5 Iono- and Osmoregulation

With the exception of some prokaryotes (e.g. halobacteria), metabolism in all cells has evolved to function optimally in a particular ionic milieu. Intracellular Na⁺ is low (~10 mM), K⁺ is high (~130 mM), and concentrations of divalent cations (Ca²⁺, Mg²⁺, etc.) are in the micro- to low millimolar range. Maintaining this ionic environment is critical because natural selection has optimized macromolecular structure and function under these conditions. Different strategies of achieving intracellular ion homeostasis have evolved to support cell function in environments with variable ion concentrations. Cells of fishes and other multicellular animals are isolated from the environment not only by their plasma membrane but also by an epithelial cell layer that separates the body fluids from the external milieu (Figure 5.1). This additional layer buffers environmental salinity fluctuations at the level of body fluids, shifting some of the energetic burden of maintaining intracellular ion homeostasis from individual cells to the level of specialized epithelial tissues that are in direct contact with the external milieu. Most fishes, except primitive taxa, pursue this strategy of osmoregulation to maintain intracellular ion homeostasis.

FIGURE 5.1 The osmotic and ionic milieu is controlled at two barriers. (a) The outer barrier (blue line) reflects epithelia that, in most fishes, maintain an osmotic gradient between the external environment and the extracellular fluid (ECF). (b) The inner barrier (red line) is the plasma membrane surrounding each cell, which maintains strong ionic (but not osmotic) gradients (e.g. [Na⁺] and [K⁺]) between ECF and intracellular fluid (ICF).

5.2 Evolutionary Strategies

Fishes encompass a spectrum of strategies ranging from osmoconformers to osmoregulators (Table 5.1; Figure 5.2). In osmoconformers, ionic homeostasis is regulated primarily at the level of cells. This strategy is evolutionarily primitive and employed by hagfish. However, lamprey (the other clade of jawless fishes) are osmoregulators, a more advanced strategy, as are teleosts (bony fish). Elasmobranchs use a third approach, being osmoconformers but strong ionoregulators. These strategies are briefly discussed in the following subsections.

Table 5.1
Ionic and Osmotic Composition of Aquatic Environments and the Body Fluids of Representative Fishes

FIGURE 5.2 Osmotic and ionic strategies of (a) hagfish, (b) lamprey, (c) elasmobranch and (d) teleost fishes in sea water, and (e) lamprey, (f) elasmobranch and (g) teleost fishes in fresh water. Grey arrows indicate obligate ion and water movements; black arrows indicate regulated responses to maintain ionic and/or osmotic homeostasis.

5.2.1 Hagfish

The body fluids of these primitive marine fish are approximately isosmotic with seawater (SW), whereas their ionic composition is similar but not identical to that of SW (Robertson 1976). Hagfish represent an early evolutionary stage at which the burden of maintaining intracellular ion homeostasis has started to shift from the plasma membrane of cells to the level of specialized epithelia. These epithelia provide a barrier between the fish and the environment, allowing the ionic composition of the body fluids to be controlled. However, ion differences across hagfish external epithelia are small because ionoregulatory mechanisms are at a primitive evolutionary stage. Specialized epithelial cells (ionocytes) probably evolved to support acid–base homeostasis and/or nitrogenous waste excretion rather than ionoregulation (Evans 1984). Hagfish have ionocytes in their osmoregulatory epithelia and may represent an evolutionary transition in which ionocytes start to be adopted for ionoregulation beyond their other roles.

5.2.2 Lamprey

Unlike hagfish, lamprey have conquered freshwater (FW), with some species remaining in FW throughout their life cycle, while others develop in FW but spend most of their life in SW. The evolutionary transition from SW to FW requires effective ionoregulation because FW ion concentrations are too low to be tolerated in the body fluids. Thus, FW invasion required the evolution of mechanisms to maintain significant ionic and osmotic gradients across external epithelia. As in teleosts and tetrapods, the body fluid osmolality of FW lamprey is 300–350 mosmol/kg. To achieve hyperosmotic body fluids relative to FW, lamprey actively accumulate NaCl and excrete water, processes that are energy dependent. Active NaCl uptake counteracts passive diffusional NaCl loss from the body fluids to FW and is achieved using ionocytes in the branchial epithelium. Thus, one evolutionary advance that appeared in the ancestor of extant lamprey was the functional adoption of branchial ionocytes for iono- and osmoregulation. A second, equally important advance was the adoption of renal glomeruli and distal tubules for selective filtration and excretion of water. These renal novelties were critical for maximizing NaCl reabsorption and retention (Beyenbach 2004).

