Elasmobranch fishes, the coelacanth, estivating lungfish, amphibians, and mammals synthesize urea by the ornithine-urea cycle; by comparison, urea synthetic activity is generally insignificant in teleostean fishes. It is reported here that isolated liver cells of two teleost toadfishes, Opsanus beta and Opsansus tau, synthesize urea by the ornithine-urea cycle at substantial rates. Because toadfish excrete ammonia, do not use urea as an osmolyte, and have substantial levels of urease in their digestive systems, urea may serve as a transient nitrogen store, forming the basis of a nitrogen conservation shuttle system between liver and gut as in ruminants and hibernators. Toadfish synthesize urea using enzymes and subcellular distributions similar to those of elasmobranchs: glutamine-dependent carbamoyl phosphate synthethase (CPS III) and mitochondrial arginase. In contrast, mammals have CPS I (ammonia-dependent) and cytosolic arginase. Data on CPS and arginases in other fishes, including lungfishes and the coelacanth, support the hypothesis that the ornithine-urea cycle, a monophyletic trait in the vertebrates, underwent two key changes before the evolution of the extant lungfishes: a switch from CPS III to CPS I and replacement of mitochondrial arginase by a cytosolic equivalent.

Evolution of urea synthesis in vertebrates: the piscine connection

The ornithinesurea cycle (OUC) is present in elasmobranch fish and many terrestrial vertebrates. Recently, a functional OUC has been reported in a few teleost species, suggesting that all teleost fish have the genes for the OUC, but expression is relatively rare. We investigated the possibility that the OUC is expressed during early development in trout as a mechanism for detoxifying ammonia produced from the catabolism of yolk protein. We followed ammonia and urea excretion rates, tissue ammonia and urea levels and OUC enzyme activities in rainbow trout up to 93 days after fertilization. Both ammonia and urea tissue concentrations increased several-fold in the first 40 days after fertilization (embryo stage), peaking at 1.7 mmol N l-1 and 2.5 mmol N l-1, respectively. Ammonia excretion could be detected in 4-day-old embryos, but urea excretion was not initiated until after hatching (day 45). Urea excretion in larval fish (days 42s93) increased several-fold and by day 93 was 14 % of total nitrogen excretion, as found in adult trout. Glutamine synthetase (GSase) and arginase activities were detected in 9whole animal9 homogenates just after hatching and the levels of activity increased markedly to day 93. Carbamoyl phosphate synthetase (CPSase) and ornithine transcarbamylase (OTCase) were first detected in 40-day-old embryos; activities peaked between days 53 and 71 and then subsequently decreased. Adult liver enzyme activity for GSase was several-fold lower than in whole larval trout and OTCase and CPSase III (glutamine- and N-acetylglutamate-dependent CPSase catalysing the first step of the OUC) activities were essentially absent in adult liver. We conclude that embryonic and larval trout are primarily ammoniotelic. Urea is synthesized immediately after fertilization, but is not excreted until after the embryo is hatched. The results provide evidence for the presence of the OUC in larval rainbow trout, since four of the OUC enzymes are induced just after hatching and the levels of activity are relatively high compared with those in adult liver tissue. Furthermore, we suggest that all teleosts have retained the OUC genes, which are expressed only during certain stages of development (embryogenesis), and in a few rare species expression is maintained throughout the life cycle to cope with unusual environmental conditions (e.g. alkaline water, air exposure).

Induction of ornithine-urea cycle enzymes and nitrogen metabolism and excretion in rainbow trout (Oncorhynchus mykiss) during early life stages

Under long-term (24 h) control measurements, significant urea was excreted (15 to 90% of excreted nitrogen) byOpsanus beta (Goode and Bean) collected in 1989 from Biscayne Bay, Florida, USA. Urea excretion rates and plasma urea concentrations were not affected by antibiotic treatments which decreased intestinal microbe populations. These results suggest that nitrogen recycling by gut microbe urease is probably not significant in this species. Urea excretion rates increased significantly following 8 h of air-exposure and in response to high levels of NH4Cl. These results suggest that urea is synthesized and excreted by the toadfish primarily in situations that limit ammonia excretion. Thus, the ornithineurea cycle in the toadfish liver primarily maintains low concentrations of ammonia within the fish. High levels of variability in urea excretion rates and variation in response to air-exposure suggest that urea synthetic rates are affected by immediate past environmental conditions.

Variation in urea excretion in the gulf toadfish Opsanus beta

Ammonia and urea dynamics in the Lake Magadi tilapia, a ureotelic teleost fish adapted to an extremely alkaline environment.

Rainbow trout (Oncorhynchus mykiss) survived in alkaline fresh water (pH = 9.50) for 72 h, although the exposure rendered the fish more susceptible to mortality from other causes. At pH 9.50 ammonia excretion ($J_{Amm}$) was initially blocked, and total plasma ammonia levels increased. However, $J_{Amm}$ steadily recovered thereafter; by 48 h control rates were reestablished and plasma total ammonia stabilized at six times the control level. The initial blockade of $J_{Amm}$ was associated with a reversal of the blood to bulk water PNH₃ gradient. Paradoxically, the continued depression of $J_{Amm}$ until 48 h occurred despite the presence of favorable blood-to-water gradients for passive NH₃ and $NH_{4}^{+}$ difusion. An increase in urea excretion helped sustain waste N excretion in the face of inhibited $J_{Amm}$. A respiratory alkalosis (decreased arterial Pco₂, increased arterial pH) occurred initially but was partially counteracted by a metabolic acidosis (decreased plasma $HCO_{3}^{-}$), which stabil...

Nitrogenous Waste Excretion, Acid-Base Regulation, and lonoregulation in Rainbow Trout (Oncorhynchus mykiss) Exposed to Extremely Alkaline Water

1. 1. Relatively high activities of all five of the ornithine-urea cycle enzymes were found in liver of the teleost Opsanus tau. 2. 2. The substrate and cofactor requirements of these enzymes appear to be the same as those of most other vertebrates. 3. 3. Concentrations of urea and ammonia in the blood of Opsanus tau are low, and the rate of urea excretion is low compared with the rate of ammonia excretion. 4. 4. Thus, there appears to be a lack of correlation between the levels of ornithine-urea cycle enzyme activity and the pattern of nitrogen excretion.

Lawrence J. Read

Papers

The presence of high ornithine-urea cycle enzyme activity in the teleost Opsanus tau