change in urea content during larval development was similar to that of ammonia content. Ammonia and urea contents were significantly different between the NB and NF embryo populations (P.5). Urea cycle enzyme activities Total CPSase activity (CPSase II+CPSase III) was relatively low in embryos (Fig. 5A). Activity generally increased during late embryogenesis, and was several-fold higher than initial levels by hatching. Total CPSase activities were significantly higher in NB embryos than in NF embryos (P.5). Activity of total CPSase in adult liver was 11-fold lower than the activity in larvae at 52 d.p.f. It should be noted that enzyme activities in embryos are underestimated prior to hatching, because the chorion and perivitelline fluid (which account for about 81 % of the total embryonic mass but probably do not contain enzymes) were included in the mass analysis. Nitrogen content (µmol N g -1 ) Hatching Newfoundland, NF New Brunswick, NB Ammonia Urea Fig. 4. Ammonia-N and urea-n contents (per gram of wet mass) in whole embryos (circles) and larvae (filled squares) of Atlantic cod up to 52 days post-fertilization. Values are means ± S.E.M. of 4 14 measurements. Ammonia-N and urea-n contents of NB embryos were significantly different from those of NF embryos (P.5). Other details are as in Fig. 1. The pattern of CPSase III expression (Fig. 5B) was similar to that of total CPSase (Fig. 5A); CPSase III activity was detected shortly after fertilization (2 d.p.f.), generally increased during late embryogenesis and was several-fold higher than initial values by hatching. NB embryos expressed CPSase III at significantly higher levels than NF embryos (P.5). CPSase III activity was not detected in adult liver (Fig. 5B). CPSase II was detected in all embryonic and larval stages examined at variable activities (data not presented). The effects of substrate and effectors on the activity of CPSase are presented for tissues of adult cod in Table 1. There was no activation of activity by AGA in any tissue, indicating the absence of CPSase III. The complete inhibition of activity in each tissue by uridine triphosphate (UTP) indicates that all of the CPSase activity was CPSase II activity (because CPSase II but not CPSase III is inhibited by UTP). CPSase II activity was highest in intestine and kidney, lower in liver and lowest in skeletal muscle. OTCase and arginase, like CPSase III, were present in all embryonic and larval stages examined. OTCase activity was detected as early as 2 d.p.f., and was expressed at variable levels during embryogenesis and larval development (Fig. 5C). NB embryos expressed significantly higher OTCase activities than NF embryos (P.5). OTCase activity in adult liver was low relative to larval levels (Fig. 5C), and was similar to levels

6 2658 T. D. CHADWICK AND P. A. WRIGHT Total CPSase activity (µmol g -1 min -1 ) A Hatching Newfoundland New Brunswick CPSase III activity (µmol g -1 min -1 ) B Adult liver Adult liver 1.8 C 4 D OTCase activity (µmol g -1 min -1 ) GSase activity (µmol g -1 min -1 ) Adult liver Adult liver 6 E Fig. 5. Activities (per gram of wet mass) of urea cycle enzymes in whole embryos (circles) and larvae (filled squares) up to 52 days post-fertilization and in adult liver tissue (open squares) of Atlantic cod. Values are means ± S.E.M. except where N=1 (indicated by the absence of error bars). Larvae used for post-hatching analysis were acquired from Mont-Joli, Québec (see Materials and methods). (A) Total carbamoyl phosphate synthetase (CPSase) activity was measured in the presence of glutamine and N-acetyl-L-glutamate (AGA) and therefore represents both CPSase II (AGA-independent) and CPSase III (AGA-dependent) activities (N=2 4). (B) CPSase III activity (N=2 4). (C) ornithine transcarbamoylase (OTCase) activity (N=2 6). (D) Glutamine synthetase (GSase) activity (N=2 6). (E) Arginase activity (N=2 6). Total CPSase, CPSase III and ornithine transcarbamoylase OTCase activities in embryos from New Brunswick were significantly higher than those of Newfoundland embryos, but arginase was not significantly different between embryo populations. Arginase activity (µmol g -1 min -1 ) Adult liver