5.2.3 Elasmobranchs

Elasmobranchs include sharks, skates, and rays, and most species are marine, although several euryhaline species complete at least part of their life cycle in FW. Marine elasmobranchs are strong ionoregulators while still being osmoconformers. They maintain an NaCl concentration of about one-third SW yet a total osmolality of ~1000 mosmol/kg, slightly hyperosmotic to SW, in their body fluid. Organic osmolytes, primarily urea and trimethylamine oxide (TMAO), bridge this ‘osmotic gap’. The passive inward diffusion of NaCl is countered by active NaCl excretion by ionocytes in the rectal gland. Marine elasmobranchs must also actively retain elevated urea and TMAO. To counter diffusional loss of these organic osmolytes to SW, they are constantly produced as by-products of nutrient metabolism.

Euryhaline elasmobranchs are iono- and osmoregulators in FW, with high body fluid osmolality (>600 mosmol/kg) due to urea and TMAO concentrations that are reduced in FW but remain considerable. The high osmolality of elasmobranch body fluids relative to FW presents a significant energetic burden; that is, in FW, elasmobranchs would have to either greatly tighten their epithelia to minimize diffusion and/or actively absorb NaCl against huge concentration and osmotic gradients. Unlike for teleosts, it is currently unknown whether euryhaline elasmobranchs regulate the leakiness of epithelial tight junctions when entering FW. Moreover, the number of euryhaline elasmobranchs able to enter FW for extended periods is small relative to their strictly marine counterparts. It appears that the energetic burden associated with maintaining elevated body fluid osmolality prevents widespread evolutionary expansion of elasmobranchs into FW habitats. The few stenohaline FW elasmobranchs (e.g. the Amazonian stingray Potamotrygon sp.) have low urea levels similar to those of FW teleosts (Table 1, Evans and Claiborne 2009).

5.2.4 Teleosts

All teleosts are osmoregulators, with body fluid osmolality of ~300–350 mosmol/kg in both FW and SW. In an evolutionary sense, this strategy shifted much of the burden of maintaining intracellular ion homeostasis from the level of the cell to that of specialized epithelia. For instance, instead of a Na⁺ gradient across the plasma membrane of ~500 mosmol/kg in SW versus 10 mosmol/kg intracellular, it becomes ~150 mosmol/kg in body fluids versus 10 mosmol/kg intracellular. However, maintaining body fluid osmolality below that of SW represents a formidable task for marine teleosts. Osmotic water loss across the external epithelia is countered by ingestion of SW. Water absorption across the intestine is driven by NaCl absorption, which adds to the NaCl load gained by diffusion. Excess NaCl is actively excreted by gill ionocytes, while the absorbed water is actively retained by reabsorption in the renal tubule, keeping urinary volume to the minimum necessary for waste and divalent ion excretion. This process yields a net gain of water that compensates for the dehydration resulting from osmosis. Drinking rates of euryhaline fishes vary with environmental salinity, being virtually zero in FW and high in SW. Maintaining body fluid osmolality above that of FW similarly represents a challenge for FW teleosts. Water gain by osmosis across the external epithelia is countered by copious production of dilute urine, which is not completely devoid of ions and aggravates ion loss. Thus, gill ionocytes in FW teleosts actively take up NaCl and Ca²⁺.

5.3 Physiology of Iono- and Osmoregulatory Tissues

The main iono- and osmoregulatory tissues are skin (especially during early development), gill, renal, and gastrointestinal epithelia. Other epithelia (e.g. those of the operculum, oral cavity, and pyloric caeca) may also have osmoregulatory functions but largely represent structural and functional extensions of gill or gastrointestinal epithelia and are not considered separately in the following overview. The elasmobranch rectal gland is an extension of the gastrointestinal tract (GIT) but functions like the gill of a marine teleost and will be discussed briefly in that context.

5.3.1 Skin

In most adult fishes, ion transport across the body surface is dominated by the gill (Evans et al. 2005), and the skin serves as a permeability barrier. The exceptions include specific skin regions that are ionocyte rich, such as the opercular epithelium (Degnan et al. 1977), and a few marine and amphibious species that have ionocytes across the body surface (e.g. Martin et al. 2019). The opercular epithelium was used as a model to investigate NaCl secretion in marine teleosts because, unlike the gill, it provided a flat epithelium that could be used for Ussing chamber analysis of electrophysiology, and ionocytes are readily accessible to electrodes (Marshall and Bellamy 2010).

In embryonic and larval fishes, ion transport is strictly cutaneous until the gill develops. Systemic regulation of ion and water balance begins during the early embryonic stages, when ionocytes appear in the skin and yolk sac epithelium. These cutaneous ionocytes increase in abundance as development proceeds, peaking as ionocytes first appear in the developing gill. As the gill develops, the abundance of branchial ionocytes increases, while that of cutaneous ionocytes falls (Figure 5.3). Correspondingly, ion transport activity shifts from the skin and yolk sac epithelium to the gill. Although the timing of this ontogenetic trajectory is species specific, cutaneous ionocyte density peaks during larval development, and by the juvenile stage, fishes are reliant on branchial ion transport (Varsamos et al. 2005; Rombough 2007). The transition from cutaneous to branchial ion transport reflects surface area-to-volume constraints (Rombough 2007). As the fish grows, the relative ionoregulatory capacity of the skin falls due to the declining surface area-to-volume ratio. Exacerbating this situation are two additional factors. First, the skin becomes thicker, making it more difficult for surface ionocytes to achieve transepithelial transport. Second, the period of rapid growth after hatching greatly increases the demand for ions that, for FW fishes, must be acquired from the environment. The transfer of ionoregulatory function from the skin to the gill overcomes these limitations. Indeed, the gill is required for ionic regulation earlier in development than it is required for gas transfer (Fu et al. 2010), which led to the hypothesis that early gill development is driven by requirements for ionic regulation (‘ionoregulatory hypothesis’) rather than gas transfer (‘oxygen hypothesis’; Rombough 2007). Importantly, the cutaneous ionocytes of larval fishes, particularly those of larval zebrafish (Danio rerio), have emerged as a valuable model for investigating mechanisms of active ion uptake in FW fishes (Evans 2011; Guh et al. 2015).

