Ionic, Osmotic, and Nitrogenous Waste Regulation

6 IONIC, OSMOTIC, AND NITROGENOUS WASTE REGULATION PATRICIA A. WRIGHT

1. Introduction 1.1. Origins in Seawater 1.2. ‘‘Parting of the Ways’’: The Move to Freshwater 1.3. Key Sites of Osmoregulation and Nitrogen Excretion in Fishes 2. Ionic and Osmotic Regulation 2.1. In Seawater 2.2. In Freshwater 2.3. Moving Between the River and Sea 3. Nitrogen Excretion 3.1. Toxic Ammonia 3.2. Synthesis of Nitrogen End‐Products 3.3. Excretion 3.4. The Challenges of Estivation 4. Concluding Remarks

Among primitive fishes, there is a diversity of strategies that have evolved to cope with ion, water, and nitrogen balance. The whole physiological spectrum is found from ionic and osmotic conformation to the regulation of body fluids distinct from the environment. The most primitive of vertebrates, the marine hagfish iono‐ and osmoconforms to its seawater environment, whereas their euryhaline relatives, the lampreys, iono‐ and osmoregulate. The gills of Agnathans contain both pavement and mitochondrial rich cells, but the arrangement of cells and structural features are unique relative to euryhaline teleosts. Coelacanths are osmoconformers but ionoregulators, maintaining high internal urea levels like the elasmobranchs. In many primitive species, ammonia is the dominant excretory product as it is in most teleost fishes. The exception is the coelacanth and estivating lungfish that synthesize urea via the urea cycle and excrete urea. Membrane transporters have been isolated in fish that regulate urea and possibly ammonia movements between tissue 283 Primitive Fishes: Volume 26 FISH PHYSIOLOGY

Copyright # 2007 Elsevier Inc. All rights reserved DOI: 10.1016/S1546-5098(07)26006-6

284

PATRICIA A. WRIGHT

compartments and to the environment. Nitrogen excretion during early life stages presents a particular challenge in encapsulated embryos dependent on yolk protein catabolism. As yet, little is known about how primitive fish embryos face these challenges. Research on primitive fish species will broaden our knowledge of the evolution of osmoregulation and excretion in fish and terrestrial vertebrates. 1. INTRODUCTION The focus of this chapter is on the Agnathans (lampreys and hagfishes), the Sarcopterygians (the coelacanth and the lungfishes), and the primitive Actinopterygians such as the Polypteriformes (bichirs and reedfish), the Chondrostean Acipenseriformes (paddlefishes and sturgeons), and the Neopterygians (gars and the bowfin). The systematics and phylogeny of these fishes is outlined by P. Janvier in Chapter 1. This chapter concerns the regulation of ions, water, and nitrogen end‐products. Although the endocrine control of these processes is important in their regulation, the current chapter focuses on the sites, structures, and mechanisms involved in iono‐ and osmoregulation. Readers are referred to two comprehensive chapters on ‘‘Peripheral Endocrine Glands’’ by J. Youson in this volume (Chapters 8 and 9) for a more detailed review of endocrine control. 1.1. Origins in Seawater Over the last 100 years or so, scientists have debated whether the first fishes evolved in freshwater or seawater (Smith, 1932, 1961; Munz and McFarland, 1964; Fa¨nge, 1998). These arguments are based on osmoregulatory structures present in extant fishes, including the most primitive jawless fishes, the Agnathans. Collective opinion weighs in on the seawater side (GriYth, 1994; Holland and Chen, 2001; Chang et al., 2006); early Agnathans are thought to have inhabited shallow seas or estuaries (Helfman et al., 1997). The living jawless hagfishes are entirely marine. They are unique among vertebrates in having plasma ion concentrations and osmolarity roughly the same as seawater, similar to marine invertebrates. 1.2. ‘‘Parting of the Ways’’: The Move to Freshwater Smith (1932) first noticed the interesting contrast between the blood osmotic concentration of the hagfish Myxine glutinosa with that of the lamprey Petromyzon fluviatilis. Hagfish iono‐ and osmoconform to their seawater habitat, whereas lamprey iono‐ and osmoregulate in either seawater or freshwater. It has taken decades to form a more complete understanding of vertebrate osmoregulation, but the following prescient statement by Smith

6.

OSMOREGULATION

285

(1932) captures so much. It is thus possible that these two groups lead back to a ‘‘parting of the ways’’ in the evolution of body fluid regulation. (The two groups he refers to are the hagfish and the lamprey.) After early beginnings in seawater, lamprey ancestors moved back to freshwater, no later than the early Cretaceous (Chang et al., 2006). Osmotic control that evolved first in lamprey and teleost ancestors has been an adaptive trait with a selective advantage, in both freshwater and marine environments (Robertson, 1963). Although the hagfish lineage has survived for millions of years, their unique form of iono‐ and osmoconformation has not appeared in any other group of aquatic vertebrate. 1.3. Key Sites of Osmoregulation and Nitrogen Excretion in Fishes The gills, kidney, intestine, urinary bladder, and integument are the key sites of ion exchange, nitrogen elimination, and osmoregulation in most fishes (for a review see Marshall and Grosell, 2006). In many of the primitive fish groups, there is limited information to verify the involvement of these sites or the importance of one tissue over another. However, it is probably safe to say that the gill is the dominant site of exchange of ions, water, and nitrogenous waste products in most of the primitive fishes discussed below. Among primitive fishes, with the exception of lungfish, the gill has a large surface area in contact with flowing water and with the aid of specialized branchial epithelium, materials are transported between the blood and water. The kidney is also an important structure, particularly in freshwater fishes where passive water gain is countered by a high urine output with reabsorption of key monovalent ions (see Section 2.2); however, renal nitrogen excretion is typically low. The intestine and urinary bladder are important for absorption of ions and water, but will not be considered further due to the lack of research on primitive fishes. The integument in most fish presents a barrier to the exchange between the internal and external environments, and may only play a critical role in a few unusual species (Wood, 1993; see Section 2.2). It should also be noted that a postanal gland, similar in structure to the elasmobranch rectal gland, has been described in the coelacanth Latimeria chalumnae (reviewed by Locket, 1980). This gland probably represents an additional site of NaCl secretion. 2. IONIC AND OSMOTIC REGULATION 2.1. In Seawater 2.1.1. Ionoconform, Osmoconform: The Hagfish Strategy Hagfish are the only aquatic vertebrate known that ionoconform as well as osmoconform to their seawater environment (Figure 6.1A) (Robertson, 1963; Evans, 1993; Karnaky, 1998). They are not found in water of low salinity,

286

PATRICIA A. WRIGHT

Fig. 6.1. Schematic representation of ionic and osmotic balance in (A) hagfish, (B) coelacanth, (C) seawater lamprey, and (D) freshwater lamprey. Total plasma osmolality (mosmol kg
1), plasma osmolality attributed to NaCl (mosmol kg
1), and plasma urea concentrations (mmol kg
1) are given inside each diagram, were appropriate. Passive fluxes are represented by a dashed line, whereas active mechanisms are shown as a solid line. [A, values from McDonald and Milligan (1992), B, GriYth (1991), and C and D modified from Bartels and Potter (2004).]

6.

OSMOREGULATION

287

contrasting sharply with their Agnathan relatives, the lampreys (see Section 2.1.3, below). They gain weight in hyposmotic water, slowly recovering after 7 days, whereas in hyperosmotic water they lose weight and fail to recover (McFarland and Munz, 1965). Smith (1932) established that the ionic composition of hagfish blood approximated the inorganic ion concentrations of seawater (Figure 6.1A), a very diVerent osmoregulatory strategy compared to osmoconforming coelacanth and elasmobranch fishes (Figure 6.1B; see Section 3.3.3). Similar to elasmobranchs, the intracellular osmotic composition of marine hagfish has a large organic component, with trimethylamine oxide (TMAO) concentrations exceeding 200 mM and one‐third lower inorganic ion levels relative to serum levels (Bellamy and Jones, 1961). There has been very little attention to the mechanisms of iono‐ and osmoregulation in hagfish, possibly because it was assumed that iono‐ and osmoconforming with the environment requires minimal eVort. Large mitochondrial rich cells (MRCs) are numerous on hagfish gill lamellae (Mallat and Paulsen, 1986; for a review see Bartels, 1998; Choe et al., 1999), but unlike seawater‐acclimated lampreys (see Section 2.1.3) the MRCs appear singly, sandwiched between gill pavement cells. A leaky paracellular junction that is so clearly observed between MRCs in marine teleosts and lampreys and allows for the passive leak of Naþ is not apparent in hagfish gills. Hagfish gill MRCs stain for Naþ/Kþ ATPase (Mallat et al., 1987; Choe et al., 1999) and Naþ/Hþ exchanger isoforms (NHE) are also expressed in the M. glutinosa gill but cell localization has yet to be determined (Edwards et al., 2001; Choe et al., 2002). Evans (1984) proposed that NHE and Cl
/HCO
3 exchangers were operating in parallel in hagfish gill epithelium for acid or base excretion, and provided a ‘‘preadaptation’’ for ion regulation in species that later inhabited freshwaters. This proposition was later supported by McDonald et al. (1991) who showed that acid–base disturbances in M. glutinosa were fully corrected by gill mechanisms, probably involving NHEs. Indeed, gill NHE mRNA is upregulated in hagfish gill tissue following an acid infusion (Edwards et al., 2001). It might be expected of the iono‐ and osmoconforming hagfish that passive water influx (found in freshwater teleosts) or passive water eZux (found in seawater teleosts) would be minimal, raising the question of kidney structure and function (GriYth, 1994; Fels et al., 1998). The hagfish kidney is unusual in having large glomeruli (500–1500 mm) and two archinephric ducts or ureters (Riegel, 1998). There have been only a few studies on kidney function (Karnaky, 1998). Overall, the hagfish kidney functions in the reabsorption of glucose and amino acids and secretion of some ions (Munz and McFarland, 1964; Riegel, 1998); however, there are some discrepancies in the literature whether reabsorption of Naþ or Cl
occurs (McInerney, 1974; Alt et al., 1981). Little progress has been made over the last 40 years or so on understanding of iono‐ and osmoregulation in hagfish. More information is required on

288

PATRICIA A. WRIGHT

the stability of plasma NaCl concentrations under diVerent physiological conditions (e.g., feeding, exercise, and acid–base disturbances) to determine if NaCl in the blood is regulated under some circumstances. This would go a long way in understanding the potential roles of gill MRCs and specific ion transporters. Osmotic disturbances may be primarily due to the composition of the diet in this stenohaline fish. Using traditional methods to study ion fluxes and water balance, it would be valuable to know how hagfish compensate for a meal with a high water content (e.g., teleost tissues) versus an isosmotic meal (e.g., invertebrate, elasmobranch tissues). 2.1.2. Ionoregulate, Osmoconform: The Coelacanth Strategy An overall understanding of ionic and osmotic regulation is lacking in Latimeria, with only two surviving species of Coelacanths, L. chalumnae (Comoro Islands and vicinity) and L. menadoensis (discovered in 1999 oV the coast of Indonesia) (Holder et al., 1999). It is well accepted that L. chalumnae plasma and tissues contain elevated concentrations of urea similar to marine elasmobranchs and they osmoconform to their seawater environment (Figure 6.1B). What is less clear is whether plasma osmolality is somewhat greater (as in elasmobranchs); equal to or slightly less than (GriYth, 1991) the local marine environment. The confusion over this issue no doubt relates to a limited number of samples that have been collected from either frozen (Pickford and Grant, 1967) or moribund specimens (GriYth et al., 1976). On the basis of available evidence, it does appear that plasma NaCl concentrations are 25% lower than values reported in marine elasmobranchs (McDonald and Milligan, 1992). If this is indeed the case, then there are several implications. First, other organic osmolytes besides urea and TMAO (GriYth et al., 1974) must be present in the plasma to add up to 1000 mosmol kg
1. Second, to maintain a combined osmolality of plasma NaCl at 350 mosmol kg
1 (Figure 6.1B), a value almost identical to stenohaline marine teleosts (346 mosmol kg
1; McDonald and Milligan, 1992), L. chalumnae, must have powerful mechanisms to secrete NaCl. Are these mechanisms partly in the gill (i.e., chloride‐type cells) or solely in the postanal gland? The structure of the kidney resembles other osteichthyes (Locket, 1980), but measurements of a single urine sample are insuYcient to understand renal function (GriYth et al., 1976). There is much to learn and physiologists await the opportunity to study multiple live Latimeria. 2.1.3. Ionoregulate, Osmoregulate: The Alternative Strategy In seawater, marine teleosts as well as lampreys and sturgeons maintain body fluid osmolality and NaCl concentrations at about one‐third of their environment (Morris, 1972; Potts and Rudy, 1972; Beamish, 1980a). The seawater origins of Agnathans [with lampreys later entering freshwater

6.