7 Urea cycle enzyme expression in Atlantic cod 2659 Table 1. Activities (per gram of wet mass) of various urea cycle enzymes in muscle, intestine, liver and kidney tissues of adult Atlantic cod Substrate and effector(s) present Muscle Intestine Liver Kidney CPSase Glutamine.14±.3 (5).55±.11 (4).28±.2 (4).45±.23 (4) Glutamine+AGA.16±.5 (5).54±.11 (4).22±.19 (4).49±.26 (4) Glutamine+AGA+UTP.4±.3 (5).1±.1 (4).1± (4) (4) OTCase.5±.8 (6).74±.2 (6).31±.9 (6).63±.12 (6) GSase.5±.1 (6) 1.87±.65 (6).35±.5 (6).26±.4 (6) Arginase.23±.1 (6).16±.4 (6) 4.2±1.34 (6) 2.76±.61 (6) CPSase, carbamoyl phosphate synthetase; OTCase, ornithine transcarbamoylase; GSase, glutamine synthetase; AGA, N-acetyl-L-glutamate. Enzyme activities (nmol product g 1 min 1 for CPSase; µmol product g 1 min 1 for OTCase, GSase and arginase); values are mean ± S.E.M. (N). CPSase activity was measured in the presence of glutamine alone, glutamine with AGA, or glutamine with AGA and uridine triphosphate (UTP), to differentiate between CPSases II and III. See Materials and methods for details. of OTCase activity in other adult tissues (Table 1). GSase activity was relatively low during embryogenesis (2 21 d.p.f.; Fig. 5D), but increased throughout larval development. GSase activity in 52 d.p.f. larvae was about ninefold higher than that in adult liver (even higher compared with adult skeletal muscle and kidney tissues), but was slightly lower than GSase activity in adult intestine (Fig. 5D and Table 1). Arginase activity was relatively low from 2 21 d.p.f. (Fig. 5E) but increased throughout larval development. Arginase activity was not significantly different between the NF and NB embryo populations. In contrast to the other enzymes, arginase activity in adult liver was higher than the levels in larvae at 52 d.p.f. (Fig. 5E). Arginase activity was also relatively high in adult kidney, but was lower in adult intestine and muscle (Table 1). Discussion The results indicate that four of the six urea cycle enzymes measured were expressed during embryonic and larval stages of the Atlantic cod at relatively high levels, and urea was excreted by embryos and larvae as a significant proportion of total nitrogen excretion (1 1 %). The expression of urea cycle enzymes in embryos and larvae is conspicuous, because activities of these same enzymes in adult cod were low or undetectable, as they are in adults of most teleost species (Huggins et al., 1969; Anderson, 198; Felskie et al., 1998). Based on these results, we reject the hypothesis that expression of the urea cycle during early life stages is unique to rainbow trout. Furthermore, the fact that two phylogenetically distant teleosts with different life histories induce urea cycle enzymes during embryogenesis provides support for the hypothesis that early urea cycle enzyme expression is universal among teleost fishes. CPSase III, GSase, OTCase and arginase activities were detected very early in cod development; this is the first report of CPSase III activity in a teleost before hatching. For rainbow trout, simultaneous expression of CPSase III, GSase, OTCase and arginase did not occur until after hatching (45 d.p.f., Wright et al., 1995). However, CPSase III mrna has been detected very early in trout development (3 d.p.f., Korte et al., 1997). In cod, activities of urea cycle enzymes were highest in larvae at 52 d.p.f. Levels of expression of CPSase III, GSase, OTCase and arginase in cod larvae are similar to the highest levels reported in the early development of rainbow trout (Wright et al., 1995), but CPSase III activity in cod larvae is two to three orders of magnitude lower than CPSase III activity in the liver tissue of elasmobranchs and ureogenic teleosts (Casey and Anderson, 1982; Saha and Ratha, 1987; Mommsen and Walsh, 1989; Randall et al., 1989). CPSase II (involved in pyrimidine biosynthesis) was expressed in cod embryos and larvae (data not presented) at levels that are comparable with those in tissues of adult cod, and to those reported in liver extracts of other teleostean species (Felskie et al., 1998), including the Pacific cod Gadus macrocephalus (Anderson, 198). In the present study, enzyme activities were measured from whole-animal preparations and expressed per gram of wet mass, so the perivitelline fluid (PVF) and the acellular chorion (representing 81 % of the embryonic mass) were included in the analysis, even though they probably did not contain enzymes. Further, the yolk sac makes up a large proportion of both embryos and larvae of cod (Morrison, 1993), and although it is not known whether urea cycle enzymes are present in cod yolk, levels of CPSase III, GSase, OTCase and arginase are low or undetectable in the yolk of rainbow trout (T. D. Chadwick and P. A. Wright, unpublished results). As a result, enzyme activities were probably underestimated prior to the disappearance of the yolk sac (i.e. 3 d.p.f.). Arginase and GSase were expressed in some adult tissues at higher levels than in early stages. This is not surprising, because these enzymes are not exclusively involved with the urea cycle pathway. Arginase is involved in the metabolism of dietary arginine, which it hydrolyzes into urea (arginolysis). GSase catalyzes the conversion of glutamate and ammonia into glutamine, which is a substrate for purine biosynthesis; purines can subsequently be broken down into urea (uricolysis). Glutamine also acts as a storage molecule for ammonia and therefore, GSase is important in ammonia detoxification (Wu,