FIGURE 5.3 (a) Light micrograph of the lateral body and yolk sac of a larval zebrafish (Danio rerio) at 4 days-post-fertilization. Immunofluorescent markers identify three ionocyte types: green cells take up Na⁺ and excrete H⁺, red cells take up Na⁺ and Cl⁻, and blue cells take up Ca²⁺. Scale bar = 1 mm. (b) Changes in cutaneous versus branchial ionocyte abundance during early development of tilapia (Oreochromis mossambicus).
(Panel a: image courtesy of E. Kunert. Panel b: figure modified from Rombough, P., Comp. Biochem. Physiol. A, 148, 732–742, 2007.)

5.3.2 Gills

The fish gill is a structurally complex, multifunctional organ that serves as the primary site of gas transfer as well as body fluid ion, water and pH regulation and nitrogenous waste excretion. Anatomically, the gills consist of paired arches on the pharynx that support rows of filaments, from which arise small, plate-like lamellae (Evans et al. 2005). Blood flowing through the lamellae is separated from water flowing over them by a branchial epithelium that provides the extensive surface area, high permeability and thin diffusion barrier necessary for efficient gas transfer. However, the structural features that enhance gas transfer also favour diffusive ion and water movements. Gill morphology therefore represents an ‘osmorespiratory compromise’ in which lamellar surface area and the thickness of the lamellar epithelium have been selected through evolutionary time to balance the minimum rate of O₂ transfer needed for metabolism against the maximum rate of passive ion and water transfer that can be tolerated (Gonzalez 2011). To manage the osmorespiratory compromise, some fish species use reversible gill remodelling. With this strategy, the lamellar epithelium is covered by a cell mass to reduce passive ion and water movement when O₂ availability is high and O₂ demand is low, and the cell mass is shed to increase O₂ uptake in response to exercise, hypoxia or warm temperatures (Gilmour and Perry 2018).

Remodelling of the branchial epithelium itself can occur to meet increased requirements for active ion transport or to respond to acid–base challenges (Perry and Laurent 1993). The ionocytes of the branchial epithelium are mitochondrion rich and relatively sparse, accounting for <10% of the epithelial surface area. Because of their spherical shape, increases in ionocyte abundance can cause thickening of the epithelium that, in accordance with the osmorespiratory compromise, impairs gas transfer and hypoxia tolerance (Perry 1997). Ionocytes exhibit a highly folded basolateral membrane enriched in Na⁺,K⁺-ATPase (NKA), and more generally, the apical and basolateral membranes of ionocytes express distinct complements of ion-transporting proteins. Broadly, the branchial ionocytes of marine fishes carry out NaCl secretion, whereas those of FW fishes actively take up Na⁺, Cl⁻ and Ca²⁺ from the dilute environment. Ionocytes also play a key role in acid–base regulation. The structural features of branchial ionocytes differ among teleost, elasmobranch and agnathan fishes (Evans et al. 2005). Teleost ionocytes have been studied most extensively and therefore are the focus of the following discussion.

5.3.2.1 Freshwater Fishes

Classic studies of ion uptake in FW fishes (reviewed by Evans et al. 2005; Evans 2011; Gilmour and Perry 2009) indicated that Na⁺ uptake is linked to H⁺ secretion and Cl⁻ uptake is linked to HCO₃ ⁻ secretion. As CO₂ crosses the gill by diffusion, some portion is hydrated to H⁺ and HCO₃ ⁻, which serve as counter-ions in ion uptake mechanisms. Due to this coupling of NaCl uptake to secretion of an acid–base equivalent, the FW gill is essential for acid–base regulation as well as ion balance (Evans et al. 2005; Gilmour and Perry 2009). Ionocyte contributions to ion uptake have been recognized since the early 1960s (Dymowska et al. 2012), but the last 15 years have yielded major advances. Through immunohistochemistry and in situ hybridization, transporters have been localized to specific cell populations. Techniques such as scanning ion-selective electrodes (Lin et al. 2006) and isolation of specific cell populations by magnetic separation (Galvez et al. 2002) or laser capture microdissection (Leguen et al. 2015) provided insight into ionocyte function. The coupling of such approaches with loss-of-function technologies proved particularly powerful (Horng et al. 2007). Across the handful of species examined to date, there are common features but also marked differences. What is clear, however, is that multiple ionocyte types differing in transporter complement and hence function are present in the branchial epithelium (Figure 5.4).