OSMOREGULATION

289

(Chang et al., 2006)], but freshwater origins of Teleostei (with subsequent forays to seawater) suggest that convergent evolution may explain the remarkable similarity of gill and renal osmoregulatory mechanisms in these two groups of fish (Bartels and Potter, 2004). Fish ion and osmoregulation have been well reviewed (Marshall, 2002; Bartels and Potter, 2004; Marshall and Grosell, 2006). Ionic and osmotic gradients result in the constant influx of NaCl and loss of body water that are counterbalanced by active excretion of NaCl across the gills and replenishment of water by drinking (Figure 6.1C). The branchial epithelium, therefore, plays an important role in ionoregulation. The structure and cellular composition of the lamprey gill have been extensively described (reviewed by Bartels and Potter, 2004). Chloride cells in lamprey gills in seawater form long rows and lack accessory cells that are associated with chloride cells in teleosts. The apical crypts so distinctive in teleost gills in seawater are absent in lampreys. Despite these small structural diVerences, the lamprey chloride cells share most of the other characteristics typical of teleost fishes and other salt‐secreting epithelia, such as a high density of mitochondria, basolateral membrane elaboration, and a leaky paracellular pathway (Laurent, 1984; Karnaky, 1986; Bartels and Potter, 2004). Furthermore, the mechanism of active NaCl secretion, involving Naþ/Kþ ATPase, Naþ/Kþ/2Cl
cotransporter, and a chloride channel (Marshall and Grosell, 2006), is assumed to be present in seawater lamprey gills (Bartels and Potter, 2004), but this has not been verified. The kidney plays a small role in lamprey osmoregulation in seawater, producing low volumes of urine as would be expected of marine osmoregulators (Logan et al., 1980). Similar to teleosts, lamprey and sturgeon kidneys preferentially secrete divalent ions in the urine (Pickering and Morris, 1970; Logan et al., 1980; Krayushkina et al., 1996). Following transfer from freshwater to brackish water, glomeruli size declines and the tubule cells and brush border were reduced in two sturgeon species, Acipenser naccarii (Cataldi et al., 1995) and Huso huso (Krayushkina et al., 1996), implying reduced function in hypersaline water. 2.2. In Freshwater In freshwater, iono‐ and osmoregulation in primitive fishes is accomplished as outlined in Figure 6.1D for freshwater lamprey, similar to freshwater teleosts. Plasma is hyperosmotic to the surrounding media and therefore passive water gain and NaCl loss must be compensated by the elimination of copious volumes of urine and the active uptake of ions via the branchial epithelium. The majority of studies on primitive fish osmoregulation have focused on lampreys and sturgeon, with far fewer on bowfin,

290

PATRICIA A. WRIGHT

gar, paddlefishes, and birchirs. Elegant studies by Bull and Morris (1967) and Morris and Bull (1968, 1970) established that freshwater ammocoete larva of Lampetra planeri carefully regulate serum and tissue water and ion content, that external calcium aVects gill permeability to ions, and that sodium influx is dependent on both internal and external sodium concentrations. The life cycle of all species of lamprey consists of a larval phase (ammocoetes) in freshwater, metamorphosis into young adults that may migrate downstream to the ocean, if anadromous, a marine trophic phase, followed by a return migration upstream to freshwater streams where they spawn and die (Beamish, 1980b). The gill cell composition of lampreys changes with life cycle and external salinity. Larval gills have both ammocoete MRCs and intercalated MRCs, as well as pavement cells. Downstream migrants (freshwater) retain the intercalated MRCs and pavement cells, and new chloride cells develop (Youson and Freeman, 1976; Bartels and Potter, 2004). Choe et al. (2004) further identified two subtypes of MRCs based on immunohistochemical staining of the gills of freshwater adult lampreys. They proposed a model of ion transport where MRC‐A that express Naþ/Kþ ATPase are responsible for Naþ uptake, whereas MRC‐B that stain for carbonic anhydrase and V type Hþ ATPase transport Cl
. Verification of this model will require sophisticated separation of MRC‐A and MRC‐B type cells, similar to isolated cell studies in freshwater teleosts (see review by Marshall and Grosell, 2006). Kidney function in freshwater primitive fishes is probably comparable to teleosts. In bowfin and lampreys, the kidney reabsorbs Naþ and Cl
, and has a relatively high glomerular filtration rate that is correlated with urine flow rate (Logan et al., 1980; Butler and Youson, 1988). The renin‐angiotensin system (RAS) has been identified in the river lamprey Lampetra fluviatilis (Cobb et al., 2002; Brown et al., 2005). In vertebrates, the RAS plays an important role in blood volume and pressure regulation, and studies in L. fluviatilis indicate that the RAS responds to external water salinity changes (Rankin et al., 2001; Brown et al., 2005). Ionic and osmotic balance in submerged African lungfish has not been well studied, but presents an interesting challenge given the reduction of gill surface area (Laurent et al., 1978) and reliance on lung respiration (Perry et al., 2005a,b). Moreover, the absence of gill convection during terrestrial episodes may further exacerbate osmoregulatory control. Wilkie et al. (2007) have discovered that Protopterus dolloi remains in ionic and osmotic balance after a 6‐month episode on moist land, partly because they exchange water and ions across their ventral body surface. In fact, their data indicates that the majority of water exchange in submerged lungfish also occurs across the ventral skin, which may play a similar role as the pelvic region in amphibians. This is a fascinating avenue for future study.

6.

291

OSMOREGULATION

2.3. Moving Between the River and Sea The life history of anadromous lampreys and sturgeon involves an initial freshwater phase, followed by migration to the sea and a later return to freshwater streams to spawn. In lamprey (Petromyzon marinus), anadromous populations are far better at maintaining plasma osmolality with rising external salinity relative to landlocked populations (Beamish et al., 1978). Plasma osmolality increases on exposure to saline water in sturgeon (McEnroe and Cech, 1985; Cataldi et al., 1995; Krayushkina et al., 1996; Altinok et al., 1998; McKenzie et al., 2001; Martı´nez‐Al´varez et al., 2002; Rodrı´guez et al., 2002) and lamprey (Beamish, 1980a). Gill chloride cell size and number increase when sturgeon are transferred to hypersaline waters (Altinok et al., 1998) accompanied by an upregulation of gill Naþ/Kþ ATPase activity in some species (McKenzie et al., 1999; Rodrı´guez et al., 2002), but not all (Jarvis and Ballantyne, 2003). These responses are comparable to euryhaline teleosts moving from fresh to seawater environments, although far more details of molecular and cellular changes have been uncovered in teleosts (for a review see Marshall and Grosell, 2006). Gill cell composition in euryhaline lamprey is distinct from teleosts. Prior to migration down river to the sea, the surface of the gill MRC of young adult lampreys is covered by the flanges of adjacent pavement cells, with only a relatively small circular area exposed covered with microvilli (Peek and Youson, 1979; Mallat et al., 1995; Bartels and Potter, 2004). After young lampreys enter seawater, the pavement cells retract revealing a larger rectangular microvilli‐free chloride cell surface area, as well the paracellular channel between adjacent cells widens. These excellent structural studies now need to be linked to functional investigations to understand the role of ion transport proteins in specific cell types. 3. NITROGEN EXCRETION 3.1. Toxic Ammonia In aqueous solution, ammonia exists both as NH3 and NHþ 4 , according to the equation: NH3 þ H3 Oþ $ NHþ 4 þ H2 O The term ammonia represents the sum of the NH3 and NHþ 4 concentrations. The pK of the reaction is about 9.5 so that at fish blood pH values (pH  8) þ  about 96% of ammonia will be in the NHþ 4 form at 25 C. NH4 is charged and larger than NH3 and therefore has a lower diVusivity compared to NH3.

292

PATRICIA A. WRIGHT

Ammonia may accumulate in fish under a variety of conditions and, if severe, can result in convulsions, coma, and eventually death (for reviews see Ip et al., 2001, 2004; Randall and Tsui, 2002). Most of the fish discussed in this chapter are primarily ammoniotelic, that is, they excrete primarily ammonia. If environmental conditions preclude normal rates of ammonia elimination (e.g., elevated water pH, air exposure, or limited access to water), then endogenously produced ammonia may accumulate in the fish. Elevated environmental ammonia occurs in natural and hatchery freshwaters. The reversal of the branchial blood‐to‐water ammonia gradient results in ammonia uptake and elevated plasma and tissue ammonia concentrations in a variety of fish species, including lungfish (Chew et al., 2005). The toxicity of elevated environmental ammonia varies with water pH, temperature, salinity, and oxygen levels (for review see Ip et al., 2001). As well, intraspecific variation, developmental eVects, nutritional status, and acute versus chronic exposure all impact ammonia toxicity. There is a paucity of data on primitive fishes. In one study by Fontenot et al. (1998), the 96‐h median‐lethal concentration (96‐h LC50) for NH3 for fingerling shortnose sturgeon Acipenser brevirostrum was 0.58  0.21 mg liter
1 (mean  SD, 18  C). As a comparison, the 96‐h LC50 value for NH3 in both rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas) was 0.37 mg liter
1 (14  C) (Thurston et al., 1981). With very little solid data on ammonia tolerance between diVerent orders of fishes, it is not easy predicting which fish might demonstrate a higher tolerance to environmental ammonia. The strongest guidelines may be environmental or ecological considerations. For example, there is evidence that freshwater fishes are less susceptible to ammonia toxicity compared to their seawater counterparts (Ip et al., 2001). The tolerance to elevated water ammonia levels may be high in fish that form aggregations, burrow into confined spaces, or encounter low water volumes. Thus, there is an apparent correlation between ammonia and hypoxia tolerance in fish (for review see Walsh et al., 2006). For example, high densities of hagfish have been observed feeding on whale carcasses in deep ocean environments (Martini, 1998). Rotting flesh combined with hundreds of relatively large (0.5 m) ammonia‐excreting hagfish may create a local environment high in ammonia. As well, hagfish normally reside in mud burrows on the ocean floor, such a confined space may also result in elevated external ammonia. Due to these circumstances, it is possible that hagfish may have evolved a high tolerance to ammonia, but this has not been tested. Another Agnathan, the lamprey burrows into soft mud for several years in the larval stage (ammocoete) and feeds on detritus (Moore and Mallatt, 1980). Depending on the rate of water exchange near the ammocoete surface, endogenous ammonia may accumulate in the local environment. In fact, the 96‐h LC50 value for NH3 was 1.7 mg liter
1 in P. marinus

6.