8 266 T. D. CHADWICK AND P. A. WRIGHT 1963). Both arginase and GSase are present in adult teleosts (in a number of tissues) in the absence of a complete urea cycle (Cvancara, 1969; Felskie et al., 1998). Ammonia excretion rates and ammonia contents of cod embryos in the present study correspond to those reported previously for Atlantic cod embryos (Finn et al., 1995a). In cod embryos, ammonia accumulated throughout embryogenesis, even though ammonia excretion rates also increased with development. Comparatively, urea content was relatively unchanged throughout embryogenesis. In several studies of nitrogen metabolism and excretion in the early life stages of fishes (including the Atlantic cod), urea excretion was assumed to be insignificant and therefore was not measured (Buckley and Dillmann, 1982; Finn et al., 1991, 1995a,b, 1996). The present study has demonstrated that urea can be a significant component of nitrogen excretion in cod. Furthermore, urea excretion has been observed in early stages of rainbow trout Oncorhynchus mykiss (Wright et al., 1995; Wright and Land, 1998), common carp Cyprinus carpio (Kaushik et al., 1982), coregonid Coregonus schinzi palea (Dabrowski and Kaushik, 1984a), whitefish Coregonus lavaretus (Dabrowski et al., 1984b) and African catfish Clarias gariepinus (Terjesen et al., 1997), constituting between 36 % of total nitrogen excretion. Thus, when determining the nitrogen budget for early development in teleosts, urea may constitute a significant portion of total nitrogen excretion. For some of the embryonic stages of cod, urea was the predominant nitrogen waste excreted. This is the first report of an oviparous teleost excreting predominantly urea during early life stages. To our knowledge, the viviparous eelpout (Zoarces viviparus) is the only other teleost that has been reported to excrete predominantly urea during early development (about 65 % of total nitrogen; Korsgaard, 1994). Korsgaard (1994) reported that urea excretion by post-yolk sac embryos of Z. viviparus decreased after acclimation to sea water, and suggested that their capacity for urea excretion may be related to the prolonged stay in ovario and the accumulation of toxic ammonia levels. However, no significant effect of external ammonia on urea excretion in embryos was observed in a later study (Korsgaard, 1996) and urea cycle enzymes have not been measured in this species. In rainbow trout, there was a significant elevation of urea excretion in free embryos (hatched) exposed to conditions that inhibit ammonia excretion (i.e. elevated ammonia or elevated ph), but not in embryos (Wright and Land, 1998). Although it is plausible that early expression of urea cycle enzymes in trout and cod relate to detoxification of endogenous or exogenous ammonia, further study is required to test this hypothesis directly. Evidence in support of an important role for the urea cycle in early cod development is also apparent when one compares the data from the two cod populations. Urea excretion rates were higher for New Brunswick (NB) embryos than for Newfoundland (NF) embryos, and this corresponds to higher activities of CPSase III and OTCase (enzymes that are exclusively involved with the urea cycle). The differences in enzyme activity between NB and NF embryos are not due to proximate changes in metabolic rate, because all embryos were obtained on the second day after fertilization and were reared under identical environmental conditions. One ecological difference that exists between the populations relates to the season of spawning: the first spawn of NB cod occurs in February, whereas the first spawn of NF cod occurs in April. Hence, population differences in the parental reproductive schedule may translate to differences in offspring metabolism. Whatever the reason for the differences, the fact that higher activities of CPSase III and OTCase correspond to higher urea excretion rates suggests that upregulation of the urea cycle resulted in higher levels of urea synthesis. Adult cod excreted 17 % of their total nitrogen as urea, this value is typical of most teleost species (Wood, 1993). CPSase III was absent from adult tissues and levels of OTCase activity were low, so urea was presumably derived from arginolysis and/or uricolysis rather than from the urea cycle. The relatively high levels of arginase activity in liver and kidney tissues indicate ample capacity for arginolysis in adult cod. In summary, this study demonstrates that the early induction of urea cycle enzymes is not unique to rainbow trout, as it is also found in Atlantic cod. Taken together, these findings support the hypothesis of Griffith (1991) that all teleosts express the urea cycle during early ontogeny. 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