FIGURE 5.4 Generalized ion uptake mechanisms for (a) four types of ionocyte found in the gill of freshwater teleost fish and (b) the NaCl-secreting ionocyte of the marine teleost fish gill. ATPases are indicated by black fill and facilitated diffusion mechanisms by white fill.
NHE: Na⁺,H⁺ exchanger; NCC: Na⁺,Cl⁻ cotransporter; CA: carbonic anhydrase.

An ionocyte capable of Na⁺ uptake linked to H⁺ excretion is present in all species studied to date and expresses apical Na⁺/H⁺ exchanger (NHE) and/or vacuolar-type H⁺-ATPase (VHA). This ionocyte increased in abundance in fish experiencing low Na⁺ or acidosis (Chang et al. 2013; Brannen and Gilmour 2018), suggesting a role in both Na⁺ uptake and acid–base regulation. A second ionocyte that contributes to Na⁺ uptake is present in several species. This ionocyte expresses an apical Na⁺,Cl⁻ cotransporter (NCC) and therefore also contributes to Cl^- uptake but probably not acid–base regulation (Kumai and Perry 2012). Knowledge of ionocytes involved in Cl⁻ uptake linked to HCO₃ ⁻ secretion (and hence acid–base regulation) remains incomplete. Ionocytes expressing anion exchangers are present in zebrafish (Bayaa et al. 2009; Perry et al. 2009). In rainbow trout (Oncorhynchus mykiss), an ionocyte type increased in response to alkalosis, suggesting a role in HCO₃ ⁻ secretion (Brannen and Gilmour 2018). Knowledge of Ca²⁺-transporting ionocytes is similarly sparse, except in zebrafish, where an ionocyte that expresses apical and basolateral Ca²⁺ transporters and increases in abundance in low-Ca²⁺ environments was identified (Liao et al. 2007). In short, despite considerable progress in identifying ionocytes, their transport proteins and their responses to ionic and acid–base challenges (Evans 2011; Dymowska et al. 2012; Kumai and Perry 2012; Takei et al. 2014; Guh et al. 2015), much remains to be learned, particularly on the molecular mechanisms of inwardly directed ion transport from the dilute external environment.

5.3.2.2 Marine Fishes

The branchial epithelium of marine teleosts contains an NaCl-secreting ionocyte. Unlike FW ionocytes, which generally occur singly in the branchial epithelium and form extensive, multi-stranded tight junctions with neighbouring cells, SW ionocytes occur in multicellular complexes with accompanying accessory cells. The apical membranes of these cells form a recessed crypt, which is a distinctive feature of the marine teleost branchial epithelium (Evans et al. 2005). Shallow tight junctions occur between ionocytes and accessory cells within the complex and are selectively leaky to cations, a structural feature that is of functional importance for Na⁺ secretion (Chasiotis et al. 2012).

Salt secretion is achieved by secondary active transport of Cl⁻ and passive diffusion of Na⁺ (Evans et al. 2005). The mechanism (Figure 5.4) relies on basolateral NKA, which keeps intracellular Na⁺ levels low to create a gradient for Cl⁻ entry coupled to Na⁺ via basolateral Na⁺, K⁺, 2Cl⁻-cotransporter (NKCC). Chloride ions exit the ionocyte apically via a cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel, while Na⁺ leaves paracellularly via the cation-selective leaky tight junction. The final component of the mechanism is the recycling of K⁺ ions that entered the cell via NKA and NKCC by a K⁺ channel. In addition to the suite of transporters for NaCl secretion, these ionocytes express apical NHE for acid–base regulation – Na⁺ entry down its electrochemical gradient drives H⁺ excretion. At the whole-animal level, acid and base secretion in marine fishes are linked to Na⁺ and Cl⁻ absorption, respectively, despite the resulting ionic and osmotic burden (Evans et al. 2005). However, the cellular and molecular mechanisms of acid–base regulation remain poorly defined, especially for base secretion. Recent progress focused on elasmobranch fishes, where two ionocytes contribute to acid–base regulation (Figure 5.5): an acid-secreting cell that expresses NHE and is enriched in NKA, and a base-secreting cell that expresses an apical anion exchanger (pendrin) and is enriched in VHA (see Wright and Wood 2015). Both cell types also express soluble adenylyl cyclase (sAC), an evolutionarily conserved cellular acid–base sensor (Roa and Tresguerres 2017). In fish exposed to alkalosis, activation of sAC by HCO₃ ⁻ ions triggers translocation of VHA from cytosolic vesicles to the basolateral membrane, thereby recycling H⁺ into the blood to counter the alkalosis, while HCO₃ ⁻ is exported across the apical membrane (Tresguerres et al. 2010). Whether comparable mechanisms are present in teleost gills remains to be investigated.