OSMOREGULATION

293

ammocoetes (Wilkie et al., 1999), fivefold higher relative to the values reported for teleosts (see above). Lungfish encounter limited water availability or a complete absence of water during estivation (see Section 3.4), and therefore may frequently encounter ammonia loads and have a correspondingly higher tolerance for ammonia. LC50 values have not been reported in the African lungfish, but this group of fish certainly appears to be ammonia tolerant. P. dolloi survives 6 days in water containing 100 mmol liter
1 NH4Cl and barely accumulates ammonia in the extracellular compartment (Chew et al., 2005). It has recently been discovered that their remarkable insensitivity to elevated external ammonia may be partly linked to the excretion of acid. Wood et al. (2005a) found that when P. dolloi are exposed to 309 mmol liter
1 NH4Cl for 7 days they excrete both CO2 and titratable acid (e.g., Hþ) into their external environment, lowering water pH to as low as pH 3.7 in one case (even with aeration). Environmental acidification ensures that the highly diVusible NH3 remains low in the external water, thereby lowering the overall uptake of ammonia by the lungfish. P. dolloi also detoxifies excessive ammonia by conversion to urea via the urea cycle (see Section 3.2.2) when confronted with exceptionally high external ammonia concentrations (Chew et al., 2005). Hence, the African lungfish may have multiple strategies of coping with this toxic compound. It is likely that African lungfish rank up there with other highly ammonia‐tolerant species, such as the mudskipper (Periophthalmodon schlosseri), that can survive also in 100‐mmol liter
1 NH4Cl and has a 96‐h LC50 for NH3 of 7.6 mg liter
1 (Ip et al., 2004)! The brain of fish, as well as all vertebrates, is the most vulnerable organ to elevated plasma ammonia levels (Felipo and Butterworth, 2002; Walsh et al., 2006). This topic has been extensively reviewed elsewhere and will only be briefly described here. In the case of high extracellular ammonia, if NH3 is the primary permeant species, cytosol pH will increase. If mostly NHþ 4 enters the cell, intracellular pH will decrease. Any pH change will influence the function of intracellular processes. Ammonia has numerous other eVects that appear to be due to the unique properties of the NH3 or NHþ 4 molecules þ þ themselves. NHþ can directly substitute for K or H in ion exchangers, 4 disrupting ion balance and nerve propagation (Cooper and Plum, 1987). Elevated brain ammonia in fish interferes with normal cell metabolism and the synthesis of the neurotransmitter, glutamate (Wicks and Randall, 2002). The most important detoxification enzyme, glutamine synthetase (GSase), catalyzes the conversion of glutamate and NHþ 4 to glutamine and is induced in the brain of some ammonia‐exposed teleost fishes (Wicks and Randall, 2002; Wright et al., submitted for publication). As well, tolerance to elevated environmental ammonia levels is correlated with constitutive activities of GSase in the brain of closely related Batrachoididae fishes (Wang and

294

PATRICIA A. WRIGHT

Walsh, 2000). Comparable data on brain metabolism in ammonia‐exposed primitive fishes would be a step toward understanding the evolution of ammonia detoxification mechanisms. 3.2. Synthesis of Nitrogen End‐Products Nitrogen end‐products are the result of protein catabolism. Ingested or cellular proteins are hydrolyzed to the component amino acids by proteolytic enzymes. Excess amino acids are catabolyzed forming ammonia, the primarily nitrogen end‐product in fishes. Ammonia may be further ‘‘repackaged’’ as urea, glutamate, or glutamine. Several reviews have been written on nitrogen metabolism and excretion in fishes (Wood, 1993, 2001; Walsh, 1998; Anderson, 2001; Ip et al., 2001; Walsh and Mommsen, 2001; Wilkie, 2002). 3.2.1. Ammonia Figure 6.2 describes the three pathways for ammonia synthesis. Amino acid transferase enzymes transfer an amino group from the L‐amino acid to a‐ketoglutarate (a‐KG) forming glutamate and a‐keto acid (Figure 6.2A). Mitochondrial glutamate dehydrogenase (GDH) catalyzes the conversion of glutamate to a‐KG and NHþ 4 . Hepatic and red muscle mitochondrial GDH activities are comparable between bowfin (Amia calva), a Holostean fish and various teleost species (Chamberlin et al., 1991; Felskie et al., 1998). In lamprey (P. marinus) GDH activities in liver, intestine, and muscle vary with developmental stage. In the parasitic phase, liver GDH activity and protein abundance were six times higher relative to the lamprey ammocoete or upstream migrant (Wilkie et al., 2006). The authors proposed that high levels of GDH in the liver of parasitic lamprey allow for rapid catabolism of amino acids when feeding opportunities arise. This supposition is further strengthened by parallel high activities of two liver transferase enzymes, alanine, and aspartate aminotransferase in the parasitic lampreys (Wilkie et al., 2006). Ammonia is also created when AMP is degraded to IMP, catalyzed by AMP deaminase (Figure 6.2B). Although present in fish liver (Casey and Anderson, 1983), it probably only makes a significant contribution to ammonia synthesis in skeletal muscle tissue after exhaustive exercise (Mommsen and Hochachka, 1988; Wright et al., 1988). Finally, the breakdown of glutamine also results in the generation of ammonia and glutamate, catalyzed by glutaminase (GLN) (Figure 6.2C). The reverse reaction, catalyzed by GSase, consumes ammonia (Figure 6.2D). Thus, the balance between the two enzymes will determine the net ammonia synthesis in fish tissues (Chamberlin et al., 1991). In bowfin, liver GLN activity is higher than GSase activities (Chamberlin et al., 1991), suggesting a net production

6.

OSMOREGULATION

295

Fig. 6.2. Pathways for ammonia synthesis in and out of the mitochondrion. (A) L‐Amino acids are transaminated forming glutamate and an a‐Keto acid. Glutamate enters the mitochondrion where the enzyme GDH deaminates glutamate forming a‐ketoglutarate (a‐KG) and NHþ 4 . (B) In the purine nucleotide cycle, the adenylate AMP is degraded to IMP and NH3, catalyzed by AMP deaminase. (C) Glutamine may enter the mitochondrion where the enzyme GLN catalyzes the reaction forming glutamate and NH3. (D) Glutamate and NHþ 4 are combined by the enzyme GSase to form glutamine. GS is typically cytosolic in ammoniotelic fish, but mitochondrial in ureotelic fish.

of ammonia by the liver typical of other ammonotelic fishes. Ureotelic species such as elasmobranchs and most likely the coelacanth (not measured) have much higher liver GSase activities because available ammonia is scavenged to form glutamine, the nitrogen donating substrate for the urea cycle (see below). 3.2.2. Urea Urea is formed by three known pathways in fish: uricolysis, arginolysis, and the urea cycle (Figure 6.3). Uric acid arises from a purine ring that is formed by a series of complex reactions involving glutamine, aspartate, glycine, HCO
3 , and phosphoribosyl pyrophosphate (PRPP) (Figure 6.3A). Uric acid degradation to urea occurs in the peroxisomes in fish (Noguchi

296

PATRICIA A. WRIGHT

Fig. 6.3. Pathways for urea synthesis in the peroxisome (A) and mitochondrion (B), (C). Uricolysis is depicted in (A), where a purine ring is formed by a series of complex reactions involving 2 glutamine, aspartate, glycine, HCO
3 , and PRPP. Uric acid is degraded by uricase (URC) to allantoin which is further degraded by allantoinase (ALN) and allantoicase (ALC) to urea. Ariginolysis (B) is the simple conversion of arginine to ornithine and urea catalyzed by arginase. The urea cycle in fish is initiated by the enzyme carbamoyl phosphate synthetase III (CPS III) that combines glutamine and HCO
3 to form carbamoyl phosphate, which in turn is converted to citrulline catalyzed by ornithine carbamoyl transferase (OTC). Citrulline is converted to arginine by two enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL), the final step is arginolysis (B) forming urea.

6.

OSMOREGULATION

297

et al., 1979) and involves three enzymes, uricase (URC), allatoinase (ALN), and allatoicase (ALC). Activities for all three uricolytic enzymes were first reported in lungfish in an aquatic habitat (Brown et al., 1966). Later studies revealed that urea synthesis via the urea cycle in estivating lungfish far surpassed the capacity of uricolysis (Forster and Goldstein, 1966). It is likely that all fish have the capacity to degrade uric acid because the pathway plays an important role in nucleic acid metabolism. It is surprising therefore that in hagfish (Bdellostoma cirrhatum renamed Eptatretus cirrhatus), no uricolytic enzymes were detected (Read, 1975), a finding that has not been confirmed in this or other hagfish species. In contrast, the full suite of uricolytic enzymes were found in liver of lamprey ammocoetes (P. marinus), and the level of activities were similar to teleost values (Wilkie et al., 1999). The first uricolytic gene, uricase (or urate oxidase), has been cloned in African lungfish (P. annectens), as well as in several teleost species (Andersen et al., 2006). Although the allantoicase gene has been identified in the puVer fish (Fugu rubripes) genome database (Vigetti et al., 2003), cloning of allantoicase and allantoinase genes in fish has not been successful to my knowledge. Given that several enzymes in the uricolytic pathway have been lost during the evolution of higher vertebrates, much would be gained from understanding the evolution of the genes coding for the uricolytic enzymes in primitive as well as other fishes. Urea can also be formed from arginine degradation in fish mitochondria catalyzed by arginase (ARG) (Figure 6.3B), independent of a complete urea cycle. ARG is widespread in fish tissues, including hagfish (Read, 1975), lamprey (Read, 1968; Wilkie et al., 1999, 2004, 2006), lungfish (Janssens and Cohen, 1966), coelacanth (Brown and Brown, 1967), sturgeon and gar (Cvancara, 1969), as well as bowfin (Felskie et al., 1998). Two mitochondrial isoforms of ARG, ARG type I and II, are coded by two genes in puVer fish (Takifugu rubripes) and four genes in rainbow trout (O. mykiss) (Wright et al., 2004), whereas ARG type I in terrestrial ureotelic vertebrates is cytosolic (Ikemoto et al., 1990). At what stage during the transition to a terrestrial habitat did ARG type I lose its mitochondrial leader sequence? Mommsen and Walsh (1989) proposed that this shift in the intracellular location of liver ARG first appeared in the lungfish. Although enzyme activity data support this notion (Mommsen and Walsh, 1989), the sequencing of lungfish ARG genes (type I and II) would provide valuable information for a more complete phylogenetic analysis and lead to a better view of the evolution of the urea cycle in vertebrates. The urea cycle is the main pathway for urea synthesis in terrestrial vertebrates (amphibians, mammals), as well as elasmobranchs (Anderson, 2001), coelacanths, lungfish (GriYth, 1991), and a few teleosts (Walsh and

298

PATRICIA A. WRIGHT

Mommsen, 2001). Five enzymes form the backbone of the pathway: carbamoyl phosphate synthetase III (CPS III), ornithine carbamoyl transferase (OTC), argininosuccinate synthetase (ASS) and lyase (ASL), and ARG (Figure 6.3B and C). CPS III requires glutamine as the nitrogen donating substrate and, therefore, mitochondrial GS is considered an important accessory enzyme. The properties of CPS III and the subcellular location of ARG distinguish the fish urea cycle from that of amphibians and mammals (Anderson, 2001). In terrestrial vertebrates, CPS I utilizes ammonia as the N‐donating substrate and ARG is cytosolic. A third CPS cousin, CPS II, is prevalent in all vertebrate tissues, is part of the pyrimidine pathway, requires glutamine as a substrate, but does not require the eVector N‐acetyl glutamate (required by both CPS I and III). Confusion in the literature arose a few decades ago when it was first reported that CPS activity was fairly common in fish (Cvancara, 1974), but subsequent work clarified the importance of careful assay technique to provide the correct conditions for the urea cycle‐related CPS III and separate it from CPS II (Felskie et al., 1998). Thus, it is now clear that hagfish (Read, 1975; Mommsen and Walsh, 1989), lamprey (Wilkie et al., 1999, 2004), and bowfin (Felskie et al., 1998) have extremely low or nondetectable levels of CPS III and lack a functional hepatic urea cycle. There is limited information on urea cycle enzymes in sturgeon, paddlefish, and bichir; however, the absence of the pathway is reported in Mommsen and Walsh (1989). Despite this, the bichir was assigned a CPS type III enzyme by Mommsen and Walsh (1989) and would be interesting to investigate further given this fish’s predilection for river margins, flood plains, and swamps, similar to lungfishes. The coelacanth, L. chalumnae, first came to the attention of urea cycle enthusiasts with the reports of Pickford and Grant (1967) and Brown and Brown (1967) that blood and tissue urea levels as well as OTC and ARG activities were similar to the ureosomotic elasmobranchs (GriYth, 1991). A number of follow‐up studies expanded on these initial findings but fascinating questions remain. For example, during early development, coelacanth pups are carried within the oviducts of the mother (Wourms et al., 1991) and obtain nutrition from large attached yolk sacs (Locket, 1980). Are these developing young ureosmotically independent as have been found in little skate (Raja erinacea) embryos (Steele et al., 2004) or is urea cycle capacity only apparent after release from internal maternal support? African lungfish express all urea cycle enzymes (Janssens and Cohen, 1966) and detoxify ammonia to urea when water supplies become limited (Janssens and Cohen, 1968; see also Section 3.4). Mommsen and Walsh (1989) classified CPS in the lungfish P. aethiopicus as type I, with a preference for ammonia over glutamine as the N‐donating substrate. This designation was later challenged by Loong et al. (2005) who claimed that hepatic CPS activities in P. aethiopicus and P. annectens were 30‐ to 60‐fold higher if

6.

OSMOREGULATION

299

glutamine was the available substrate rather than ammonia. It is possible, however, that both subtypes of CPS exist, but that the developmental stage or previous environmental conditions of the lungfish play a role in determining whether CPS I or III dominates (P. Walsh, personal communication). Clearly, sequence information on lungfish CPS genes would help to illuminate the controversy. 3.3. Excretion Aquatic animals tend to be ammoniotelic, whereas terrestrial animals excrete mostly urea (ureotelic) or uric acid (uricotelic) (reviewed by Wright, 1995). The key nitrogen excretory product in fish is ammonia, but elasmobranchs (and probably the coelacanth) release primarily urea (ureotelic) as a by‐product of their ureosmotic strategy (Table 6.1). Other nitrogenous end‐ products including amino acids, proteins, creatine, creatinine, and unknown substances, which may account for 20–40% of the total nitrogen excreted in teleost fishes, are less well understood (Kajimura et al., 2004). 3.3.1. Ammonia Fish excrete ammonia mostly as NH3 down the blood‐to‐water partial pressure gradient (Wilkie, 2002; Evans et al., 2005). To determine if NH3 diVusion dominates at the gill, researchers have manipulated external water pH (Wright and Wood, 1985). The diVusion trapping model predicts that NH3 excretion will increase in acid environments and decrease in alkaline environments (Wright and Wood, 1985; Wilkie and Wood, 1991). This phenomena is nicely illustrated in sturgeon (Acipenser ruthenus) fingerlings acclimated gradually for 1 week to water pH values ranging from pH 4.0‐ to 9.4 (fish died at pH 9.6) (Figure 6.4). If one takes 500 mmol kg
1h
1 as the control excretion rate (pH 7–8.4), then there is an 80% increase in Jamm at pH 4.0 and a 34% decrease in Jamm at pH 9.4, with virtually no change in Jurea. The very high rate of Jamm under acid conditions would be hard to maintain and it is not surprising that some mortalities were noted after 2 days at pH 4.0. þ There is evidence for some branchial NHþ 4 eZux possibly linked to Na influx in fish (Wilkie, 2002; Evans et al., 2005). Interestingly, in the hagfish M. glutinosa, Evans (1984) proposed that branchial ammonia excretion was dominated by NH3 diVusion, with no evidence for Naþ‐dependent NHþ 4 transport. In general, gill NHþ 4 diVusion may be more important in seawater compared to freshwater fish. In seawater lamprey and sturgeon, leaky paracellular junctions adjacent to gill MRCs (see Section 2.1.3) may provide a passageway for NHþ 4 . In contrast, in the hagfish gill MRCs are tightly associated with pavement cells and this arrangement probably prevents NHþ 4 diVusion between cells.

Table 6.1 Ammonia (Jamm) and Urea (Jurea) Excretion Rates and the Percentage of Nitrogen Wastes Excreted as Urea (% urea) in Hagfish, Lamprey, Elasmobranch, Lungfish, Sturgeon, and Bowfin Species

Species

300

Hagfish Eptatretus stoutii Myxine glutinosa

Lamprey Petromyzon marinus

Entosphenus tridentatus Elasmobranch Squalus acanthias Raja erinacea Lungfish Protopterus dolloi

Jamm (mmol N kg
1h
1)

Jurea (mmol N kg
1h
1)

Urea (%)

Comments

References

73 218 200

5 – –

6 – –

SW SW SW

Walsh et al., 2001 Evans, 1984 McDonald et al., 1991

50 100

9 15

18 13

Wilkie et al., 1999 Wilkie et al., 2004

2500

200

7

119

0

0

FW (ammocoetes) FW (adults) fasted FW (adults, fed trout blood) FW

Read, 1968

11 150 124

547 411 111

98 73 47

SW SW (adult) SW (embryo)

Wood et al., 1995 Steele et al., 2005 Steele et al., 2004

170

21

11

2‰

Wood et al., 2005

Wilkie et al., 2004

Protopterus aethiopicus Protopterus annectens Protopterus dolloi

63 96 133 265

66 50 42 221

51 34 24 46

FW FW FW (fasted) FW (fed)

Loong et al., 2005 Loong et al., 2005 Lim et al., 2004 Lim et al., 2004

469

–

–

FW (juvenile)

208

16

7

FW

142

15

10

9‰ SW

Acipenser oxyrhynchus

438

–

–

FW (juvenile)

Acipenser brevirostrum

381

–

–

FW (juvenile)

Acipenser gueldenstaedti

724

95

12

FW (juvenile)

594

130

18

10‰ SW (juvenile)

Dabrowski et al., 1987 Altinok and Grizzle, 2004 Altinok and Grizzle, 2004 KieVer et al., 2001 KieVer et al., 2001 Gershanovich and Pototskij, 1995 Gershanovich and Pototskij, 1995

607

60

9

FW

Sturgeon Acipenser baeri Acipenser oxyrinchus desotoi

301

Bowfin Amia calva

McKenzie and Randall, 1990

302

PATRICIA A. WRIGHT

Fig. 6.4. Ammonia (Jamm) and urea (Jurea) excretion rates (mmol kg
1 h
1) in Sturgeon fingerlings A. ruthenus in response to 1 week of exposure to water of varying pH. [Reprinted from Gershanovich and Pototskij (1995) with permission from Elsevier.]

Gill ammonia transporters have been isolated in two teleost species, Rivulus marmoratus (Hung et al., 2007) and O. mykiss (Nawata, et al., submitted manuscript), which share sequence similarities to the Rhesus‐associated glycoproteins (RhG) (Huang and Peng, 2005). There is an ongoing controversy whether RhG proteins transport NH3 (Ripoche et al., 2004), NHþ 4 (Nakhoul þ et al., 2005), and/or mediate NHþ /H exchange (Handlogten et al., 2004) in 4 mammals. In crab gills, an Rh‐like protein (RhCM) is thought to actively transport NHþ 4 across the cuticle (Weihrauch et al., 2004). Huang and Peng (2005) have reported the presence of ‘‘genuine Rh genes in hagfish’’ and suggest further characterization of these genes may shed light on CO2 conductance across red blood cell membranes. Thus, across the spectrum of organisms and tissues many putative roles have been associated with RhG genes and the path forward in fish will be clearly very interesting. The major site of Jamm is typically the gills in fish (Smith, 1929). Divided chamber experiments performed by Read (1968) on the lamprey Entosphenus tridentatus demonstrated that 87% of the ammonia was eliminated across the gills, 8% across the skin, and 4% via the kidneys. Little attention has been paid to cutaneous ammonia excretion, but premetamorphic lamprey larvae have a thinner integument (Youson, 1980) and possibly rely less on branchial excretion while in burrows compared to adults; however, this has not been tested. In the submerged lungfish P. dolloi, ammonia and urea excretion are almost equally partitioned between the anterior (internal and external gills) and posterior end (most of the skin and urinary opening) (Wood et al., 2005).

6.

OSMOREGULATION

303

What percentage of the posterior excretion is attributable to the skin versus urine is unknown; however, the skin may be the key site of urea elimination following prolonged terrestrialization in lungfish (see Section 3.4). Feeding has a profound eVect on nitrogen excretion rates in fish, but most Jamm values in the literature are for starved fish (Wood, 2001). One of the most dramatic examples of the influence of nutrition is found in the parasitic lamprey (P. marinus) (Wilkie et al., 2004), where postprandial Jamm was 25 times higher after a blood meal (Table 6.1). Parasitic lampreys may consume up to 30% of their body weight/day and are very eYcient at assimilating the energy in a blood meal (Farmer et al., 1975). The African lungfish (P. dolloi) appears to take a diVerent approach to a surfeit of amino acids following feeding. Although Jamm does increase significantly after a meal, urea synthesis is stimulated to a greater extent resulting in higher tissue urea contents and a greater proportion of nitrogen excreted as urea (Lim et al., 2004). 3.3.2. Urea In jawless and ray‐finned primitive fishes, Jurea constitutes 6–18% of total ammonia and urea excretion (Table 6.1). Jurea depends on both the simple diVusion of urea as well as facilitated transport (Walsh and Smith, 2001). The first fish urea transporter (UT) was isolated and characterized in the dogfish shark (Squalus acanthias) kidney (ShUT; Smith and Wright, 1999). ShUT, homologous to the mammalian facilitated transporter UT‐A2 family, shares sequence similarity with UTs cloned from the kidneys of other elasmobranch species (Janech et al., 2003, 2006; Morgan et al., 2003; Hyodo et al., 2004). A survey of several marine fish indicates that UT gill expression may be fairly widespread, although no signal was detected in hagfish (Eptatretus stoutii) (Walsh et al., 2001). This negative result requires further investigation because a teleostean UT probe was used. Other UT isoforms may be present in hagfish or branchial urea transport may be solely dependent on simple diVusion. Due to the unique position of hagfish in the fish evolutionary tree, a more complete picture of Agnathan urea transport would be valuable. There have been only a few studies in the primitive fishes that report fluctuations in Jurea with changing physiological conditions. Jurea is not particularly sensitive to changes in the external water pH (Figure 6.4) or salinity (Altinok and Grizzle, 2004). Feeding enhances Jurea, just as it does Jamm (see above). In parasitic lamprey feeding on rainbow trout, Jurea was elevated by 15‐fold initially and remained elevated for 8 h after the meal (Wilkie et al., 2004). More remarkably, two lampreys (P. marinus) were caught parasitizing basking sharks and Jurea was as high as 9000 mmolNkg
1h
1 after the meal (Wilkie et al., 2004). This surge in Jurea is presumably necessary to clear the lamprey body fluids of the excessive

304

PATRICIA A. WRIGHT

urea concentrations taken in with the shark blood. The rapid eZux of urea under these conditions may be dependent, in part, on the upregulation of gill UTs (Wilkie et al., 2004). 3.3.3. Retention of Urea in Coelacanths and Elasmobranchs The coelacanth, holocephalans (chimaeras), and marine elasmobranchs evolved a strategy of osmoregulation that sets them apart from all other known fish species, namely the retention of urea to counterbalance the osmotic strength of seawater. Like many other evolutionary experiments, it is not apparent why an excretory waste product (urea) would be retained at relatively high concentrations as an osmolyte (for a discussion see Walsh and Mommsen, 2001). Kirschner (1993) compared the energetic costs of hyposmotic regulation by marine teleosts with ureosmotic regulation by marine elasmobranchs and concluded that they were similar. Urea is less toxic than ammonia at the same concentration, but urea at high concentrations denatures proteins (Yancey, 2001). In elasmobranchs, the destabilizing eVects of urea are counterbalanced by methylamines or other organic osmolytes (Yancey, 2001). In some marine elasmobranchs, the ratio of urea to TMAO concentrations is 2:1, whereas in other species a variety of counteracting osmolytes along with TMAO have an additive eVect (Steele et al., 2004, 2005). Embryos of the oviparous little skate, R. erinacea, have a urea: TMAOþ other stabilizing osmolytes ratio of 2.3:1 (4 months) and 2.7:1 (8 months) (Steele et al., 2004), whereas the ratio was 1.68:1 in the skeletal muscle of adult skates (Steele et al., 2005). These findings imply developmental changes in osmolyte regulation. Coelacanth hemoglobin is unaVected by urea concentrations >3 M, similar to elasmobranchs (Mangum, 1991). Are other coelacanth proteins sensitive, insensitive, or urea‐requiring, as has been found in some elasmobranchs? In the coelacanth, skeletal muscle TMAO was 300 mmol liter
1 (Lutz and Robertson, 1971) considerably higher than the little skate value of 50 mmol liter
1 (Steele et al., 2005) or 180 mM in the shark Scyliorhinus canicula muscle (Robertson, 1989). These diVerences raise many interesting questions about the coelacanth counterbalancing osmolyte strategy. For example, do coelacanth pups within the mother (Wourms et al., 1991) retain the same organic osmolyte ratios as adults? Are these osmolyte concentrations sensitive to external salinity changes, as reported in elasmobranch embryos (Steele et al., 2004) and adults (Steele et al., 2005)? Maintaining elevated urea concentrations in body fluids is a considerable challenge for coelacanths and elasmobranchs, given the large blood (400 mmol liter
1)‐to‐water (0 mmol liter
1) gradient across the gills and the obligatory release of more or less isosmotic urine. In marine elasmobranchs, renal reabsorption of urea has been long established, but the

6.