FIGURE 5.5 Mechanisms of base and acid secretion by ionocytes of the elasmobranch gill. White arrows indicate the sequence of events. Although sAC is present in the acid-secreting cell, its role in activating cell responses remains to be determined. sAC: soluble adenylyl cyclase; CA: carbonic anhydrase; NHE: Na⁺,H⁺ exchanger.

Key to NaCl secretion is the elaboration of the basolateral membrane, which houses the NKA that is the driving force for NaCl secretion. Similarly, some FW ionocytes exhibit an elaborate basolateral membrane enriched in NKA that helps drive ion uptake. At the molecular level, however, there are differences in NKA between marine and FW ionocytes (McCormick et al. 2009) and even among FW ionocytes (Guh et al. 2015). When euryhaline teleosts move between FW and SW, not only does NKA isoform switching occur, but so also must the complement of branchial ionocytes. How this conversion occurs is not yet well understood, although it seems to involve both transformation of ionocytes from one type to another and differentiation of new ionocytes (Hiroi and McCormick 2012; Inokuchi et al. 2017).

5.3.2.2.1 The Elasmobranch Rectal Gland

The branchial ionocytes of elasmobranch fishes contribute to acid–base regulation, to active ion uptake in species that tolerate or inhabit FW (Piermarini and Evans 2001), and probably to NaCl secretion in SW (see Wright and Wood 2015). In marine elasmobranchs, the rectal gland also secretes NaCl by producing a fluid that is iso-osmotic with plasma but only contains NaCl (Wright and Wood 2015). The rectal gland is a small, digitiform organ near the distal end of the intestine. It consists of secretory tubules that drain into a central canal and from there, to the intestine. Making up the secretory tubules are ionocytes that secrete Na⁺ and Cl⁻ using the mechanism described earlier. Although loss of the rectal gland under normal conditions may have little impact on ion balance, rectal gland NaCl secretion contributes to clearing salt and/or volume loads such as those that accompany ingestion of SW during feeding. The secretory activity of the rectal gland is under both nervous and hormonal control (Anderson 2015).

5.3.3 Kidney

In fishes, the gill is responsible for the majority of active transepithelial ion transport, but the kidney contributes to osmoregulation as well as waste excretion, detoxification, hormone production and acid–base regulation. The two main osmoregulatory functions of fish kidneys are water excretion (urine production) and the secretion of divalent ions (chiefly Mg²⁺, Ca²⁺, PO₄ ³⁻ and SO₄ ²⁻).

5.3.3.1 Freshwater Fishes

FW fishes actively counter the passive diffusion of water into the body by excreting large volumes of hypoosmotic urine. For instance, FW-acclimated European eels (Anguilla anguilla) excrete 1.1–3.5 ml urine/kg/h compared with only 0.25–0.6 ml/kg/h in SW-acclimated conspecifics (Hickman and Trump 1969). FW lampreys have even higher urinary excretion rates of 15–20 ml/kg/h (Logan et al. 1980). Urine production reflects high rates of glomerular filtration and ion reabsorption. Glomerular filtration is size selective, with larger molecules such as organic ions being retained. This property affords a mechanism for selectively retaining organic osmolytes to balance diffusional loss of ions. In addition, FW fishes actively reabsorb most of the smaller ions that enter the glomerular filtrate; for example, only 5–20 mM sodium is lost in the urine (Evans and Claiborne 2009). Reabsorption across the epithelial cells of the nephron (and the urinary bladder) relies on basolateral NKA, which generates a Na⁺ gradient that drives Na⁺-coupled transepithelial movement of Cl⁻, divalent ions and organic solutes. For example, glucose is reabsorbed via Na⁺-glucose cotransporters.

5.3.3.2 Marine Fishes

Hagfish are essentially isosmotic to SW but regulate the concentration of divalent ions. In particular, Mg²⁺ and SO₄ ²⁻ are actively excreted by myxinid kidneys (Beyenbach and Liu 1996; Beyenbach 2004). Hagfish have well-developed renal glomeruli used for selective filtration of water and small molecules and retention of larger, energy-rich molecules. Marine lamprey (P. marinus, euryhaline species in SW) and teleosts, which osmoregulate in SW, have kidneys that are functionally similar. Divalent ions are excreted, and water is reabsorbed along with NaCl to minimize urinary water loss. Water conservation is facilitated by reduced glomerular filtration rates. For example, when the euryhaline Lampetra fluviatilis was acclimated to SW, urine volume fell by 95% compared with FW-acclimated conspecifics; this was accomplished through increased water (and NaCl) reabsorption and decreased glomerular filtration rate (Logan et al. 1980).