OSMOREGULATION

305

mechanisms involved have not been resolved. The passive countercurrent reabsorption of urea by the kidney (Boylan, 1972; Lacy et al., 1985) is probably dependent, in part, on UT proteins (Smith and Wright, 1999) with diVerent functional characteristics and regional heterogeneity (Walsh and Smith, 2001; Janech et al., 2003, 2006; Morgan et al., 2003; Hyodo et al., 2004). Although sequence and tissue distribution information on UTs in elasmobranchs is valuable, what is now needed is a more complete understanding of the functional role of various tubule segments. This work has been impeded by the complexity of the elasmobranch nephron structure. On the other hand, the coelacanth kidney is thought to be more similar to the Osteichthyes (see Section 2.1.2); a more detailed structural analysis needs to be performed for comparisons with the elasmobranch nephron. GriYth (1991) postulated that coelacanths do not reabsorb urea from the renal filtrate based on samples collected from the urinary bladder of a moribund specimen (GriYth et al., 1976). Urea is energetically expensive to produce and, therefore, it is hard to imagine how the coelacanth could manage such a high rate of urea loss via the urine unless urine flow is remarkably low. A secondary active Naþ/urea antiporter in the gills of the dogfish shark is thought to transport urea out of the gill epithelial cells back into the blood against the urea concentration gradient (Fines et al., 2001). The gene coding for this Naþ/urea antiporter has not been isolated in elasmobranchs, nor in the mammalian kidney where active Naþ‐coupled urea transport is thought to play an important role (Kato and Sands, 1998). Dogfish gill basolateral membranes have a very high cholesterol content (Fines et al., 2001) that decreases the permeability to urea (Pugh et al., 1989). Hill et al. (2004) reported that apical and basolateral gill membrane vesicles in dogfish and marine flounder share relatively low permeabilities to water and urea. A picture of branchial urea retention in elasmobranchs is starting to emerge, but no comparable data is available for coelacanth. Even though it is unlikely that physiologists will obtain live coelacanth for whole‐animal experiments, fresh gill and kidney tissue could provide a wealth of information on ultrastructure and molecular composition. 3.3.4. The Developmental Perspective An understanding of nitrogen excretion during early development in teleost fishes (Korsgaard et al., 1995; Wright and Fyhn, 2001) may shed some light on unstudied ammoniotelic primitive fishes (hagfish, lungfish, and Actinopterygii fishes) or provide comparisons to the one group that has been examined, the lampreys. Ammonia excretion (Jamm) can be detected in many teleost embryos very early after fertilization and depending on the species, Jurea can account for a significant fraction of total nitrogen excretion (Wright and Fyhn, 2001). GriYth (1991) proposed that urea synthesis via

306

PATRICIA A. WRIGHT

the urea cycle arose in early gnathostome fishes as a protective mechanism to ensure low tissue ammonia levels during a long embryonic development phase solely dependent on yolk proteins and amino acids for energy. Indeed, urea cycle enzymes, including the key enzyme CPS III, are expressed in freshwater and marine teleosts embryos encompassing a variety of early life histories (Wright et al., 1995; Chadwick and Wright, 1999; Terjesen et al., 2001; Barimo et al., 2004). Interestingly, in most of these species the urea cycle is not operational in the adult stage. The exception is the embryos of the gulf toadfish, Opsanus beta, which develop into ureagenic adults, turning on urea synthesis under stressful conditions (for a review see Walsh, 1997). An alternative route for ammonia detoxification during early development is glutamine synthesis, which may or may not feed into the urea cycle. Early induction of GSase genes in rainbow trout embryos and subsequent formation of the active enzyme before hatching may be necessary to prevent excessive accumulation of ammonia (Essex‐Fraser et al., 2005). Given this background on teleost early development, it would be fascinating to know more about the ontogeny of nitrogen excretion in the oldest living vertebrate, the hagfish. Female hagfish are thought to produce 20–30 yolky encapsulated embryos varying in size between 20 and 70 mm (Martini, 1998). In large embryos such as these, ammonia diVusion to the surrounding seawater would be comparatively slow, and therefore detoxification pathways such as the urea cycle and/or glutamine synthesis might be imperative. Embryonic hagfish have rarely been found over the last 100 years (Martini, 1998). The picture is brighter for the lampreys. Larval ammocoetes synthesize low levels of urea via uricolysis, not the urea cycle (Wilkie et al., 1999). When exposed to elevated external ammonia, plasma and tissue ammonia levels increased in ammocoetes without changes in urea or glutamine (Wilkie et al., 1999). Premetamorphic lampreys have a depressed metabolic rate compared to postmetamorphic stages (Wilkie et al., 2001), partly explaining the low rates of nitrogen excretion relative to postmetamorphic stages (Table 6.1). After metamorphosis, parasitic and upstream‐migrant lampreys express only very low or nondetectable levels of the urea cycle enzymes, CPS III and OTC (Wilkie et al., 2006). Is the urea cycle expressed in embryonic lamprey? Embryos of P. marinus are about 1–2 mm in diameter (Richardson and Wright, 2003), have a relatively large yolk sac, and hatch after 20 days (22  C) in the nest (Applegate, 1950). One might predict that lamprey embryos consuming yolk proteins may induce urea cycle enzymes prior to hatching, but later repress the activity of urea cycle enzymes when metabolic rate and protein intake is low when larvae inhabit mud burrows and feed on detritus.

6.

OSMOREGULATION

307

3.4. The Challenges of Estivation The three families of lungfish tolerate terrestrial conditions to varying degrees. Some species of African lungfish Protopterus estivate (i.e., survive the dry season by forming a cocoon or a protective layer of dried mucus and reduce metabolic rate), whereas the South American Lepidosiren will partially estivate in a moist environment, and the Australian Neoceratodus is completely aquatic and does not estivate. GriYth (1991) and Graham (1997) presented excellent reviews of the older literature on nitrogen metabolism and excretion in the three families of lungfish. There have been several new studies on the African lungfish Protopterus, mostly due to the ease of shipping these animals to laboratories far away from their native habitat. P. dolloi form dry, brown mucus cocoons in the laboratory when water is removed and animals are kept slightly moist for 30 or 40 days (Chew et al., 2004; Wood et al., 2005). During estivation, Protopterus and Lepidosiren maintain low tissue ammonia levels, but accumulate urea in order to avoid ammonia toxicity (Janssens, 1964; Carlisky and Barrio, 1972; Chew et al., 2004; Wood et al., 2005). Urea synthesis in these air‐exposed lungfish occurs via the hepatic urea cycle (see Section, 3.2.2). The activities of urea cycle enzymes were enhanced by approximately twofold in P. dolloi, although the increase is remarkably modest given that these fish were without water for 40 days (Chew et al., 2004). Moreover, the data suggest that P. dolloi maintains a high reserve capacity for urea synthesis under control or immersed conditions, even when they are ammoniotelic (Chew et al., 2003). This may be beneficial if low water or impending drought is not readily anticipated by the fish. In the nonestivating Neoceratodus forsteri, the rate of urea synthesis is 100 times lower than that in Protopterus (Goldstein et al., 1967). A ‘‘washout’’ of urea was observed in Protopterus when estivating lungfish were returned to water (Smith, 1930; Janssens, 1964). An initial pulse of Jurea (peak 0–1 h) was followed by a second pulse (peak 12 h) of greater magnitude (Wood et al., 2005). When lungfish were placed in divided chambers to separate the anterior (gills) from the posterior (most of the skin and urine/feces) end of the fish, more Jurea occurred from the posterior end in the second phase of the urea ‘‘washout’’ (Figure 6.5). Further characterization of the second pulse of Jurea suggested that facilitated type UTs may have been mobilized to accommodate the enormous flux at this time (Wood et al., 2005). Isolation of lungfish UTs and their tissue distribution will be an important next step. The environmental or endogenous signal(s) that stimulates estivation has not been identified in lungfish. Ip et al. (2005) hypothesized that a subtle increase in external salinity as the river water evaporates prior to a drought

308

PATRICIA A. WRIGHT

Fig. 6.5. Nitrogen excretion in the African lungfish (Protopterus dolloi) submerged (A) and after 21 days of terrestrial conditions (skin remained moist), (B) 0–3 h and (C) 12–13 h. Nitrogen

6.

OSMOREGULATION

309

may be one instigator of estivation in P. dolloi. Multiple factors are probably involved and identification of changes in hormone‐signaling pathways would be valuable information toward a more thorough understanding of the control of estivation. Bowfin is tolerant of air‐exposure and there were suggestions in the literature that Amia estivates. McKenzie and Randall (1990) attempted to induce estivation in A. calva in the laboratory by gradually air‐exposing fish over a 10‐day period, or elevating external water ammonia or decreasing water oxygen levels. None of these treatments induced estivation, and A. calva died following 3–5 days in air. 4. CONCLUDING REMARKS In the area of ionic, osmotic, and nitrogenous waste regulation in primitive fishes, there are as many gaps in our knowledge as there is detailed information. For example, considerable research has focused on gill morphology, cell type, and ultrastructure of the Agnathans, but limited work has been directed toward the functional role of gill subtypes and the expression of ion transporters. Likewise, an explosion of papers on lungfish nitrogen excretion has unveiled fascinating responses to environmental perturbations but much less is known of ionic and osmotic regulation. As outlined in specific sections above, molecular approaches may provide the missing links in a number of cases (e.g., the urea cycle enzyme CPS in lungfish, expression of UTs and Rh‐factor ammonia transporters in gills). Finally, we have negligible data on any aspect of osmoregulation and nitrogen excretion in birchir, gar, bowfin, and paddlefish (not to mention coelacanth!). Studies of extant primitive fishes may provide more than just data on another fish species, but lead to a broader understanding of how early vertebrates evolved under changing external conditions (e.g., ions, salinity, water availability).

excretion was partitioned between the anterior (i.e., head) and posterior (i.e., body) compartments by placing lungfish in divided chambers under (A) control aquatic conditions (N ¼ 10), (B) after return (0–3 h) to aquatic conditions following 21 days of terrestrial conditions (N ¼ 5), and (C) 12–13 h after return to aquatic conditions following 21 days of terrestrial conditions (N ¼ 9). Asterisks indicate significant diVerence ( p  0.05) from the aquatic rates in panel A, whereas crosses indicate significant diVerence ( p  0.05) from the corresponding rates in the anterior compartment. [After Wood et al. (2005b) with permission from University of Chicago Press.]

310

PATRICIA A. WRIGHT

ACKNOWLEDGEMENTS The author wishes to thank Drs. Wilkie, Wood and Terjesen for access to unpublished material, Tom Binder for helpful discussions on lamprey development, Ian Smith for graphics, Kim Ong for digging up references and Lori Ferguson for skilled clerical help. The comments of two anonymous reviewers are very much appreciated. Financial support was provided by the Natural Sciences and Engineering Research Council.

REFERENCES Alt, J. M., Stolte, H., Eisenbach, G. M., and Walvig, F. (1981). Renal electrolyte and fluid excretion in the Atlantic hagfish Myxine glutinosa. J. Exp. Biol. 91, 323–330. Altinok, I., and Grizzle, J. M. (2004). Excretion of ammonia and urea by phylogenetically diverse fish species in low salinities. Aquaculture 238, 499–507. Altinok, I., Galli, S. M., and Chapman, F. A. (1998). Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon, Acipenser oxyrinchus de sotoi. Comp. Biochem. Physiol. 120, 609–616. Andersen, Ø., Aas, T. S., Skugor, S., Takle, H., van Nes, S., Grisdale‐Helland, B., Helland, S. J., and Terjesen, B. F. (2006). Purine‐induced expression of urate oxidase and enzyme activity in Atlantic salmon (Salmo salar). Cloning of urate oxidase liver cDNA from three teleost species and the African lungfish Protopterus annectens. FEBS J. 273, 2839–2850. Anderson, P. M. (2001). Urea and glutamine synthesis: Environmental influences on nitrogen excretion. In ‘‘Fish Physiology Vol. 20, Nitrogen Excretion’’ (Wright, P., and Anderson, P., Eds.), pp. 239–278. Academic Press, San Diego. Applegate, V. C. (1950). Natural history of the sea lamprey Petromyzon marinus, in Michigan. US Department of Interior, Fish and Wildlife Service, Special Scientific Report: Fisheries. no. 55. Barimo, J. F., Steele, S. L., Wright, P. A., and Walsh, P. J. (2004). Ureotely and ammonia tolerance in early life stages of the gulf toadfish. Opsanus beta. J. Exp. Biol. 207, 2011–2020. Bartels, H. (1998). The gills of hagfishes. In ‘‘The Biology of Hagfishes’’ (Jørgensen, J. M., Lomholt, J. P., Weber, R. E., and Malte, H., Eds.), pp. 205–222. Chapman and Hall, London. Bartels, H., and Potter, I. C. (2004). Cellular composition and ultrastructure of the gill epithelium of larval adult lampreys. J. Exp. Biol. 207, 3447–3462. Beamish, F. W. H. (1980a). Osmoregulation in juvenile and adult lampreys. Can. J. Fish. Aquat. Sci. 37, 1739–1750. Beamish, F. W. H. (1980b). Biology of the North American anadromous sea lamprey, Petromyzon marinus. Can. J. Fish. Aquat. Sci. 37, 1924–1943. Beamish, F. W. H., Strachan, P. D., and Thomas, E. (1978). Osmotic and ionic performance of the anadromous sea lamprey, Petromyzon marinus. Comp. Biochem. Physiol. 60A, 435–443. Bellamy, D., and Jones, I. C. (1961). Studies on Myxine glutinosa. I. The chemical composition of the tissues. Comp. Biochem. Physiol. 3, 175–183. Brown, G. W., Jr., and Brown, S. G. (1967). Urea and its formation in coelacanth liver. Science 155, 570–573. Brown, G. W., Jr., James, J., Henderson, R. J., Thomas, W. N., Robinson, R. O., Thompson, A. L., Brown, E., and Brown, S. G. (1966). Uricolytic enzymes in liver of the Dipnoan Propterus aethiopicus. Science 153, 1653–1654. Brown, J. A., Cobb, C. S., Frankling, S. C., and Rankin, J. C. (2005). Activation of the newly discovered cyclostome rennin‐angiotensis system in the river lamprey Lamprey fluviatilis. J. Exp. Biol. 208, 223–232.

6.

OSMOREGULATION

311

Boylan, J. W. (1972). A model for passive urea reabsorption in the elasmobranch kidney. Comp. Biochem. Physiol. 42A, 27–30. Bull, J. M., and Morris, R. (1967). Studies on freshwater osmoregulation in the ammocoete larva of Lampetra planeri (Bloch). J. Exp. Biol. 47, 485–494. Butler, D. G., and Youson, J. H. (1988). Kidney function in the bowfin (Amia calva L.). Comp. Biochem. Physiol. 89A, 343–345. Carlisky, N. J., and Barrio, A. (1972). Nitrogen metabolism of the South American Lungfish Lepidosiren paradoxa. Comp. Biochem. Physiol. 41B, 857–873. Casey, C. A., and Anderson, P. M. (1983). Glutamine‐ and N‐acetyl‐glutamate‐dependent carbamoyl phosphate synthetase from Micropterus salmoides. Purification, properties and inhibition by glutamine analogs. J. Biol. Chem. 258, 8723–8732. Cataldi, E., Ciccotti, E., di Marco, P., di Santo, O., Bronzi, P., and Cataudella, S. (1995). Acclimation trials of juvenile Italian sturgeon to diVerent salinities: Morpho‐physiological descriptors. J. Fish Biol. 47, 609–618. Chadwick, T. D., and 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. Chamberlin, M. E., Glemet, H. C., and Ballantyne, J. S. (1991). Glutamine metabolism in a holostean (Amia calva) and teleost fish (Salvelinus namaycush). Am. J. Physiol. 260, R159–R166. Chang, M., Zhang, J., and Desui, M. (2006). A lamprey from the Cretaceous Jehol biota of China. Nature 44, 972–974. Chew, S. F., Ong, T. F., Lo, L., Tam, W. L., Loong, A. M., Hiong, K. C., Wong, W. P., and Ip, Y. K. (2003). Urea synthesis in the African lungfish Propterus dolloi—hepatic carbamoyl phosphate synthetase III and glutamine synthetase are upregulated by 6 days of aerial exposure. J. Exp. Biol. 206, 3615–3624. Chew, S. F., Chan, N. K. Y., Loong, A. M., Hiong, K. C., Tam, W. L., and Ip, Y. K. (2004). Nitrogen metabolism in the African lungfish (Protopterus dolloi) aestivating in a mucus cocoon on land. J. Exp. Biol. 207, 777–786. Chew, S. F., Ho, L., Ong, T. F., Wong, W. P., and Ip, Y. K. (2005). The African lungfish, Protopterus dolloi, detoxifies ammonia to urea during environmental ammonia exposure. Physiol. Biochem. Zool. 78, 31–39. Choe, K. P., Edwards, S., Morrison‐Shetlar, A. I., Toop, Tes, and Claiborne, J. B. (1999). Immunolocalization of Naþ/Kþ‐ATPase in mitochondrion‐rich cells of the atlantic hagfish (Myxine glutinosa) gill. Comp. Biochem. Physiol. 124, 161–168. Choe, K. P., Morrison‐Shetlar, A. I., Wall, B. P., and Claiborne, J. B. (2002). Immunological detection of Naþ/Hþ exchangers in the gills of a hagfish, Myxine glutinosa, an elasmobranch, Raja erinacea, and a teleost, Fundulus heteroclitus. Comp. Biochem. Physiol. 131A, 375–385. Choe, K. P., O’Brien, S., Evans, D. H., Toop, T., and Edwards, S. L. (2004). Immunolocalization of Naþ/Kþ‐ATPase, carbonic anhydrase II, and vacuolar Hþ‐ATPase in the gills of freshwater adult lampreys,. Geotria australis. J. Exp. Zool. 301A, 654–665. Cobb, C. S., Frankling, S. C., Rankin, J. C., and Brown, J. A. (2002). Angiotensin converting enzyme‐like activity in tissues from the river lamprey or lampern, Lampetra fluviatilis, acclimated to freshwater and seawater. Gen. Comp. Endocrinol. 127, 8–15. Cooper, A. J. L., and Plum, F. (1987). Biochemistry and physiology of brain ammonia. Physiol. Rev. 67, 440–519. Cvancara, V. A. (1969). Studies on tissue arginase and ureogenesis in freshwater teleosts. Comp. Biochem. Physiol. 30, 489–496. Cvancara, V. A. (1974). Liver carbamyl phosphate synthetase in the primitive freshwater bony fishes (Chondrostei, Holostei). Comp. Biochem. Physiol. 49B, 785–787.

312

PATRICIA A. WRIGHT

Dabrowski, K., Kaushik, S. J., and Fauconneau, B. (1987). Rearing of sturgeon (Acipenser baeri Brandt) larvae. III. Nitrogen and energy metabolism and amino acid absorption. Aquaculture 65, 31–41. Edwards, S. L., Claiborne, J. B., Morrison‐Sheltar, A. I., and Toop, T. (2001). Expression of Naþ/Kþ exchanger mRNA in the gills of the Atlantic hagfish (Myxine glutinosa) in response to metabolic acidosis. Comp. Biochem. Physiol. 130A, 81–91. Essex‐Fraser, P. A., Steele, S. L., Bernier, N. J., Murray, B. W., Stevens, E. D., and Wright, P. A. (2005). Expression of four glutamine synthetase genes in the early stages of development of rainbow trout (Oncorhynchus mykiss) in relationship to nitrogen excretion. J. Biol. Chem. 280, 20268–20273. Evans, D. H. (1984). Gill Naþ/Kþ and Cl
/HCO
3 exchange systems evolved before the vertebrates entered fresh water. J. Exp. Biol. 113, 465–469. Evans, D. H. (1993). Osmotic and ionic regulation. In ‘‘The Physiology of Fishes’’ (Evans, D. H., Ed.), pp. 315–341. CRC Press, Boca Raton. Evans, D. H., Piermarini, P. M., and Choe, K. P. (2005). The multifunctional fish gill: Dominant site of gas exchange, osmoregulation, acid‐base regulation, and excretion of nitrogenous waste. Physiol. Rev. 85, 97–177. Fa¨nge, R. (1998). Introduction: Early hagfish research. In ‘‘The Biology of Hagfishes’’ (Jørgensen, J. M., Lomholt, J. P., Weber, R. E., and Malte, H., Eds.), pp. 13–19. Chapman and Hall, London. Farmer, G. J., Beamish, F. W. H., and Robinson, G. A. (1975). Food consumption of the adult landlocked sea lamprey, Petromyzon marinus L. Comp. Biochem. Physiol. 50A, 753–757. Felipo, V., and Butterworth, R. F. (2002). Neurobiology of ammonia. Prog. Neurobiol. 67, 259–279. Fels, L. M., Kastner, S., and Stolte, H. (1998). The hagfish kidney as a model to study renal physiology and toxicology. In ‘‘The Biology of Hagfishes’’ (Jørgensen, J. M., Lomholt, J. P., Weber, R. E., and Malte, H., Eds.), pp. 347–363. Chapman and Hall, London. Felskie, A. K., Anderson, P. M., and Wright, P. A. (1998). Expression and activity of carbamoyl phosphate synthetase III and ornithine urea cycle enzymes in various tissues of four fish species. Comp. Biochem. Physiol. 119B, 355–364. Fines, G. A., Ballantyne, J. S., and Wright, P. A. (2001). Active urea transport and an unusual basolateral membrane composition in the gills of a marine elasmobranch. Am. J. Physiol. 280, R16–R24. Fontenot, Q. C., Isely, J. J., and Tomasso, J. R. (1998). Acute toxicity of ammonia and nitrite to shortnose sturgeon fingerlings. Prog. Fish Cult. 60, 315–318. Forster, R. P., and Goldstein, L. (1966). Urea synthesis in the lungfish: Relative importance of purine and ornithine cycle pathways. Science 153, 1650–1652. Gershanovich, A. D., and Pototskij, I. V. (1995). The peculiarities of non‐faecal nitrogen excretion in sturgeons (Pisces: Acipenseridae). 2. EVects of water temperature, salinity and pH. Comp. Biochem. Physiol. 111A, 313–317. Goldstein, L., Janssens, P. A., and Forster, R. P. (1967). Lungfish Neoceratodus forsteri: Activities of ornithine–urea cycle and enzymes. Science 157, 316–317. Graham, J. B. (1997). ‘‘Air‐Breathing Fishes.’’ Academic Press, San Diego. GriYth, R. W. (1991). Guppies, toadfish, lungfish, coelacanths and frogs: A scenario for the evolution of urea retention in fishes. Environ. Biol. Fish. 32, 199–218. GriYth, R. W. (1994). The life of the first vertebrates. Bioscience 44, 408–418. GriYth, R. W., Umminger, B. L., Grant, B. F., Pang, P. K. T., and Pickford, G. E. (1974). Serum composition of the coelacanth, Latimeria chalumnae Smith. J. Exp. Biol. 187, 87–102. GriYth, R. W., Umminger, B. L., Grant, B. F., Pang, P. K. T., Goldstein, L., and Pickford, G. (1976). Composition of bladder urine of the coelacanth,. Latimeria chalumnae. J. Exp. Zool. 196, 371–380.

6.