The kidney of marine elasmobranchs reabsorbs almost all urea, TMAO, and other organic osmolytes (e.g. free amino acids) lost from the plasma by glomerular filtration (Hickman and Trump 1969). Such reabsorption is critical to maintain the high concentrations of these compounds needed to achieve isosmolality with SW at a greatly reduced plasma NaCl concentration. Urea transporters and presumably sodium-coupled amino acid transporters facilitate the reabsorption of these organic osmolytes (Schmidt-Nielsen and Rabinowitz 1964; Friedman and Hebert 1990).

Marine teleosts lack much of the distal tubule that is responsible for NaCl reabsorption in FW fish. The functional priority of the kidney in marine teleosts is water retention, which has promoted the evolutionary loss of the glomerulus in several species. Unexpectedly, some aglomerular species are euryhaline, such as the oyster toadfish Opsanus tau (Lahlou et al. 1969), although these species may not tolerate dilute environments for extended periods. Because hagfish exhibit glomerular kidneys, the aglomerular kidney of some marine teleosts represents a secondary, derived trait rather than an ancestral condition (Ditrich 2005). Water reabsorption is regulated by aquaporins (water channels), with salinity-dependent changes occurring in the expression of aquaporin isoforms in specific nephron segments (Lignot et al. 2002). The kidney in marine teleosts must also excrete excess divalent ions. This is accomplished by active transport processes driven primarily by NKA and VHA with secondary active Na⁺- and H⁺-coupled exchangers facilitating the movement of divalent cations (Evans and Claiborne 2009). These transporters concentrate divalent ions into the small volume of urine excreted by marine teleosts; for example, 80 mM for SO₄ ²⁻ and 140 mM for Mg²⁺ (Marshall and Grosell 2006).

5.3.4 Gastrointestinal Tract

The fish GIT consists of the oesophagus, stomach and various segments of the intestine, each of which contributes to osmoregulation. The function of the GIT differs between FW and marine fishes. In FW fishes, which avoid drinking, the GIT functions to absorb ions along with nutrients taken up with the diet. The dietary availability of osmolytes is variable, and the uptake of essential ions via the diet probably represents a feedback parameter for controlling appetite in FW fishes. Thus, feeding and ionoregulation are intimately linked in FW fishes because the diet represents a major source of ions in the dilute aquatic environment; dietary uptake of ions reduces the need for branchial ion uptake. Dietary ion uptake may be particularly important in FW carnivores that ingest prey rich in organic and inorganic ions.

5.3.4.1 Marine Fishes

Marine osmoregulators (lamprey and teleosts) drink up to 1% of their body weight in SW per hour, with about 75% of the ingested water being absorbed via GIT epithelia to counteract passive dehydration (Rankin 1997). Active absorption of NaCl from ingested SW generates an osmotic gradient that is harnessed to move water from the GIT into the body. Basolateral NKA generates a gradient for Na⁺ entry into the cell, and secondary active transporters use this gradient to shuttle NaCl from the GIT lumen into the epithelial cells (apical NKCC, NCC), and then to the plasma (basolateral K,Cl-cotransporter, Cl⁻ channels). Water follows NaCl passively by osmosis (Figure 5.6). Aquaporins are expressed in GIT epithelia, including oesophagus and intestine, to increase the (transcellular) water permeability of these epithelia (Lignot et al. 2002). In addition, paracellular water movement occurs across tight junctions (Evans and Claiborne 2009). An apical anion exchanger also contributes to Cl⁻ uptake, and the HCO₃ ⁻ secreted into the GIT lumen combines with divalent cations (Ca²⁺, Mg²⁺) to form insoluble carbonate precipitates. Precipitate formation in the GIT lumen is enhanced by increases (~4×) in divalent cation concentrations, as water is absorbed but the divalent cations are not. Excretion of these carbonates by marine teleosts is sufficient to contribute substantially to the global ocean carbon cycle (Wilson et al. 2009).

FIGURE 5.6 Ion transport mechanisms involved in water absorption across the intestine of the marine teleost. NCC, Na⁺,Cl⁻ cotransporter; CA, carbonic anhydrase; NBC, Na⁺,HCO₃ ⁻ cotransporter; KCC, K⁺,Cl⁻ cotransporter.

5.4 Euryhalinity

It is evident that FW and marine fishes face different osmoregulatory challenges. Energy-dependent water and salt transport rely on disparate sets of proteins, molecular transport mechanisms and cellular adaptations in FW versus marine fishes. Ionocytes differentiate into distinct populations depending on the external salinity. Furthermore, intercellular junctions, epithelial morphology, blood circulation, drinking rates and endocrine functions are markedly different in FW and marine fishes. Stenohaline fishes are limited in their ability to overcome these differences; they are adapted to either FW or SW and cannot tolerate salinity deviations that exceed 15 g/kg from the norm (Figure 5.7). In contrast, euryhaline fishes tolerate both FW and SW. These species are fewer in number than stenohaline species and have multiple independent evolutionary origins (Schultz and McCormick 2013). Even within families and genera, a mosaic phylogenetic pattern of euryhalinity is evident (Kültz 2015).