OSMOREGULATION

313

Handlogten, M. E., Hong, S.‐P., WesthoV, C. M., and Weiner, I. D. (2004). Basolateral ammonium transport by the mouse inner medullary collecting duct cell (mIMCD‐3). Am. J. Physiol. 287, F628–F638. Helfman, G. S., Collette, B. B., and Facey, D. E. (1997). ‘‘The Diversity of Fishes.’’ Blackwell Science, Malden, Massachusetts. Hill, W. G., Mathai, J. C., Gensure, R. H., Zeidel, J. D., Apodaca, G., Saenz, J. P., Kinne‐ SaVran, E., Kinne, R., and Zeidel, M. L. (2004). Permeabilities of teleost and elasmobranch gill apical membranes: Evidence that lipid bilayers alone do not account for barrier function. Am. J. Physiol. 287, C235–C242. Holder, M. T., Erdmann, M. V., Wilcox, T. P., Caldwell, R. L., and Hillis, D. M. (1999). Two living species of coelacanths? Proc. Natl. Acad. Sci. USA 96, 12616–12620. Holland, N. D., and Chen, J. (2001). Origin and early evolution of the vertebrates: New insights from advances in molecular biology, anatomy, and palaeontology. Bioessays 23, 142–151. Huang, C.‐H., and Peng, J. (2005). Evolutionary conservation and diversification of Rh family genes and proteins. Proc. Natl. Acad. Sci. USA 102, 15512–15517. Hung, C. Y. C., Tsui, K. N. T., Wilson, J. M., Nawata, C. M., Wood, C. P., and Wright, P. A. (2007). Rhesus glycoprotein gene expression in the mangrove killifish kryptolebias marmoratus exposed to elevated environmental ammonia levels and air. J.Exp.Biol. (in press). Hyodo, S., Katoh, F., Kaneko, T., and Takei, Y. (2004). A facilitative urea transporter is localized in the renal collecting tubule of the dogfish Triakis scyllia. J. Exp. Biol. 207, 347–356. Ikemoto, M., Tabata, M., Miyake, T., Kono, T., Mori, M., Totani, M., and Murachi, T. (1990). Expression of human liver arginase in Escherichia coli. Purification and properties of the product. Biochem. J. 270, 697–703. Ip, Y. K., Chew, S. F., and Randall, D. J. (2001). Ammonia toxicity, tolerance, and excretion. In ‘‘Fish Physiology Vol. 20, Nitrogen Excretion’’ (Wright, P., and Anderson, P., Eds.), pp. 109–148. Academic Press, San Diego. Ip, Y. K., Chew, S. F., and Randall, D. J. (2004). Five tropical air‐breathing fishes, six diVerent strategies to defend against ammonia toxicity on land. Physiol. Biochem. Zool. 77, 768–782. Ip, Y. K., Peh, B. K., Tam, W. L., Lee, S. L. M., and Chew, S. F. (2005). Changes in salinity and ionic compositions can act as environmental signals to induce a reduction in ammonia production in the African lungfish Protopterus dolloi. J. Exp. Zool. 303A, 456–463. Janech, M. G., Fitzgibbon, W. R., Chen, R., Nowak, M. W., Miller, D. H., Paul, R. V., and Ploth, D. W. (2003). Molecular and functional characterization of a urea transporter from the kidney of the Atlantic stingray. Am. J. Physiol. 284, F996–F1005. Janech, M. G., Fitzgibbon, W. R., Nowak, M. W., Miller, D. H., Paul, R. V., and Ploth, D. W. (2006). Cloning and functional characterization of a second urea transporter (strUT‐2) from the kidney of the Atlantic stingray, Dasyatis sabina. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R844–R853. Janssens, P. A. (1964). The metabolism of the aestivating African lungfish. Comp. Biochem. Physiol. 11, 105–117. Janssens, P. A., and Cohen, P. P. (1966). Ornithine‐urea cycle enzymes in the African lungfish, Protopterus aethiopicus. Science 152, 358–359. Janssens, P. A., and Cohen, P. P. (1968). Nitrogen metabolism in the African lungfish. Comp. Biochem. Physiol. 24, 879–886. Jarvis, P. L., and Ballantyne, J. S. (2003). Metabolic responses to salinity acclimation in juvenile shortnose sturgeon Acipenser brevirostrum. Aquaculture 219, 891–909. Kajimura, M., Croke, S. J., Glover, C. N., and Wood, C. M. (2004). The eVect of feeding and fasting on the excretion of ammonia, urea and other nitrogenous waste products in rainbow trout. J. Exp. Biol. 207, 1993–2002.

314

PATRICIA A. WRIGHT

Karnaky, K. J., Jr. (1986). Structure and function of the chloride cell of Fundulus heteroclitus and other teleosts. Am. Zool. 26, 209–224. Karnaky, K. J., Jr. (1998). Osmotic and ionic regulation. In ‘‘The Physiology of Fishes’’ (Evans, D. H., Ed.), 2nd edn., pp. 157–176. CRC Press, Boca Raton. Kato, A., and Sands, J. M. (1998). Active sodium-urea counter-transport is inducible in the basolateral membrance of rat renal initial inner medullary collecting ducts. J. Clin. Invest. 102, 1008–1015. KieVer, J. D., Wakefield, A. M., and Litvak, M. K. (2001). Juvenile sturgeon exhibit reduced physiological responses to exercise. J. Exp. Biol. 204, 4281–4289. Kirschner, L. B. (1993). The energetics of osmotic regulation in ureotelic and hypoosmotic fishes. J. Exp. Zool. 267, 19–26. Korsgaard, B., Mommsen, T. P., and Wright, P. A. (1995). Adaptive relationships to environment ontogenesis and viviparity. In ‘‘Nitrogen Metabolism and Excretion’’ (Walsh, P. J., and Wright, P., Eds.), pp. 259–287. CRC Press, Boca Raton. Krayushkina, L. S., Panov, A. A., Gerasimov, A. A., and Potts, W. T. W. (1996). Changes in sodium, calcium and magnesium ion concentrations in sturgeon (Huso huso) urine and in kidney morphology. J. Comp. Physiol. 165B, 527–533. Lacy, E. R., Reale, E., Schusselburg, D. S., Smith, W. K., and Woodward, D. J. (1985). A renal counter‐current system in marine elasmobranch fish: A computer aided reconstruction. Science 227, 1351–1354. Laurent, P. (1984). Gill internal morphology. In ‘‘Fish Physiology’’ (Hoar, W. S., and Randall, D. J., Eds.), Vol. 10A, pp. 73–183. Academic Press, Orlando. Laurent, P., Delaney, R. G., and Fishman, A. P. (1978). The vasculature of the gills in the aquatic and aestivating lungfish (Protopterus aethiopicus). J. Morphol. 56, 173–208. Lim, C. K., Wong, W. P., Lee, S. M. L., Chew, S. F., and Ip, Y. K. (2004). The ammonotelic African lungfish, Protopterus dolloi, increases the rate of urea synthesis and becomes ureotelic after feeding. J. Comp. Physiol. B 174, 555–564. Locket, N. A. (1980). Some advances in coelacanth biology. Proc. R. Soc. Lond. B 208, 265–307. Logan, A. G., Morris, R., and Rankin, J. C. (1980). A micro‐puncture study of kidney function in the river lamprey Lampetra fluviatilis adapted to sea water. J. Exp. Biol. 88, 239–247. Loong, A. M., Hiong, K. C., Lee, S. M. L., Wong, W. P., Chew, S. F., and Ip, Y. K. (2005). Ornithine‐urea cycle and urea synthesis in African lungfishes, Protopterus aethiopicus and Protopterus annectens, exposed to terrestrial conditions for six days. J. Exp. Zool. 303A, 354–365. Lutz, P. L., and Robertson, J. D. (1971). Osmotic constituents of the coelacanth Latimeria chalumnae Smith. Biol. Bull. 141, 553–560. Mangum, C. P. (1991). Urea and chloride sensitivities of coelacanth hemoglobin. Environ. Biol. Fishes 32, 219–222. Mallat, J., and Paulsen, C. (1986). Gill ultrastructure of the Pacific hagfish Eptatretus stouti. Am. J. Anat. 177, 243–269. Mallat, J., Bailey, J. F., Lampa, S. J., Evans, M. A., and Tate, W. (1995). Quantitive ultrastructure of gill epithelial cells in the larval lamprey Petromyzon marinus. Can. J. Fish Aquat. Sci. 52, 1150–1164. Mallat, J., Conley, D. M., and Ridgway, R. L. (1987). Why do hagfish have gill ‘‘chloride cells’’ when they need not regulate plasma NaCl concentration? Can. J. Zool. 65, 1956–1965. Marshall, W. S. (2002). Naþ, Cl
, Ca2þ and Zn2þ transport by fish gills: Retrospective review and prospective synthesis. J. Exp. Zool. 293, 264–283. Marshall, W. S., and Grosell, M. (2006). Ion transport, osmoregulation, and acid‐base balance. In ‘‘The Physiology of Fishes’’ (Evans, D. H., and Claiborne, J. B., Eds.), 3rd edn., pp. 177–230. CRC Press, Boca Raton.

6.

OSMOREGULATION

315

Martı´nez‐Al´varez, R. M., Hidalgo, M. C., Domezain, H., Morales, A. E., Garcı´a‐Gallego, M., and Sanz, A. (2002). Physiological changes in sturgeon Acipenser naccarii caused by increasing environmental salinity. J. Exp. Biol. 205, 3699–3706. Martini, F. H. (1998). Secrets of the slime hag. Sci. Am. 279, 70–75. McDonald, D. G., and Milligan, C. L. (1992). Chemical properties of the blood. In ‘‘Fish Physiology’’ (Hoar, W. S., Randall, D. J., and Farrell, A. P., Eds.), Vol. XIIB, pp. 55–133. Academic Press, San Diego. McDonald, D. G., Cavdek, V., Calvert, L., and Milligan, C. L. (1991). Acid‐base regulation in the Atlantic hagfish. Myxine glutinosa. J. Exp. Biol. 161, 201–215. McEnroe, M., and Cech, J. J., Jr. (1985). Osmoregulation in juvenile and adult white sturgeon, Acipenser transmontanus. Environ. Biol. Fishes 14, 23–30. McFarland, W. N., and Munz, F. W. (1965). Regulation of body weight and serum composition by hagfish in various media. Comp. Biochem. Physiol. 14, 383–398. McInerney, J. E. (1974). Renal sodium reabsorption in the hagfish, Eptatretus stouti. Comp. Biochem. Physiol. 49A, 273–280. McKenzie, D. J., and Randall, D. J. (1990). Does Amia calva aestivate? Fish Physiol. Biochem. 8, 147–158. McKenzie, D. J., Cataldi, E., Di Marco, P., Mandich, A., Romano, P., Ansferri, S., Bronzi, P., and Cataudella, S. (1999). Some aspects of osmotic and ionic regulation in Adriatic sturgeon (Acipenser naccarii). II. Morpho‐physiological adjustments to hyperosmotic environments. J. Appl. Ichthyol. 15, 61–66. McKenzie, D. J., Cataldi, E., Romano, P., Owen, S. F., Taylor, E. W., and Bronzi, P. (2001). EVects of acclimation to brackish water on the growth, respiratory metabolism, and swimming performance of young‐of‐the‐year Adriatic sturgeon (Acipenser naccarii). Can. J. Fish Aquat. Sci. 58, 1004–1112. Mommsen, T. P., and Hochachka, P. W. (1988). The purine nucleotide cycle as two temporally separated metabolic units—a study on trout muscle. Metabolism 36, 552–556. Mommsen, T. P., and Walsh, P. J. (1989). Evolution of urea synthesis in vertebrates: The piscine connection. Science 243, 72–74. Moore, J. W., and Mallatt, J. M. (1980). Feeding of larval lamprey. Can. J. Fish Aquat. Sci. 37, 1658–1664. Morgan, R. L., Ballantyne, J. S., and Wright, P. A. (2003). Regulation of a renal urea transporter with reduced salinity in a marine elasmobranch, Raja erinacea. J. Exp. Biol. 206, 3285–3292. Morris, R. (1972). Osmoregulation. In ‘‘The Biology of Lampreys ’’ (Hardisty, M. W., and Potter, I. C., Eds.), Vol. 2, pp. 193–239. Academic Press, London. Morris, R., and Bull, J. M. (1968). Studies on freshwater osmoregulation in the ammocoete larva of Lampetra planeri (Bloch). II. The eVect of de‐ionized water and temperature on sodium balance. J. Exp. Biol. 48, 597–609. Morris, R., and Bull, J. M. (1970). Studies on freshwater osmoregulation in the ammocoete larva of Lampetra planeri (Bloch). III. The eVect of external and internal sodium concentration on sodium transport. J. Exp. Biol. 52, 275–290. Munz, F. W., and McFarland, W. N. (1964). Regulatory function of a primitive vertebrate kidney. Comp. Biochem. Physiol. 13, 381–400. Nakhoul, N. L., DeJong, H., Abdulnour‐Nakhoul, S. M., Boulpaep, E. L., Hering‐Smith, K., and Hamm, L. L. (2005). Characteristics of renal Rhbg as an NHþ 4 transporter. Am. J. Physiol. 288, F170–F181. Noguchi, T., Takada, Y., and Fujiwara, S. (1979). Degradation of uric acid to urea and glyoxylate in peroxisomes. J. Biol. Chem. 254, 5272–5275. Peek, W. D., and Youson, J. H. (1979). Ultrastructure of chloride cells in young adults of the anadromous sea lamprey, Petromyzon marinus L., in freshwater and during adaptation to sea water. J. Morphol. 160, 143–164.