FIGURE 5.7 Tolerance limits of stenohaline marine (blue line), stenohaline freshwater (FW, green line) and euryhaline fishes (red line). Euryhaline species typically exceed the upper salinity tolerance limit of marine fishes, tolerating up to 2–4× sea water.

How did euryhalinity evolve, and what are the key physiological innovations that permitted elaborate switches from active water retention/salt secretion in SW to active water excretion/salt absorption in FW? The multiple independent evolutionary origins of euryhalinity suggest that fishes are inherently equipped with the molecular mechanisms to support both hyper- and hypo-osmoregulation. These mechanisms are likely pleiotropic, and other functions (e.g. acid–base regulation, nitrogenous waste excretion, cellular and neuroendocrine stress responses) originally drove their evolution. To understand the evolutionary and physiological processes that facilitate euryhalinity, we must ask how fishes perceive changes in environmental salinity, and how these osmotic signals are processed to direct active ion transport according to the external salinity.

5.4.1 When Does Natural Selection Favour Euryhalinity?

Euryhaline fishes evolved in environments that experience large temporal or spatial salinity gradients. Whereas stenohaline species are limited to behavioural avoidance, euryhaline fishes can respond physiologically to salinity fluctuations rather than being forced out of particular environments. If behavioural avoidance is not possible, then extreme selection pressure for euryhalinity may arise. For example, rainfall during low tide can trap marine fishes in tide pools, where they must cope with low salinity. Desert fishes that live in FW creeks and ponds may encounter the opposite scenario. If the underlying bedrock or sediment contains large amounts of salt, then evaporation during the warm season can temporarily increase water salinity even above SW. In these examples, salinity fluctuations are stochastic and can be sudden. Fishes must rapidly sense, process and respond to salinity fluctuations in these environments.

Contrasting with such temporal salinity fluctuations are the spatial salinity gradients encountered by migratory species. In this case, euryhaline fishes voluntarily migrate between FW and SW habitats (diadromy) and can anticipate and pre-acclimatize to salinity changes via dedicated developmental processes (e.g. smoltification; see McCormick 1994). Two forms of diadromy are common. Anadromous fishes begin their life cycle in FW, migrate to SW as juveniles and return to FW as adults to reproduce. Examples include salmonids (Salmo sp. and Oncorhynchus sp.), some populations of three-spined sticklebacks (Gasterosteus aculeatus) and several sturgeon (Acipenser sp.). Catadromous fishes hatch and complete early development in SW. They enter FW or low-salinity brackish water as juveniles to grow out before returning to the ocean to reproduce. Examples include eels (Anguilla sp.), mullets (e.g. Mugil cephalus) and milkfish (Chanos chanos).

Both temporal and spatial salinity gradients exist in estuaries, especially those that are subject to large tidal currents. The salinity gradient can shift horizontally, depending on whether incoming tidal flow or river-runoff prevails, and seasonally with rainfall, floods and droughts. Vertical gradients also exist because water density is affected by salinity. Stenohaline fishes in these environments are restricted to habitat within their physiological capacity, whereas euryhaline species inhabit a much larger realm. For example, the Salou estuary (Senegal) exhibits an extremely large and dynamic salinity gradient, ranging from FW to 130 g/kg, and euryhaline species such as the blackchin tilapia (Sarotherodon melanotheron) use habitat over this entire salinity gradient (Tine et al. 2011).

Despite the evolutionary advantage of increased habitat use provided by euryhalinity, most fish species are stenohaline. The energetic cost of keeping osmosensory surveillance and signalling mechanisms poised to trigger salinity acclimation processes when needed may select against euryhalinity. The following section briefly reviews these osmosensory and signalling processes.

5.4.2 Cellular Mechanisms of Osmosensing and Signal Transduction

Transitions between FW and SW environments necessitate a switch in osmoregulatory mode that is completed in several stages (Foskett et al. 1981). The first stage occurs only with acute transfer between salinity extremes and is limited in duration to approximately 1 day. It consists of rapid termination of the prevailing active ion transport mechanisms, which are now operating in the wrong direction, tightening of epithelia to minimize passive water and ion movements, and activation of cell volume regulatory and cellular stress responses to overcome temporary dysregulation of ionic/osmotic homeostasis. Such dysregulation is evident in transiently altered plasma osmolality when euryhaline fishes are acutely transferred between salinities (Pavlosky et al. 2019). In the second stage, effector mechanisms (e.g. secondary active ion transporters) that are constitutively expressed at a minimum level are activated rapidly to operate in the proper direction and support the new osmoregulatory demand. This stage is completed within approximately 3 days and is characterized by altered expression and posttranslational regulation of osmoregulatory genes and their corresponding proteins as well as adjustments of epithelial and ionocyte morphology (Foskett et al. 1981). The third stage takes up to a week and consists of changes in cell proliferation and differentiation, giving rise to distinct populations of ionocytes and altered leakiness of tight junctions. The extent to which each of these stages is activated depends on the magnitude and acuteness of the salinity change encountered by the fish (Kültz 2015).

Euryhaline fishes must be able to sense environmental salinity to induce qualitatively and quantitatively appropriate responses. This is achieved using osmosensors, which trigger signalling cascades that activate appropriate effector mechanisms (Figure 5.8). Fishes monitor external salinity using both direct and indirect osmosensory input from multiple proteins (Fiol and Kültz 2007). With indirect input, environmental salinity changes are recognized by monitoring corresponding changes in plasma osmolality. Osmotic imbalances in body fluids are detected at the level of individual cells, by the epithelial cells that face the external environment apically and the internal milieu basolaterally and by pituitary cells and neurons. The epithelial cells of osmoregulatory tissues also use apical, basolateral and intracellular proteins as direct molecular osmosensors. Plasma membrane tension, plasma membrane ion transporters and water channels, cell volume and cytoskeletal organization, protein and chromatin structure, and macromolecular crowding all may serve as osmosensors because they are directly impacted by changes in intracellular ionic strength (Kültz 2001). In some cases, activation of osmoregulatory effectors is very direct. For instance, the enzymes myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1.1 (IMPA1.1) combine osmosensory and effector functions in a single molecule. Abnormally high intracellular inorganic cation levels stimulate MIPS and IMPA1.1 activity and induce a feedback loop that replaces excess cations with the compatible organic osmolyte myo-inositol (Villarreal and Kültz 2015). However, this very short chain of events from sensory input to effector seems to be the exception, and most effector mechanisms depend on signalling networks.

FIGURE 5.8 Combinatorial input into osmosensory signalling networks of fishes. Multiple molecular osmosensors perceive osmotic stress in fish cells, including steroid hormone receptors (SHR), arachidonic acid-regulated Ca²⁺ selective channels (ARC), annexin A11 (ANXA11), arachidonic acid (AA), phospholipase A2 (PLA2), calcium sensing receptor (CaSR), adenylate cyclase (AC), aquaporins (AQP), transient receptor potential vanilloid 4 cation channel (TRPV4) and cytokine receptors (CR).
(Reproduced from Kültz, D., Physiology, 27, 259–275, 2012.)

Examples of the osmosensory proteins of euryhaline fishes are depicted in Figure 5.8 (Kültz 2012). Osmotic and ionic changes alter the conformation and activity of these proteins, in turn activating common intracellular signalling pathways. The combination and relative degree of activation of these signal transduction pathways forms a signalling network that adjusts osmoregulatory effector mechanisms to the extent needed. The activity of osmoregulatory effector proteins may be adjusted directly through posttranslational regulation. For example, both NKCC and CFTR are phosphorylated by focal adhesion kinase in euryhaline killifish (Fundulus heteroclitus) exposed to salinity stress (Marshall et al. 2009). Increasing mRNA and protein abundance is another common mechanism for regulating ion transporters and organic osmolyte-producing enzymes. For example, increased salinity induces transcription of MIPS and IMPA1.1 in tilapia and eels (Gardell et al. 2013; Kalujnaia et al. 2010). These enzymes produce the organic osmolyte myo-inositol, which compensates for disturbances of plasma osmolality, cell volume and intracellular ion homeostasis during a switch from FW to SW.

Recently, the cis-regulatory element (OSRE1) responsible for induction of MIPS and IMPA1.1 was identified (Wang and Kültz 2017). However, the transcription factor that binds to OSRE1 and links the osmosensory signalling network to transcriptional induction of effector genes is not yet known. Two candidates include nuclear factor of activated T cells 5 (NFAT5), which binds to a similar cis-element in osmoresponsive genes of mammals (Lee et al. 2011), and osmotic stress transcription factor 1 (OSTF1), which has been identified in euryhaline fishes (Fiol and Kültz 2005; Tse 2014). Linking osmoresponsive transcription factors to effector genes, on the one hand, and osmosensory signalling networks, on the other hand, remains a formidable task for the future.

5.5 Conclusion

Natural selection has optimized the macromolecular structures and function of cells to operate in an ionic milieu in which intracellular Na⁺ concentrations are low, K⁺ concentrations are high, and the concentrations of divalent ions (e.g. Ca²⁺, Mg²⁺) are in the micro- to low millimolar range. In multicellular animals, the cells are exposed to extracellular fluids that are isolated from the external milieu by an epithelial cell layer. Different strategies of achieving intra- and extracellular ionic and osmotic homeostasis have evolved to support cell function across a wide range of environmental ion concentrations. Fishes exhibit a spectrum of strategies ranging from iono- and osmoconforming to iono- and osmoregulating. The present chapter presented an overview of the ionic and osmotic strategies of the major fish groups, reviewed the structure and function of iono- and osmoregulatory tissues, and considered the challenges faced by fishes that encounter environmental salinity fluctuations in space and/or time.

Acknowledgements

Research of the authors was supported by NSF grant IOS-1656371 (DK) and NSERC DG RGPIN-2017-05487 (KMG).

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