316

PATRICIA A. WRIGHT

Perry, S. F., Gilmour, K. M., Swenson, E. R., Vulesevic, B., Chew, S. F., and Ip, Y. K. (2005a). An investigation of the role of carbonic anhydrase in aquatic and aerial gas transfer in the African lungfish Protopterus dolloi. J. Exp. Biol. 208, 3805–3815. Perry, S. F., Gilmour, K. M., Valesevic, B., McNeill, B., Chew, S. F., and Ip, Y. K. (2005b). Circulating catecholamines and cardiorespiratory responses in hypoxic lungfish (Protopterus dolloi): A comparison of aquatic and aerial hypoxia. Physiol. Biochem. Zool. 78, 325–334. Pickering, A. D., and Morris, R. (1970). Osmoregulation of Lampetra fluviatilis L. and Petromyzon marinue (Cyclostomata) in hyperosmotic solutions. J. Exp. Biol. 53, 231–243. Pickford, G. E., and Grant, F. B. (1967). Serum osmolality in the coelacanth, Latimeria chalumnae: Urea retention and ion regulation. Science 155, 568–570. Potts, W. T. W., and Rudy, P. P. (1972). Aspects of osmotic and ionic regulation in the sturgeon. J. Exp. Biol. 56, 703–715. Pugh, E. L., Bittman, R., Fugler, L., and Kates, M. (1989). Comparison of steady‐state fluorescence polarization and urea permeability of phosphatidylcholine and phosphatidylsulfocholine liposomes as a function of sterol structure. Chem. Phys. Lipids 50, 43–50. Randall, D. J., and Tsui, T. K. N. (2002). Ammonia toxicity in fish. Mar. Pollut. Bull. 45, 17–23. Rankin, J. C., Cobb, C. S., Frankling, S. C., and Brown, J. A. (2001). Circulating angiotensins in the river lamprey, Lampetra fluviatilis, acclimated to freshwater and seawater: Possible involvement in the regulation of drinking. Comp. Biochem. Physiol. 129B, 311–318. Read, L. J. (1968). A study of ammonia and urea production and excretion in the fresh‐water‐ adapted form of the Pacific lamprey, Entosphenus tridentatus. Comp. Biochem. Physiol. 26, 455–466. Read, L. J. (1975). Absence of ureogenic pathways in liver of the hagfish Bdellostoma cirrhatum. Comp. Biochem. Physiol. 51B, 139–141. Richardson, M. K., and Wright, G. M. (2003). Developmental transformations in a normal series of embryos of the sea lamprey Petromyzon marinus (Linnaeus). J. Morphol. 257, 348–363. Riegel, J. A. (1998). An analysis of the function of the glomeruli of the hagfish mesonephric kidney. In ‘‘The Biology of Hagfishes’’ (Jørgensen, J. M., Lomholt, J. P., Weber, R. E., and Malte, H., Eds.), pp. 364–377. Chapman and Hall, London. Ripoche, P., Bertrand, O., Gane, P., Birkenmeier, C., Colin, Y., and Cartron, J.‐P. (2004). Human Rhesus‐associated glycoprotein mediates facilitated transport of NH3 into red blood cells. Proc. Natl. Acad. Sci. USA 101, 17222–17227. Robertson, J. D. (1963). Osmoregulation and ionic composition of cells and tissues. In ‘‘The Biology of Myxine’’ (Brodal, A., and Fa¨nge, R., Eds.), pp. 503–515. Scandinavian University Books, Oslo. Robertson, J. D. (1989). Osmotic constituents of the blood plasma and parietal muscle of Scyliorhinus canicula (L.). Comp. Biochem. Physiol. 93A, 799–805. Rodrı´guez, A., Gallardo, M. A., Gisbert, E., Santilari, S., Ibarz, A., Sa´nchez, J., and Castello´‐ Orvay, F. (2002). Osmoregulation in juvenile Siberian sturgeon (Acipenser baerii). Fish Physiol. Biochem. 26, 345–354. Smith, C. P., and Wright, P. A. (1999). Molecular characterization of an elasmobranch urea transporter. Am. J. Physiol. 276, R622–R626. Smith, H. W. (1929). The excretion of ammonia and urea by the gills of the fish. J. Biol. Chem. 81, 727–742. Smith, H. W. (1930). Metabolism of the lung‐fish, Protopterus æthiopicus. J. Biol. Chem. 88, 97–130. Smith, H. W. (1932). Water regulation and its evolution in the fishes. Q. Rev. Biol. 7, 1–26.

6.

OSMOREGULATION

317

Smith, H. W. (1961). ‘‘From Fish to Philosopher.’’ American Museum of Natural History, New York. Steele, S. L., Yancey, P. H., and Wright, P. A. (2004). Osmoregulation during early embryonic development in the marine little skate Raja erinacea; response to changes in external salinity. J. Exp. Biol. 207, 2021–2031. Steele, S. L., Yancey, P. H., and Wright, P. A. (2005). The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low‐salinity challenge. Physiol. Biochem. Zool. 78, 216–226. Terjesen, B. F., Chadwick, T. D., Verreth, J. A. J., Rønnestad, I., and Wright, P. A. (2001). Pathways for urea production during early life of an air‐breathing teleost, the African catfish Clarias gariepinus Burchell. J. Exp. Biol. 204, 2155–2165. Thurston, R. V., Russo, R. C., and Vinogradov, G. A. (1981). Ammonia toxicity to fishes. EVects of pH on the toxicity of the un‐ionized ammonia species. Environ. Sci. Technol. 15, 837–840. Vigetti, D., Binelli, G., Monetti, C., Prati, M., Bernardini, G., and Gornati, R. (2003). Selective pressure on the allantoicase gene during vertebrate evolution. J. Mol. Evol. 57, 650–658. Walsh, P. J. (1997). Evolution and regulation of urea synthesis and ureotely in (Batrachoidid) fishes. Annu. Rev. Physiol. 59, 299–323. Walsh, P. J. (1998). Nitrogen excretion and metabolism. In ‘‘The Physiology of Fishes’’ (Evans, D. H., Ed.), 2nd edn., pp. 199–214. CRC Press, Boca Raton. Walsh, P. J., and Mommsen, T. J. (2001). Evolutionary considerations of nitrogen metabolism and excretion. In ‘‘Fish Physiology Vol. 20, Nitrogen Excretion’’ (Wright, P., and Anderson, P., Eds.), pp. 1–30. Academic Press, San Diego. Walsh, P. J., and Smith, C. P. (2001). Urea transport. In ‘‘Fish Physiology, Vol. 20, Nitrogen Excretion’’ (Wright, P. A., and Anderson, P. M., Eds.), pp. 279–307. Academic Press, San Diego. Walsh, P. J., Wang, Y., Campbell, C. E., DeBoeck, G., and Wood, C. M. (2001). Patterns of nitrogenous waste excretion and gill urea transporter mRNA expression in several species of marine fish. Mar. Biol. 139, 839–844. Walsh, P. J., Veauvy, C. M., McDonald, M. D., Pamenter, M. E., Buck, L. T., and Wilkie, M. P. (2006). Piscine insights into comparisons of anoxia tolerance, ammonia toxicity, stroke and hepatic encephalopathy. Comp. Biochem. Physiol. Part A (in press). Wang, Y., and Walsh, P. J. (2000). High ammonia tolerance in fishes of the family Batrachoididae (toadfish and midshipman). Aquat. Toxicol. 50, 205–219. Weihrauch, D., Morris, S., and Towle, D. W. (2004). Ammonia excretion in aquatic and terrestrial crabs. J. Exp. Biol. 207, 4491–4504. Wicks, B. J., and Randall, D. J. (2002). The eVect of sub‐lethal ammonia exposure on fed and unfed rainbow trout: The role of glutamine in regulation of ammonia. Comp. Biochem. Physiol. 132A, 275–285. Wilkie, M. P. (2002). Ammonia excretion and urea handling by fish gills: Present understanding and future research challenges. J. Exp. Zool. 293, 284–301. Wilkie, M. P., and Wood, C. M. (1991). Nitrogenous waste excretion acid‐base regulation, and ionoregulation in rainbow trout (Oncorhynchus mykiss) exposed to extremely alkaline water. Physiol. Zool. 64, 1069–1086. Wilkie, M. P., Wang, Y., Walsh, P. J., and Youson, J. H. (1999). Nitrogenous waste excretion by the larvae of a phylogenetically ancient vertebrate: The sea lamprey (Petromyzon marinus). Can. J. Zool. 77, 707–715. Wilkie, M. P., Bradshaw, P. G., Joanis, V., Claude, J. F., and Swindell, S. L. (2001). Rapid metabolic recovery following vigorous exercise in burrow‐dwelling larval sea lampreys (Petromyzon marinus). Physiol. Biochem. Zool. 74, 261–272.

318

PATRICIA A. WRIGHT

Wilkie, M. P., Turnbull, S., Bird, J., Wang, Y. S., Claude, J. F., and Youson, J. H. (2004). Lamprey parasitism of sharks and teleosts: High capacity urea excretion in an extant vertebrate relic. Comp. Biochem. Physiol. 138A, 485–492. Wilkie, M. P., Claude, J. F., Cockshutt, A., Holmes, J. A., Wang, Y. S., Youson, J. H., and Walsh, P. J. (2006). Shifting patterns of nitrogen excretion and amino acid catabolism capacity during life cycle of the sea lamprey (Petromyzon marinus). Physiol. Biochem. Zool. 79, 885–898. Wilkie, M. P., Morgan, T. P., Galvez, F., Smith, R. W., Kajimura, M., Ip, Y. K., and Wood, C. M. (2007). The African lungfish (Protopterus dolloi): Ionoregulation and osmoregulation in a fish out of water. Physiol. Biochem. Zool. 80, 99–112. Wood, C. M. (1993). Ammonia and urea metabolism and excretion. In ‘‘The Physiology of Fishes’’ (Evans, D. H., Ed.), pp. 379–425. CRC Press, Boca Raton. Wood, C. M. (2001). Influence of feeding exercise, and temperature on nitrogen metabolism and excretion. In ‘‘Nitrogen Excretion’’ (Wright, P. A., and Anderson, P. M., Eds.), pp. 201–218. Academic Press, San Diego. Wood, C. M., Walsh, P. J., Chew, S. F., and Ip, Y. K. (2005a). Ammonia tolerance in the slender lungfish (Protopterus dolloi): The importance of environmental acidification. Can. J. Zool. 83, 507–517. Wood, C. M., Walsh, P. J., Chew, S. F., and Ip, Y. (2005b). Greatly elevated urea excretion after air exposure appears to be carrier mediated in the slender lungfish (Protopterus dolloi). Physiol. Biochem. Zool. 78, 893–907. Wourms, J. P., Atz, J. W., and Stribling, M. D. (1991). Viviparity and the maternal‐embryonic relationship in the coelacanth Latimeria chalumnae. Environ. Biol. Fishes 32, 225–248. Wright, P. A. (1995). Nitrogen excretion: Three end products, many physiological roles. J. Exp. Biol. 198, 273–281. Wright, P. A., and Fyhn, J. H. (2001). Ontogeny of nitrogen metabolism and excretion. In ‘‘Nitrogen Excretion’’ (Wright, P. A., and Anderson, P. M., Eds.), pp. 149–200. Academic Press, San Diego. Wright, P. A., and Wood, C. M. (1985). An analysis of branchial ammonia excretion in the freshwater rainbow trout eVects of environmental pH change and sodium uptake blockade. J. Exp. Biol. 114, 329–353. Wright, P. A., Randall, D. J., and Wood, C. M. (1988). The distribution of ammonia and Hþ between tissue compartments in lemon sole (Parophrys vetulus) at rest, during hypercapnia and following exercise. J. Exp. Biol. 136, 149–175. Wright, P. A., Felskie, A. K., and 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. Wright, P. A., Campbell, A., Morgan, R. L., Rosenberger, A. G., and Murray, B. W. (2004). Expression of arginase type I and II genes in rainbow trout: Influence of fasting on liver enzyme activity and mRNA levels in juveniles. J. Exp. Biol. 207, 2033–2042. Yancey, P. H. (2001). Nitrogen compounds as osmolytes. In ‘‘Fish Physiology Vol. 20, Nitrogen Excretion’’ (Wright, P. A., and Anderson, P. M., Eds.), pp. 309–341. Academic Press, San Diego. Youson, J. H. (1980). Morphology and physiology of lamprey metamorphosis. Can. J. Fish Aquat. Sci. 37, 1687–1710. Youson, J. H., and Freeman, P. A. (1976). Morphology of the gills of larval and parasitic adult sea lamprey Petromyzon marinus L. J. Morphol. 149, 73–104.